S E V E N T H E D I T I O N

c4n, anthanarayan
and

^^
Textbook
miker ' s

Microbiology

Edited by
C K J Paniker

Copyrighted material
 Dedicated

to

..
Dr R Afunthanarayin
17.6/1913-&2.1998

ORIENT [ ( WOMAN PRIVATE LIMITED

Ofik
-- *
3 6 751 Hiniuysitnsftgir. Hyderabad 5CKH 029 IA . PK India
-
u - maiI: hjd2 oifl.nin.gCQ ^anclh arnet m

£M«r {Jffitrv
KangjiluA/Bhiifhkl/ BImhanehhwjr/irhenikaj/FfnaXalam/GuwAliaci/
I lytkriLbMJtV'ljiipv Kci'lik -aiLi/ LuLknLnvyM.umkhiii/Nc- w Delhi/Paina
^
0 Orient Lmmunn Private Limited I 9TH. MI, I9H
*. LWO. 1W6. 2DM . 1005
)

first pviblUhHl im
Reprinted 1979, lW]
SecDnd Edition 1941
Reprinrted 19®3, 1983, ] 9KJ, tOK 5
Third Edmgn 1WJ6
Fourth Edition 1990 7y/*r .urjt ii-
.
Reprinted IWl 1992 (iwke% 1994, 199S (iwkt) Ashwiiii Sjiteiu,
fifth Edition 19M Chennai 600 014
Reprinted 19%, 1997 (thrice) _
MlW MV /tfe/jOj #W
Reprint with levisjMi IOTH
SS Colour [impression Private Limited
Reprinted IOT9 Chennai 600 J 06
Sixth Edition 2000
Reprinted 2001 { tvdosk 2002 (twktX 2003 (twk©),. 2004 {ihriuv ! Pufr / tslred &y
Seventh Edition 2005 Uncnl Longman Private Ltd.
Reprint 2005 (fifth! £006p
160. Anna SaJ&i
Chennai 600 002
1SBIS 01i 290 2808 Q e - mail : ullmad @ nnl4. vinl.DeLm

Every eflfori Hmis been mads in ensure anoncy of maierial . hue the publisher, primer and editor wil] not he held responsible for
way hudvwlHl enura.

opy righted material

SEVENTH EDITION

C no nthana ra yan
anci
Moniker's
Textbook
of
Microbiology

This OUB

LFQT - E 11- E09J

CoDvriarited material
Copyrighted material
 SEVENTH E D I T I O N

C'^Dnanthanarayon
and m
-A
«
.
niker
]
,
s
Textbook
of
Microbiology

(Late) ft Ananthanarayon
.
SA , MBBS I Madrail . 06. PhD \ ionion )
Formerly . FWrfeiuar of Micrptwlogy .
Medmol Colleges, Cd»cut, Koftnyom . Triwondhuiii and Gulbarga
raid Adviser sig tbe DHS, Kerala. on Laboratory Service
*

CK Jayaram Poniker
MD
Formerly , PirfdQrFrEifiHor of Microbiology and Principal,
Medmdl College, Calicut, Kerala

This edition edited by

CK Jayaram Poniker

0
Orient Longman

Copyrighted material
Hainr
.
F
Trinity
r i|r
^ . . '.
lil rtuili i r 1 t JiliTiriWiimS flihJPH

Make you future-readw

English 15 tlie acirngwledged passport to
b e t t e r education and e m p l o y m e n t
opportunities worldwide, In such a scenario
the need for proficiency in tenin
and ^
skills in F.ng li s h
" JJ

h a & go ne up tre mendously

To help you in rhis, Urient Longman has joined

hands with Trinity College London to bring
I n t e g r a t e d Skills In English
examinations ( ISE ) to India . The EJE is
Many international institutions accept Trinity aligned with the Cornmor European
ISE for admissions. Some of them are the Framework ( CEF ) a n d i s a n
University of Brighton, Cornwall College, Internationally -' recognised standard of
Cranfietd University, the FaLmouth College of proficiency lithe English language.
Arts, the University of the West of England and
the University of Derby ,

Trinity makes available downloadable digi-

bnoks a n d syllabus i n f o r m a t i o n a t *
www.lrinitycoUege. co . uk

.
4
To orrrt yourself with cutting- edge certification in English,
( or for a full list of universities that accept Trinity ISE for admissions,
write to us at exams@orientbngmari. com

0
Orient Longman
3- 6- 752, Himayatnagar, Hyderabad 5 CH) 023
Tel; (040 ) 2766 5446 /7, 2761 0893. E mail: Ettiiros orierittong man .com
^
Copyrighted material
Preface to the Seventh Edition
Only fnuryrars have pawed since the sixth edition of TbriboakafMkmtoiakjgjim published , bur rapid devetopirienrs
LEI die subject have made a new edition necessary. During this short period, new infectious diseases have emerged

-
nr rc cmcrged in new forms. For example* Severe Acu.ce Respiratory Syndrome (SARS) virus appeared suddenly
causing death and panic in many countries, and the Bird Flu virus posed repeated pandemic threats.
,

Microbiology has become an increasingly important discipline, set to (face new challenges- Exciting advances in
diagnostic microbiology using sophisticated techniques can help in the rapid identification of new pathogens and
serve to contain them.This was shown by the idendfLcation. of the new SA RS vims within weeks by concerted multi ”
discipl i nary international effons. While such scienri fie progress is a boon., some of it can be potentially dangerous, as
lor instance in the recent case of chemical synthesis of a complete pathogenic poliovirus in the laboratory.

In rhU edition., [he seventh, relevant new information has been added and all chapters revised and updated,
,

maintaining the general format of the book. The Textbook nfMicmhiofogy has been in use now for more than a
q mirier of a century. It has bent fired greatly from the comments and suggestions from students, teachers and other
reader?.
Their help is gratefully acknowledged.

C . K. Jayaram Paniker
ShantUi * 1/SSM, Eui Ml Rcrad ,
Cjflkifl, Kcndu 63? OOfr.
'

Preface to the First Edition

Many of the health problems tn developing countries like Tndia arc different from those of developed countries.
Bacterid diseases still play a considerable role in diseases in our country. Topics such as cholera and enteric diseases
,

are important to us though only of less or academic interest to the developed countries. The Increasing importance
of the newer knowledge in immunology to health and disease is not adequately stressed in most of the extant
textbooks. Vi ms- disease* whic h are responsible for nearly 60 per cent of human illness require wider coverage. The
general approach_ to the tnLfun ofmkrobioloj - Inmraiuntryhas also been rather static. All these factors cjJlcd for
^ ^
a icxrhook of mu:mbi.o]ogy nrirore ir d cci L^uuinriv like lodil.
^^ ^
We therefore undertook th is endeavour based on mar experience of reaching undergraduates and postgraduates for
over two decades. We omitted the discipline of paroritolagy from our book since we already have an excellent
,

textbook on the subject published in India.

Th is book has taken us over three years co wrire and over a year in publication . N aturally we would be out of date to
a certain and inevitable extent. We do not claim any perfection. On the contrary, we have reqtbCSted medical
students and teachers all over the country to write tn us about any shortcomings and give us suggestions as to how
to improve- the hook. We shall spare no pains in seeing that their valuable suggestions are given effect to in our
-
second edition.

R. Aiwthanarayan
C. K. JayaLram Paniker

Copyrighted material
 Acknowledgements
For kind perm i 5- FLon to UBC iilusf rations , supply of photographs, help lid suggestions and advice, the author is
indebted to the following civilisations and persons:
Wirld Health OipniiilMm; Indian Council of Medical Research; National Institute of Viralogy; Pune; Dr. G.
Baikrith Nsur, Du A.N. Ghosh, Dr. TN. Nsik and Dr Triverti Krishnan of National Institute of Cholera and
Enteric diseases, Calcutta, Prof. M. Maihui and Piol. T. Jacob John i >t Christian Medical College , Wllore, Prof ,
Anin Ch i tale,JailoJt Hospital Mumbai; Dr SJM.Sirsat, Canter Research 1 nstitute, Mumbai; Prof M.D, Mathur,
Maulana Azad Medical College, New Delhi, Ptof. J. Shanmugham, SCT Medical Centre, Trivandrum;
Dr. Girija G - Rao, Medical College, CaJicur; Dr. G .M. Warfce, TIiMedia Laboratories, Mumbai.

u opy
y righted materia
 Contents

Purl I
1 lisitorical Introduction 1
2 . Morphology and Physiology of Bacteria 7
3 St ^iril isariun and 13is.infectii0iH 24
nlnire Media
"
4 (' 34
Culture Methods 39
6 Identification of Batttria 44
7, Racrenial Taxonomy 48
fi , Bacterial GcilCtifl 51

[]
9, Infection 64
10. Immunity 71
11 , Antigens SO
12. Antibodies-ImmunoglobuJ ins 94
13 , Antigen-Antibody Reactions 92
14. Complement System 110
15 , Structure and Functions of the Immune System 117
16, Immune Response 133
17. Immunodeficiency Diseases 152
IS. Hypersensitivity 159
19. Autoimmunity 169
20. Immunology of Transplantation and Malignancy 176
21 , Immunohematology 184

I'ilTl HI
22 , Staphylococcus 192
23 . Streptococcus 202
24. Pneumococcus 216
2L_ Neisseria 222

opyrigmea nr na
26. Corynebacterium 231
27 Bacillus 241
?R -
O rEftfri Hli LI rtii 24fi
29. Ncnsporing Anaerobes 266
in RntnmliafferiaiiMr TJ Cotifrtfms-Prrtteiis 271
31 . Enterobacteriaceae 11: Shigella 285
32. Enterobacteriaceae III: Salmonella 290
31 . Vihfift ins
34
35 .
^
Pwn nmnnas
Yersinia. Pasteurelk. Frandsella 324
36 . Haemophilus 333
37 Hoidetella n <j
38 Brucella
39. Mycobacterium 1: Tuberculosis 351
40. Mycobacterium II: Atypical Mycobacteria 36S
41 . Mycobacterium Ill: M, Leprae 370
42 . Spirochetes 377
43 . Mycoplasma 395
44, Actmomyoetes 400
45. Miscellaneous Bacteria 403
46, Ricketirdaceae 412
47 , Chkmydiae 422

Part IV
4S, General Properties of Viruses 430
49
50.
—
VirtiS HoAt I nTeraLtinns : Viral Tnfectinns
Bacteriophage
444
461
51 . Poxviruses 460
52. Herpesviruses 474
53. Adenoviruses 4fl £
54. Picomaviruses 490
55 . Orthomyxovirus 501
56 , Ikramyxoviruses 512
57. Arboviruses 521
5$, Rhabdovi ruses 535

.1 , i
J nanie ate nai
 59, Hepatitis Viruses 547
fin Miscellaneous Viruses 563
61, Oncogenic Viruses 574
62, Human Immunodeficiency Virus: AIDS 582

T V
^63
rt
, Norma] Microbial Flora of the Human Body 599
64, Bacteriology of Water, Milk and Air 603
65, Medical Mycology 610
66 . Laboratory Control of Antimicrobial Therapy 628
67. Immunoprophylaxis 631
68, Hospital Infection 634
1 ndex 639

Copyrighted material
Copyrighted material
 Historical Introduction
McdLuad microbiology is the study cst microbes that
infect humans , the diseases they cause , Their
, Leeuwenhoek llie world of liJtlc'
diagrams, premonition smd treatment , It also deals
with the response of the human hosr ro microbial
and other antigens.
Disease and death have always held the
act ^ ndoii of the human mind. Ancient humans
ascribed them to divine wrath and other supcm&tuial
fuit-es. Later, other concepts such as the effect of
'
the environment, of bodily constitution and of faulty
diet were proposed. There have been, from very
early ti mes, occasional suggestions that diseases may
result from invasion of the body by external
contagion. Vsno and Columella in the first century
ftC postulated that diseases were caused by invisible
beings { Animalia minuta ] , inhaled nr ingested -,
Fracastoirius of Verona ( 1546 ) proposed a
corttagium vivum as a possible cause of infectious
disease and von Pterion ( 1762} suggested that each
disease was caused by a separate agent. Kireher
( 1659 ) reported finding minute worms in the blood
of plague victim s, but wi th the equipment available
to him it is more likely that what he observed were
on ]v hlofx) cells,
As miernhes are Invisible to rhe unaided eye ,
defiodvc knowledge; about them tW to await the
development of microscopes, llie credit for having
first observed and reported bacrem belongs to
An tor v van Leeuwenhoek, a draper in Delft r
Holland, whose hobby was grinding lenses and
observing diverse materials through them. In 1 b83
he made accurate descriptions of various types of
bacteria and communicated them to the Royal
Society' of London . The significance nl these
observations was not realised then * n d t(J
ajlnBaktJfS ' fli
he called Them , represented only a curiosity of
nature . ][ was only some [ wo centuries 1 a[er that
f
their importance in medicine and biology as a whole
came to be recognised .
The curliest discovery of a pathogenic
microorganism was probably made by Augnstino
hassi ( 1S. 5 ) . who showed that the muscardinc
^
disease of silkworms was caused by a fungus.
Davuine and PollenJer [1850) observed anthrax
bacilli in the Mood of animals dying of the disease.
In fact , even before the microbial etiology of
infections had been established , Oliver Wendell
Holmes in the OSA ( 184.1) and Iguaz Sem muhveis
in Vienna (1846) bad independently concluded that
puerperal sepsis was contagious. Semmelweis also
identified its mode of transmission by' doctors and
medical students attending on women in labour in
tilt hospital and had prevented it by the simple
measure of washing hands in un antiseptic solution ,
for which service to medicine and huminin', he
was persecuted by medical orthodoxy and driven
insane .
The development ofniiicmhjoktgy as a scientific
discipline dates from Louis Pasteur ( 1822- 5 ). ^
Though trained as a chemist , bis studies on
fermentation led him ro take an interest in
inkfOoganisms . He eytablished that fermentation
was the result of microbial activity and that different
types of fermentations were associated with different
types of microogan i sms ( 1857 ) . The bask pri nciples
and techniques of microbiology were evolved by
Pasteur during his enquiry into the origin ot
Copyrighted materi r_ l
2 4 Texlbcc * al Microbiology *

microbes. This was then the subject of much
controversy. Needham, an Irish priest , had m 1745
published experiments purporting the spontaneous
generation (abiogeneri -s ) of microorganisms in
putrescible fluids . This view was opposed by
Spallanzani, an Italian abbot (1769). In a series of
classic experiments, Pasteur proved conclusively that
all forms of life , even microbes, arose only from
their like and not tie novo. In the course of these
studies, he introduced techniques of steriliRation
and developed the steam steriliser, hot air oven and
autoclave. He also established the differ mg growth
needs of different bacteria. E lis work attracted such
attention and he attained such eminence In the
world of science that not only France hut all Europe
looked to him to solve mo jot problems in various
fields. Thus starred Ins studies on pebrine, anthrax ,
chicken cholera and hydrophobia. An accidental
observation that chicken cholera bacillus culture*
left on the bench for several weeks lost rheir
pathogenic property but returned their ability to
protect the birds against subsequent infection by
Contributions and simitar institutions were
established soon in manv other countries for the
preparation -of vaccines and lor tlue investigation of
infectious diseases.
An immediate apphcacLon ofRasteur’s work was
the introduction of ant isepliC techniques in surgery
by I . if ter (1867) effecting a pronounced drop in
mortality and morbidiry due tu surgical sepsis
Lister's antiseptic surgery involving the use of
carbolic add was cumbersome and hazardous but
was a milestone in the evolution nfsurgicalpradke
from the era of 'laudable pus’ to modern aseptic
techniques-.
While Pasteur in France laid the foundations
-
of microbiology, Robert Koch (1843 1910 ) in
Germany perfected bacteriological techniques
during his studies on the culture and life cycle of
the anthrax bacillus (1876 ). He introduced staining
techniques .mel method * of obtaining bacteria ill
pure culture u -lng solid media. He discovered the
bacillus of tuberculosis (1882 ) and the cholera
vibrio (18S3 ) r

them , led to the discovery of the process of
attenuation and the development of Live vaccines.
He attenuated cultures of the anthrax bacillus bv
incubation at high temperature ( 42-43 °C ) and
proved that inoculation oJ such cultures in animals
induced specific protection against anthrax .. [’ he
success of such immunisation was dramatically
demonstrated by a public experiment on a farm at
Pbuilty- le - Fort ( 18 B1 ) during which vaccinated
sheep, goats and cows were challenged wirh a
virulent anthrax bacillus culture. All the vaccinated
a nil u ills survived the challenge , while an L- qu.d
Pasteur and Koch attracted many gi tted dm i pics
who discovered rhe causative agents of several
r
bacterial infections and enlarged the scope and
content of microbiology by their labours- In 1974
Hansen described the leprosy bacillus; in 1879
Neisaer described the gonococcus; in 1881 Og ton
discovered the staphylococcus; in 1384 Loefflcr ^
isolated the diphtheria bacillus; in 1884 Nicolaicr
observed the tetanus bacillus in soil ; in 1886
Fracnkcl described the pneumococcus; in 1887
11 nice identified the causatu'e agent of Malta fever,
in 1905 Schaudmn and Hoffmann discovered the

nu mber of unvacc i nated control ani mils succumbed

to it . it was Pasteur who coined the term valine
for soich prophylactic preparations to commemorate
the first of such preparations , namely cowpox,
employed by Jenner for protect i on against smallpox.
The greatest impact in medicine was made by
Pasteur's development of a vaccine for hydrophobia.
This was acclaimed throughout the world. The
l flute nr Institute , Pans wan built by public
-
spirochete of yphilis.
Roux and Versin (1883) identified a new
mechanism of pathogenesis when they discovered
the diphtheria toxin. Similar toxins were Identified
in tetanus and some other bacteria. The toxins were
found to he specifically neutralised b > their
antitoxins, Ehrlich whostudied toxins and antitoxins
m quantitative terms laid the foundations of
biological standardisation .

pyrignte<
—-
i. P ria
iLI 1
 * HiglOlkfil IrttrotiUCtinn
The causative agents of various infectious
diseases were being reported by different
iiivc ^ ri tutors nsuch prolusion that i t was necessity
'
.
to introduce criteria for proving the claims that a
niLcifi ( iri ;,mh: ii iliolited from a disease was indeed
causally related to if . These criteria, first militated
by Hctilc, were enunciated by Koch and ate known
as Koch 's postulates . According to these , a
microorganism can be accepted as the causative
agenr of an infectious disease only if the follow ing
conditions arc satisfied:
1 . The bacterium should be constantly associated
with the lesions of the disease .
1 . It should be possible tu isolate the battenLiin in
pure culture from the lesions.
3 . Inoculation of such poire culture into suitable
laboratory animals should reproduce the lesions
ot the disease .
4 . It should be possible to nsisolite the bacterium
in pure culture from the lesions produced in
the experimental animals.
An additional criterion introduced subsequtndy
requires that specific; antibodies to the hacteri . jjn
should be demonstrable in the scrum of patients
suffering from the disease . Though it may not
always he possible to satisfy all the postulates in
every ease, they have proved extremely useful in
sifting doubtful claims made regarding the causative
agents of infectious dj - eases.
By the beginning of the twentieth century, many
infectious diseases had been proved to be caused
by bacteria . But there remained a large number of
diseases such as smallpox , chickenpOJt , measles,
ridiirm and the common cold for which no
ci . utcri . il cause could he established. During his
l Oves Ligation of rabies in dog , Pasteur had suspected
^
that the disease could be caused hv a miemhe too
small to be seen even under the microscope . The
existence of such ultramicroscopic microbes was
proved when Ivanovslqr ( L 892 l TV produced mosaic
disease in the tobacco plant, by applying to bealtby
leaves juice from the diseased plants from which
all bacteria had been removed by passage through
3
fine filters . Beijerinck ( 1898 ) confirmed these
findings and coined the term virus for such filterable
infectious agents . Locfiler and Frosch ( 1898)
observed rhat the foot and mouth disease of cattle
wan caused by a similar filter - passing virus . The
first human disease proved to have a viral etiology
was yellow fever, T*hc US Army Commission under
V a
Walter Reed , invesrigating yellow fever in Cuba
i. 19 Q 2 ) established not only that it was caused by a
filterable virus but also that ir was transmitted
through rhe bhe of i i lfocted mosquitoes. 1 -andstc 11 rtt
and Popper (1909 ) showed that poliomyelitis was
caused by a filterable virus and transmitted the
disease experimentally to monkeys. Investigation of
viruses and the diseases caused by them wasr
rendered difficult as viruses could not be visualised
under the light microscope or grown in culture
media . Though the larger viruses enutd be seen
after appropriate staining under the light
m icroscope, det .i i led study of their morphology had
to wait till the introduction of the electron
microscope by Ruska ( 1934) and subsequent
refinements in electron microscopic techniques.
Cultivation of viruses was possible only in animals
or in human volunteers [ ill the technique of
growing them on chick embryos was developed by
Goodpasture in the 1930s. The application of tissue
culture in virology expanded the scope of virological
techniques considerably.
The possibility' that virus infection cnuld lead
to malignancy was first put forth by KHerman and
Bang ( 1908 ), Peyton Rous ( 1911 ) isolated a virus
causing sarcoma in fowls. Several viruses have since
been isolated which cause natural and experimental
tumours in animals and birds . Viruses also cause
ma!ignant transformation uf infected cells in tissue
culture . The discovery of viral and c e l l u l a r
oncogenes has shed light or the possible
mechanisms of viral oncogenesis . After many
decades of futile search , positive proof of a virus
-^
i ; j'. j . i :i human malignancy wafi established when
the virus of human I - ceil leukemia was isolated in
1980.
W opy righted material
4 * Tcodbook
Twt » rt ( ISIS ) and d ' Hercllc ( 1917 )
independently dermic red a lyric phenomenon in
bacterial cultures. The , Lt;enL > responsible were
termed bacteriophages — viruses that attack bacleri . L.
Early hopes that bacteriophages may have
therapeutic appl :icafions had to be abandoned but
these viruses have paid unexpected scientific
dividends. The essentia ] part of viruses is rheir core
of nucleic acid which acts as the Cartier of genetic
:. nformation in the same manner as in Higher
organisms. The discipline of molecular biology owes
its origin largely rn studies or the genetics of
bacteriophage;* and bacteria .
It had hern noticed from very early days [hat
persons surviving an attack of smallpox did not
develop the disease when exposed to the infix non
subsequently. This observation had been applied
for the prevention of the disease by producing a
mild form of smallpox intentionally (variolation ).
This practice , prevalent in India , China and other
, of phagocytosis and proposed the phagocytic
am lent civilisations from time immemorial , was
introduced in England by Lady Mary Worthy
Montague ( 1718) who had observed the custom
in Turkey. Variolation was effective but hazardous,
lenner , observing the immunity to smallpox in
milkmaids who were exposed to occupational
cowpox infection , introduced the technique of
vaccination using cuwpux material ( 1796 ) , This was
the first ' ustance of scientific immunisation and,
though introduced empirically, has stood the test
of time limners vaccination paved the way for the
, oppo'- Lte effect. Kjoch ( lE90) had noti - ned tbit when
ultimate eradication tif smallpix .
The next m;iinr discovery in immunity was
1
Pasteur's development of vaccines for chicken
cholera, anthrax and rabies. While the techniques
introduced by him were successful , the mechanism
of protection afforded by them remained obscure .
The cxplanat I on of the underlying mechanism came
from two sources. Nuttall ( 13SS ) observed that
defibrinated blood had a bactericidal effect , and
Buchner ( 1889) noticed that this effect was
abolished by beating the sera for one hour at 55 °C.
The heat labile bactericidal factor was termed
Mjcmbiatogy
'aicxitw*.A spe ific humoral (actor or 'antibody*
was described by voci Behring and Kitasato (1890 )
in the serum of animals which had received
sublethal doses of tetanus torin. Pfeiffer ( 1893)
demonstrated bactericidal effect in vivo by injecting
live cholera vibrios irtraperitoneally in guinea pigs
previously Injected with killed vibtios. The vibrios
were shown to undergo lysis. The humoral nature
of such lytic activity was proved by Bordet ( 1895 },
who defined the two components participating in
the reaction, the first being heat stable and found
in immune sera (antibody or substance
scnsrfijitca trice) and the second being heat labile
aod identical with Buchner 's aJudne , subsequently
named 'cnmplancfi t \ Soon a number nf other ways
were demonstrated in which antibodies react with
an11 JI ILS , such as agglutination , precipitation ,
"
complement fixation and neutralisation ,
MctchmkolT ( 1883) discovered the phenomenon
response as the prime defence against the microbi . il
invasion of tissues. This led to the cellular concept
of immunity. Polemics regarding the significance
of the cellular and humoral mechanisms of
i mmunity were largely put to rest with the discovery
by Wright ( 1903) of optimisation , in which
antibodie* and phagocytic cells act :.n conjunction.
Pi i.or expeiience with a microorganism or other
notion d: d not always result in the beneficial effect
ot immunity or protection. At times it: caused the
the tuber . ILL bacillus or its protein was injected into
'
a guinea ;rig already infected with the bacillus, an
exaggerated response took place - a hypcisensidvity
reaction known as Koch’s phenomenon fbrtirt ,
and Richet ( 19U2) , studying the effect of the luric
extracts of sea anemones in dogs made the
paradoxical observation that dogs which had prior
contact with the toxin were abnormally sensitive
to even minute quantities of it subsequently. This
phenomenon was termed anaphylaxi* . Later, many
similar reactions were observed, both
experimentally and in nature, of injury, disease or
Copyrighted materi3
 * historical Introduction
even death resuming from repeated contacts wirh
antigens . 11 M. importance of this phenomenon, in
the pathogenesis of many human diseases, led to
the development of the discipline of allergy.
The characteristic feature of immunity, whether
if is protective or destructive (as in allergy), is its
specificity. As the mediators of humoral immunity
(an til Jodies) are globulins, the explanation for the
exquisite spedGdty ol the immunological reaction
had to await the advances iin protein chemistry, The
pinneeri ng work of I ,andsteincr laid the foundations
of immunochen ilstry Chen i ists dom 11 rated the study
of immunity for several decades , and theories of
anl. i body synthesis were postulated by them , will i ch
sometimes ran counter to biological laws. In 1955,
ILTUC proposed the natural selection theory of
antibody synthesis which attempted to explain the
chcmit al specificity and HologjraJ basis of antibody
synthesis, signifying i return ro the original views
of antilxxdy formation proposed bv Ehrlich (1 S9E ).
Burnct ( 1957 ) modified thi ? into the clonal
selection 1 he ore, a concept which, with minor
alterations, holds sway even now. The last few
decades have witnessed an explosion of conceptual
and technical advances in immunology .
Immunological processes; in health and disease are
now better understood follow!ng the identification
of the two components of irnmuni fy - the humoral
or andbody mediated processes and the cellular or
manilest in separate
—
cell medi . Lted processes which develop and are
pathways.
Til ] recently, a teleological view of immunity
prevailed. It was considered a protective mechanism
designed ro defend the body against invasion by
microorganisms . Based on the original suggestion
f Thomas- ( 1959) , Burnet ( 1967 ) developed the
conceptof immvnaingiczl survriifancc , according
to which the primary function of the immune
system is to preserve the integrity of the body,
seeking and destroying all ‘foreign’ amigenS ,
whether autogenous or external in origin .
Malignancy was visualised as a failure of this
function, and the scope of immunity was enlarged
* 5
to include natural defence against cancer Another
aspect of tti i s role of i mnrauniTy is in the reiec c i nn of
homografts. Understanding of the immunological
basis of transplantation, largely due to the work of
Medawar and Burnet , made successful transplants
possible by elective immunosuppression and proper
selection of donors based on hbtooomparibility. The
history of transplantation thus runs parallel to the
history of blood transfusion , which was
unsuccessful and even fatal before the discovery of
blood groups by Landsteiner ( 1900).
In the early twertliertth century, attempts were
made to exploit the immunological information
available by the development of vaccines and sera
for the prophylaxis and treatment of infectious
diseases. Till Domagk ( 1935 ) initiated scientific
chemotherapy with the discovery of prontosil ,
antisera were the only specific therapeutic agents
available for the management of infectious diseases,
Fleming (1929) made the accidental discovery that
the fungus Fcnicilliurn produces a substance wludi
destroys Etaphylococvi. Work on this at Oxford bv
Florey, Chain and their team during the Second World
War led to the isolation of the active substance
penicillin and its subsequent mass production. This
was the begi nni ng of the antihiotk: era - Other sii n ilar
antibiotics were discovered in rapid mcecssion. With
the sudden availability of a wide range of antibiotics
with potent antibacterial activity, it was hoped that
bactcn.i] iidecbonii wmild he controlled within . i ,- hort
per,ixl. But soon the development oJ drug resistance
in bacteria presented serkius difficulties.
With the development of a wide variety of
antibiotics active against the whole spectrum of
path* ’jgenic bacteria, and of effective vaccines against
most viral ibseases, expectations were rinsed about
the eventual elimination of alt infectious, d Meases.
The global eradication of smallpox inspired visions
of similar campaigns against other major pestilences.
However, when new infectious diseases began TO
appear , caused bv hitherto unknown micro'
organisms, or bv known microbes producing novel
manifestations, it was realised that controlling
opyrighted materia
i
6 r Te ^ lbcck o1 Micros

microbes was a far more difficult task than was 1901 Emil A Von Behnng
imagined . The dimax came in 1901 when AIDS 1902 Ronald Ross
was identified in the USA and began its pandemic 1905 Robert Koch
spread. Unceasing vi ii appear? essential to protect 1907 CIA Lavcran
^
humans from microbes .
1900 Paul Ehrlich and Elie Metchnikoff
1913 Charles Richet
Apart from the obvious benefits such as specific
methods of diagnosis, prevention and control of
infectious diseases, medical microbiology has
^
1919 ulcs Bordet
1926 Johannes Fibiger
1920 Charles Nicoflc
contributed to scientific knowledge and human 1930 KaH Landsteiner
1939 Gerhardt Domagk
welfare in many other ways . Microorganisms 1945 Alexander Fleming, Howard Finney and EB
constitute the smallest forms of lining beings and, Chain
therefore, have been employed as models of studies 1951 Max Theiler
dl gentries and biochemistry. As nature's laws are 1952 SeLman A Waksman
universal in application, information derived from 1954 JF Endens, FC Robbins and TH Weller
the investigation of m icrobcs holds true, in the main, 1958 GW Beadle,Joshua Lederbergand EL Tatum
1960 MadarJane Burnet and Peter Brian Medawar
for humans as well. 1965 Francois Jacob, Andre Lwoff and Jacques
Studies on microorganisms have contributed, Monod
more than anything else , to unravelling the genetic 1966 Peyton Rous
code and other mysteries of biology at the molecular 1969 Max Delbruck, AD Hershey and Salvador
level. .Bacteria and their plasmids , yeasts and viruses Luna
are mutinely employed M vectors in recombinant
1972 Gerald Edelman and Rodney Porter
1975 David Baltimore, Renato Dulbeoeo and
DNA technology. They have made available Howard M Tcmin
precious information and powerful techniques for 1976 S Baruch , Blumbcrg and Carleton
genetic manipulation and molecular engineering. Gajdusek
They need to be used wisely and well for the benefit 1978 W Arber, D Nathans and HO Smith
of all living beings. I 960 Banjj Benacmaf, Jean Dausset and George
The number of Nohel laureates, in Medicine
Snell
1984 Niels Jcrne, Cesar Milstein and Georges
and Physiology awarded the prize for their work Kohler
in microbiology, listed below, is evidence o f the 1987 Susurnu Toregawa
positive contribution made to human health by the 1989 J . Michael Bishop and E Vaimus
science of microbiology. 1996 Paul Doherty and Rolf Zinkemagel
1997 Stardev PrusiTier

Further Reading
Btuaotmf B et al . I 960. A History oFBactscr.- niogy and Jmmunotogy, London; William Heinemann .
Clark PF 1 %1 . PTuueer JVfjtnjfrwk r rs of Amelia. Madison: University of Wisconsin Pftti.
'

^ ^
Collard P 1976. The Development of Mictoi' iohgy, Cambridge University Press .
deKri - f P 1958 . Microbe !iuntets. London: Hutchison.
'

fo' ii ' T WI ) 1970. A History of Medial Bacteriology and Immunology, London; Cox and Wyman .
Lethfivaller HA unci M Solotorovsky 1974 , Three Centuries of Microbiology'. frovtr Publications.
Marquardt M 1951. Aiuf Ehrlich N*w York; bchuman ,
[hripb HJ i 960. Victory with Van vies - The Story of Immunisation , Loudon: Livingstone .
Vail cry Radot R 1948. The I .: fe ofPaattut . i r u = : . by ftL Devonshire. London: Constahbe.
1

Wiicrsnn AP . mil L Wilkinson 1978. An Introducoon to the History of Virology. London: Cambridge University Preas.
Williams G i 960. FJrus Hcijinfrtf . London: Hutchison .

opyrigniec maiterial
 Morphology and Physiology
of Bacteria

Microarguuims in J. heterogeneous group of SIZE OFBACTBKIA

several distinct classes of living beings. They were 'Hie unit of measurement used in bacteriology is
originally classified under the plant and animal the micron ( micrometre, pm )
kingdoms. As this proved unsatisfactory, rhcyr were
classified under a third kingdom , thejnotHti. Based
on differences in cellular organisation and
1 micron ( p ) or micrometre ( pm )
thousandth of a mill ] metre.
one -
hiocheTnistry, the kingdom protista has been divided 1 millimicron ( m|i ) or nanometre (nm ) = one
thousandth of a micron or otic millionth of a
into two groups prokaryotes and eukaryotes.
Bacteria and blue green algae arc prokaryotes, millimetre .
while fungi, Other al ae , ‘lime moulds and 1 Angstrom unit ( A ) tenth of a nartume tre .
protozoa are eukaryotes . ^ One

The limit of resolution with the unaided eye is

Bacteria are prokaryotic microorganisms that about 200 microns . Bacteria, being much
smaller,
do out contain chlorophyll . They are unicellular can he visualised only under magnification.
Bacteria
and do not show true branching, except in the so-
called higher bacteria ' ( ActinomyccraJes).
of medical importance generally measure 0.2 1,5
pm in diameter and about 3-5 im in length .
—
^
Table 2.1 Some differences between prokaryotic and eukaryotic cells
Character Prokaryotes EtiiaiyofeG
Nucleus
Nuclear membrane Absent Present
Nucleolus Absent Present
Deoxyt i bonfucItopro^e a TI Abie n r Present
Chromosome One (eitcnlar ) More rhm one ( linear)
MirorJc division Absent Present
Cytoplasm
Cytoplasm ic streaming Absent Preient
Pinncytosis Absent Prcsenr
Mitochondria Absent Present
Lysosomes Absent Present
Golgi apparatus Absent Present
Endoplasmic reticulum Absent Present
Chemical composition
Sterols Absent Present
Muramic acid Present Absent

Copyrighted material
 S * lEfK:bM (t (tf M < r.' Qb \Q
MICROSCOPE
The mcjrpht ]]( JL;itnl study of bacteria requires the
use of microscopes. Microscopy lus come a long
wav since Leeuwenhoek fiTsr observed bacteria over
three hundred years ago using hand - ground lcnscSr
The following types of microscopes ire employed
now,
Optic III or 11. ;Kt ihiontUDpC: Bacteria may
he examined under the compound microscope,
either in the Living state or after fixation and staining.
Examination of wet films or ' hanging drops
indicates the shape, arrangement, motility and
approximate -iic of the cells. But due to lack of
contrast , dcra: H cannot be appreciated.
Phase contrast microscopy : improves the
contrast and makes evident the structures within
cells that differ in thLckness or refracri vv i ndex. Also,
the differences in refractive index between bacterial
cells and the surrounding medium make them
clearly visible . Retardation, hy a fraction of a
wavclengrb, of rhe rays of light tint pass through
the obiecr, compared to the ravs pacing through
the Surrounding medium, produces 'phase
differences between the two types of rays. In the
phase contrast microscope, 'phase differences are
1
converted : nto differences in intensity of light,
producing Light and dark contrast in the image.
Dark field t Dark ground microscope:
Another method of improving the contrast is the
dirk field ( dark ground ) microscope in which
reflected light is used instead of the transm itted
light used in the ordinary microscope. The essential
parr of the dark field microscope is the dark held
condenser with a central circular stop , which
illuminates the object with a cone oflight, without
letung my ray of Light to fall directly on the
objective lens. .1 .l.nht rays falling on the object are
reflected or scattered on to the objective lens, with
the result that the object appears self- Tumi -lOUS
against a dark background, The contrast gives an
illusion o: increased resold ion, so- that verv slender
organisms, such as spirochetes, nor visible under
ogy
ordinary illumination , can be clearly seen under the
dark field microscope.
Thc resolving power of the Light microscope is
limited by the wavelength of light- Tn order to be
seen and delineated (resolved), an objiXt has to hive
a size of approximately half the wavelength of the
light used. With edible light, using the best optical
systems, the limit of resolution is about J500 nm . Tf
light of shorter wavelength is employed, as in the
ultraviolet microscope, the resolving power can be
1
proportionately extended,
r\vO specialised types of microscopes arc 1) the
interference microscope winch not only reveals cell
organelles bur also enables quantitative
measurements of the chemical consritucnts of cells
such as lipids, proteins and nucleic acids, and 2 }
the polarisation microscope which enables the Study
of intracellular structures using differences in
birefringence .
Electron mioroaoope: Ln the electron
mcroscope, a beam of electrons is employed instead
of the beam oflight used in the opritaJ microscope.
'
The electron beam is focused hy circular
electromagnets, which arc analogous to the lenses
in the Light microscope. The object which is held
i n the path of the beam scatters the electrons and
produces an image which is focused on a fluorescent
viewing screen. As the wavelength of electrons used
is approximately 0,005 nm, as compared to 500 nm
with visible light, rhe resolving power of the
electron microscope should be theoretically
*
times that oflight microscopes but in
practice the resolving power is about 0.1 nm.
I he technique of shadow - casting with vapori ^cd
heavy metals has made possible pictures with good
contrast and three - dimensional effect. Another
valuable technique in studying fine structure is
negative staining with phosphotuilgstic acid.
Gas molecules scatter electrons , and it LS
therefore necessary to examine the object in a
vacuum. Hence, only dead and dried objects can be
examined in the electron microscope. This may lead
Copyrighted materia
 « Morphology and Physiology of Bacteria *
to considerable distortion in cell morphology, A
method introduced to overcome this disadvantage
.
is freeze- etching involving the deep- freezing of
specimens in a liquid gas and the subsequent
formation of carbon-platinum replicas of the
material. Since such fnJ7ei«l!s may remain viable ,
it is claimed that (reeze-erehing enables the study
of cellular ultrahtructure as it appears in the living
state. The recent development of very high voltage
electron microscopes may render possible the
eventual examination of live objects. The scanning
electron microscope is a useful in novation which
permits the study of cell surfaces with greater
contrast and higher resolution linn with the
shadow -easting technique .
S I AIMKII PHKFAK
1 AVC bactc ri a do not show much strue tu ral dctiiI
under the light microscope due to lack ol contract.
Hence ir is customary to use staining Techniques to
.
produce colour contrast Bacteria may be stained
in the living state, hut this type of staining is
employed only for special purposes Routine . resist decolonisation and retain the primary ttain,
methods fer tfainiDg of bacteria involve drying and
fixing smears, procedures that kill them. Bacteria
have an affinity for basic dyes due to the acidic
nature of [ heir protoplasm. The following are
staining techniques commonly used in
bacteriology.
Niimph; ‘t uns: Dyes such as methylene blue or
basic TLICLLSIRI are used for simple staining. '1 hey
provide colour contrast , but impart the same colour
tn bacteria.
all
Negative aldirling: Here, bacteria are mixed
wich dyes such as Indian ink or nigrosin that provide
a uniformly coloured background against which the
unstained bacteria stand out in contrast. This is
particularly useful in the demonstration of bacterial
capsules which do not take simple stairs . Very
slender bacteria such as spirochetes that arc nor
demonstrable by simple staining methods can be
viewed by negative staining.
impTugimlinn methods; Cells and structure ?
9
too rhin to he seen under the ordinary microscope
may he rendered visible if they are thickened by
.
iinprcgtiatieinofsilvcron the surface Such methods
are used for the demonstration of spirochetes and
bacterial flagella.
Differential atainst These stains impart
different colours to different bacteria or bacterial
structure'-. The two most widely used differential
stairs arc the Gram stain and the acid fast stain.
The Gram slain was originally devised by the
histologist Christian Gram (1&84) as a method of
staining bacteria in tissues. The staining technique
consists of JIJUT steps:
1 . primary staining with a pararosanilinC dye such
as crystal violet, methyl violet or gentian violet;
2 . application of a dilute solution of iodine ;
.v decolouriisariuii with an organic solvent such as
ethanol , acetone or aniline;
J. counters!aining with a dvr * ft contrasting Colour,
such as carbo ] tuebsin , sutranine or neutral red.
The Gram stain differentiates bacteria into two
broad groups. Gram positive bacteria arc those that
appearing violet . Gram negative bacteria are
decolourised by Organic sohcntl and, therefore, take
the counremain, appearing ted, The exact
mechanism ofthe Gram reaction is not understood.
The G rjrn positive cells have J more acidic
protoplasm, which may account for their retaining
the basic primary dye more strongly than the Grain
negative bacteria. DeeolourisaiLon is not an all-or -
ioftC phenomenon. Even Gram fjositive cells may
*
he decolourised bv prolonged treatment with the
organic solvent . Conversely, inadequate
dccolouiisation may cause all «11 H to appear Gram
positive,I’hc Gram reaction may be related to the
permeability of the bacterial ceil wall and
cytoplasmic membrane to the dye - iodine complex,
the Gram negative, but not the Gram positive cells,
permitting the outflow of the complex during
dccolorisstion. The Gram positive bacteria become
Gram negative when the cell wall is damaged.
Gram staining is an essential procedure used
Copyrighted material
10 * Textbook of Microbiology *
in the identification nf bacteria and frequently is
the only method required for studying cheit
morphology. Gram reactivity is of considerable
importance as the Gram positive and negative
bacteria differ rn > t mertlv in staining thanuteristhin
and in structure but also in several other properties
HILL IL as growth requirement ;; , susceptibility to
'
antibiotics and pathogenicily
The iCtd fast stain was discovered bv EhrticlL,
whra found that after staining with aniline dyieSs
tubercle bacilli resist decolouri -sarion with acids.
The method, a & modified by Ziehl and Neelsen , is
in common use now. The smear is grained bv a
,
j
strong solution of tarhol fuchsin with the
application of heat . It is then decolourised with
20 per cent sulphuric arid and enunterstained with
a contrasting dye such as methylene blue. " Hie acid
fast bacteria retain the fiachsin ( red ) colour , while
the others take the countenstain . Acid fastness has
heen ascribed to the high content and variety of
lipids , fatty acids and higher alcohols found in
tubercle bacilli. A lipid peculiar to acid fast bacilli,
i high molecular weight hydroxy acid wan
containing carboxyl groups ( mycolic acid ) is acid
fast in the free state . Acid fastness is not a property
of lipids alone but depends also on the integritv of
the Cell wall.
SHAPE OF BACTERU
Depending on their shape, bacteria are classified
into several varieties ( Fig . 2.1 ):
1. Cocci {from kvkkot meaning berry ) are spherical
or oval cells.
2, Bacilli ( from bacuhi ? meaning rod ) arc rod
shaped cells.
2. Vibrios are comma shaped , curved rods and
derive the name from their characteristic
vibratory motility .
4. Spirilla are rigid spiral forms.
5 . Spirochetes {from speira meaning coil and chaffie
meaning hair) are tlexunus spiral forms ,
b . AcdDODiyccttH are branching filamentous
bacteria , so called because of a fancied
resemblance to the radiating rays of the sun when
seen Jo tissue lesions ( from acras meaning ray
and myites meaning fungus).
7 . Mycoplasma* are bacteria that are cell wall
deficient and hence do not possess a Stable
morphology;They occur as round or oval bodies
and as interlacing Filaments . When cell wall
synthesis becomes defective , cither
spontaneously or as a result nf drugs like
penicillin, bacteria lose their distinctive shape.
Such cells are called protoplasts, sphenopla &ts
or L forms.
Bacteria sometimes show characteristic cellular
arrangement or grouping ( Fig - 2.2 ', . Thus, cocci
may be arranged in pairs (diplococd ) , chains
(streptococci ) , groups ol four ( tetrads ) or eight
( sarcina) , ot a grape - like dusters (sraphylococd) .
Some bacilli too may be arranged in chains
( streplohacilli ). Others are arranged at angles to
each other, presenting a cuneiform 01 ChiriCK letter
pattern ( curyoebacteria ) . The type of cellular
arrangement is determined by Lhc plane through
which binary fission takes place and bv the tendency
of the daughter cells to remain attached even after
division .
•
>
1
3
4
5
. . .
Fig 2.1 Shapes of bacteria : 1 coccus 2 bacillus
. . .
3 vibrio 4 spirillum 5 spirochete
Copyrighted material
 * Morphology ano Physiology -
oF BacCe i LI n

T
i

O to
"•
'£V i

<#% $
3
4
/\
2
m
5 7
n
«- am W
5 < k
/ >
* A
*a
^ 3

Fig . 2.3 Arrangemenl or bacilli i . bacilli In cluster

Fig. 2.2 Arrangement cf cocci: 1. ilroptocoeel
.
2. pneymocMol J. qon«Mcl J meningococci 1 bacilli In chains IS. anltaM) 3 dtplotecWI
6. Neisseria oteffMlJ E Gattiiy iefra errs [K . pnetnuomM)
7. sarclna 13 staphylococci * ^

w~?
c 1C
1
H

f r

11

.
Fig 2.5 Diagram ol an ideatlsed bacterial ceil :
. .
I Capsule 2 . PIN 3 Outer membrane 4 Division .
seplum & . Ribosome 6 ONA 7 Granular inclu-
Fig. 2.4 Arrangements a I curved bacteria : l vibrio sion; 3 . Flaoel - a 9. Fat globules 10 Mesoaome
2. spirilla 3. spirochetes II. Cytoplasmic membrane 12. Paptldoglycan

Copyrighted material
12 * Textbook of Microbiology
BACTISKIAI ANATOMT
Fig . 2.5 shows the stractuic of an idealised bacterial
cell . The outer layef or tell envelope CtiJftei& tS of
rwo components - ;L rigid t ell wall and beneath it ;L
cytoplasmic nr plasma membrane, The cell envelope:
encloses [ fie protoplasm, comprising tfie cytoplasm,
cytoplasmic inclusions siifb as ribosomes and
mesosomes, granules, vacuoles and rhe nuclear body.
Besides these essential components, some bacteria
imav iwssess additional structures. The cell may be
enclosed in a viscid layer, which may he a loose
slime layer, or organised is a capsule. Some bacteria
CJirTy filamentous appendages protruding from tlir
cell surface - rhe flagella which are organs nt
locomotion and the ilmbriae which appear io be
organs for adhesion.
The cull wall: The cell wall accounts for the
shape of the bacterial cell and confers OIL it rigidity
and ductili ty. T he cel] wall cannot he seen by d ireel
light microscopy and docs not stain with simple
stain* - lr mjy he demonstrated by plasmolvsis.
When placed in a hypertonic Solution , the
cytoplasm loses water by osmosis and shrinks, while
the cell wall retains its original shape and sfv.c
( bacterial glidEi ) . The cell wall may also he
demonstrated hv mierodissectiou, reaction with
specific antibody, mechanical rupture of the cell ,
difiiientiil sraining procedures or by electron
tniavnopfr Bacterial cell walls are about lCb-25
nm thick and account for about 20-50 per cent of
the dry weight of the cells. Chemically the eel!
wall is composed of mucopeptidr (pcplidnglycan
oi mu rein ) scaffolding formed hy N icetyl
glucosamine and N acetyl mumnic acid mnleailcH
alter na ring in chains, which arc cross!inked hy
Table 2 ,2 Comparison of cell walls ol Gram positive and Gram negalivs bacteria
Thickness
Variety of aminoadds
Aromatic and sulphur cemtaininp aminnadds
Lipids
Tocknc seid
s
peptide chains (Fig- 2 , h ) , The interstices of this
scaffolding contain other chemicals, carving io the
different species . In general, the waits of the Oram
positive bacteria have simpler chemical nature than
those of t iram negative bacteria [Table 2 ,2 ). Tire
cell wall Carrie * bacterial antigen * that are
important in virulence and immunity.
The lipopoLysaeehflrida (LPS) present on the
eel! walls of ( !ram negative bacteria account for
"
their cndotoxic activity and O antigen specificity.
They were formerly known as the Soivin anitgen.
The LPS consists oi three regions. Region T is the
polysKcharide porrion determining the O antigen
specificity. Region II is tire core polysaccharide .
Region III is the glycolipid portion (lipid A ) and
is responsible for the cndotnxk activities, that is ,
pyrogenicitv, Lethal effect , tissue necrosis ,
.
anticamplcmcntaiy activity', & cell mitogenicily,
ioiioiiiLoadjuvant property and antitumour activity.
The outermost layer of CJram negative bacterial
cell wj.ll j* called rhe outer m«nbriiwT which
contains various proteins known as outer membrane
proteins (OMP). Among these are porins which
tor in trans- membrane pores [ bat serve as ditfusior
channels tor small molecules- They also serve as
specific receptors for some bacteriophages.
Cell wall synthesis may be inhibited by many
fimttfa. Lysozyme, an en/yrtlC MtnuJjy present in
many tissue fluids, lyses susceptible bacteria by
split Ling the cell wall inucopeptide linkages. When
lysoTvrne acts on a t iram positive bacterium in a
hypertonic solution, a protoplast is formed ,
consisting of [ be cytoplasmic membrane and its
With Gram negative bacteria, the result
contents.
is a spbemplust which differs from the protoplast
Gum jPOjj five Gram ncjifirt
Thicker Thinner
Few Several
Absent Present
Absent or scant Present
Present Ahsem
k _J opy righted materia
 * yorphclogy ar- d
i t
t
Wr =
* *tFi
'J 1.'. n "- na
^ -
#wur
^ ir .n
rwfl ifl®
'
I +g?ltfCP«H ± 7
^ chtm
tX 'C'jT
.
Fig 2.6 Chemical situelure nl bacterial cell Mall
in that some cell wall material n retained.
ProtopLuts and spheroplasts art spherical ,
regardless of the original shape of the bacterium.
Celt Will deficient forms of bactEria may probably
hive a role in the persistence of certain chronic
infections such as pyelonephritis,
f Jytopl:] mmc mtilihrjini." The cytoplasmic
( plasma ) membrane is a thin (5-10 nm ) layer lining
the inner surface of the cell wall and separating it
from the cytoplasm. JT aers as a semipermeablc
membrane controlling the inflow and outflow ot
metabolites to and from the protoplasm - Passage
through the membrane is nut suldv a function of
the molecular siise of the particles but depends, in
many eases, on the presence in the membrane of
specific enzymes ( pomatset ). Electron microscopy
shows the presence of three layers constituting a
' unit membrane ' structure. Chemically, the
membrane consists of lipoprotein with small
amounts of carbohydrate. Sterols are absent, except
in mycoplasma.
Cytoplasm The bacterial cytoplasm is a colloidal
system ut a Variety of organic and inorganic solutes
in a viscous waters solution . It differs from
Physiology pi Bacteria L3
eukaryotic cytoplasm in not exhibiting internal
mobility ( protoplasmic streaming) and in the
absence of endoplasmic reticulum or mitochondria.
Hie cytoplasm stains uniformly with bade dyes in
young cultures but becomes increasingly granular
with ige. The cytoplasm contains rihnsonncs ,
mesostJines, inclusions and vacuoles ,
Ribosomes are centres of prolein synthesis . They
arc slightly smaller than the ribosomes of eukaryotic
cell? (sedimentation constant 70 S) and arc seen
integrated in Linear strands of mRNA to form
poLymmei.
MfrSOSOltlCB ( c h o n d r o i d s ] are v e s i c u l a r,
convoluted or nmlriEammated structures formed as
invagmuions of t h e plasma membrfin -C i ^ t ^ i the
cytoplasm . They arc more prominent in Gram
positive bacteria. They are the principal sites of
respiratory enzymes in bacteria and are analogous
to the mitochondria ot eukaryotes. JVfBOSCVTJCJ ate
often seen in relation to the nuclear body and the
site of syn thesis of cross wall septa , Suggesting that
they coordinate nuclear und cytoplasmic division
during binary fission .
Inlnioytoplasnitu inclusions mav be of
various types, the chief of which arc volutin ,
polysaccharide, lipid and crystals. They are
characteristic for different species and depend on
the age and condition of the culture. Volutin
granules (iTHtlchlHnatlC Of flilbes- Efflir gTUIllfar)
are highly refractive, strongly basophilic bodies
consisting of poly me raphosp hate . They appear
reddish when stained with polychrome methylene
blue or tohudine blue (ilietHchmmaFia ). Special
seaming techniques Such as Albert’s or Neisser’s
demonstrate rhe granules more dearly . Volutin
granules any charactvnsricallv present ill diphtheria
bacilli , Their function is Uncertain. They have been
considered in represent a reserve ot energy and
phosphate for cell metabolism but they arc most
frequent in cells grown under conditions of
nutritional deficiency and tend to disappear when
the deficient nutrients are supplied. Polysaccharide
granules may be demonstrated by staining with
Copyrighted material
14 « Textbook ol Microti . olQQy *
iodine, and lipid inclusions wkh fat soluble dyes
siu h as Sudan bl . uk . They Appear to be s l u r . i e
'
products . Vacuoles are fluid-containing cavities ^ ,
separated from the cytoplasm by a membrane . ThciT
function and significance arc uncertain.
Nucleus: Bacten ^ il nuclei can be demonstrated
by and or riImnuciease hydrolyse and subsequent
staining for nuclear material - They maybe seer by
electron microscopy. They appear as oval or
elongated bodies, generally one per cell. Some cells
may possess two or more nuclear bodies due To
asynchrony between nuclear and cytoplasmic
division.
Bacterial nuclei have no nuclear membrane or
nucleolus , The nuclear deoxyribonucleic acid
( DNA ) is not associated with basic protein. The
genome consists of a si nub - molecule of double -
stranded DNA arranged in the form of a circle,
which may open under certain conditions to form
a long chain, about 1 mm in Length. The bacterial
chromosome is haploid and replicates hy simple
fission instead of hy mitosis as in higher cells . The
differences between the nucLei of bacteria and higher
organisms form the main basis for classifying them
as prokaryotes and eukaryotes .
Bacteria may possess extranuclear genetic
elements consisting of DNA. These cytoplasmic
carriers of genetic i nformati^ n are termed plasmids
or epfsomes [see Chapter 6). Besides being
transm i tted to daughter cells during binary fissh >n,
they may be transferred from one bacterium to
another either through conjugation or the agency
of bacteriophages, They are not esserri ;:] for the
life of the cell they inhabit but may confer on it
certain properties like tux igeni city and drui;
resistance which may constitute a survival
advantage.
Siimu layer and capsule: Mary bacteria
secrete a viscid material around the cell surface.
When this is organised into a sharplv defined
structure , as in the pneumococcus, it is known as
the capsule . When it is a loose undemarcared
scenerion, as in leuconostoc, it is called the slime
layer- Capsules too thin to be seen under the light
microscopes are called m rmcajuules. The slime
^
is generally, but not invariably, polysaccharide (for
example pneumococcus ] or polypeptide (for
example anthrax bacillus) in nature. Some bacteria
may have both a capsule and a slime layer (for
example . Streptococcus silivirius ) . Bacteria
seonermg large amounts of slime produce mucoid
growth on agar, which is of a stringy consistency
when couched with the loop.
Slime has little affinity for basic fives and is not
visible in Gram stained smears. Special capsule
staining techniques are available, usually employing
copper salts AS mordants. Capsules may be readily
demonstrated by negative staining in wet films with
India ink . when they are seen as dear halos around
the bacteria, against a black background ( Fig, 2.7).
Capsular material is antigenic and may be
demonstrated by serological methods. When a
suspension of a capsulated bacterium is mixed with
its specific anti -capsular serum and examined under
the microscope , the capsule becomes very
prominent and appears ’swollen ' due to an increase
'
in its reftactivity. This: capsule swelling or Qiidlung
reaction, described by Ncufcld (1902) was widely
employed for the typing of pneumococci in the pre-
SLilphonamide days when lobar pneumonia used to
be treated with specific anticapsular sera. Capsules
protect bacteria bum deleterious agents such as lytic
enzymes found in nature. They also contribute to
the virulence of pathogenic bacteria by inhibiting
phagocyrosH. Loss of the capsule by mutation may
render the bacterium avirulent. Repeated
subcultures in vitro lead to the Joss of capsule and
aisu of virulence.
Mage ) In: Motile bacteria, except spirochetes ,
possess "tie or more unbranched, long, sinuous
filaments called flagella, which arc the organs of
locomotion. Each flagellum consists of threedistinct
parts, the filament , the hook and the basal body.
The filament is external to the cell and connected
to the hook at the cell surface.
-
The hook basal body portion is embedded in
opyrighted n iteria
 * Morphology and Physiology ol Bacteria
the cell envelope . The huok and basal body Are
antigeoicaily different. Mechanical detachment of
the fJimtnt does not impair the viability of the
cell. The flagella are 3-20 pm long and arc of
-
uniform diameter (0.01 0.013 pm ) and terminate
in a square dp. The wavelength and thickness of
the filament are characteristic of each species but
some bacteria exhibit hipliclty, that is, they have
flagella of two different wavelengths ( Fig . 2 , 8) ,
Flagella are made up of a protein (flagellin ) similar
to keratin or myosin - Though flagella of different
genera of bacteria have the same chemical
composition ,, they are antigenic ally different .
Flagellar antigens induce specific antibodies in high
titres. Flagellar antibodies are not protective hut
are useful in serndiagnosis.
The presence or absence of flagella and thei r
number and arrangement are characteristic of
different genera of bacteria ( Fig. 2.9 ), Flagella may
he arranged all round the cell ( peritridirius} as in
typhoid bacilli, or situated at one or both ends of
the cell ( pulaf ) . Polar flagella irtuy be single
( monorrlcltous) as in cholera vibrios, in tufts
%i
JESjjp * T- -
‘
W
, A* * "• r^ mjm ^
'
i" m the cell membrane. Fimbriae can be seen only under
'
%: '
.
Fig 2.7 Pnfcumocacd negatively stained with India
ink to show capsule
* 15
(lophotxichous) AS in spirilla or wirh flagella at
both poles (amphiitrichcnis).
Flagella arc less than 0-02 pm in thickness and
hence beyond the limit of resolution of the light
microscope . They' may, in some instances, be seen
under dark ground illumination. They can be
visualised by special staining techniques in which
their triiclcntsh is increased by mordanting , or by
electron microscopy ( Fig. 2.10 ) . Due to the
difficulty of demonstrating flagella directly, their
presence is usually inferred from the motility of
bacteria. Motility can be observed by noting the
spreading type of growth on a semi -solid Agar
.
medium. Under the microscope Active motility has
to be differentiated from the passive movements of
the cells , either due to air currents or due to
Brownian movement. Bacterial motility may range
from the slow 'stately ' motion ofpcriirichatc bacteria
[ for example, Bacillus ) to the darting movement of
polar flagellated vibrios . The cholera vibrio may
move as fast as 200 pm per second .
Fimhnw Some Gram negative bacilli carry very
fine , hair -like surface appendages called fimbriae
or pili. They arc shorter and thinner than flagella
( about 0.5 pm long and less than 10 nm thick ) and
project from the cell surface as straight filaments.
At least eight morphological types of pili are
known , classifiable as cither common or sex pili
on the basis of their function. Pili comprise self-
aggregating monomers of piiin , They originate in
the electron microscope. They are unrelated to
motility and are found qn motile as well as
nanmotilc cells. 'Hiey are best developed io freshly
isolated smirii and in liquid Cultures. They tend to
disappear following subcultures on solid media -
Fimbriae function as organs of adhesion, helping
the cells to adhere firmly to particles of various
kinds . This property may serve to anchor the
bacteria in nutritionally favourable m i c r o
environments. Fimbriated bacteria form surface
pellicles in liquid media. Many fimbriated cells (for
example Fscherichia , Klebsiella ) agglutinate rod
Copyrighted material
16 + Textbook of Microbiology

bluud tells of gu i Licit filers, fowl, horses and pigs

Strongly, human and. -sheep cells weakly, arid ox
fells scarcely at all .
Hemagglutination provide* a simple method for
detecting the presence of such fimbriae. The
hemagglutination is Specifically inhibited by D-
marnose (mannose sensitive ).
lumbriae are antigenic . As members ofdifferent
genera may possess the same timbrial antigen, it is
necessary to ensure that the bacterial antigens
employed for serological tests and preparation of 4
antisera are devoid of fimbriae.
A specif type nf fimbria arc the sent pilbThese
are longer and fewer in number chan other fimbriae.
They are found on ’male' bacteria and help in the
attachment of those cells to ' female ' bacteria, 5

Fig. 2.9 Types of llagellar arrangement: 1. Singhs

.
flagellum 2 single llagallum at each pole 3. tuft
of flagella at one pals 4 . tuMA ol flagella at both
.
poles 5 peritrichous flagella

forming hollow conjugation rubes through which,

ir is assumed , genetic material is trainferred from
the donor to the recipient cell. PUi are classified
into different types (for example , E I) based on
susceptibility to specific bacteriDphages.
Spu - .- : Some hacteriii, particularly members of
j

z
l
J
the pentn 11 aciHus and C1«rrldium have rhe ubili ty
I 5 to form highly rc ^isLuni refit rig Stages called spores
] ,

f frch bacterium forms one spore, which nn

Fig. 2.8 Flagellum and Its parts l. Arrow shows the
,
junction ol (ha hook and filameni. 2 , Ring for
attachment ID outer lipopolysaccharide 0 antigsn
compiei membrane of the cell wall 3 Rod . .
. .
connecting cop and bottom rings 4 King for fts
association wth the pepildogtycan layer of Ihe
.
call wal 5. Ring located jusl above cytoplasmic
membrane M ring Tor attachment io Ihe cytoplasmic
membrane.
germination forms a single vegetative cell .
SpOfubtion in bacteria, therefore, Is not A method
.
of reproduction As bacterial spores arc fanned
inside the parent cell, they are called endofpORs.
While the exact stimulus for sporulatiot) is not
known, it occurs after a period of vegetative growth
- and is presumed to be related rn the depletion of
exogenous nutrients. Spoiulation is ini tinted by the
appearance of a. clear area, usually near one end of
the cell, which gradually becomes more opaque tn
form the 'forespnre’. The folly developed spore has

Copyrighted materia
 i Marpnatagy and P^iysioiocjy of Idadu^. f
at its core rhe nuclear body, su mounded by the spore
wall, a delicate membrane from which the cell wall
of the future vegetative bacterium will develop, Outside
this LS the thick spore COXtCX, which in turn i.s enclosed
by 2 multiJayered tough spore coat. Some spores have
an additional outer covering called exDSfJorium, which
may have distinctive ridges and grooves (Fig- 2, 11) .
New amij trts appear on sporulatum.
^
Young s[K>res we seen attached to the parent
cell. The shape and position of the spore and its
si ye relative to the parent cell ;Lre species
characteristics . Spores may be central
(equatorial), terminsil or suhtrrminnl . They may be
aval [ ]r spherical. Ifocy may or may not distend the
bacillary body (Fig. 2.12 ).
Bacterial spares constitute some of the most
resistant forms of life. They may remain viable for
centuries,I ' ’hey are cxtrenvcly resistant to desiccation
and relatively so to chemicals and heat . Though
some spores may resist boding for prolonged
periods , spores of all medically important species
are destroyed by autoclaving at L 20 for 15
minutes- Methods of sterilisation and disinfection
J 3
Fig , 5.1Q Electron micrograph of E . coli I . FlageNa
2, F - pili 3. ordinary piM or fimbriae
17
should ensure that spores also are destroyed.
Spoliation helps haetcria survive for long periods
under unfavourable environments .
When transterred to conditions conducive for
growth* spores germinate. The spore loses its
refractiliiy and sw ells. The spore wall is shed and
the germ cell appears by rupturing the spore coat
and elongates to form the vegetative bacterium.
Spores maybe seen in unstained preparation as
refract!le bodies. The fcrespore stains intensely,but
once the spore envelope is laid down , the spore
.
d<ies not stiiin readily. Spores appear as unstained
areas in Gram stained preparations, hut being more
acid fast than the vegetative cell-s, they can be stained
by A modification of rhe Ziehl- Neclsen technique,
Pluouflorphiim and involution forms;
Some species of bacteria exhibit great variation In
the shape and size of individual cells 1 his is known
AS pltomorph ism. Certain species ( for example,
plague bacillus, gonococcus ) show -swollen and
Aberrant forms in ageing cultures, especially in the
presence of high salt concentration. These arc
known as involution forms . Many -of the ceils may
H
..
:
g 5.11 Oisgremnurtit representation erf a oacteha!
spore. 1. Germinal gmove 2. Outer comcal layer
3 . Cortei 4. Internal (pare CC- .H! S. Subtree : m.lieflal
6 Outer spots coal 7. Cytoplasmla membrane
8, Cell wall prirnonlium
 * TesIbdAk a* MiCfObiClotjy *

be nonviable Pleomorphkm and involution forms

* The cell divides by a eoustrietive or pinching
arc often Jut to defective cell tv a 11 synthesis
Involution forms may also develop due ro the activity
of aucoiytit enzymes.
L forms KJeinebecget- Nobel, studying cultures
nf Strrptnhat.illui; tTUffiiiifbttiiis* observed swollen
cells and other aberrant morphological forms and
named them L forms, after Lister Institute:, London,
where the observation was made . L forms arc seen
in several species of bacteria , developing either
spontaneously or in the presence of penicillin or
other agents that interfere with cell wall synthesis.
L forms may be unstable in that the morphological
abnormality' 1? maintained only in the presence of
penicillin or other inducing agents, or stable, when
the uherrant form becomes the permanent feature
of the strain and is retained in serial subcultures, L
-
form : resemble mycoplasma in several ways ,
including morphology, type of growth on agar and
filter ability. It is possible that mycoplumis
re] tneseiu stable I „ forms nf us vet iiniden ti bed pare nr
bacteria.
GROWTH AMJ MULTIPLICATION OV
BA^ TRUI A
Bacteria divide by binary fission. When i bacterial
cell reaches a certain si / e , it divides to form two
daughter cells . Nuclear division precedes cell
division and , therefore* in a growing population *
many cells carrying two tiuck-ar bodies can be seen.
* process, or by the ingrowth of a transverse septum
across the cell In some species, the daughter cells
may remain partially attached after division.
The interval oftime between two cell divisions,
required for a bacterium to give rise to
or the time
two daughter cells under optimum conditions, is
known as the gene rat ion time or population
doubling time. In coliform bacilli and many other
medically Important bacteria, the generation time
is about 20 minutes. Some bacteria are slow
giowing; the generation time in tubercle bacilli is
-
about 20 hours and in lepra bacilli as lung A - about
20 days. As bacteria reproduce so rapidly and by
-
geometric progression , a single hacterial cell can
theoretically give rise ro 1(P progeny in 24 hours,
wi rh ,t mass of approximately 4 ,000 tonnes!1 n actual
practice, when bacteria arc grown in a vessel of
liquid medium ( batch culture . multiplication is
arrested alter a few cell divisions due to depletion of
nutrients nr accumulation of MMC products , By the
use of special devices for replenishing nutrients and
removing bacterial cells ( cheirostat or turbidisrat ), it
is possible to maintain continuous vulture of bacteria
tor industrial or research purpoen. Whcn pathogenic
bacteria multiply in host tissues, the situation may be
intermediate between a I utvh vuln i re .ind a am r i n u < ws
culnire ; the source of numerics mtn be inexhaustible
but the parasite has to contend with the defence
mechanisms of (he bodyt Bacteria growing on solid

1 1 12
3 E*
51 6

.
Fig. 2.12 Types of bacterial spores. 1. central, bulging , 2 central , not bulging. 2. suSterminaE. bulging,
4. subliminal . rwl bulging, 5. terminal, spherical 6. terminal, oval.

Copyrighted material
 H Morphology and Physiology ol Bacteria
media form colonic . Each colony represents a done
of cells der. ved from a single - urcuC cell. In liqLud
media , growth is diffuse .
Bactef d growth may be con > idered at two lcvds,
increase in the size of the individual odl and increase
,:i the number of cells. The former is ordinarily limited
and when the critical si * is reached , the cetl divides,
Except when cetl division is inhibited bv substances
like pci lictQin or ac riflaii nc or bygrowth in magnesi um
deficient media . Growth in numbers can be studied
hv bacterial counts. Twii types ofhactcri.il counts can
be Tuade— total counl and viable count .
'Ihe total count gives the tota ] number of cells
in the sample , irrespective of whether they are living
or not. It can he obtained by
1 . direct counting under the microscope u - ing
counting chamber:,
2. counting in an electronic device as in the Coulter
counter,
3 - direct counting u^ing stained smears prepared
by spreading a known volume of the culture
over a measured area of a slide ,
4. comparing relati ye numbers i 1 i smears ofthc culture
mixed with known numbers of other cells,
5 . by opacity measurements using an absnrptfo
meter or nephalometer,
6 . by separating the cells by centrifugation or
filrration and measuring their wet or dry weight ,
and
7 . chemical assay of celt components such as
nitnngcn -
The viable count measures the number oriiving
cells, LII .U is, dells capable of multiplication. Viable
counts are obt .LI ued by diluti < m or plaf : ng methods .
In the dilution method, the suspension is diluted
to a point beyond which unit quantities do not yield
growth when inoculated into suiLible liquid media
(extinction ) . Several tubes are moculated with
varying dilutions and the viable count calculated
statistically from the number of tubes showing
growth . The method docs not give accurate values
but is used widely in water bacteriology for estimation
of [ lie 'presumptive coliform count’ in drinking water.
» 19
In the plating method, appropriate dilutions are
inoculated on solid media, either on the surface of
places or as pour plates. The number of colon ics that
develop after incubation give? an estimate ofthc viable
count . The method commonly employed is that
described by Miles and Misra. [1938 ) in which serial
dilutions are dropped on the surface of dried plates
and colony counts obtained.
ftxct HRIAL GROWTH CURV E
When a bacterium is seeded into a s - iirablc liquid
'
medi um and i ncuhated , its growth follows a defini:c
course, If bacterial counts are made at intervals after
inoculation and plotted in relation to rime, a growth
curve is obt . Lined [Fig. 2.13). The curve shows the
following phases:
Lug phase: Immediately following the seeding
of a culture medium , there is no appreciable
increase in numherH , though there mav be an
increase in the size of the cells . This initial period
is the time required for adaptation to the new
environment , during which the necessary en-zymcR
and metabolic intermediates are built up in adequate
quantities for mult ip lie at ion to proceed . The
dura non of the lag phase varies with the species,
size of inoculum , nature of culture medium and
environmental factors such as temperature .
I ( ) J£ ( LOGFLRILHMIC ) OT exponential! plnisel
H
Following the Lag phase, the cells start dividing
and their numbers increase exponentially or by
geometric progression with time. If the logarithm
of the viable count is. plotted ag unst lime , a straijfot
line will be obtain Led . ^
Stationary phase; After a varying period of
expouenfial growth, cell division stops due to
depletion of nutrients and accumulation of toxic
products. The number of progeny cells formed i s
j list enough to replace the number of oellfi that die.
The viable count remains stationary as an
equilibrium exists between the dying cells and the
newly formed cells.
E^ ILUHU « * j dvuliiiu: Tim is the phase when the
population decreases due to cell death , Besides
Copyrighted material
20
i
I
LOG 1 There arc. however, some differences which are
PHASE
i S TA ' lOftlAR -f
LAfn PHASE
PHASE! I source of carbon, a source of nitrogen and some
! I'
TWE
.
Fig 2.13 Bacterial growth curve. The viable count
.
shows tag. log slationary and decline phases In
the total count, ihe phase ol decline Ls not evidenl-
nulritionfll exhaustion and toxic accumulation , LL !1
death may also he caused :iuit ( i]ytir enzymes .
by
When the total ex Hint is plotted , it parallels the
viable count up to the stationary phase hut it
continue* steadily without any phane of decline.
With autolytic bacteria, even the total count shows
a phase of decline.
The various stages of the growth curve are
associated with morphological and physiological
alterations of the cells. Bacteria have the maximum
cell size towards the end of the lag phase. In the
log phase, cells are smaller and stain uniformly. In
the stationary' phase , cells frequently are Gram
variable and show irregular staining due ro the
presence of intracellular storage granules.
Sporulation occurs at this stage. Also, many bacteria
produce Secondary metabolic products such as
exofnjtins and antihintics. Involution forms are
common in the phase of decline .
HACTRRIM Ml TRTTION
I'hc bacterial cell has the same general the mi cal
pattern as the cells of higher organisms. The
principal const ] ruent of bacterial cells is water,
^ Te * tbooK of Microbiology *
which represents about SO per cent of the total
weight. Proteins^ polysaccharides, Lipids, nucleic
acids, rmteopeptidcs and low molecular weight
compounds make up the rest . Bacterial metabolism
TOTAL
CQL
*Jr also iu closely similar to the metabolism of die higher
*
organisms, exemplifying the ' unity of biochemistry '.
VIABLE
exploited in selective toxicity and chemotherapy.
*
COUNT for growth and multiplication of bacteria, the
[i i ini mum nutritional requirements are water, a
RHASF inorganic salts. Water is the vehicle for the entry
ff D CL « of all nutrients into the celli and lot the elimination
of all waste products lit participates in the me tain die
,
reactions and also forms an integral port of the
pUltOplilHlTL.
Racteriit L-an he classified nutritionally, based on
. rheir energy requirements and on their ability to
synthesise essential metabolites . Bacteria which
derive their energy from sunlight are called
piotofrophs and those that obtain energy from
chemical reactions arc called chcmorrophs. Bacteria
that can Synthesise all their organic compounds are
called autotrophs - Those that are unable to
synthesise their own metabolites and depend on
preformed organic compounds are called
, hercrofropfrs. Autotrophs arc able to utilise
atmospheric carbon dioxide and nitrogen. They are
capable of independent existence in water and soil
and are of no medical importance, though they are
of vital concern in agriculture and the maintenance
of soil fertility- Meterotrophic bacteria arc unable
to grow with carbon dioxide ax the sole source of
carbon . The nutritional requirements of
heterotrophs vary widely. Some may require only a
single organic substance such as glucose , while
others may need a large number of different
compounds such as ami noacids , nucleotides, lipids,
carbohydrates and coenzymes.
Bacteria require a supply of inorganic salts,
particularly the anions phosphate and sulphate* and
the cations sodium , potassium , magnesium, iron,
manganese and calcium . These are normally present
pyrighted materia
k. l* l El
 H Morphology and Fhy oiogy ol Bacteria
in the natural environments where bacteria live but
will have to be supplied in culture media. Some
ions such as cobalt maybe needed in nacr amounts-
Sume bacteria require certain organic
compounds in minute quantities . These are known
as growth factors or bacterial vitamins. Growth
factors are called «»ntuf when growth docs not
occur in their absence, or accessory when they
enhance growth, without being absolutely necessary
with the vitamins necessary. for mammalian
-
for i t. In many cases, bacterial v i t am in - are i Jenti cal
nutrition, particularly those belonging to the B
group, thiamine , riboflavine , nicotinic acid,
pyridoxine, folic acid and vitamin B12,
If a microorganism requiting an essential growth
factor is inoculated into a medium containing an
excess of all Other nutrients, it ? growth will be
proportional to the amount of the limiting substance
added. Within a certain range, the concentration
of the growth factor will bear a linear relationship
to the amount of growth of the organism. This is
the principle id microhiuLogical assays, which
provide a very sensitive and specific method for
estimation of many aminoacids and vitamins, as in
the determination of vitamin Bl 2 using
LactofwcitfufT fckhnwnnil
D\
^3 EN REOUJKEJUENT AINU
M i -. r A i K i i . i s M
Depending on the influence of oxygen on growth
and viability bacteria arc divided into aerobes and
anaerobes. Aerobic bacteria require oxygen for
growth. They may be obligate aerobes like the
CII^ -JCNL vihrii :., which will jp-fra- i >:ilv in ihc ptencriCr
of oxygen , or facultative anaerobes which are
ordinarily aerobic but can also grow in the absence
of oxygen, though less abundantly. Most bacteria
of medical importance are facultative anaerobes.
Anaerobic bacteria, such as Clostridia , grow in the
absence of oxygen and the obligate anaerobes may
even die on exposure to oxygen. Microaerophilic
bacteria arc those that grow best in the presence of
a low oxygen tension.
> 2\
The reason for the apparent toxicity of oxygen
-
lor anaerobic bacteria i i not well understood. If
has been suggested that in the presence of oxygen,
hydrogen peroxide and other toxic peroxides
accumulate . The enzyme catalase which splits
hydrogen peroxide is present in most aerobic
bacteria but is absent in the anaerobes . Another
reason might be that obligate anaerobes possess
essential enzymes that are active only in the reduced
state.
The influence of free oxygen is related to the
metabolic character of the bacterium . Aerobic
bacted ; ii obtain their energy and intermediates only
through oxidation involving oxygen as the ultimate
hydrogen acceptor , while the anaerobes use ,
hydrogen acceptors other than oxygen. Facultative
anaerobes may act in both ways. In the case of
aerobes , where the ultimate electron acceptor is
atmospheric oxygen (aerobic respiration ), the
carbon and energy source may be completely
oxidised to carbon dioxide and water. Energy is
provided by the production of energy rich
phosphate bonds and the conversion of adenosine
-
diphosphate ( ADP) to adenotine triphosphate
( Ad P ). This process is known as nxidptive
.
phosphorylation Anaerobic bacteria use as electron
acceptors compounds such as nitrates or sulphates
instead of oxygen (anaerobic respiration ), A more
common process in anaerobic metabnli ^ m may be
a series of axidoreduccions in which the carbon
and energy source acts as both the electron donor
and electron acceptor. This process is known as
fermentation and leads to the formation of several
^ 11 1:11:11 end prutiucl- K such as .11 i 4 - :LHIL .11 . phitif , .i -,
^ as of gas (carbon dioxide and hydrogen ).
' '
L
well
During the process of fermentation , energy-rich
phosphate bonds are produced by the introduction
of organic phosphate into intermediate metabolite *.
This process is. known ay substrate - level
-
phosphorylation. The energy rich phosphate
groups so formed ate used tor conversion of ADP
-
to ATP
In determining rhe growth of aerobic and
opy righted material
8 dr w
 22 4 Ty .Kttxrak a 1
anaerobic bacien .i, what Is more important L 11-.11:
the presence or absence of oxygen is the state of
oxidation of the environment , 'fhc oxidising or
reducing condition of a system is indicated by the
net readiness of all the components in that system
GO take up, oe parr with electrons. This is known as
— -
the oxidxtinn reduction \ redox ' potential of the
system. The redox potenti .d of a medium is best
estimated try measuring the elccliical putenti J
difference set up between the medium and an
unattackable electrode immersed in it . This
electrode potential ( Eh ) car be measured in
millivolts.The more oxidised the system, the higher
the potential. A simpler , though less accurate,
method of measurmg the redox potential is the use
of oxidation-reduction indicators such as methylene
blue, and noting the change in colour.
CARBON DIOXIDE
All bacteria require smalt amounts of carbon
dioxide for growth.Tics requirement is usually met
by the carbon dioxide present in the atmosphere,
or produced endogenously by cellular metabolism.
Some bacteria like BtuCella abottui require much
higher levels of carbon dioxide (5-10 per cent) for
growth, especially on fresh isolation (capnophilic).
TBMhfiftATt itH
Bacteria vary in their requirements of temperature
for growth. For each species, there is a 'temperature
'
range and growth does not occur above the
,
ma’i i 1 num or below the mini mum of the range. The
temperature at which growth occurs best is known
,
as the "optimum temperarurr winch in the case of
F protoplasm and hence drying i * lethal to cells.
most pathogen- -.. bacteria is 37 VC , Batten :* which
,
'
grow best at temperatures of 25^40 “ C are called
mesophihe. All parodies of warm blooded animals
are mesophilic Within the group of mesophuc
bactcri i, some like Pseudomonas aeruginosa have
1 wider -
range (5 43 *C ) t while others like the
-
: gonococcus have a restricted range (30 39 *C ).
Paychruphilic hacteria are those that grow best
at temperatures below 20 aC , some of them even
Micrgtuology *
growing at temperatures as low as -7 °C. They arc
soil and water saprophytes and though not of direct
medical i mpoitance, may cause spoilage of refrigerated
food. Another group of nonpathogenic bacteria, the
thcimophrfcs, grow best at high temperatures, 55
90 UC, They may cause spoilage of under processed
-
canned food - Some thcrmophilcR [ like Baiv / Zus
STCAROTHERMOPHJ J U F ) form spores that are
'
'
exceptionally thermores istant. Extremely
thermophilic bacteria have been identified which
can grow at temperatures as high as 250 flC -
Bacteria also differ in the effect of temperature on
viability. Heat is an important method for the
destmcri'- in of microorganisms (sterilisation), moist
heat causing coagulation and denafluratfon of proteins
and dry heat causing oxidation and charting. Moist
heat is more lethal than drv heat . The lowest
temperature that kills a bacterium under standard
conditions in a gircn lime is known as the themed
deathporrtf. Under moist conditions most vegetative,
mesophikc bacteru have a thermal death point
,
between 50 and 65 JC and most spores between
100 and 120 *C .
Ai low temperatures some species die rapidly
but most survive well. Storage m the refrigerator
- -
(3-5 "C) or the deep freeze cabinet ( 30 to 70 “ C)
is used for preservation of cultures. Rapid freezing
as wi tii solid carbon J iodide or the use of a stabil i her
such as glycerol, minimises the death of cells on
freezing,
Moira Dm INC
UK AND
Water is an essential ingredient of bacrenal
However, the effect of drying varies in different
species. Some delicate bacteria like Treponema
pallidum arc highly sensitux, while others like
staphylococci withstand diving for months. Spores
are particularly resistant to desiccation and may survive
ill the dry state for several dccadiB. Drying in vacuum
111 thecold ( &W 3rdiyitigar haphils$aQnn) is a method
for the preservation ofbscteria , viruses and many labile
biological materials.
Copyrighted material
 * Mofphafogy and Pfiy$i$logy o( Bacteria * 23

It - ION CONCENTRATION only on exposure to light and not when incubated

Bacteria acu sensitive to variations in jsH , Etch
species has a pi I range, above or below which it
does nor survive, and an optimum pH at which it
grown bent . The majority of pathogenic bacteria
grow best at neutral or slightly alkaline reaction
(pH 7.2 — 7.6) . Some acidophilic bacteria such as
lactubacilli grow under acidic conditions . Others,
such as the cholera vibrio , arc very enfirrvr tn acid,
but to ] erare high degrees. of alkalinity. Strong
solutions of acid or alkali (5% hydrochloric acid
or sodium hydroxide) readily kill most bacteria,
though mycobacteria arc exceptionally resistant to
them.
in the dark.
OSMOTIC EFFECT
Bacteria are mure tolerant fo osmotic variation than
most other cells due to the mechanical strength of
their cell walls , budden exposure to hypertonic
solutions may cause osmotic withdrawal of water
and shrinkage of protoplasm - pbatBoty$& This
occurs more readily in Gram negative than in Gram
positive bacteria . Sudden transfer from a
concentrated solution to distilled water may cause
phtsmopiytii (excessive osmotic imbibition leading
to swelling anti rupture of the cdl).

IxIfjHT MRCU \ M C A I. \ NIJ SONIC STRHSS

Bacteria ( except rhe phototrophic species) grow well
in the dark. They are sensitive to ultraviolet Light amt
driver radiations. Cultures die if exposed to snnlight.
Exposure- m light msy influence pigment production,
Photochromogenic mveobaL-ieria form a pigment
Though bacteria have tough eel! w iiu , they may
*
be ruptured by mechanical stress such as grinding
or vigorous shaking with glass beads. They may
also he disintegrated by exposure to ultrasonic
vibration ,

Further heading
.
Kl iir DF 1995. How bacteria
tease and swim. Anmrad flrv Mirrnbioi tf lN.
^
Dawes |W -snd W SudKrlupd LVNI . Mfcmbiat Phyjkrftjfi ) „ 2^ Ed. UnlunL: EJIKILWCLL
CiuuLd. GW and A Him£ |WJB Thf Rorlrrial Spprf . Um m: Acadflnic PtCH.
Mmat AG and JW Fcwter 19-95. jWirnpJwcj/ Phyiinlofry ,
'
* -
edn. NEW Vcirfc ; Wikv Liss,
Sanief RY e ( * \ IM Thr Microbial World* 5* edn. Ne Jersey: PrcrUke-HaJI.
*

Copyrighted material
 Sterilisation and Disinfection
Microorgamitny are ubiquitous. Since they cause
COntaiflination , infection arid decay, i [ becomes
nccessuy to remove or Jestrov them from iQdfleflAh
cir from ureas. This is the obicct of sterilisation.
The process of sterilisation is used in microbiology
for preventing cun laminar ion h y ext raucous
organisms , in surgery for maintaining asepsis, in
food and drug manufacture for ensuringsafely from
contaminating organisms, and in many other
situations . The methods of sterilisation employed
depend on the purpose for which it is carried out ,
the material which has to he sterilised and the
nature of the microorganisms that arc to he removed
( jr destroyed.
or
Sierilhaiiwi is defined as the process by which
an article, surface or medium is freed of all living
microorganisms cither in rhe vegetative or spore
state. Djarlfcftl'o/) rnflttU the destruction or rcntdval
of all pathogenic organisms , or organisms capable
of giving rise to infection. The term dfltrfqxu is
used to indicate the prevention of infection, usually
by inhibiting the growth of bacteria in wounds or
tissues. Chemical diisintectants which can lie safeiv *1
applied to shin or mucous membrane and are used
to prevent infection by inhibiting the growth of
bacteria are called anfjstynfjcs. agents
or art; those which are able to kill
{
bacteria . facte rjfwtxnr agents only prevent the
multiplication ot bacteria which may, however,
remain alive, A chemical which is bactericidal at a
particular concentration , may only be bacteriolttatic
at ahigher dilution.
L leaning, though a routine nontechnical
procedure , plays an important preparatory role
before sterilisation or disinfection, by removing soil
and ocher dirt and reducing rhe microbial burden ,
nukiiig seeriEisadon more efTaiiw- Deconianiinarian
refers to the process of rendering an article or area
free of danger from contaminants, including
microbial , chemical, radioactive and other hazards.
The various agents used in sterilisation cun be
classified as follows :
A . Physical a fit ms 1
1 . Sunlight ,
2. Diyi ng.
3. Dry heat - flaming, incineration, hot air.
4. Moi st hea r : pasteurisation, boiling, steam u nder
normal pressure, steam under pressure.
5. Filtration: candles , asbestos pads, membranes .
6. Radiation.
7 . Ultrasonic and sonic vibrations -
B Chemiunls
,
1 . Alcohols: ethyl , isopropyl , irichlorobutand .
2. Aldehydes: formaldehyde, glutaraldchydc,
3. Dyes.
4. Halogens.
5. Phenols,
6. Surface-active agents.
7 . Metallic salts .
H . Gases: ethylene oxide , formaldehyde, beta
propinlaclone.
Si NLIQHT
Sunlight possesses appreciable bactericidal activity
and pi ays an important role in the spontaneous
sterilisation that occurs under natural conditions,
fht action is primarily due to its content of
Copyrighted materia
 * Steri Ionian and DiF. miRr.rian
ultraviolet rays , most of which , however, are
screened out by glass and the presence of ozone in
the outer regions of rhe atmosphere. Under natural
circumstances.
-
conditions, its sterili. ing power varies according to
Direct sunlight , ns in tropical
countryside where it is not filtered off by impurities
i:i the atmosphere, has an active germicidal effect due
In the comhined effect of ultraviolet and heat rays.
Semple and Grieg showed LII . U in Lidia, typhoid bacilli
exposed to the sun on pieces of white drill doth were
killed in two hours, whereas controls kepr in, the dark
were still alive after six days- Bacteria suspended in
water are readily destroyed by exposure to sunlight .
I ) \< 'i i :s t i
Moisture is esSenlial for the growth of haute tin.
Four-fifths of the weight of the bacterial cell is
due ro water . Drying in air has therefore , a
deleterious effect on many bacteria . However, this
method is unreliable and is only of theoretical
interest. Spores are unaffected by drying.
I l l-
Heat i -; the most reliable method of iterilisarion
and should be the method of choice unless
contraindicated. Materials char may be damaged
by heat can be sterilised at lower temperatures, lot
longer periods or by repeated cycles. The factors
influencing sterilisation by heat are:
—
1 - nature of beat CITY heat or moist heat,
2. temperature and time,
2. number of microorganisms present *
4. character I its of the organisms* such as, species*
^
strain , sparing capacity, and
5. type of material from which the organisms have
to be eradicated .
Tbe killing effect of dry heat is due Bo prate in
dcnatur .ition, oxidative damage and LIIC toxic effeci
of elevated levels of electrolytes. The lethal effect
of moist heat is due to the denaluration and
coagulation of protein. The advantage of steam lies
in the latent heat liberated when it condenses on a
cooler surface , raising the temperature of that
25
surface. In the case of the spore, steam condenses
on it, increasing : ts water content with ultimate
hydrolysis and breakdown of the bacterial protein.
-
I n a completely moi ture-ffee atmosphere, bacteria ,
like many proteins, sir more resistant to heat. They
are killed when ax ids Lion of the cell constituents
occurs and this requires much higher temperatures
than that reeded for coagulation of proteins .
The time required for sterilisation is inversely
proportional to the temperature of exposure and
can be expressed as 'thermal death time', which is
the minimum time required ro kill a suspension of
organisms at a predetermined temperature in a
specified environment. The stei disarm time is
related to the number of organisms in the
suspension, presence or absence of Spores, and the
strain and characteristics of organism . The
recommended minimum sterilising times do not
include the lime taken tu reach the specified
temperature. 'I'he nature of the material in which
the organisms are heated affects the rate of killing.
The presence of organic substances, proteins, nucleic
.iciils , starch, gelatin, sugar, fats and oils, increase
the thermal death time. The presence of
disinfectants and high acid or alkaline pH hasten
bacterial killing.
DRY HEAT
Flaming: Inoculating loop or wire , the tip of
forceps and searing spatulas are held in a bunsen
flame till they become red hat. Inoculalion loops
canrying infective material may be dipped in a
disinfectant before flaming to prevent spattering.
Incineration: This is an excellent method for
safely destroying materials such as contaminated
clorh, animal carcasses, and pathological material*.
Plasties such as PVC and polythene can be dealt
with similarly bur polystyrene materials emit clouds
of dense black smoke and hence should be
autoclaved in appropriate containers.
Elm tiir oven: This is the most widely used
method of sterilisation by dry heat, A holding pen uni
of 160 °c far are hour is used to sterilise glassware .
Copyrighted material
2 f>
1 Eflr
t
1 into thioglycollitc or Cooked meat media and
3 incubated for sterility test under strict anaerobic
r - j
ct L
j
i .' I
j
Ftg . 3.1 Ho! air awn
forceps, , udpdj, all-gloss syringes , svrabi,
JcnoR
some pharmaceutical products such as liquid
paraffin , dusting powder. fats and grease. Hot air LS
a had conductor at heat and Ltn penetrating power
15 |tr/v- The OKU is usually li elite L! hy dectxidty
with hearing elements in the wall of the chamber.
It must be fitted with a Fid to ensure even
distribution a I air Sind elimination of air pockets
( Fig. 3.1), It should not be overloaded . The [ lucerial
should he arranged so as to allow free circulation
of air in between the objects. Glassware should be
perfectly dry before being placed in the oven. Test
tubes and flasks should he wrapped in paper .
Kubbcr materials, except silicon rubber, will not
withstand the temperature. At 18041 C cotton plugs
may get chaired . Cutting instruments such as those
u&ed in ophthalmic imgrry, should Ideally he
sterilised for two hours at t SO ' C . The British
"
PfadftKHtopotH recommends a holding time ot one
hour at 150 “ C for oils , glycerol and dusting
powder. The oven must be allowed to coo! lowly
for about two hours before the door is opened, sin re
the glassware may crack due to sudden or JiiL- vcn
cooling
* Textbook of Microbiology
SttiriLiaatior control; The spores of a
nontoxigenic strain of Ooftrrdnuzi tetam are used
as a microbiological test of dry heat efficiency. Paper
strips impregnated with 101' spores are placed in
envelopes and inserted into suitable packs. After
sterilisation , the strips are removed and inoculated
Lurulitiuns for five days at 37 ,:Ca
J
A Brownes tube (green spat ) is available For
dry heat and is- eemvenient for routine use* After
proper sterilisation a green colour is produced (after
60 minutes at 160 UC or 115 minutes at 150 °C).
Thermocouples may also be used periodically.
MOIST HEAT
Temperatures below LOO *C
For pasteurisation of milk : Jht milk is ,
heated at either 63 °C for 30 minutes ( the holder
method ) or 72 JC for 15-20 seconds ( the flash
process ) followed by cooling quickly to 13 °C or
lower. By these processes all iionsporing pathogens
such as mycobacteria bruceliac and salmonellac
arc destroyed- CojfjeWa humeri/ is relatively heat
resistant and may survive the holder method.
Vaccines at non sparing bacteria ate heat
-inactivated jjp special vaccine baths at 60 :IC tar
one hour- Set lien or body fluids containing
cnagulable proteins can he sterilized by hearing for
one hour at 56 *C in a water bath on several
successive dlavs
Jr
.
Media auch as Lowcnstcin — Jensen and
LocftlcTs scrum are rendered sterile by hearing at
DC lor half m hour on three successive davs
in an inspissator.
Though practically all mesophilic nonsporing
bacteria are lolled by exposure to moist beat at (iQ “ C
for 3 U minutes, Staphylococcus aureus and
Strcjiwocccuf facetlit require f > 0 m inn ten . A
- temperature of SO °C for 5-10 minutes destroys
the vegetative forma of all bacteria , yeasts and
monMs. Among the most heat resistant cells are
the spores of CfoytriJiurn botulinum which require
Copyrighted materia
 * Sterilisation and Disirof ration » 27

120 *C for fot i r tm Inures , or 100'C tor 330 mi mites

for their destruction. Most virustH are inactivated
Victy rabidly at fcti “ C, but some art relatively
resistsmt, for example , poliovirus and hepatitis 13
virus which may survive for 30 minutes and over
10 hours respectively, it this temperature.
Temperature m HID "{’
IVtiling: Vegetative haeteria me killed almost
immediately ar 90-100 UC, bur sporiog bacteria
require prolonged periods of boiling Hulling in nor
,

'
recommended I D: Sterilising of instruments lined
for su rgicftl procedures and should be regarded only
as a means of disinfection. Nothing short of
autoclaving it high pressure Cad destroy spores and
ensure sterilisation. Hard water should not be used.
Sterilisation may be promoted b) the addition of
29f» sodium bicarbonate to the water.
In rases where boiling is considered adequate,
the material should be immersed in the water and
boiled for 10-30 manures. The lid of the steriliser Fig.. 3.2 Steame#
should not be opened during the peri -. hi. in a favourable medium, will germinate and be killed
Steam til slmosphuric pressure on the subsequent occasions. Though generally
tlGO k An atmosphere of free steam is used to adequate * this method may tail with spores oi
^
' l
sterilise culture media which may decompose if certain anaerobes and thermophiles..
subjected to higher temperatures . A Koch or Arnold
,
Steam under pressures I he principle ot the
steamer is usually used . ( Laboratory autoclaves cart autoclave car steam steriliser is that water boils when
also be used for this purpose.) This is an itsvapour pressure equals that of the surrounding
inexpensive method . I’hc container and medium are atmosphere. Hence when pressure inside a closed
simultaneously sterilised , evaporation from the vessel increases* the temperature at which water
medium Is prevented and the apparatus requires boils also increases Saturated steam has penetrative
little or mi attention. power. When steam comes into contact with a
The steamer consists of a tinned copper cabinet cooler surface it condenses to water and gives up
with the walls duirahlv lagged . " I "he lid is conical , its latent heat to that surface ( 1600 ml steam at
enabling drainage of condensed steam , and a 100 “ C and at atmospheric pressure condenses into
perforated trav fitted above the water level ensures one ml of water at 100 *G and releases 518 calorics
that the material placed on it is surrounded by steam of heat). The large reduction in volume sucks in
( Fig , 3.2 ), A tingle exposure of ninety minutes more steam to the area and the process continues
usually ensures sterilisation hut for media containing till the temperature of that surface is raised to that
sugars or gelatin an exposure of 100 ,:C for 20 of rhe steam . The condensed water enwnes moist
minutes on three successive days is used . This is conditions tor killing the microbes present.
known as tvjuJai riarj'oji oi infemjTtrejj; sreriisaffon.
T
^
The principle is chit the first exposure kills all
Sterilisation by steam under pressure is carried
out at temperatures between 10B UC and 147 ^C.
vegetative bacteria , and the spores, rincc they are By using the appropriate temperature and time, a

Copyrighted material
28 < Textbook oi Microbiology *

variety of material such as dressings instruin^Lnts ,

Laboratory wire, cue tin tod phirtnireLiiicil
products can be sterilised. Aqueous solutions arc
sterilised between IQS 'C and 126 rC . Heat is
conducted through the wills of the sealed
containers until the temperature ol the fluid inside
is the same as that of the steam outside.
Several types of steam sterilisers ire in use:
1 . laboratory autoclaves,
.
2 hospital dressing sterilisers,
3 . bowl and instrument sterilisers , and
4 . rapid cooling sterilisers.
DQQQ
Even the domestic pressure cooker can be used
as a steriliser.
In irs simplest farm, the laboratory autoclave
nimiiii
consists of a vertical or horizontal qdhudeT ofgunnietai
or stainless steel, in a Supporting sheet iron case. The
lid or dour is Mastered hv screw clamps and mg;ic
airtight bv a suitable washer The autoclave IULK on its Fig. 3,3 A simple autoclave
lid or upper side a diR<-hat|£ tap for air and steam, a tend to hoil violently and spill from the container
pressure gauge cod a safety valve that tan be set to and some rimes an explosion may Occur. If opened
blow off ar any desired pressure. I {eating is by gas or after the pressure inside II .L ^ fallen below
electricity (F g. 3.3 ).
^
Sufficient water is pur in rhe cylinder, the
an nospheric pressure, an otce sivr amount of water
*
would have evaporated and lost from the media.
material ro he sterilised is placed on die tray, and The detects in this type o| autoclave arc:
the autoclave is heated. The lid is screwed tight 1 . The method of mr discharge is ineffioent, and
with the discharge tap open. The safely value In it is difficult to decide wln-ii the discharge is
adjusted to the required pressure. The steam - air Complete. If die air is nor comp letch removed,
mixture is allowed to escape freclv rill all the air the desired temperature will nor be attained.
has been displaced . This cun be tested by leading 2. There is no facility for drying the toad after
the escaping steam into a pail of water through a sterilisation ;L[ ]d before t.IIRIINLT ir out.
rubber tubing. When no more air bubbles come The domestic pressure ookcr serves as a
'

out in the pail the discharge tap is closed. The
steam pressure rises inside and when it reaches the
desired set level, the salety valve opens and the
excess steam escapes. From tliis point, the holding
period is calculated. When the holding period is
over, the heater is turned off and the aui^lave
allowed to cool till the pressure gauge indicates
that the pressure inside is equal to the atmospheric
pressure . The discharge tap is opened slowly and
air is let into the autoclave. If rhe cap is opened
when the pressure inside is high, Liquid media will
mininrure autocUre and maybu used for srerilnirLg
small articles in climes and similar csublishmcNh.
A ivude variety of JLIRNCFIVES have been
manufactured incorporating carious devices fur
overcoming these defects and other difficulties in
working.
Sterilisation cofttrab Far determining the
efficacy af moist heat sterilisation,spores offiflerffus
stcurothamephilur are used at the ctsi arganisio,
I his LS a thermophilic otpuniui wirh nn optimum
growth temperature of 55-60 C and irn spores

Copyrighted material
 * Sterilisation and Disinfection »
Table 3.1 Recommended temperature and duration lor heat sterilisation
Method Temperature ( C )
Autoclave 121
1 26
134
Hot air oven 160
170
100
1 90
require nn exjwKLLre if 12 minutes at 121 *C to be
[ potential of Asbestos has discouraged their une.
kilted. Paper strips impregnated with 10* spores
are dried at room temperature and placed in pa ]K- r
envelopes . These envelopes an- inserted in different
p.u'Isi jf tilt luad . After stcrili satin n, the Htripn ire
( Thcv have Low absorptive property and can be
inoculated intti a suitable recovering medium and
incubated for sterility test at 55 aC for five days .
Chemical indicators , autoclave rapes and
thermocouples may also be used instead,
F I LTR VTION
Kilfruritm help* to remove hai teri .i from heat Labile
'
liquids such as sera and solutions of sugars or
anribinth- H LLS«I tor preparation of culture media.
As viran pass through ordinary filters, filtration
can be used to obtain bacrcria-fice filtrates ofdinicaJ
samples for virus isolation, hiker discs also help to
concentrate bacteria from liquids as for example in
testing water aainpLes for vhulera vibrinfR or typhoid
bacilli. Bacteria ] toxins can be obrai]ied by passing
cultures through filters.
The following types of filters have been used.
( Randle filters ; These arc manufactured in
different grades of pnrositv anti have been used
widely for purification of water for industrial and
drinking purposes. They are of two types: (a) Un-
glued ceramic filters (for example, Charnberbm!
and Dimlton lliter ; 0> ) Dialnmaceoti* earth filters
( for example, Berlucfcld and Mandler filters).
They have high adsorbing capacity and tend rn
-
Asbestos tillers arc ditpottblc, single use discs.
alkalinisc filtered liquids . The carcinogenic
29
Holding rime fin mjnufesJ
15
10
3
45
10
7.5
15
Examples arc Seitz and Stcrimat fibers .
Sintered glass filters arc prepared by heat-
fusing finely powdered glass panicles nfgraded sizes,
deafleJ easily but are brittle and expensive .
Membrane fillers made of cellulose esters or
other polymers hive largely replaced other types
of filters. They are routinely used in water
purification and analysis, sterilisation and sterility'
testing, and for the preparation of solutions for
parenteral use. They cwtL in a wide range of average
pore diameters ( APDfi the 0 J 2 mm size being
most widely used for sterilisation.
r
RADIATION
Two types of radiation art used for sterilisation,,
noniunismg and ionising. Infrared and ultraviolet
,
says ate of die rtonionisiuig low energy type* while
gamma rays and high energy electrons are the high
energy" ionising type.
Monionising radiation: Here electromagnetic
rays with wavelengths longer than those of visible
light are used. These are to a large extent absorbed
as heat. Hence infrared radiation can be considered
as a form of hot air sterilisation. Infrared radiation
is used for rapid mass srerilisation of prepacked
items: such as syringe* and catheters , Ultraviolet
radiation is used for disinfecting enclosed areas such
,
as enlryways,. operation theatres and laboratories.
Ionising radiation: X - rays, gamma rays and
cosmic ray? are highly lethal to UNA and other
,
Copyrighted material
- 30 * Textbook
vital constituents . They have very high penetrative
power. Since there is no appreciable increase in
temperature in this method, it is referred to as cold
sterilisation , Commercial plants use gamma
radiation far sterilising irons like plastics, syringes,
swabs* catheters , animal feeds ^ cardboard , oils,
greases , fabrics anti metal toils.
1 ligh energy electron radiation ns a method of
sterilisation in not widely used in medicine .
ULTRASONIC AND SONIC VIBRATION
Ultrasonic and sonic waves arc credited, with
bactericidal powers but the rcs- ults have been
variable. Mirftnrg niuiu vary in their sensitivity
^
tn them and survivors have been round after such
.
treatment Hence this method is of no practical
value in sterilisation and disinfection.
AtJEXTtt
( llTKM U L A l .
Several chemical agents are wed as antiseptics and
disinfectants. However little is known about the
mechanism of action of many of these agents. An
ideal antiseptic or disinfectant should :
have i wide spectrum of activity and must be
effective against all microorganisms , that is,
bacteria including spores, viruses, protozoa and
fungi;
* be active in tile presence of organic matter;
"
be effective in acid -as well , LH alkaline media;
* have speedy action;
* have high penetrating jHivver:
* be stable;
* be compatible with other antiseptics and
disinfectants;
* not corrode metals;
* not cause local irritation or sensitisation;
* not interfere with healing;
* not be toxic if absorbed into circulation;
* be cheap and easily avail able; ard be safe and
easv to use.
Such an ideal chemical is yet to he found .
The factors that determine the potency of
disinfectants arc :
or M crobiotogy
concentration of the substance;
* time of action;
pH of the medium;
* temperature;
'
* nature Ot rhc organisms; and
presence of extraneous material;
Chemical agencs act in various ways. The main
modes of action arc :
1. protein coagulation;
2, disruption of cell membrane resulting in
exposure, damage or Id&J of the Contents;
3, removal of free sulphydryt groups essential for
Line functioning of the enzymes; and
4 . substrate competition : A compound resembling
the essential substrate of the amine diverts or
r
misleads rhe tnymes necessary for the
metabolism of the cell and causes cell death.
Ai . ciiuni s
Ethyl alcohol (ethanol) and isopropyl ulcoltol are
the most frequently used . They sire used mainly as
skin antiseptics and act by denaturing bacterial
proteins. They have no action on spares, To be
effective, they must be used at a concentration of
6( b-9fl per cent in water. Protein slows its action
whereas 1% mineral acid or alkali enhances the
action. Isopropyl alcohol is preferred as it is a better
fit solvent, more bactericidal and less volatile . It is
used far the disinfection of clinic , LI thermometers .
Methyl alcohol is effective against fungal spores
and is used for treating cabinets and incubators
affected by them. The insides of the chambers arc
wiped with liberal amounts of methanol A pad
moistened with rue than > 4 and a dish of water ( to
ensure hi h humidity ) are kept insidiCj and rhe
^
incubator is left at working temperature for several
hours. Methyl alcohol vapour is roxic and
inflammable.
AUD - ll V lilts
Fbimidchj/de is active against the amino group in
the protein molecule . In aqueous solutions, it is
markedly bactericidal and sponodol and also has a
Copyrighted material
 * Stanlisancm ana Disinfection
lethal effect on viruses . It is used to preserve
:i n ; Lh > iiiLL - J sped mens. and for destroying anthiU*
sj>ores in hair and wook 10 formalin containing
0.5% sodium tetraborate is used to sterilise dean
metal iiLHTnjmenrh.
Formaldehyde gas is used for sterilising
instruments and beat sensitive catheters and for
fumigating wards., sick rooms and laboratories.
Uradi i proper!v controlled Conditions, clothing,
'
bedding, forniture and books can he satts.factcariLi'
disinfected.
Tic gas is irritant ami toxic when inhaled.
Surfaces which haw been disinfected by [ his agent
may give off an irritant vapour for some time after
.
disinfection Ill in can be nullified by exposure tea
ammonia vapour after disinfection has been
completed ,
fillutanaldchyde: This has an action similar to
formaldehyde . It is specially effective against
tubercle bacilli , fungi and viruses. It is less toxic
and irritant to the eves and skin tk.ni formaldehyde.
It has no deleterious effect on the cement or lenses
of instruments such as cyito &copes and
bronchoscopes, li can be safely used to treat
corrugated rubber anesthetic tubes and lace mask -- ,
pbstk endotracheal lubes, metal instruments and
pulythene tubing.
I1 v \: s
Two groups of dyes , aniline dyes and acridine dyes
are used extensively as skin and wound antiseptics.
Both are bictfirioftalic in high dilution but are of
low bactericidal activity. The aniline dyes in use
are brilliant tfreen, malachire green and crystal violet.
They are more active agains- l Gram positive
organisms than against (dram negative organisms.
They have no activity against tubercle bacilli , and
fie nee i fie use of malachite green in rite
LoWCt Lite in-Jen sen medium. Though they are
nonirritant to the tissues and nuntoxic, lliev are
considerably inhibited hv organic material such at;
'
pus. Their lethal effectR on bacteria are believed to
be due to their reaction with the acid groups in the
> 31
cell . I he sc dyes arc used in the microbiology
htontny as selective agents in culture media.
The acridine dyes ate mote active against Gram
positive organisms than against Gram negative but
tire not av selective as the aniline dies . They are
affected very Little by the presence of organic matter.
The more important dyes arc proflavine, acriflavine,
cuflavint and aminicrinc. They show no significant
di fferences in potency. J f impregnated in gauze, they
are slowly released in a moist environment , and
hence their advantage and use in clinical medicine.
They ini pair the DNA complexes of the organisms
and thus kill or destroy the reproductive capacity
of the cell.
HALOOFXS
Iodine in aqiKOUl and alcoholic solution has been
used widely as a skin disinfectant . It is actively
bjlCttfitidal, with modflltt action against spores.
Ir is active against the tubercle bacteria and viruses.
Compound* of iodine with nonitmic welting or
surface active agents known HR iodophores arc
claimed to be more active than the aqueous or
alcoholic solutions of iodine.
Chlorine and its compounds have been used as
disinfectants for many years- Water supplies,
swimming pools , food and dairy industries use
chlorine for disinfection . Chlorine is used
commonly as hypochlorites . Chlorine and
hypochlorites are markedly bactericidal. They have
a wide spectrum of action against viruses . The
organic chloramine* are u*ed an antiseptics for
dressing wounds,
PH K .M t l L S
These are obtained by distillation of coal tar
between temperatures of 170 "C and 270 G , 1 ,ister,
the father of antiseptic surgery, first introduced their
usv in surgery [1365). Since then A wide range of
phenolic compounds hare been developed as
disinfectants [ fie Lethal effect of phenols is due to
,
their capacity to cause cell membrane damage,
releasing cel] contents and causing lysis. Low
Copyrighted material
n 4 Texlhook of Microbiology
concentrations ol phenol precipitate proteins .
-
Membrane Wund aakhici and dehydrogenases arc
in activated by concentrations of phenol that arc
rapidly bactericidal for microbes.
Phenol ( carbolic acid ) is a powerful
microbicidal substance , This ami other phenolic
disinfectants derived from coal tar arc widelv used
as disinfectants for various purposes in hospitals,
Lynol anil -L TCHOI are active against a wide range of
organisms. They are not readily inactivated by the
presence of organic miner and are thus good general
disinfectants. These are toxic To humans. Various
proprietary preparations or turmularinns ot phenol
are in wide use . The related chlorophemola and
chlorojtyphenois, though less toxic and irritant, are
less active and more readily inactivated by organic
matter. fforh these gn >ups of substances arc relatively
inactive Against Pseudomonas . Various
combinations of these are used in the control of
pyogenic cocci in mrgicfll and neonatal units in
hospitals. Hcxachtorophene Is potentially toxic and
should be used with care . Chlorhexidine ( Hibitane)
is a relatively non toxic skin antiseptic most active
against Gram positive organism* and fairly effective
against Gram negative ones. A Ueuus solutions are-
used in the treatment of wounds. ^
G \ s i -. s
Ethylene nxidc: Th]^ is a colourless liquid with
a boiling point of l 0.7 *C+ and ar normal temperature
.and pressure is a highly penetrating gas with a sweet
ethereal smell. It in highly In flammable and in
concentrations in a :ir greater than d % highly h
explosive. Ely mixing it with inert gases such is
carbon dioxide or nitrogen, to J Concentration ot
10%, its explosive tendency is eliminated.
It* action is due to its alkylating tint amino,
carboxyl, hydroxyl and stilphydryl groups in protein
molecules . In addition , ir reacts with [ ) N A and
RN A, Its use as a disinfectant presents a potential
1
toxicity rn human beings, including mutagenicity
and carcinogen i dry, Tr is effective against all tvpcs
of microorganisms including viruses and spores.
»
It diffuses rhrnugh many types of porous
materials and readily penetrates some piastres. It is
Specialty used for Sterilising heart— lung machines,
respirators, sutures, dental equipment, hooks and
clothing. It is unsuitable tor fumigating rooms
because ot its explosive property. It has been
successfully used to sterilise a wide range of
materials such as glass , metal and paper surfaces ,
clothing, plastics , soil, some foods and tobacco. It
is Lin [ ant, and personnel working with it have to
take strict precautions,
I' nrniuIds: hyde gusj This is widely employed
tor fumigation of operation theatres and other
rooms. After sealing the window* and other outlets,
,
formaldehyde gas is generated by adding 150 g of
KMnO, to 2E0 ml formalin for every 1000 LLL. ft
.
[ 2 S. 3- cu . ]Ti ) of ruom volume The reaction produces
considerable heat , and so heat resistant vessels
should be used. Alter starling generation of
tormaldrhyde vapour, the doors should be scaled
,rnd left
unowned fur 4S lumrs.
BetapropiuEnctone ( BPL ): This is a
condensation product of kerane and formaldehyde
with a boiling point of 163 *C, Though as a gas it
lias low penetrating power, it is said to be more
efficient tor fumigating purposes than formaldehyde.
It has a rapid biocidal action but unfortunately has
Carcinogenic activity. Tor sterilisation of biological
products 0, 2% BPL is used , It is capable of killing
all microorganisms and is very active against viruses.
-
Si HL ACB ACTIVE AGE: \ TH
Substances which alter energy relationship at
INTERFACEH , producing a reduction of surface or
intei-facial tension nrc referred to as surface active
agents - 'I'hey are widely used as wetting agents,
detergents and emulsifiers . Thcv are classified into
four mid ii groups, anionic, cationic, nonionic and
amphoteric. The most important antibacterial
agents are the cationic surface active agents. These
act on the phosphate groups of the cell membrane
and ako enter the cell . The membrane loses its
sc mi permeability and the cell protein ? are
Copyrighted material
 SierilisaliQP
denatured. The cationic compounds in the form of
quaternary ammonium compounds are markedly
bactericidal , bung active against Gram positive
.
-
organ i ms and ro a lesser extent on Gram negative
ones They haw no action on spores, tubercle bacilli
and most viruses. The common compounds arc
acetyl tr: methyl ammonium bromide (Cetavion or
cetrimide) and benaalfcoftiuin chloride . These me
most active at alkaline pH. Acid inactivates them .
Organic matter reduces their action and anionic
surface active agents, like ordinary soaps , render
them inactive . The anionic compounds, c g, , . determine efficiency of a disinfectant. This is due
common soap , have moderate action - Soaps
prepared from saturated fatty ac kin (such as coconut
oil ) arc more effective against Gram negative bacilli
while those prepared from unsaturated fatty acids
(oleic acid ) have greater action against Gram
positive and Nci -;5cri :i group of organisms . The
amphoteric or ampholytlc compounds, known *4
'
JL ' UO ' compounds, are active agsains-t a wide range
of Gram positive and Gram negative organisms and
some v: Tuses. These , however, are no! in general
use .
METALLIC SALTS
Though all salts have a certain amount of germicidal
action depending on their concentration, salts of
heavy metals have a greater action - The salts of
i I wr, copper and mercury are used as disinfectants.
They are protein Coagulants and have the capacity
to combine with free sulphydryl groups of cell
enzymes, when used at appropriate concentrations.
Further Reading
Block 55 1 :. ! 1991 . Chemical Disinfection in Hospitals, 2 cdn . London, E 'u'. - lu Health Labcuutiiry Service .
'
Gardner JK. and MM - Pcd 1991 . Irrmxknr .' tJil JU i> (trM .irjtjon End Disinfection* !?'1 cdn . Edinburgh ; Churchill Livingstone .
Russell AD er a]. 1992 . I '.- iiiLijikx aid Ptuciiix nl ' Di.^ i '. .- itL-titm , SteriH -ntiitn und PJT*TTVJI 1. . n . 2^ edn . Oxford: Blackwell
Si i -c nr fi < .
and D\ s > 33
Mercuric chloride , once used as a disinfectant is
highly toxic. The <*rg& nic compounds, tfliinmeisal,
phenyl mercury nitrate and mercurochrome, are less
toxic and are used as mild antiseptics and have a
marked bacteriostatic, hut a weak bactericidal and
limited fungicidal action , blivet salts in aqueous
solution have a limited use. Copper salts arc used
as. fungicides.
TESTING OP DISINFECTANTS
There is no single reliable test available to
to the number of parameters which influence
disinfectant activity. Traditionally In such tests
phenol is taken as the standard - In the Rideal
Walker ttM , suspensions containing equal number^
nf typhoid bacilli are submitted to the action of
varying concentrations of phenol and of the
disinfectant be tested. The dilution of the rest
disinfectant which sterilises the suspension in. a
gLven nine, divided by the corresponding dilution
of phenol is stated as the phenol coefficient ( Phenol
.
= 1 ) of the disinfectant This test does not reflect
natural conditions as the bacteria and the
disinfectant react directly without any organic
matter being present . MudificiTumK have therefore
been suggested - In the Chick Martin test, the
disinfectant acts in the presence of organic matter
(dried yeast Or feces). Even this modification falls
short of dinul ? ring naturalconditions . Various other
modifications have been introduced, but no test is
entirely satisfactory.
r
Copyrighted material
 Culture Media
Bacteria haw KJ l >e j invn (cuJnireJj for Them iu
^
be identified , a ? imlv rarely can they he recognised
hy rheir morphology atone. 'Hit study of bacteria
involves the ;tudy of bacterial popukioiu rather
than of single bacterial cells. In tlLt. IUIIILATL and
1
animal bodies, as well as in dthcr natural sounxa ,
bacteria occur as mixed populations. By appropriate
procedures they have to he grown separately
(isofoted } on cufrufif media and obtained as pure
cuftirre? lor study.
Numerous culture media have been devised. The
originLil media used by Luuii PaBttUJ weft liquids
such as urine or meat broth. Liquid media have
many disadvantages. Bacteria grow mg in liquid
media may not exhibit specific characteristics for
their identification. Jt is also difficult to isolate
different types of bacteria drum mined populations,
using liquid media . However, ] ic|UIL ) mediia have
their uses, for example, tor obtaining bacterial
growth from blood water when large ^ illumes
have to be tested , and for preparing hulk cultures
of antigens or vaccines.
While bacteria grow diffusely in liquids , lliev
produce discrete visible growth on solid media . 11
inoculated in suitable dilutions, bacteria form
monies, which are clones of cell * originating from
a single bacterial cell . Oil solid media , bacteria have
distinct colony morphology mul exhibit many other
characteristic features -mil as pi grit en tar ion ur
hemolysis, making identification easy 'flic earliest
, such as riboflavin . Diiterenl brands of peptone show
solid medi nm was c < mktd ,mt pntuti > \ i -^d I'' k nbtrt
'
Kocb Later ho introduced gelatin to solidify liquid
. promoting properties. There may be variations
media but it was pot- satisfactory as gelatin is
liquefied at 24 "C and also by mult proteolytic
bacteria . The use of agar to solidify culture media
was suggested by Frau Hesse , the wile ul one of
tbe investigators in Kocb’s laboratory, wko had seen
her mother using agar for making jellies!
Agar (or agar-agar ) is now universally used
for preparing solid media. Agar is obtained from
some types uf seaweeds , its chief constituent is a
long chain pripardiuidt- It also contains varying
amounts of inorganic Milts and small quantities of a
-
protein like substance. lt has virtually no nutritive
value and is rot affix ted by the growth of bacteria .
Ag ; u is hydrolysed ac high temperatures, at high
acid i > r alkaline ; ill . Irs unique property is that it
melts LIL 98 -'C and usually sets Lit 42 "iS depending
on agar concentration Approximately 2% agar is
employed for solid media. The irl lining property
varies in different brands < if agart for example, New
Zealand agar has more jellifying capacity than
Japanese agar. Agar is manufactured either in long
shreds nr as powder . There may be variations
berween different hatches nf the same brand, apart
from rhi. differences due to a different source of
agar.
Another almost universal ingredient > > I common
media is jjeptune. It is a complex mixture of partially
digested proteins . Its constituents arc proteoses ,
pnlyi >epiiJcs and aminoacids, ; L variety of inorganic
salr * including phosphates , potassium and
magnesium acid certain accessory growdi factors
appreciable differences in romposirion and growth
between different batches of the same brand.
''necial brand? of peptone such as ueopeplone
Copyrighted materia
 •r Cullure Media * 35

In
%
¥ y

Ui

. -
E'ood agnr ; STfiep pyagorr es
showing beti hemolysis

w
Blood agar . Strap pyogenes magnified lo snow
small colonics surrounded try zones of dear hemolysis

m. 'VI
T

&V f
'W f - T_
r

* >

i
i * - _
r *
* *
* -#-
. S *

r w .

r:

0 lwd agar wdh alpha hEmolyltC Blood (eFIunte n;.

: ir showing black colonic
*
vlrkJanS streptococci of diphtheria touiri

Copyrighted material
36 4 TexflDflofe aJ Microbiology

_•

fw/
r
*

P
,
r
f
r
,- . A

/r
nr
^ iv
*vl .{1*^ *%
* i * ,* * * ** * -
i

V 5^ — *
*
*• *
t
\ Ai
Wf * * y

V?
#1 *

' -
f
4
^
X
*
1+
A
B*,
?
* *X
l\ - - *
Vi
\ W5
^
\ MacConkey agar KI 111 Idftji mucoid
Kfefi.pneumortjac colonies

MaeConkey agar wrth smocih pm colonies .

of f scfiwff /ifi coti *
.

/ & i
' v
*
r,/ ar *
H -
-~
-
-
*' > .
I
-- A>
:
j

V
*
* j
i

l
A
j4 r*
- - !- i "
" V'
* , -
r
"l

1
v
- * V
I
. <s 1
"
Si'
Vi
-

TCBS aganvilh green colonies

ft at )i parahemolyTicus
f

Maetonksy agar wilh colonic ?

of £ toff | pmkf and 5 ryui'n i colourless i

Copyrighted material
 * Culture Media *
and proteose peptone- arc available tar special UKS-
be utdL
- Meat
CoTHitK rfially available or digest broth can
jieptunes
extract Ls also available OOmnierdillr
and is known as I J^ Lcmco. Blood, scrum and yeast
extract arc other common ingredients,
Ti V K S
Media have bee it classified in many ways :
1 . Solid media, liquid media, scmisolid media .
2 . Simple media , complex media * synthetic or
defined media, semidefined media, special
media. Special media arc further divisible into:
enriched media , enrichment media, selective
media , indicator or differential media , sugar
media, and transport media.
3. Aerobic media, anaerobic media.
Simple media ( basal mudiab An example
is nutrient broth . It Lunisistsof pep; ' me, meat extract ,
SOdiutn chloride and water. Nutrient agar, made by
adding 2% ugar to nutrient broth is the simplest
and most common medium in routine diagnostic
laboratories . If the concentration of ipr is reduced
to 0.2-0.5%, semisolid nr sloppy agar is obtained
which enables motile organisms to spread. Increasing
the uonue otration of agar to 6% firevents spreading nr
swanning by organisms such as Ptotcus.
Complex rued in: Ine have added ingredients
for special purposes or for bringing our certain
characteristic nr providing special nutrients required
for the growth of the bacterium under study.
Synthetic Or defined (tied in: These media
are prepared from pure chemical tuibfttanoes and
the exact composition of the medium is known.
These are used for various special studies such as
metabolic requirements. Simple peptone water
medium , 1% peptone with 0.5% N »< ’ l in water,
may be considered a scnndcfincd medium since its
composition is approximately known..
Enriched media: In these media, substances such
as hl ( K >d, scrum , W Cgg are added to A basal medium .
They are used to grow bacteria which are more
exacting in their nutritional needs, Examples arc blood
agar, chocolate agar and egg media.
37
Enrichment media: In mixed cultures or in
materials containing more than one bacterium, die
bacterium to be isolated is often overgrown hv the
unwanted bacteria . Usually the ntmpathogenic or
commensal bacteria tend fo overgrow the
pathogenic ones , for example S . typhi being
overgrown by E . coll in cultures from teccs. In such
situations, substances which have a stimulating
effect on the bacteria to be grown or an inhibitory
effect on those to he suppressed are incorporated
in the medium. If such substances art added to a
liquid medium, the result is an absolute increase in
the numbers of the wanted bacterium relative to
the other bacteria . Such media are called
enrichment media, for example tetrathinnate broth
where rhe tetrathlonirc inhibits colilbrm < while
allowing tvphnLd - paratyphoid bacilli to grow freelv.
and Selenite Fbroth tar dysentery bacilli.
Selective media : As in the above case , it rhe
inhibiting substance is added to a solid medium, it
enables a greater number of the required bacterium
to form colonies then the other bacteria, tar example,
desoxychofatc citrate medium for dysentery bacilli.
Such solid media are known as selective media.
Indicator media: These media contain an
indicator which changes colour when a bacterium
grows in them , for example incorporation of sulphite
in Wilson and Blair medium. S, typhi reduces
sulphite to sulphide in the presence of glucose and
the colonies of £. typbi have a black metallic sheen,
Rttassimn tellurite in MtUod 'n medium is reduced
to metallic tellurium by the diphtheria bacillus to
produce black colonies.
Differential media: A medium which has
substances incorporated in it, enabling it to bring
out differing characteristics of bacteria and thus
helping to distinguish between rhem . is called a
differential medium. For example, MacConkey’s
medium which consists oi peptone, lactose, agar,
neutral red and taurochoiate shows up lactose
fermenters as pink colonics, while nonlactosc
fermenters arc colourless or pale . This may also be
termed indicator medium.
Copyrighted material
IS i Textbook of Microbiology *

Some of These terms for medi .L arc
interchangeable. For example , the blood agar
medium i& 10 enriched medium but bacterii ly- mg
nod cells show a clearing around their colonies.
Thus, d is an indicator medium .LS well. There are
many special inedi .L for demonstrating particular
characten -tLCH, like Nagler 's medium which enables
view letirhimase activity.
us to
Sugar media: ' fhc term 'sugar In microbiology
denotes any fermentable substance. They may be:
1. Monosaccharides — a) pentoses, e.g. , araKinose,
xylose, b) hexoscs, c .g,, dextrose, mannose
2. Disaccharide& t e.g., saccharose, lactose
3 - FblyMCL - luridcs, e .g., starch , innIin
4. Trisaccharides, e.g., raffmose
5 . Alcohols, crg„ glycerol, sorbitol
b . Glucostdes, e.g., salicin, aesculln
7 . Noncarbohydrare substances, e.g., inositol.
( such as dysentery or cholera organisms in feces),
special media are devised for transporting the
specimens. These are termed transport media, for
example, Stuart '& medium— a non-nutri ent soft agar
gel cont.1ini:ig1reducing agent to prevent oxidation,
and charcoal to neutral ir- e certain bacterial
inhibitors— for gonococci , and buffered glycerol
saline for enteric bacilli .
Anaerobic media: These media arc used to
grow anaerobic organisms, for example, Robertson's
cooked meat medium.
Various media to test special properties like
urease production, and composite media for
simultaneous demonstration of different features
have been devised . They are dealt with in the
appropriate chapters.
For identifying prepared media, a colour code
i s usually adopted. This depends on the laboratory

The usual sugar media consist of 1% nfthe sugar
iii peptone water along with an appropriate
indicator. A small tube (Durham's tube) is kept
inverted in the sugar tube to detect gas production.
For organisms which arc exacting in their growth
requirements ( for example pneumococcil , Hiss
'
serum sugars arc used . They contain 3% serum.
Transport media: In the case of delicate
organisms (like gonococt D which may not survive
the time taken for transporting the specimen to the
laboratory or may be overgrown by nonpathogens
or group of laboratories, One colour or a mixture
of colours is used on the cotton stopper, or colour
prints are used on the caps.
Culture media used TO be prepared in
laboratories themselves , starting w i t h b a s i c
ingredients. Not only was this laborious but it also
led to considerable batch variation in the quality of
media, With the ready availability of commcrci d .
dehydrated culture media, the process of media
making has become Simpler and its quality more
uniform.

Further K end in a.
Atlaa PM . I 1.C Plaits 1999 . iVfnTofiwfcypni? Media, Maryland: CRC Pews,
Crci .:Wiaflt ti * 1 aL. 1975. Malicil MiiTvbihhjry Vol. II, 77« Practice afMedicai MiirobiologyA 12"' edn. London:
t hun- hill I .iv: MptHinj;.
AfjflUjt? tti f>*frnMiwd CWturc Media and ffej n-Ty, 1977, 9* h edn, Michigan: Difco Laboratories.
^
Oxoid Manual 1976.3rd edn. Basingstoke: Ojtoid [ h i .
Murray PR et a] 1999. AfanuaJ of Opnical Aftcmbtcdcg}-: edn. Washington I>C: ASM Press.

Copyrighted materia
 to Culture Methods
Culture methods employed depend on the purpose
fix which they are intended. In ihn clinical laboratory,
the indications -for culture are mainly to:
1. isolate bacteria in pure culture;
2. demonstrate their properties;
3. oh ruin sufficient growth fur preparation oi
antigens and tor other tests;
4 . type isolate* hy methods HiK h as bacteriophage
'
and haercriocin susceptibility;
5. determine sensitivity to antibiotics;
6 . estimate viable counts; and
7 . maintain stock Cultures.
The methods of culture used ordinarily in the
laboratory are the streak lawn , stroke, stab, pour
plate and liquid cultures . Special methods are
employed for culturing anaerobic bacteria
The jTreaJ; cuff tin? ( surface plating} method is
routinely employed for the isolation of bactena I:I
pure culture tnorn climca] Specunens. A platinum
loop is charged wirh the specimen to be cultured.
Owing to the high cost ot platinum* loops for
routine work are made of Nichrome resistance wire
{ 24 SWG size ) . One loopful of the specimen is
transferred onto the surface of a well dned plate ,
on which it is spread ovci a small area at the
periphery . The inoculum is rhen distributed thinly
'
IWIT the plate hy -streaking it with tile loop in 1
series of parallel lines-, in different segments ot the
plate . The loop should be flamed and cooled
between the different w:ts of streaks . On i neubitton,
growth may be confluent at the site of original
inoculation , but becomes progressively thinner, and
well separated colonics are obtained over the find
scries of streaks.
The hwn or carpet cu turr provides a uniform
^
surface growth of the bacterium and is useful tor
bacteriophage typing and antibiotic sensitivity
testing (disc method ). It may also be employed
when a large amount [if growth is required on Solid
media as, for instance, in the preparation of bacterial
antigens and VUdlKL Lawn cultures are prepared
by flooding the surface of the plate with a liquid
culture or suspension of tbe bacterium, pipetting
off the excess inoculum and incubating [ be plate.
Alternatively, the surface of the plate may be
inoculated by upplyi ng a swab staked i n the bacterial
culture or suspension,
Tbe stroke ciJnire is made in tubes containing
agar atnpe (slant) and is employed for providing A
pure growth of the bacterium tor slide agglutination
and other di agnostic tests.
htifh cultures arc prepared hy puncturing a
suitable medium such -,is nutrient gelatin or glucose
agar with a long, straight , charged wire , The
medium is allowed to set, with the rube in the
upright position, providing a flat Surface at the top
of the medium. Stab cultures are employed mainly
tor demonstration of gclati n 1 iquefactinn and oxygen
requirement of the bacterium under study. They
ate also used in rhe maintenance of stock culturcs.
For preparing pour plate culture , tube*
containing 15 ml of the agar medium are melted
and left to cop] in a water bath at 45- 50 :: C,
Appropriate dilutions of the inoculum (of I ml )
'
are added to the molten agar, mixed well and the
concent* of the rubes poured into sterile Pfctri dishes
and allowed to set. Alter incubatioin, colonies will
be seen well distributed throughout the depth of
Copyrighted materia
40 i Textbook d Microbiology »
i In-medium and can be enumerated U - . MIL' colony
counters. The pour plate method gives an estimate
of the viable bacterial con nt in a suspension and is
the recoin mended method for quantitative urine
cultures.
In the sweep plate method, the edges of the
Petri dishes containing the culture medium arc
robbed over the fabric, with the medium ft- ing it.
'HlC dust
particles shired up Irom the cloth settle
on the culture medium , and colonics develop on
incubation. They can he counted and estimates
made .
Liquid cu/rtm*s in tubes, bottles ot flasks may
he inoculated bv touching with a charged loop or
by adding the inoculum with pipettes or syringes.
Large lima:l.i can be employed in /.quid Cultures
and hence this method is adopted lor blood culture
and for sterility tests, where the conccntrcuion of
bactcria i n the i -locul i is expected to be sm all. Liquid
cultures are preferable for inocula containing
antibiotics and other inhibitory' substances, as these
arc rendered ineffective by dilution in the medium .
Liquid cultures are also preferred when large v n I
are desired, the yield being enhanced by agitation,
acral inn, midi Mon of nutrients and removal of toxic
metabolites (contnuous culture methods) . 'lTic
maim disadvantage of liquid culture i. s that :l does
not provide a pure- culture from mixed inocula.
AN II -. HCUIIC CUI Tl METHODS
RE
Anaerobic bacteria d:irter in their requirement of
and sensitivity to oxygen. Some , such as Cl .
Ajjtujyfjcrml, are aenotoletant and may produce
some growth on the surface of aerobic plates, while
others such as Cl- temni, are strict anaerobes and
form surface growth only if the oxygen ter -inn is
less than 2 mmHg. A number of methods have
been described for achieving an aerobic -- is, hv
exclusion of oxygen or production of a vacuum ,
displacement of oxygen with other gases, absorption
of oxygen by' chemical or biological means, and
reduction of oxygen.
1. CulLvation in vacuum was attempted by
incubating cultures in a vacuum desiccator, but
the method is unsari -. factory as some oxygen is
always left behind. Fluid cultures may hoi I over
and the media mav get detached from the plates
in the vacuum produced- This method is not in
use now.
.
2 Displacement of oxygen with gases such as
hydrogen, ni ' togcn, helium or carbon dioxide
is somerimcH employed, hilt this method rarely
produces complete anaeiobiosis. A popular, but
ineffective method is the candle jar . Here
inoculated plates are placed inside largea
airtight container and A lighted an idle kept in
it betore the lid is sealed. The bum : ng candle is
expected touse up all the oxygen inside before
:l is extinguished, hut some oxygen is always
left behind . The candle iar provides a
concentration of carbon dioxide wh : ch
stimulates the growth of most bacteria.
3 . In the chemical or biologic J method, alkaline
pyrogallol absorbs oxygen . Thi - method, first
introduced by Buchner ( ISflE ) has been .
. employed vnh different modifies irons for
providing anaerobinsis . Pymgallic acid added
to a solution of sodium hydroxide in a large teat
tube placed inside an airtight jar provides
anaernbiosis hut a small amount of carbon
mnnnridc, which L formed do ring the reatriun,
mav be inhibitory to some bacteria . The method
m a!
h. i- I 'i.- '.Tl applied to single tube and pla te cuitures.
The Spray anaerobic dish is a glass dLh with
its bottom partitioned into two halves, tlie cop
accommodating hall :rf a lhetri dish carrying the
medium. Pyrngallic acid and sodium hydroxide
arc placed in the separate halves at the bottom
of the dish . The inoculated culture plate is
inverted on the top of the dish and LK sealed
completely. The dish is [hen rocked to mix the
reagents, producing anaerobinsis . The anaerobic
dish is not in use now.
A si mplc modihcation eons Lfs of a Pctn dLh,
between the two halves of which is inserted a
metal disc of slightly larger diameter , wnh a
LJ opy righted materia
 * Culture Methods *
hole in the centre, The metal disc in attached to
tbe bottom lialfof the Pfeiri dish with plubdne.
Through the -central hole , i few j'cllets ol sodium
hydroxide and 10 nil of . 1 10% solution of
H
pyrogallic acid -are added. The inoculated half
of the Petri dish ! H then inverted < m the metal
disc and sealed tightly.
The method in common use employs A disc
of filter paper having the same diameter as a
lVtrii dish. If is placed on top of one half ot the
dish and a mixture of pyrogallol and sodium
carbonate, in dry powder form, is spread on it.
The inoculated plate is inverted over the filter
paper and scaled tight with molten wax . The
dry pyrogallol mixture in activated by the
moisture within the closed syFCfim, and complete
macrobiosis develops within about two hour^ ,
[ plates, and the lid damped tight. The outlet tube
Instead of alkaline pyrogallol, anaerobioau
has been produced within jars with ,1 mis hire
ot chromium and Sulphuric acid ; Rosenth .il
method ) or with vtdlow phosphorous.
Absorption of oxygen from small closed
systems has been attempted by incubation along
with aerobic bacteria , germmating >eeds or
•t A l Y f
in . FT
woer
FL FllTF cj
ltHMlUftLS CXIAiVST
.
Fig 5.1 Mclntesh-FUdes jar
41
chopped vegetables. Aimerobiosis produced by
such biological methods is slow and ineffective.
The most reliable and widely used anaerobic
-
method is tlte Mclntosh Filded MiiMemhie jar
( Fig. !i. l ) . It consists of a stout glass Dr metal
jar with a metal lid which can be damped air
tight with a screw. The lid has two tubes with
taps, one acting as the ga* inlet and the other «
tlie outlet. The lid also has two terminals
which can be connected to an electrical supply.
Leading from the terminals and suspended by
stout wires on the underside of the lid is a small
GROOVEL! porcelain spool around which is
wrapped a layer of palladiniscd asbestos.
Inoculated culture plates are placed inside the
jur, with the medium in the bottOfll half of the
is connected to a vacuum pump and the air
inside is evacuated. The nutlet tap is then closed
and the inlet tube connected to a hydrogen
supply After the j .u i -s filled with hydrogen, the
electric terminals arc connected to a current
supply so that the ptlhdmised asbestos is heated.
This acts as a catalyst for the combination of
hydrogen with the residual oxygen present in
the jar. This method ensures complete
anacrobiosis but carries the risk of explosion,
which may rarclv occur. This risk can he
eliminated by modification of the catalyst .
Alumina pellets coated with palladium Ju a
gauze sachet suspended tnom the lid of the jar
act as a catalyst at room temperature, at long as
the sachet Ls kept dry.
Tbe Gisynk. is now the method of choice
for preparing anaerobic jars . The tiaspik. is
commercially available as a disposable envelope,
Containing chemicals which generate hydrogen
and carbon dioxide on the addition of water.
After the inoculated plates are kepi in tlte jtt,
rhe Oaspak envelope , with water added , is placed
inside and the lid screwed right . Hydrogen and
carbon dioxide are liberated and the presence
of 1 cold catalyst in the envelope permits the
Copyrighted material
42 i Textbook of Microbiology *
combination of hydrogen and oxygen to produce
an anaerobic environment . The Gaspak is
simple and effective, eliminating the need for
drawing a vacuum and adding hydrogen .
An indicator should be employed for
vet dying the anaerobic condition in the jars.
Reduced methylene blue is generally used for
this purpose . It remains colourless anaerobically
but turns blue on exposure to oxygen.
4. Reduction of oxygen in the medium is achieved
by the use of various reducing agents, including
1% glucose, 0.1% thinglyoolate , 0.1% ascorbic
a < id and 0,05% cysteine , Broth is an easily
prepared anaerobic medium imo which pieces
of . red hot mcCallii: iron are introduced , It is
then fctvertd over with sterile vaseline - Rrnth
containing fresh animal tissue, such as rabbit
medium ) r supports the growth of many
-
kidney, spleen, testes or heart (Smirh Noguchi
anaerobes.
Thiogiycolatc broth with hemm and vitamin K
is an enriched liquid medium for culturing
anaerobic and microaemphi I ic bacteria .
Addition of a small quantity of agar enhances
the anaerobic capacity of the medium byslowing
the diffusion of oxygen in it.
Robertsons cooked meat medium is probably
the most widely used fluid medium for the culture
of anaerobes. It consists of fat - free minced cooked
meat in broth, with a layer of sterile vaseline over
it . lr permits the growth of even strict anaerobes
and indicates rheir saccharolytic or proteolytic
activities, b ^ the meat being turned fed or blade,
respectively.
For fastidious anaerohes , particularly for
quantitative cultures, pre- reduced med: . L and an
anaertibic chamber ('glove box’ ) may be used . The
anaerobic chamber is an airtight, glass - fronted
cabinet filled w:lh inert gas, with an entry luck fur
the introduction and removal of materials, and
gloves for die bands.
METHODS bt ISOLATING Puna
Cl LTURES
The following methods may be employed for
isolating pure cultures of bacteria fmm mixtures:
1. Surface plating is the method routinely
employed in clinical bacteriology and enables
the isolation uf distinct colonics which may he
picked out, :.f necessary' for further purifieation
and study .
.a
2 Enrichment, selective and indicator medin are
widely used for the isolation of pathogens from
specimens such as feces, with varied flora.
d. Pure cultures may be obtained by pre-treatment
of specimens with appropriate bactericidal
suh stances which destrw the unwanted bactcria.
This method is the standard practice for the
isolation of tubercle bacilli from sputum and
other clinical specimens, by treatment with
alkali, acid or orher Substance* to which must
commensals- ate suucepLible but tubercle bacilli
-
are rc istant.
4 Obligate aerobes and anaerobes may be
.
separated by cultivation under aerobic or
anaerobic conditions. Shake cultures in Veillon
tubes were in use formerly but are now obsolete.
This consists of a glass tube open at both ends.
One end is closed with a rubber stopper and
molten glucose agar in which the inoculum is
evenly dispersed is poured into the tube and
allowed to set in a vertical position. The top of
the tube is closed with a cotton plug . On
incubation , the bacteria in the inoculum
differemiace depending on their oxygen
requirement - The obligate aerobes grow at the
top and the anaerobes at the but tom . while the
facultative hactefia grow throughout the
column . The entire medium can be extruded on
to a plate and the different colonics fished out.
5. Separation of bacteria with different
temperature optima can be effected by
incubation at different temperatures. Only
Copyrighted materia
 * Culluro M -alhods * 43

thermophilic bacteria grow at 60 “ 'CL A mixture
containing N* meningitidis and iV.
can be purified by incubation at 22 *C vdhen
only the latter gram.
A. Ry hearing a mixture containing vegetative and
spore forming bacteria, at 80 *G the former can
be eliminated . This method is useful for the
isolation of tetanus bacilli from dust and similar
sources.
7 Separation of motile from nonmotile hacteria
r ,
can be effected using Cnigie s tube. Tlxis conriits
of a tube of scrnisolid agar,. with a narrow tube
open at both ends placed in the centre of tile
medium in such a way that it projects above the
bacteria alone traverse the agar and appear at the
top of the medium outside the central tube.
A IJ - tuhe also serves the same purpose, inoculation
being perfumed in one limb and the subculture
taken from the other. This method can also be
used to obtain phase variants in Satmundla species.
8. Pathogenic bacteria may be isolated from
mixtures by inocidatian into appropriate animals -
Anthrax bacilli can be distinguished from other
aerobic speculating bacilli by inoculation into
mice or guinea pigs. Anthrax bacilli produce a
fatal septicemia and may be cultured pure from
the bean blood.
9 Bacteria of differing sizes may be separated by
?

Level of die medium. The mixture is incwrulated the use of selective filters. Fdters are widely used
into the central tube. On incubation, the motile for separating viruses from bacteria .

Further llvudmji
CoILceJG *
ei il . 19%. PlracticjJ Medical Microbiology. 4 edit. , Edinburgh: Churchill Livingstone .
Murray PR ct d . 1999. Manual of Clinical Microbiology. 7" cdn. Washington DC: American 5-ociety of Microbiology
*

Copyrighted material
 Identification of Bacteria
Once a bacterium has l >ren obtained in pun.' culture
it has- to be identified.The following characteristics
have to he studied in the proccss.
, microscopy all help in these studies,
MLIRPHOI nf . l
The morphology of the bacterium depends on a
number of factors auch at- the st ^iin studied , nature
of the uilmre medium, temperature and time of
incubation, age ot the culture and the number of
subcultures it htis undergone , The characteristics
noted arc shipe, size, arrangeme nt, mot i I i ry, fl agdin ,
sports and capsules. All these cannot he made our
in a single medium. The shape may he spherical ,
filamentous, rod shaped, comma shaped or spiral.
The axis of the organism may be straight or curved .
The length and breadth may vary. The sides of the
organism may be parallel , eonvex, concave or
irregular. The end* may be cut straight rounded or
, characteristics helps in preliminary idcJltiticiLtion
tapering. Considerable variations in shape iind size
leading to club, navicular and swollen < > r shadow
or giant forms may be seen. They may be arranged
singly. ; u pairs, in tetrads or in packets of eight , or
in chains, shaft or lung , in the ease uf cocci; bacilli
may he arranged at random, in short long chains ,
in Chinese letter patterns, .is palisades or in bundles ;
vibrios may he single or in or spiral fbrrn ^ They
,
may be uonmotile, sluggishly motile, actively motile
O r may exhibit darting motility. They may be
without flagella, that is atfichitc, or raonotrichiite,
lo]ihotrichutcr littphi trie hate or peritrichane. The
spores, when present may be oval or spherical or
ellipsoidal and may be of the same width or wider
th n that of the badllary hrxly. The spores may he
^
equatorial , suhtcrminal or terminal t \ipsulcs may
, papillate
, or may rot he present . Hanging drop preparations ,
, lark gnou nd i I Lum i na tinn, phase eon trust or electron
S H I M M . Kb U T I O I M S
The age of the culture is important . In older
aiImres , staining characteristics eichei vary or ate
nol hroughr out well . Simple stains bring out the
morphology best - Differential and special stains ait
necessary to bring out characteristics like flagella ,
capsules, spores and metachromatic granules. The
( i ram stain divides bacteria Into linam positive and
( Jnnn negative; the Zichl-Neclscn stain into acid
fast and non-add fast . The fluorescent antibody P
technique enables one to identify them according
to their surface antigens.
1 'ic study of morphology and staining
of the isolate .
Clit .i l : itAL C n AH v c r i '. K i s I i c s
These provide additional information for the
identification of the bacterium. The characteristics
re veiled i n dlffene nt type > of media are noted. Wh i le
studying colonies on solid media, the following
features nrc noted ;
—
1 . shape circular, irregular, or rhtzoid ;
2 . size itt millimetres;
—
3- elevation ettosc , elevated , convex, concave,
umhonate or nmbilieate;
—
4 . margins bevelled or otherwiset
—
i . surface smooth , wavy, rough , granular ,
or glistening;
Copyrighted material
 H Identification d Bacteria
6. —
edges cut Ire, undulate, erenatedf fimbriate or
curled ;
7 . colour;
—
S . structure opaque, translucent or transparent
9 . consistency— membra nous , triable , butyfOUJ or
viscid;
LII.emubiliability; and
1 Lwhether rhev are differentiated into a central
and a peripheral portion.
In a stroke culture, note
—
1. the degree of growth scanty, moderate, or
profuse;
—
2 their nature discrete; or confluent, filiform,
. 0.5 ml KOVILS reagent and shake gently. Red colour
spreading or rhizoid;
.3 . their elevation , surface , edges , colour, structure ,
"
odour, cnni I stability, consistency and changes
in the medium.
In , L fluid medium , the decree of growth ,
presence of turbidity and iti nature, presence of
deposit and its character , nature of surface growth
such as pellicle and irs quality and case- r > t
disintegration, and odour arc noted.
Ill - SISTAMCK
The resistance of the organism lo heat and to
disinfectant is tested, both for vegetative and spore
forms. The resistance of S . faecalis beat at 60 'C
for half nr hour and of clostridial sports to I Milling
for various periods arc examples . Resistance to
antibiotic and chemotherapeutic agents and
.
h LL tcnocins would also help in ditfrrenriatinn and
identification .
M MUKOI ISM
The requirements of oxygen , the need r < > r carbon
dioxide , the capicitv to form pigments, and the
production of hemolysis help in classification .
KlUtHKMA'lUW Ol' Hbh
-
Ii 11 L : 111. MJCAI . PmiritHTlhH
The more important and widely used lests are
described below:
I . Sugar fermentation: This is rested in sugar
45
media . Acid production is shown by change in the
colour of the medium to pmk or ted , and the gas
produced collects in Durham’s tube.
2 . Litmus milk: There may he no change in
the medium, or acid oi alkali maybe produced;
Hotting of milk, peptortisabort or s .Lpmnficul n ML may
occur . The clot may be disrupted by the gas
produced {stormy fermentation ).
3. Indole production: This is tested in a
peprone water culture after 48 or 96 hours
incubation at .17 IC, This rest demonstrates the
|
production of indole from tryptophane. Add
indicates a positive reaction. Kovac 's reagent
consists of
PartdimerhvlamiTiuben / aldehycle 10
Amyl or isoamyl alcohol ^
ISO rnl
Concentrated HCI 50 ml
.
iTkis is prepared small quantities and stored
in
in the refrigerator.
4 . Methyl red lest ( MR ): This test is
employed to detect the production ot acid during
the fermentation or glucose and the maintenance
of a pH below 4, 5 in an old culture. Five drops of
0,Cl 4'hi tulutiuii of methyl red are added to rhe
culture in gfuCn-se phosphate medium which had
been incubated at 30 “ C for five days, mixed well
and read at once . Red colour is positive while yellow
signifies a negative test.
-
5. Voges Prishatter test ( V J 3 >: This test
depends on the production of acetyl
jnethyleirbinol from pyruvic acid , as an
intermediate stage in us conversion to 2:5 butylene
glycol. In the prc^- nce of alkali and atmospheric
oxygen, the small amount of acetyl rncthyl carhuiul
present itl chi; medium is oxidised to diucetyl which
reacts with the peptone of the hmth to grit a red
colour.
I he test is performed by adding O.n ml of a 5%
'’
solution of < r - miphthol in ethanol and 0.2 ml of
40% K.OH to one ml of a glucose phosphate
medium culture of the organism incubated at 30 't.’ '
fur five days or 57 H C I nr 48 bouts. In a positive
|
Copyrighted material
46 H Taktbo& k o\ Microbiology
reaction, .i pirtk cal Du r appears in 2 -5 RliAUieE,
(tccpcniriff fo magenta or crimson in half an hnur.
J ]i a negative reaction, it remainscolourless for half
in hour. Traces of pink colon ration should he
ignored,
fr. Citrate utilisation: Koser 's citrate medium
has citrate as the sole source of carbon . Abilin to
use this substance is indicated hv the production nt
turbidity in the medium.
J
Indole f MR „ VP and ci trace Cents are very uncl d
'
in the identification and classification of enteric
Grim negative bacteria. These rests are commonly
referred to by the sigfa IMVJC' tests.
7 . \ itralt reduction: This is rested after
growing the bacterium for five days at 37 aC in a
broth containing 1% KNOv The test reagent
consists of J mixture of equal volumes of solutions
of sulpharilii acid and n - naphthylaminc in 57 Not
'
acetic acid mixed just before use. 0.1 nil of the rest
reagent is added to the culture. A red colour
developing within a few minutes siginiiles a
positive reaction ,, while absence of colour indicates
a negative reaction. This i.s a test fat the presence
of the HByme nitrate reductase which reduces
nitrate to nitrite .
8 . Production of ammonia - To a peptone
water culture grown for five days nr 17 ®C, Messier 's
reagent is added brown colour is positive and Hiint
, area turns dark in 10 seconds. The solution should
yellow colour negative .
.
9 Urease lest : This test is done in
Christensens urease medium . Inoculate the slope
heavily and incubate ^ t 37 "C , Examine after four
hours and after overnight mentation. The lest
should not he considered negative till after four
days of incubation. Urease positive cultures produce
a purple pink colour. Urease producing bacteria
reduce urea to ammonia which is responsible for
the colour.
1 (1. IlydrogcTL sulphide pn nluetii m: Some
organisms decompose sulphur - containing
aminoacids producing H S among the products .
When cultured in media containing lend acetate,
they turn them black or brown , Instead of lead
feme ammonium citrate or ferrous acetate
jeetite ,
can be used. The organisms can he grown in
culture tubes. Between the cotton plug and the
tube insert a filter paper strip soaked in 1 Q% lead
acetate Solution and dried. Browning of the paper
indicates H;S production.
31 . Methylene blue reduction: One drop
of 1% aqueous methylene blue in added to the
broth admix, and incubated at 37 “ C . Complete
deculourisation ia strongly positive , while green
colour is wealdv positive .
12 . Catalase production: Place a InopfuL
11, 0 , on colonies on nutrient agar. Prompt
effervescence indicates catalase production .
Culture media containing blood are unsuitable For
rhe test as blood contains catalase.
Id . Oxidase reaction: This reaction is due
to a cytochrome oxidase which catalyses oxidation
of reduced cytochrome by oxygen . A 1.0- 1.5%
solution of retramethyl p - phenylcnc diamine
hydrochloride is poured over the colonies. Oxidase
positive colonies become maroon, purple and black
L ] i 10-3(1 minutes. The test can also be dime by
Kovac's method. A strip of filter paper, soaked in
rhe oxidase reagent is placed in a Petri dish and the
colony io be tested is smeared on the paper in a
line about 5 mm long. In a reaction the smeared
be freshly prepared .
14. Jigg ynlk read ion: Organisms producing
Lecithinase { for example, CL pcrjringena ) when
grown on a solid egg yolk medium, form colonies
surrounded by a /due of clearing.
15. Growth in presence of K(!MJ. Buffered
Liquid medium containing KCN in a final
Concentration of about 1 /13,000 is used r-n identity
sonic KUN tolerant enteric bacilli .
-
lb . Comp oiite media are being used
increasingly for the identification of isolares. These
are convenient and economical , as a single composite
medium inti Lea res different properties of the
bacterium which otherwise would have required
rhe LISC of many separate media . A popular
Copyrighted material
 * idem INCH lion oJ Bacteria > 47

composite medium is the Triple bugar Iron (TSl )
medium which indicates whether a bacterium
ferments glucose onlvi or lutOK and sucHrae also,
with or without gas formation , besides indicating
HjS production as well . The medium is
distributed in Cubes, with a hurt and slant. After
inoculative]., it the slant remains red and the butt
becomes yellow, all die sugars - glucose, lactose
and sucrose - arc fermented , Bubbles in the butt
indicate gas production und blackening of the
medium shows formation of 11 5 , The TSI
^
medium facilitates prelim in an identification
' qf
L Irani negative bacilli .
Other tests such as fermentation of organic acids,
oxidation of gluconate, ammoacid decaHtoaylation,
and hydrolysis of sodium hippumre arc sometimes
employed . With increasing knowledge of the
metabolic processes in the growth of various
bacteria , the number of rests too is on the increase .
Special manuals have to be consulted for the derails
and utility of these tests.
Antigenic Structure; FSy using specific sera
we can identify organisms by agglutination or other
suitable serological reactions. Immunofluorescence
test is useful ill SOfOt CUtJ
- for identifying microbes.
Bacterinphage and bacterincin typing:
These enable intnuspedes typing of some bacteria ,
Pathogen icily : Ptthogentcity teats by
inoculation of the test organism into laboratory
animals like the guinea pig, rabbit, rat ud mouse
by intraderina ], subcutaneous , intramuscular,
intraperituneul. intracerebral ur intravenous, Or by
oral or nasal spray were common procedures for
identification nf isolates in the past , They arc rarely
used now because simpler in virro tests arc available.
RAPII> IDENTIFICATION METHODS
While classical phenotypic characterisation of isolates
takes days , auromared merhods are now available
which only take hours. liLenrificariun iv simplified
by the detection of specific enzymes, H>x»isf antigens
or metabolic end products of the isolates. For
example , many obiigate anaerobes can be identified
rapidly by jps liquid chrumaloi raphy of tlie short
^
chain fatty acids pr-twluced bv ibem during glucose
fermentation. Molecular methods such as polymerase
chain reaction and other amplification procedures
coupled with nucleic acid probes carrying specific
DNA or RNA base sequences arc now widely tised

Further heading
Cowtn ST ed, 1978. f VtnMfcy fifMcdictl 'ft aotnji London Oxford l Inivttiiiy Ptuxx ,
^
Cowan ST and KJ Srcd 19fi 5. AJanuaJ /ur the Identification of MotfkxJ Bacteria . London . Cambridge UnntrsiTV Press .
Flnegold SM and EJ baron 19 B 6-. BJUJCJ and Sojff 's Zftj nosrjL’ Bacterioiogi . 7* edn Si . Louis: C Vr Mosby.
^
'

{'ruodfetlmv M and RG board cd . 1 60 . M o n M o J u i o i J m d l i l e i i l i S l i r j d n. London: Academic Frws.

^
Murray P tr al . eds. 1999. Manual o f Clinical Mienobiok y. 7"* edn . Washington: American Society for Microbiology.
^

Copyrighted material
 Bacterial Taxonomy
-
Bacterial taxonomy or t vstematic - comprises three
1
components :
1. Classification , or The orderly arrangement ol'
units. AgroupofunifsiscaHei J t^.vriiT (pl r. .vaj,
j
irrespective < >r its hie muchic level .
'
2 . Idrn[ificar ion of an unknown L w i r h a defined and
named unit . And
3 .
Nomenclature, or the naming ot units .
For purposes of classification, ii is necessary to
determine ah many characteristics of bacteria as
possible. Such characteristics may be weighted,
greater importance luring given to some than to
others, or they may he assigned et|uaJ importance,
depending on the method ot dassilical . m. Or ( he
'i
contrarv, fur purposes ol iden titling bacterial
isolates, IT LS important to devise a ket using the
minimum number of important characteristics
which can be easily tested.
IWlcnal claBliIlealion: The first attempts
aT bacterial classification ( Mueller 178b ;
iihrenberg 1838 ) were made when little was known
about bacteria . Haeckel (186b ) classified ail
unicellular organisms as Protista. Cohn (1872 75) - higher forms id life , a species uftil constitutes a
made a morphological classification, integrating
-
bacteria with the hlne grcen algae in the class
Schixopbyta. A detailed system of classification hvas
-
projxjsed by Mignis ( L 89I) . As more information
became available on the pbvsiologLc .il and
biochemical properties of bas teria , those were
employed in proposing new systems of bacfrrial
classideation by Knight ( 1936 ) , KJuyver and van
Niel (1936 ) and others.
Bacterial classification, presents special pnohlcms .
Linnaeus (173 ? ) divided ail living beings inLo two
kingdom?, plant and animal . Bacteria bad been
placet! in the plant kingdom and designated as
Schizomycctcs ( fission fungi ). But as bacteria
present feahI res common to btJfb pin nts 5 m! a rumal S,
it has been proposed that a new kingdom, \ loners,
he created to accommodate .LLI microorganisms
without true nuclei , pi as rids and sexual
reproduction (Stanicr and van Niel 1541) , This
proposal has not met with universal acceptance.
K i ngdoi ns are diw!«l tueccuively i ntn * tivi sion,
class , order , family tribe, genus and species. For
example , ihe full taxodomical position of the
typhoid bacillus is as follows:
Division Prott .yph f-ta
Class Schiznmyccrcs
r
Order £3rbirterr /ftf
Ffluilv
*
En fcrtjfHtCfcriaCCae
Tribe Salmonc UtC
frcmu Salmonella
Specie s Salmonella typhi
The species concept in bacteria; Species
is the standard taxonomical unit in biology. With
stage of evolution , ivitli a characteristic morphology,
and is delimited by the failure of interbreeding
uuls idc llle u i Lit - IIu ( in bacteria, tile spedH coi Luept
is vagu L- and i11 dei i ned. 1 hit to the Lihsence of fnssiI
remains in bacteria , the evolutionary status ot
species cannot be established . Morphological
differences are insufficient fui the definition of
bacterial species. The general absence of sexual
reproduction in bacteria prevent ? the use ot
inbreeding as a test tot differentiation between
species .
Copyrighted materia
 1 Bacter'ial
In spite of these difficulties, the concept of
specIcs provides a convenient unit in bacterial
taxonomy. Betide* morphological features, criteria
useful for the definition of bacterial species arc
physiological , biochemical , antigenic and
pathogenic properties . As Species' is a genetic
concept, definitive information can be obtained by
comparison of the nucleotide base ratios, which
are constant far ary OAC species but may he difiercnt
in different species. Generic homology can be
demonstrated by 1> NA hybridization between
different individuals of the same species .
Comparison of rRNA sequences helps ro arrange
bacterial species into a phylogene tic tree. 16SrRNA
sequencing ha* emerged as useful tool for
identifying many new unculturahle pathogens (e g .
BartnneHa henselae ) . cited , is Hot an essential and permanent
An important difference between tbe
classification of bacteria and that of higher
organisms is that in the former, the properties of a
population am studied , and not of an individual. A
population derived by binary fission from a single
cell is called a clone . A single bacterial colony
represents a done . Though all the cells in ; L clone
arc expected to be identical in all respects, a few of
them mav show differences due to mutation. A
Jr
population of bacteria derived from a particular
source, such as a patient, is called a strain.
The general absence of sexual reproduction in
bacteria serves to maintain their character constant -
But bacteria possess several features that contribute
to some degree of heterogeneity in then populations.
Their short generation rime and high rate of mutation
lead to tile presence, in any j'opulatLun, of cells with
altered character^. Methods ofgenetic exchange such
is transit in i tar mi i , transduction and conjugation cause
diflerertces; in cliarucDer. Propliage and plasmid DNA
can induce new properties.
Phylogenetic classification: There are two
approaches to bacterial classification . The
iLKTarLhical i - iaasification represents a branching
tree like arrangement, one characteristic being
employed for division at each branch ui level. Tliis
’Trixpr'
ipmy » 49
system iscalled phylogenetic because it implies an
evolutionary arrangement of species. Here some
characteristics are arbitrarily giver special
weightage . Depending on the characteristic so
chosen, the classification would give different
patterns. Far example, the intestinal Gram negative
bacilli have been traditionally classified depending
on whether they ferment lactose or not. While this
provides a useful distinction between the pathogenic
and non pathogenic groups of these bacilli, a
different hut usdiil classification could be obtained
ufing fermentation of sucrose as the criterion .
While classification based on weighted
characteristic is a convenient method, it has the
serious drawback that the characters used may not
he valid . Fermentation of lactose, in the example
characteristic. It nuv be acquired nr lost, upsetting
the system of arrange me nr.
Adontonian classification : Fhf Adansortian
rlanifiration , so called after Michael AdflUOIl who
introduced it in the eightenth century, avoids the
use of weighted characteristics . It makes no
phylogenetic assumption but merely takes into
account all the characteristic* expressed at the time
of study. Hence it is called a phciietic system - It
gives equal weight to all measurable features, and
groups organ is ms on the ba -si ^ of similarities of
several characteristics. Thc availability of computers
has extended [ lie scope of phenetfc classification
by permitting comparbons of very large number*
of properties of several organisms at the same rime.
This is known a*. numericri tnccf >OWy
M o L t c u E t i r n r gefleLic uliissifitillictn : T h i s
is baaed on the degree of genetic nelatedtlCSS of
different organisms Since all properties arc
,
u l t i m a t e l y hased on the genes present, this
classification is said to be the most natural or
tundamental method . ON A nelatedness can he
tested by studying the nucleotide sequences of ] 3 NA
andby DN A hybridisation or recombination methods.
The nucleotide base composition and base ratio
—
( Adenirie-ThymlniciGuanLne CvtuiLne ratio.) varies
Copyrighted material
so * Textbook of Microbiology *

widely among different groups of microorganism ;, 1

though it is constant for members of rhe same
species. Molecular classification h.Lsbeeu employed
more with viruses than with bacteria .
, bactenal nomenclature is ensured by the Code of
No method of bacterial claiiifiCation in
universally accepted . The method most widely
adopted is presented in Siuectejive editions of
ffergev 's AfamiaJ of Dctcmun4tn,t BKimal .
Intmspeoies olnssificalion: For diagnostic
nr epidemiologic purposes, it often necessary ro
for bacterial species is self-evident Chaos will result
if the same bacterium is referred toby different names
by different workers. International agreement on
Nomenclature which has the authoiity of the
International Association of Microbiological Societies .
Two kinds of names are given to baictcria . The
first is the casus/ or common name which varies from
^ country ro country and is in the local language . Names
such is nphoid bacillus' and 'gonococcus' arc casual
'

subclass ] ly bacrctial species. I 'his may he based on

'
tijnchcmiL- . Ll properties ( biotypes), antigenic features
(serotypes ), barterinphage susceptibility ( pluigu t > f *> )
or pnKiuction Dfbadmorins ( coliein types ) - A species
may he divided dint into groups and rbun into tifiev .
as for example , in. streptococci .
Much greater discri ini nation in intnasperies
taping has been achieved by the application of
newer techniques from Lmrmmology, biochemistry
unri gciKtict- Investigations ol epidemiology and
pathogenesis using these techniques have been
collectiveLv referred to as mofetOlEir epj'demioJcjri .
The methods used are of two types: phenotypic
( study of expressed characteristics ) and genotypic
( direct analysis of genes , chromosomal and
crtrachroiiwsoiTliJ DNA ). Molecular phenotypic
methods indude electrophoretic typing of bacterial
proteins and immunoblotting. Genotypic methods
include plasmid profile analysis, restriction
endonuclease atialvsis of chromosomal DNA with
i
Southern blotting , Pt_’ K and nucleotide sequence
analysis. Some of these techniques are considered
in the chapter B&cttTui Genetics .
NOMENCLATIVE
The nerd tor applying generally accepted names
names. Such names are useful lor L -ommutiiCalion at
the local level. The second is the scientific or
international name which is the Mine throughout the
world. The scientific name consists usual lv H » t two
words, die first being the name of the genus and the
second the specific epithet ( for example BtcilhlS
stjhrjfts}. The generic name i. s usually a Latin noun .
The specific epithet is an adjective on noun and
indicates some property of the species ( for example
a/biis, meaning white) , the animal in which it is
found ( for example sms, means pig), the disrate it
causes ( tetani , of tetanus ), the person who
discovered it ( weJcJtu, after Welch ) or the place of
its isolation ( londoo ) , The generic name always
begins wirh a capital letter and the specific epithet
with ,1 small letter, even ii it refers to a person or
place ( (or example Salmonella Jondort ),
TYPE CLJLTI RES
As a point of retere- noe , type cultures of bacteria are
maintained in intematHinal reference LaboratorieSrThe
type cultures contain representatives of all established
species. The originjil cultures of any new species
described are deposited in type colfcrtions- 1’hey are
made available by the reference laboratories to other
workers fur study and comparison.

Further Reading
Cowan ST ed . 1978. DidJOtfLuro/Medical Tamoinr. London: Oxford Uditttttar Press.
r p r

Cowan ST and KJ Strel 196:5 .. AJkouaJf for the Identification of Medical Bacteria London; Cambridge University Press.
Finegold SM and EJ Baiun Bailey and Scott s Dhtgoostk Bacteriology, 7* edn, St. Louis: CV. Mosby
Goodiellmv M jnd RG Board ed. 1980 Microbiological Classification and Identification. London: Academic Press.
.

Murray P ea: ilF ds. 1999. Manual of Clink dl Microbiology' . 7' edn. Washington: American Sociery for Microbiology.
1

Copyrighted material
 Bacterial Genetics
Genetics, i name coined by the llnrish biologist
William Bateson in 1906, is the study of heredity
utd variation , seeking to understand the causes of
the resemblances and differences between parents
and their progeny. Like other ogmnnu, Wteria
^ IR £ 1 breed true and maintain their characteristics
from generation to generation , yet at the same time,
exhibit variations in particular properties ir> a smaEI
proportion of their progeny. Though heritabiliry
and variation in bacteria had been noticed from
the early days of bacteriology, it was not realised
then that bacteria too obey the Laws of gtiwtics.
G c
T
# lilOl . OCY
* c
T ri
Fig . 9.1 DNA double helix
Even the existence of a bacterial nucleus was a
subject of controversy. The differences in
morphology and other properties were attributed
by Nagcli ( 1 £77 ) to bacterial pltotaorphitm,which
postulated the existence of a single , or perhaps a
tew species, of baCieria , whieh possessed a protean
capacity for variation , With the development and
application of precise methods df pure culture , it
became apparent that different type* of bacteria
retained constant form and function through
successive gene rations. This led to rhe concept of
monomorphim, proposed by Cohn and Koch,
which admitted of tittle potential for variation and
separated bacteria into species based upon single
character differences.
It was ( mlr - 11 icc the 1940s that principles of
genetics were applied to bacteria and their viruses.
ITihas led not merely to a better understanding
of the generic processes but also to fundamental
advances in biology ; ul biochemist tv and to the
hjrrh of a new h ranch of science , molecular biology.
BASIC rhismrLES OF MOLECULAR
The 'central dwgma * f molecular hiolngy is that
deoxyribonucleic acid ( DNA ) carries genetic
information, winch i - [inscribed onto ribonucleic
acid i RN -\ ) and then translated as the particular
polypeptide ( D\ A -> RNA — > polypeptide). As
the nature and timer ions of a cell .ire basically
determined by the pecific polypeptides that
constitute it - proteins and enzymes , it is evident
that the essential m .itcrial of heredity is I 3 N A
which is the storehouse of all information for
Copyrighted material
52 < Te *tbook of M cfobiology *
protein synthesis. ( An exocpticm exists in rhc case unwinding at one end to form a fork, each strand
of some viruses in which the genetic material is of the fork acting as a template for the svm thesis of
RNA instead of DNA ) a complementary strand, with which it then forms
The DNA molecule is composed of tvifl chains a double helix.
of nucleotides wound together in the form of a Basically, RNA is structurally similar in DNA,
'double helix’ ( Pig . 8.1). lunch chair has a backbone except for two major differences. It contains the
of deoxynbose and phosphate residues arranged sugar I ibose ( instead of deoxyribose which is
'

alternately. Attached to each deuxjfibose is one of
four nitrogenous bases , the purines, adenine ( A)
and guanine ( G ), arid the pyrimidines, thymine (T)
and cytosine {G ) , The cWbic‘stranded nature of
the molecule is stabilised by hydrogen bonding
between the bases on the opposite strands in such
a manner that adenine is always linked to thymine,
and guanine to cytosine ( Fig. S.2 ) .
Adenine and thymine thus form a
complementary base pair , and guanine and cyltisiiLL'
form another. A molecule of DNA will, therefore,
contain as many units of adenine as thymine, and
of guanine as cytosine but the ratio of each pair of
bases [A + T) /(G + O , though constant for each
species, varies widely from one bacterial species TO
another. The DNA molecule replicates by first
present in DNA) and ( he base uracil ( instead of
thymine which is present in IJNA). Three distinct
types of RNA can be distinguished on the basis of
structure and (unction: messenger RNA ( mRNA ),
ribosomal RNA ( rRNA ) and transfer RNA
(iKNA ). DNA acts as the template far the synthesis
of ni RNA and, therefore , the bases in the two will
.
be Complementsiy to ouch Other Adenine, guanine,
cytosine and uracil in mRNA will be
complementary to thymine, cvlosine, guanine and
adenine, respectively, in the DNA -
Geneiie information is stored in the DNA as a
code, the unit of the code consisting of a sequence
of three bases, that is, the code is triplet. Each triplet
( codon ) transcribed un mRNA sjtecifies for a single
aminoacid hut the code is ^ degenerate so that more

BACKBONE SIDE (_’ HAJ !V etc .

DEOXYR1BOSE ADENINE * - a
THYMINE DEOXYRIROSEi
/ \
PHOSPHATE PHOSPHATE
\ /
DEOXYRIBOSE
/
GUANINE E
CYTOSINE — DEOX YRIBOSE
\
PHOSPHATE PHOSPHATE
\ /
DBOXYRIBOSE CYTOSINE m GUANINE [ JEOXYRIliOSE
/ \
etc .
ONE STRAND

DOUBLE-STRANDED DNA

Fig . a .2 A teamsni ol aouole stranded DMA Illustrating its chemical structure

opyrighted material
 * Bacterial Geneves
Than cme codon may exist for the uirc immoadd.
Thus, the triplet AG A codes for arginine but the
triplets AGO, CGU, CGC, CGA and CGG also
code for the same a mi noacid . The code is non -
overlapping, each triplet being a distinct euiiiy, and
no base in one endun is employed as part nf the
message of an adjacent codon.Three codons (UAA,
UAG and UGA) do not code for any iminoadd
and arc called ' nonsense codons\ They act as
punctuation marks (stop codons) termi ranting the
message for the synthesis ot ' a polypeptide .
A segment of DNA carrying cottons specifying
for a particular polypeptide is called a A UNA
molecule consists of a large number of genes* each
ol which contains hundreds of thousands of
nucleotides. The bacterial chromosome consists of
-
a double stranded molecule of DNA arranged in a
circular form. When straightened * ir is about 1,000
jim in length . The length of DNA is usually
expressed is kilubases ( 1 kb 1 ,00i ) base pairs).
linctcrini DNA Is about 4*000 kb and the human
genome about 3 million kb long.
In higher forms of life, several stretches of DNA
that do not appear to function as codons occur
between the coding sequences of genes . These
Apparently useless noncoding intrusions are caller ]
mfrcuis, while the stretches of coded genes are
called exons. During transcription * the genome is
copied in its entirety, both introns and crons. The
irtrons arc then excised from the RNA copy before
being translated by the ribosome* Into proteins.
E \|l t \ t:H l t O.M ( > K ( > M V I G l i l V l i T t C
J
E I F M l? N T S
In addition to chromosomal DNA .. most bacteria
possess extrachromosomal genetic elements . These
are not essential for the normal life and functioning
of the host bacterium but may confer on it
properties such as drug resistance and trudgen icity
leading to survival advantage under appropriate
conditions. PlAtmidt are circular DNA molecule*
present in the cytoplasm of bacteria , capable of
autonomous replication ( iraJepemJenf nepheorw). By
53
their ability to transfer genes from one cell to
another, plasmids have become important vectors
in genetic engineering. Plasmids may also be seen
in yeasts.* which are eukaryotes. Plasmid DNA
same times may be integrated with chromosomal
DNA* The name epifinme employed for such
integrated dorms * though this distinction is not
usually made now.
Plasmids have been classified .in many ways ,
depending on whether they arc self ^ tmnsmissiblc
or non transmissible ( nonccmjugating ) , ora the
property encoded (sex , drug resistance, etc.) * by
restriction endonuclease fingerprinting or other
criteria . An important method of plasmid
classification in by imornp:itihilinr typing,- Clofely
related plasmids do nor coexist stably in the same
bactcri al cell, wli 11c unrelated plasmids can . On this
basb, plasmids have been classified into different
incoapttibiHty groups. They have alio been
clashi bed based on the types nt conjugation tuhe
induced, which determine the susceptibility of the
host bacterium to lysis by some virulent
bacteriophages.
GENlVTiPHJ AMI PHKNtlTVPIC
V M t i Ml I O N S
The sum Loial of the genes that make up the genetic
apparatus of a cell (genome ) establishes its genotype,
which is rhe hereditary constitution of the cell that
is transmitted TO its progeny. The genory ]>c includes
the Complete generic potential Ol the cell, all of
which may or may not be expressed in a given
environmental situation .
Die phenotype [ piiaenv meaning display ) is the
physical expression of tbe genotype in a given
environment . It billows, therefore , that cell mav
exhibit different phenotypic appearances in different
si tuitions; fur exam pic , the typhoid bacillus is
normal ly flagellated fejt when grown in phenol agar,
the flagella ate not synthesised. This is only i
phenotypic variation determined by the environ men!
and is reversed when cubailtured from phenol agar
into broth. Another example ol environmental
Copyrighted material
54 i Tyxlt?OQk
influence .* the synthesis by E, coil of the enzyme
-
beta galactosid ase , necessary for lactose
fermentation. JlTic bacillus possesses rhe genetic
information for the svnthcsls
.11
of the enzvme Hut
Dill
the actual nyntlutit takes place only when it is
grown in a medium containing lactose . When
grown in a medium containing glucose only, the
enxyme is not synthesised. Such enzymes which
are synthesized only when induced by the suhstrate
are called induced enzymes * as opposed to
cspjfjfijtjvf enzymes , which are synthe -used
irrespective of the presence or absence of the
substrate.
Phenotypic variations are influenced bv the
environment, Limited in range by the genotype,
temporary and not heritable. Variations are s::. id to
be genotypic when they arc due to alterations in
the genome . Genotypic variations arc stable,
heritable and not influenced by the environment.
They may occur by mutation or by one of the
mechanisms of genetic transfer or exchange, such
as transformation , trjinsducti -. m , lysogenic
conversion and conjugation.
MUTATION
Mutation is a random, undirected, heritable variation
caused by an alteration in the nucleotide sequence
at some point of the DNA of the cell. It may be
due to addition, deletion or substitution of one or
more bases ( point mutation ). Multiple mutations
cause cjitcnrvc chromosomal rearrangements . A
misienre mutation is one in which rhe rripler code
is altered so as to specify an .linin ' u , id different
from that normally located at a PARLI. LII .LT position
in the protein. l>cletion of a nucleotide within a
gene may cause premature polypeptide chain
term mar ion by generating a nonsense cudon , i.e . r
nonsense umtitiosi. Irailsvcrsiun is substitution of
a purine for a pvrami . hne and vice veiss. i. IL base
pairing - ifcippmsormutiarion is reversal of a mutant
phenotype by another mutation at a position on
the DNA distinct from that of the original mutation.
Ah genes are susceptible to mutational events but
Ol MiCrOOiUlogy
not* all mutations are - - Some
TL > CL!. mutat iiurts
involve vital functions , and such mutants arc
notifiable { lethal mu ran on ) , A type of lethal
mutation which is of great interest is 'conditional
mutatiim’- A conditional lethal mutant may be able
to live under certain contritions ( permissive
conditions). The commonest type of conditional
mutant is the temperature sensitive ( nt) mutant,
which is able to live at the permissive temperature
( say, .35 JC), but not at the restrictive temperature
,
( say, 39 C ).
L
Each gene undergoes mutation with a fixed
frequency. Mutation rates ol individual genes m
bacteria range from 10^ to 10 ]U per bacterium per
"
division. The molecular mechanism of mutation is
rhat during DNA replication, some 'errori creeps
in while the progeny strands are copied. For
-
i n lance, i i istcad of thym i ne bond i ng wii h adenine,
it may, due to tautomerism, sometimes bond with
guanine. Though mutation occurs Spontaneously,
: rs frequency can be . nercased by Ftcveral agents
'
( Mutagen* ) such is UV rays, alkylating agents,
- -
acridine dves, J bmrnnuracit and 2 ami.nopurine.
Mutation is a natural event, taking place all the
lime at its particular frequency in all the dividing
forms of life . Most mutants , however, go
unrecognized as the mutation maybe lethal or mav
affect some minor function that may not be
expressed. Mutation is best appreciated when It
involves a function which can be readilv observed.
IT
For example, an E. coli mutant that loses its ability
to ferment Lactose can be readily detected oil
MacConkey agar but is unrecognisable on nutrient
agar. Mutation is of vital importance when it
confers a survival advantage. If a streptomycin
resistant mutant of the tubercle bacillus develops
in a patient under treatment with the drug, it
multiplies selectively and ultimately replaces the
original drug sensitive population of bacteria. But
in a patient who is not on treatment, the mutation
confers no survival advantage and , therefore,
preferential multiplication of the mutant docs not
occur. Such changes in the character of bacterial
Copyrighted materia
 1 Bacterial Genetics *
populating, observed in the presence of a selective
environment , were formerly rnriRidercd to be
'adaptations'. By a post hoc, ergo propter hoc
reasmiing, the environment and the variation were
believed to have a ciuse-ind-eflfeet relationship,
bin ll induced variations were considered heritable
' MATERIAL ( GENF. TRANSFER )
in the lamarckian sense, k was the demolition of
this concept of adaptation in the 1940s that
established bacteria] genetics oil , L firm scientific
basis.
The proof that bacteria undergo spontaneous
imitation in Jejscndcnt of the environment was- tirsl
provided he l . uria and De lb ruck (1943) bv the
' fluctuation trsr ' , They found that very wide
fluctuations occurred in the numbers of
bacteriophage resistant E . coli colonics when
samples were plated from several separate small
volumecultures* as compared tri samples tested from
a single large volume culture . Statistically, this
indicated that mutations occurred randomlv in the
separate small volume cultures, some early and some
late, resulting in the Wide fluctuation. In the targe
volume cultures, fluctuations were within limits of
sampling errorr However, the logic of [ Iris
experiment was not widely appreciated by
iHObiologitfl) probably due to the complicated
statistical interpretation . Ir was the simple but
elegant 'rcplica-platlng’ technique ofLcderbcrg and
1-edeiberg (1952) that proved the point beyond doubt.
Using i velvet template, they were able ro transfer
inocuta from colonies on a master plate, onro a number
of other plates, retaining die relative positions of [ he
colonies in all the plates. By such replica^pbling on _ occasiu nally. A phage particle may have at: its core,
vulture plates with and wbrltnut baercrinphuges, [1 LL V
were able to show that bacteriophage resistant mutants
appeared without the bacteria ever having had contact
with a selective agent.
Mutation may affect any gene and hetiee may
modify BnydiuBCteriEtk of the bacterium . Mutants
may varv in properties such as nutritional
requirements , biochemical reaction , stitigenic
structure, morphological features, colony form, drug
susceptibility, virulence and bust range . The
ss
practical importance of bacterial mu radon lies
mainly in the field of drug resistance and the
development of live vaccines.
TRANSMISSION OF CFNRTIC
Traasfumialmn: Transformation is the transfer
of genetic information through the agency of free
UNA. 1 r was the first example of genetic exchange
in bacteria nt have been discovered, Griffith in 1928
found that mice died when injected with i mixmre
of live noncapsulated ( R ) pneumococci and beat
killed capsulatcd ( 5} pneumococci * neither of
wllicb separately proved fatal. If in the experiment,
the live ( R ) pneumococci weft derived from
capsul .tr type II and the killed ( S ) strain from type
II 1, from blood cultures of the mice that had died,
live type III capsulatcd pneumococcus could be
isolated, showing that some factor in the heat killed
type 11 ] pneumococcus hud transferred the
information tor capsule synthesis to the live rough
-.train. Such transformation was subsequently
demonstrated in vitro also . The nature of the
transforming principle was identified as DNA by
Avery, Mavleod and McCarty ill 1944.
IVUIIIKDLICLEOII: The transfer of a portion of the
DNA from one bacterium to another by. a
bacteriophage is known as transduction *
Bacteriophages are viruses that parasitise bacteria
and consist of a nucleic acid core and a protein
coat. During the assembly of bacteriophage progeny
inside infected bacteria * ' packaging errors' may occur
betide* its (I'VTI nucleic idd, a segment of the h*»l
DNA. When this particle infects another bacterium,
DNA transfer is effected and the recipient cell
acquires new characteristics coded by the donor
DNA. Transduction may be ‘generalised ", when it
involves xnv segment tfthfi ilonor UNA it random,
or it mav be restricted ' , when a specific
'
bacteriophage transduces onlv a particular genetic
trait . Restricted transduction has been studied
intensiveK in the ’ lambda ' phage of E. coli. The
"
Copyrighted material
56 * TexlDook of Microbiology »
prophage Lambda is inserted in iHt bacteria!!
chmrnoso-mq only between the genes d-Ct crrnin. iirig
'
galactose utilisation (jja!) and. biotin synthesis { bio)
and. therefore it transduces unlv either of these.
TnnsdLKriQin is not confined to transfer of
chromosomal DNA , Epi - omes and plasmids may
a I - be transduced . The plasmids delermi ni n r
pcnicillin resistance nt staphylococci. arc transferred^
from cell to cell hy transduction.
'
I’ransduction appears to be the most widespread
mechanism of gene transfer among prokaryotes and
provides an excellent fool tor the genetic mapping
of bacteria . Any group of bacteria for which
bacteriophages exist can be subject to transduction.
It has been reported that transducrion may
occasionally be effected in eukaryotic cells also.
Transduction has been proposed as a method of
gene t ic engi need rj: i n the treatment of some i nborn
errors of metabolism .
Lysogenic conversion: Ratter in phages
exhibit two types of lifecycle. In the virulent or lytic
cycle, large numbers of progeny phages arc built
up inside the host bacterium , which ruptures to
release them. In the remans re or nuriiWe cycle h
the host bacterium is unharmed. The phage DNA
becomes integrated with the bacterial chromosome
as the yrrvjphagie, which multiplies F Tchtonouslv
^
'
with rhe host DNA and is transferred to the
daughter oelll. This process is called Ivsngcny and
bacteria harbouiing prophages are called iysogenic
bactena Lysogeny IS extremely frequent in nature.
In lysogenic hacteris , the prophage behaves as
an additional scgmcjtt of the bacterial chromosome
-
Coding for new characteristics . Thi . process by
. portions of the host DNA also are sometimes
which the prophage DNA confers genetic
infotmari "n to a bacterium is called Jvsc emc or
phage eanversfon. In transduction , the phage acts^
only as a vehicle carryirig baCtenal genes Ircim one
cell tO another but in lysogenic conversion the
phage DNA itself is the new genetic element .
Lysogenic conversion influences susceptibility to
bacteriophages (immunity to supcrinfcction with
the same or related phages ) and antigenic
character] sties. Of great mcd: cal importance is the
lysogenic conversion in diphtheria bacilli , which
acquire roxiger. i . ity {and therefore virulence ) by
lysogcnisation wi. rh the phage beta . Elimination of
the phage from a toxigenic strain renders it
rontuxigenic.
f .onj u g a t i n n : Conjugation is the process
wherein a ‘mule’ or 'donor ' bacterium 'mates Of
makes physical contact with a ‘female or 'recipient'
bacterium and transfers genetic elements into if .
This has been considered to be rhe bacterial
equivalent of sexual mating in higher organisms
bur rhe analogy is irrelevant as , following
conjugation , the female bacterium is in turn
converted into a male cell ! Bacterial conjugation
was first described hy Lederherg and 1:itum (194b )
in a stni ! n of £ , coYs called K12 and has been most
extensively studied in this strain.
Conjugation takes place between a male cell and
- -
a female cell. The malcncss or donor C .L I U of a
cell is determined by the presence in it of a plasmid
which codes for specialised fimbria { sex pihi* }
which proiects from the surface of the cell. The
plasmid DNA replicates and a copy of ir passes
from the donor to the recipient cell, probably along
the sex pilus (cwy rogation tube). As a result, the
recipient attains donor status and can in turn
conjugate with other female cells. The maleness in
bacteria is thus a transmissible or ‘infcctioni’
characteristic , Along with the plasmid DNA .
transferred to the recipient. The donor DNA then
combines with the DNA of the recipient, effecting
genetic recombination. It was in E. coli K 1 2 that
the role of plasmids in conjugation was first
necogn i sed. The plasm id responsible was termed
the 'stx factor or 'fort liry ( F) factor '. When other
1
similar plasmids were also discovered, the term
' transfer factor came to he used lor .ill such plasmids
which conferred on their host cells the ability to
act as donors in conjugation .
CoDvriahted materia
 * Bacterial Genetics »
Thu l: factor: The F factor is i transfer factor
cLi .Lt contains the genetic information necessary for
the synthesis of the sex pilus and for seif- transfer
hut is devoid of other identifiable genetic markets
such as drug resistance. Cells canying the F factor
( F* cells ) hove DO distinguishing fcarutes other than
~
their ability to mate with F cells and render them
F. The F factor is actually an eplsome and has the
jhility to exist in some cells in the ' integrated state’
or inserted into the host chromosome . Such cells
arc able to transfer chromosomal gen to recipient
Ceils with high frequency and are known as Hrr
cells. Following conjugation with an Hfr «11, an
F only rarely becomes F*, though it receives
chromosomal geues from the donor.
This conversion of an FH cell into the Hfr state
is reversible. When the F fector reverts from the
integrated state to the free stare* it may sometimes
carry with ] L some chromosomal genes from near
its site of attachment . Such an F factor
incorporating some chromosomal genes is called
an F prime ( V ) factor. When an F cell mates with
' transfer* and a resrsrajicr determinant ( r ) for each
a recipient ., it transfers* dung with the F factor, the
host genes incorporated with it . This process of
transfer of host genes through the F factor
resembles transduction and has therefore been
called jenAlrtfojl ( Fig. B. 3).
Guildno eaic ( Col ) factor; Several strain*
^ —
of coliform bacteria produce colicins ancibiutic-
Lilte substances which are specifically and selectively
lethal to other enterobacteria. As similar substances
are produced by bacteria other than culiforms also
( pyoein hy PseudomotiMt pyveynne*, diphihcrkm
by Corvneriaofcrrirm (Jj/rhrbcrj ac ) , the name
' '
bucteriocin has been given to this group of
substances . The spcci fidty of action of bacteriucins
enables mtnupedes classification of certain bacteria
[ for example ShigtRt soiwei , Ps . aerugiitoan ).
*
Coliem production is determined by a plasmid
called the Co! factor, which resembles the F factor
in promoting conjugation leading to self ^ runsfer
and, at times, transfer of chromosomal segments .
57
Resistance transfer factor ( RTF It This
plasmid is of great medical importance as it leads
to the spread of multiple drug resistance among
bacteria .
This extrachromosomal mechanism of drug
resistance was first reported by Japanese workers
( 19? ) investigating the sudden increase in
^
infections caused hy the Shigella strains* resistant
simultaneously to SulphonaiiudeS, streptomycin ,
chloramphenicol and tetracycline . They observed
that patients excreting such Shigella strains also
shed in fheir fcecs £, coli strains resistant to the
same drugs. Transfer of multiple drug resistance
was demonstrated between E. eoti and Shigella
strains both in vitro and in vivo. The resistance is
plasmid mediated and is transferred hy conjugation .
[ JILS mechanism of drug resistance is known as
transferable, epi tnal or infectious drug resistance.
^
This plasmid consists of two components the-
transfer factor called the RWfiHXV transier factor
( RTF ) which is responsible for conjugation ]
'
^
of the several drugs. The whole plasmid ( RTF + r
determinants ) is known as the R factur. An R factor
can have several r determinants, and resistance to
as many as eight or more drags can be transferred
simultaneously ( Fig. 8.4) . SometinteH cite RTF may
dissociate from [ he r determinants, the two
components existing as separate plasmids. In such
cases, though the host cell remains drug resistant ,
the resistance is not transferable - The RTF can have
attached to it determinants other than those lor
drug resistance. EntrrotOiin and hemolysin
production in some enternpathogenk F. call arc
transmitted by this transfer factor.
Transferable drug resistance is seen widely in
various pathogenic and commensal bacteria of man
and animals, such as Enfeiutacteriicn Vibrio,
^
Pseudomonas-, FasiettreJ/a . The transfer can be
effected readily in vitro but in the normal gut, if is
inhibited by several factors such as anaerobic
conditions, bile salts, alkaline pH and the abundutce
of anaerobic: Grain positive bacteria minimising tile
Copyrighted material
58 * Textbook ol Microbiology *

IwCTEItlAL GENETICS

bxciiiun

F
+
MATING

F F

o# ^ o
Fig. 8.3 The integrated F liclur ot n Hfr cell may revert to ihe cytoplasmic slate. During excision
Sejtduclion .
some host genes may be incorporated in the F factor F \ Whan an F cell males wilh an F cell the host gene-
'
,

ts transferred to rfoe recipient

chances of contact between donor cells and suitable GENETIC M P C H A M S M S OP 1> RI C

recipient cells, but in the intestines of persons on RRHIKTAIVCR IN BACTRKIA
oral antibiotic therapy, transfer occurs readily due Bacteria mnvacquire drug resistance hv mutation
to the destruction of the sensitive normal flora and
r by one of the method;; of genetic trafufer. The
the selection pressure produced by the drug. biochemical mechanisms of instance mav . be
J

Transferable drug resistance in now universal several, including decreased permeability to the drug,
in distribution and involves all antibiotics in development of altenative mctabolic pathways, altd
common use. IK incidence is directly proportional production of enitymes inactivating the drugs .
to the frcqueocy of use of antibiotics in the area ,

bacteria carrying H factors can He transmitted from
animals to mao . Hence indiscriminate use of
antibiotics in veterinary practice or m animal feeds
can also Lead to an increase of multiple drug
resistance in the community- The addition of
m LL > ne-slep' mutation, as seen with streptomycin,
antibiotics in animal feeds has for this reason been
prohibited by legislation in some countries .
Widespread resistance has considerably diminished
the clinical efficacy of most antibiotics.
Mutational resistance is mainly of two types:
(1) the stepwise mutation , as seen with penicillin,
where high levels of resistance arc achieved only
hy a series of small- step mutations; and (21 the
where the mutants differ widely in the degree of
resistance , some exhibit lot; low resistance , while
others may be highly resistant, and some even
streptomycin dependent.

Copyrighted material
 * Bacterial GenelioS S9

In clinical practice, mutational resistanceis of penicillinase plasmids , which are transmitted by

great importance in tuberculosis . If a patient is transduction , may also carry determinants for
,

treated with streptomycin alone, initially the bacilli
die in large numbers but soon resistant mutants
appear and multiply unchecked, IF two or more
antituh ^ rculnus dnigb arc used for combined
treatment , rcpopulafion by resistant mutants docs
not occur, as a mutant resistant to one drug will be
destroyed by Tin other drug. The pKissibilitv of a
1
mutant exhibiring resistance TO multiple drills
simultaneously is so remote as to be virtually
J V
lumoasteoL This Is the talkMile behind combined
fre tment in tuhcrculnsifi . However,, inspire of this
^
knowledge, inadequate or inappropriate treatment
"
resistance to mercuric chloride and erythromycin.
Transferable drug resistance mediated by the R
factor is the most important method of drug
resistance, Acquisition of an R factor simultaneously
confers resistance to several drugs and therefore
treatment with a combination of drugs is not usefu l .
The resistance is due to die production of degrading
enzymes, and the level of resistance is usually high.
Resistance mav be transferred between bacteria of
different taxonomic groups, While resistant mutants
usually hive a lower growth rate and reduced

virulence a & compared to tile wild Strain*, bacteria

over the years has caused extensive resistance in
tubercle bacilli , leading to a pandemic ofmultidmg
resistant luberculusis ( MDR TB ) across the world .
Res Lb fancr transfer bv Transformation can be
J
demonstrated exptrimentalty but its significance in
nature is not known . Acquisition of resistance by
transduction is common in staphylococci . The
carrying K factors me uppj. pe; rstLv normal in other
respects . R factors in some cases may even lead to
enhanced virulence . Multiple drug resistance was
initially seen In bacteria causing diarrhea and such
other mild infections that did not call for antibiotic
treatment as a routine. RUT subsequently n has
spread to virtually all pathogenic bacteria affecting

3 (
c
T

Su
s +
MATING
1
(3)

HTF RTF
T c T C
<u S
Su S

: Fig . B .4 Transferable drug resistance * The R cell carries the R lactor , consisting of RTF and r determinants
1

Its transfer to a sensitive Ft bacterium converts the recipients Into a resistant Ft * cell.
60 < TeiftbooK Of WnCr &biaJ& Sy
humans; mid animals, making antibiotic therapy ot
infections ineffective.
In the laboratory, R factors may sometimes be
eliminated by treating bacteria with acridine dyes
or etliidium bromide. Bur m rhe communin ', tltc
Dnjy wav to prevent widespread dissemination ul
-
multiple resistance in to resti-ict the use of ultibiotuis.
to the essential minimum.
THARUSPUS ANT . K GHNP.TH : KI . KMENTS
Certain structurally and genetically discrete
segments (>f DKA have been identified that have
the ability to move around in a Yul- aild- pasic '
'
manner between chromosomal and extrachio
mosomai DNA molecules within cells, 'lltcsc DNA
- recipient molecule becomes heavier.
-
molecule ire called ( jumping genes’)
and this mode of genetic transfer, rmrjijsosirio.tf - The
earliest of such mobile genes WUs discovered by
Barbara McClintock in plants during work in the
1940s and 50s , tor which she was awarded the
Nobel Pri ^ e for Medicine in 1933. A transposon is
a segment of DNA with erne or more genes in the
centre, and the two ends carrying ‘inverted repeat '
—
sequences ol nucleotides nucleotide sequences
complementary to each oilier but in the reverse
order. Because of this feature, each strand of the
tmn &poson can form a singie -STnmded loop carrying
the gene, and a double-stranded stem formed hy
hydrogen bonding between the terminal inverted
repeat sequences ( Pig. S..5) . Small transposon *
(1-2 kb) are known as 'insertion sequences' or IS.
Table B.l Comparison ol mutational and transferable drug resistance
iVTunmuraJ drag resijnnee
One drug resistance nr a time
Low degree resistance
Ll’ a.ii overcome by high drug dose
Drug combinations can. prevent
Resistance does mot spread
Mutants mar be detective
Virulence may be low
*
Transposons attach at certain regions of
chromosomal , plasmid or phage DNA . Insertion
of a transposon lead ?; to the acquisition of new
characteristics by the recipient DNA molecule.
Unlike plasmids, transposons are not self replicating
.
and depend or chromosomal or plasmid DNA for
replication
bv transposition, a segment of the DNA can be
transferred from a molecule to another molecule
that has no genetic homology wit ft either the
tnmpoubk clement or with the donor DNA. Jn
this it differs from recombination . As. sizeable
,
chunks of DNA. arc added by transposition, the
Characteristics transferred by transposons may
onetii i ret confer survival advantage under appropriate
environmental conditions. It has been Suggested that
the resistance -determinant segments of the R factors
may hwc evolved as collections of transposons, each
oaming a gene: that confer; resistance to one nr several
antibiotics.
Transposition is a mechanism for amplifying
genetic transfers in nature and ha-H been identified
in microorganisms, plants and animus, Transposons
appear to accomplish in nature , gene manipulations
similar to the laboratory manipulations that have
been called genetic engineering . 1
MOLECULAR GENETICS
1 ) iscovcries in microbial genetics have provided the
basis for the discipline of molecular genetics, which
Transferable resistance
Multiple drug ruiituia
Uij h naimnoe
^
E ] Lj h dost inc- tfccrive
^
Combinations cannot prevent
Spreads to same or different species
Nor defective
VimLence not decreased
Copyrighted m ria
 * Bacterial Genetics
* 61

is cocicetTrtd with the maiyju and ourripuJatitm CJI
UNA u^ing biochemical and microbialogiaJ
technique!*. 1r lias been staled that these techniques
have revolutionised the study of bid logy rtn d
medicine f probably more than any technique since
the development of the light mi otoscopes. Some
techniques and applications of molecular genetics
are discussed beluw.
Genetic engineering: The most important
application of molecular genetics in biotechnology'
, endonucleases and the fragments containing the
is genetic engineerlug or recombinant DNA
( rDNA ) technology. This consists of imitation of
the genes ending for any desired profcin from
microorganisms or tinm cells "t higher forms of
'
-
Life including human hi;in:: and their Introduction
, A DNA copy :s made from the mRNA using the
in Co suitable mlcrcjtn anismSj in which [he genes
^
would be functional, directing the production of
the specific protein, buch cloning of genes in
microorganisms enables the preparation of the
desired protein in putt form, in large quantities
and at J reasonable coat.
Different strategics have been employed for
- obtain ing the dosi red genes , For very small protci ns,
such as the pituitary hormone somatostatin whose
complete aminoarld sequences are known, [be genes
can be synthesised in the laboratory. With larger
proteins ^ this is not possible . The DNA can be
cleaved by specific enzymes called res friction
desired genes isolated. This does not work with
DNA of higher OTganisms as they contain i citrons.
In such eases, rln - messenger RNA concerned can
be isi < h trd fit ' ni eel I s pr < iducing the dedred protein.
enzyme ttverse transcriptase. The double-stranded
DNA gene is then prepared using DNA polymerase.
This it incorporated into suitable vectors or currier
such as plasmids or temperate bacteriophages, for
^

o
OTSOTET * O T C C A r

©
I C_5 M
V
T A C C 7 G A
~ V V, Ji * '
§ 6 ©

A
CL

—-
—
——
A

T
C
o— Ol
T A
C =G
— c
-
m
~

-©
®

5
T“ A
IL
C

T st
A
—— r
A ^ -

Fig.i.S Diagrammatic representation. The figun above shows a iransposon along 1he course of a DNA
molecule. consisting of a gene in the middle and inverted repeal sequences of rtud&olidtai at either end, The
1

figures below represent me stem and loop structure (armed by each strand of the iransposon, The loop
consists of the gene and the steinm 1$ formed by hydrogen bonding between the terminal repeal sequences.
The slem and loop form can attach to Insertion silos on recipient DNA. 1 Transposon 2. Inverted repeat . .
-
.
of gene. 7. Double stranded stem formed by bonding of terminal Inverted tequmeft
- .
sequences . 3. Gene. 4. Inverted repeat sequences. 5 Double stranded DNA R fHngto ilrandtd loop consisting -.
62 * Textbook od
insertion Into microorganisms. The microorganism
commonly employed is E. coli Kl2h though many
other bacteria and yeasts have also been used.
Genetic engineering has become an established
branch of biotechnology with great scope for
commercial exploitation. Cloned human insulin ,
interferons, somatostatin, growth hormones and
many other biological!have already been marketed.
Safer vaccines can be produced by cloning the
protective antigens of pathogens, as has already been
done, as in the case of foot and mouth disease, and
hepatitis B and rabies viruses . This versatile
technique ins many extramedical application* also.
Restriction endonucleases: [restriction
enzymes ) are microbial enzymes which cleave
-
double stranded DNA at specific ohjrpmicleotide
sequences. Many such enzymes which act at
different nucleotide sequences (for example, Ecu
RI , Hind III, Taq I ) have been recognised . The
natural function of restriction enzyme* in bacteria
may be the destruction of foreign DNA that may
enter the bacterial cell
Restriction enzymes split DNA strands into
fragment ^ of varying lengths . These can be
separated by gel electrophoresis and stained with
ethydium bromide and photographed.
I ) N . \ probes The specificity of the Interaction
in base pairing during DNA or RNA synthesis
enables the production of specific DNA probes.
These art radioactive, biotinylated or otherwise
labelled copies of cloned single-stranded DNA
-
fragments, usually 20 25 nucleotides long and
containing unique nucleotide sequences which can
be used (hr the detection of homologous DNA by
hybridisation . DNA probes are being used
increasingly in the diagnose of infectious diseases.
Probes cont & i n ing sequences un i que to the microbc
( 5tm i n, species or group ) to be detected can be added
to microbial cultures, body fluids, tissues at other
materials suspected to cont .tin the microbe nr its
DNA, The DNA probe hybridises with the
complementary specific sequences on the microbes
DNA.The advantages of DNA probes for diagnosis
Microbiology »
are their high degree of specificity, ability to derect
minute quantities of complementary DNA even in
the presence of other microbes, and the capacity to
recognise microbes that arc either difficult or
impossible to culture- DNA probes for the detection
of many pathogens ate now commere ially available.
Blottii 4 techniques DNA fragments obtained
by restriction enzyme digestion and separation on
gel can be transferred from the gel by blotting to
nitrocellulose or nvlon membranes that bind the
DNA . The DNA bound to the membrane is
denatured {convened to the single-stranded form )
-
and treated with radioactive single stranded DNA
probes - These will hybridise with homologous
-
DNA To form radioactive double stranded segments,
-
which can be detected on X ray film. This highly
sensitive technique for identifying DNA fragments
by DNA : DNA hybri Libation is called 5ourficro
blotting, after EM Southern who devised it. This
technique has very wide applNation in DNA
analysis.
An analogous procedure for the analysis of
RNA has been called northern blottiOg ( a opposed
to southern blotting!). Here the RNA mixture is
separated by gel electrophoresis , blotted and
identified using labelled DNA of RNA probe;.
A similar technique for the identification of
proteins (antigens) is called immuitoblorfriig [or,
in conformity with other blotting techniques,
.
western blotting ) Here the protein antigen mixture
-
is separated by SDS PAGE (sodium dodecyl
—
sulfate polyacrylamide gel clectrophotesi s), blotted
on to nitrocellulose strips and identified by
radiolabelled or enzyme- labelled antibodies as
probes. The western blot test has received wide
publicity as the confirmatory test for the diagnosis
of HIV antibody in sera. The specificity of the test
depends on its ability to separately i d e n t i t y
antibodies d irocted agai nst different antigens of the
pathogen ( for example, against the surface, core
and reverse transcriptase antigen* of HIV ).
Polymerase chain reaction ( PCR ): This
is a rapid automated method for the amplification
Copyrighted materia
 < Bacterid Genetics 63

of specific DNA sequences {or genes), invented by diseases , in forensic in vesti gallons , L:I
Kary B Mullis in 19B3, for which he wnn the Nnhd arch cubic I logical studies of ancient specimens
Pri. iL in Chemistry in 1993 . L(_ R consists of several
1

B
cycles nf sequenrial DNA replication where the
products of the first cycle become the template for
the next cycle . IT makes available Abundant quantities
of speed ic DNA sequences starting from sources
containing minimal quantities of the same.
The technique LS as follows TWO oligonucleotide
primers complementary to the flanking region of the
DNA sequence to be amplified are incubated with
the target DNA, nucleotides and DNA polymerase.
The reaction consists of three essential steps:
1. heat demtuiat i Dn of the sample DNA to si n L I L
^ -
strand;
2. annealing of sequence-specific oligonucleotide
primers Co the boundaries nfthe DNA segment;
and
3. extern- ion of the primers by DNA polymerase
and i n the exam i nation ui phylugeiietic relatioi:sllips
in evolution.
Based on the principle of PCR, other target
amplification systems have been developed . One
-ucli, Transcj i pi ion mediated j my I i i i cal ion i Tb1A )
which amplifies ribosomal RNA instead of DNA
has been applied as a rapid diagnostic technique
for infections such as tuberculosis where cultures
are difficult or delayed .
MbletiuLer epidemiology: One offshoot of
molecular genetics is molecular epidemiology Here
molecular methods such as plasmid profile analysis,
genomic fingerprinting and PCR are used for the
idcnriiiLdrinn and matching of microbial isolates
for epidemiological purposes.
Genetic mappinj: As a result of the
remarkable advances in molecular genetics it has ,

to form new double-stranded DNA across the
segment by sequential addition of
deoxvnuclcutides.
"
These three steps constitute one cycle of the
been possible to delineate the complete genomic
sequences of bacteriophages and other viruses,
bacteria and their plasmids, and even of some
eukaryotes including mammals. Quite- apart from
,

merlon. These cycles arc repeated several times,
usually for 20-50 cycles in the thermocytler, at the
end of which hundreds of thousands of copies of
the original target sequences are available. AH the
reaction steps take place at high temperature [50-
heat -stable polymerase, such as Taq 1 has
to be employed.
With its enormous capacity to . Lmplitv DNA,
PCR is a versatile tool useful m diverse areas such
as diagnosis, of infectious, genelic or neoplastic
the useful information it has provided in
microbiology ^ its success emboldened the
international scientific community to venture on the
" human genome project', the most expensive and
ambitious scientific project so far undertaken in
biology. The results of this mammoth study became
available by the dawn of the rwenty -first centuiy
and have opened vistas in human biology and
medianeh as well as controversies and dilemmas
that transcend medicine.

Further Reading
Hardy K . 19fJ 6 . KacIVraiJ Plasmids- 2nd cdn . W* : n-bi^ld. .
Innis MA it 1 1990. PC’R IVotorak, San Disf i Academic £*TVH -
MulLin KH . 1 99 ) The pilymrme chain n- . u lion ^. Nitl'i- l Lecture , SiuckhciLm.
Sambroak J er aL 19119. Mofcaifar Cloning. Cold Spring Harbour Laboratory
Tompki HE LS. 1992 . The use of molecular methods in infocticniE diBcaaet , .Vav EnglJ Med. 127:1290.
Towner KJ and A Cockayne 1993. MftfecLilir AfedioiJs for Microbe ] JtfejiriificaTran and Typing. London: Chapman - Hall .
V/arson JD et a]. 19H3. Rtcomh i lanr DJVA. New York: Scientific American BookE.

Copyrighted material
 05 Infection
InfecEion ind Imununiiy involve interaction
between the animal body ! host ) and the infecting
microorganism. Based on their relationship to
their hosts, miaooigajiisms can be classified a.s
saprophytes ( from Creek sapros decayed; and
phvtftn plant ) and parasites. Saprophytes are free-
living microbes that subsist on dead or decaying
organic matter They are found in soil and water
and play an important role in the degradaTLon of
organic materials in nature. They arc generally
incapable of multiplying on living tissues and
therefore are of little relevance in infectious
disease. Exceptionally, hewerer, some saprophytes
like B. iubalin may infect devitalised hosts whose
natural re id stance i s greatly reduced ( npportvin i she
infection ) . Parasites are microbes that can establish
themselves and multiply in hosts . Parasitic
microbes may be either pathogens ( horn Greek
pathos suffering, and gen produce, that is, disease-
producing) or oormnemais (from Latin com with;
and men&t, table, i.e. , living together ). Pathogens
are microorganisms that arc capable of producing
disease in the host. Commensal microbes live in
Complete harmony with the host without causing
any damage to it. The normal bacterial flora of
the body consist largely of commensals. Many
commensals behave as facultative pathogens in
that they can produce disease when the host
-
rcRii lance is Inwered -
It is necessary to di s r i ngu i sh between the term
‘infection ' and infectious disease '. The
"
lodgement
and multiplication of a parasite in or on the tissues
of a host constitute infection. It docs not invariably
result LH disease . In fact, disease is but a rare
consequence of infection, which is a common
natural event .
Infections may be classified in various ways.
Initial infection with a parasite m s host :S termed
primaly infection. Subsequent infections by the
same pamsite the host are termed rejrtfec.n'ojis.
in
When a new parasite sets up an infection in a
host whose resistance is lowered by a preexisting
infectious disease, this is termed secondary
ijiftct/ on. The term focal infection ( more
appropriately focal sepsis) indicates a condition
where, due to infection or sepsis ar localised sites
such as appendix or tonsils , generalised effects
are produced. When in a patient already suffering
from a disease a new infection is act up from
another host or another external source, it is
termed cross-infection. Cross-infections occurring
in hospitals are called nosncutnial infections [from
Greek nosocomjon hospital ). The term iatrogenic
wiicctiii.' j refers co physician - induced infections
resulting from investigative, therapeutic or other
procedures. Depending on whether rhe source of
infection is from the host 's own bodv or from
jt
external sources, infect ion s arc classified as
cndt eiious or otogenous, respectively. Based on
^
the clinical effects of infections, they may be
classified into different varieties Inapparent
,
infection is one where clinical effects arc not
apparent. The term subclinical infection is often
used as a synonym . Atypical infection is one in
w h i c h the typical or characteristic clinical
manifestations of the particular infect! i ms disease
are not present. Some parasites , following
infection, may remain in the tissues in a latent or
LJ opy righted materia
 i infection *
hidden form proliferauiig .inJ producing- dmLeal
di- >icuHe when the host resistance is lowered. Thi5
is termed Intent infection.
S * * i RCBS UJP INFECTION
I Inmans: The commonest source of i nicer ion
for huituiti at* hmmni themalvts Tht partite
may originate from a patient or a carrier. A earner
in a person who harbours the pathogenic
microorganism without suffering from any |
effect because of it. Several types of carriers have
i. l
been identified. A healthy carrier is one who
liarboui $ the pathogen bait lias never > uffered from
the disease caused by the pathogen , while a
CUJTVXJESCERF tamer is one who has recovered
from the disease and continue? to hwbour the
pathogen in hi? body. Depending on the duration
of carriage , carriers are classified as temporary
and chronic, J’he temporary' carrier state last ? less
than six months , while chrome carriage may last
L
-
for several V:; LT and sometimes even for the rest
of one’s life , llic term contact carrier is applied
to a person who acquires the pathogen from i
patient, while the term paradoxi.- al carrier refer?
to a earlier who acquire? the pathogen from
another carrier.
Animals: Many pathogen ? arc able to infect
both human beings and animal?. Animal? may,
therefore, act as sources of human infection. In
some instances, the infection in animals may be
asymptomatic. Such animals serve to maintain the
parasite in nature and act as the reserve nr of human
infections. Thcv .ire, therefore, called reservoir
hosfr . Infecti ' vs diseases transin itted I n in i an i mals
to human beings are called zoono e*. Zoonotic
^
diseases may be bacterial ( plague from mrs ) , viral
( rabies from dogs), protozoal ( toxoplasmosis (tom
cats), helminthic ( hydatid disease from dogs ) or
fungal (aoophilic dermatophytes from cats and
dogs).
In sects; Blood sucking insects may transmit
pathogens to human beings . The diseases so
65
caused am called arthropod - home diseases. 1 nsects
such as mosquitoes, ticks, mite?, flics, flea? and
lice that transmit infectiiins are called vectors.
Transmission may be mechanical (for example,
transnlira. ion of dysentery or typhoid h& L ills bv
'
the domestic fly ) . Such vectors arc called
mechanical vecrors. In other instances , the
pathogen multiplies in the body of the vector, often
undergoing part oi its developmental cycle in if .
- Such vector? are termed biological vectors (for
example, Aedes aegypri mosquito in yellow fever .
Anopheles mosquito m mataiLa ). Biological
vectors transmit infection only after the pathogen
has mulLiphed in them sufficiently or has
undergone a developmental cycle. I'hc interval
between the time of entry of the pathogen into
the vector and the vector becoming infective is
called the exmraic incubation period ,
Besides acting as vectors, some insects may
also act os reservoir hosts (for mm pic , ticks in
relapsing fever and spatted fever) . Infection is
maintained in such insects by transovarial or
uansstadial passage.
Soil and water: Some pathogens can survive
in the soil for very long periods. Spores of tetanus
bacilli may remain viable in the KCMJ for several
decades and serve a ? the source of infection. Fungi
( Histopiasma capsuhtuio , Nocardia astcroides )
and also parasites such as roundworm and
hookworm survive m the soil and cause human
infection.
\ V. Lcer may set is the source ot infect ion ei ther
due to contaminaiion with pathogenic
microorganisms ( cholera vibrio , infective heparin ®
virus ) or due to the presence of aquatic vectors
(cyclop^ m guineiworm infection ).
Ftuni: Cun turn mated food may act as a source
of infection. The presence of pathogen? in food
may be due to external contamination ( food
porioiuiLg by staphylococcus ) or due to pre -
existent i nfoctii > n i n meat or other am mol product
(salmonellosis ).
opy righted material
66 « TexlEook ol Microbiology
MBTMODS ( IP THANSMESSIOIV UK
INFECTION
Contact Infection may be acquired by contact,
which may be direct or indirect . Sexually
trantmiiried diseases such as syphilis and
gonorrhea illustrate spread by direct contact . The
term ccrt £i£ii < HJSdisease ILJI.J been used KJT diseases
transmitted hy direct contact, distinct from
infection disease signifying ail other modes of
*
transmission.This distincrior is now not gcncraUv
employed. Indirect contact may be through the
agency of tomitez, which are inanimate objects
such as clothing* pencils or toys which mav be
contaminated by a pathogen from one person and
act is a vehicle for its transmission to another .
Pencils shared by school children may act as
fomites m the transmission of diphtheria, and face
towels in trachoma .
Inhalation: Respiratory infections such as
influenza and tuberculosis are transmitted by
inhalation of the pathogen. Such microbes are
shed by the patients into the environ men t, in
secretions from the m >se or throat during SflC«ingh
speaking or coughing. Large drops of such
secretions fall to the ground and dry there .
Pathogens resistant ro drying may remain viable
in the dust and act at sources of infection. Small
droplets* under 0.1 mm in diameter, evaporate
immediately to become minute particles or dropJc T
-
nuclei ( usually 1-1 Q pm in diameter ) which
remain suspended in rhe air for long periods,
acting as Sources of infection .
Ingestion: Intestinal inlecriors arc generally
acquired hy the ingestion of food Or drink
contaminated by pathogens. Infection transmitted
by ingestion nuv be waterborne { cholera ) ,
foodborne ( food pOLSonLng} or hartJborne
(dysentery ). The importance of fingerborne
transmission is being increasingly recognised , not
only in the case of pathogens entering through
the mouth, but also those that enter through the
nose and eves.
Inoculation: Pathogens, inhume instances, may
be inoculated directly into the tissues of the host.
Tetanus spores implanted in deep wounds, rabies
virus deposited subcutaneously by dog bite and
arboviruses injected by ] nsectvectors are examples.
Infection by inoculation may be iatrogenic when
unstetile syringes and surgical equipment are
employed . Hepatitis 13 and the Human
Immunodeficiency Virus ( HIV ) may be
transmitted through transfusion of infix-red blood,
or the use of contaminated syringes and needles,
particularly among addicts of injectable drugs.
Insects; Insects may act as mechanical or
r
biological vectors of infectious diseases.
Congenital: Some pathogens are able to cross
the placental barrier and infect the fetus in utcro.
This is known as vcrT?W transmission This may
,
.
result in abortion , miscarriage or stillbirth. Live
infants may be born with manifestations of a
disease , as in congenital syphilis . Intrauterine
P
infection with the rubella virus, especially in the
first trimester of pregnancy, may interfere with
organogenesis and lead to congenital
malformation. Such intections are known as
tmtqgenic infections,
Ifltrogeriic amt laboratory infection ;
*
Infection may sometimes be transmitted during
administration of injections, lumbar puncture and
CltheterigltUHl , il meticulous care in asepsis is
lacking. Modern methods of treatment such as
exchange transfusion, dialysis, and organ transplant
surety have i ncreased the possibilities for iatrogenic
infections. Laboratory peraannet bundling infectious
material are At risk and special care should be taken
to prevent laboratory infection.
The outcome of an infect ion will depend on
die interaction between microbial fector? which
predispose to pathogenicity and host fact ora which
contribute to resistance .
FACTORS PREDISPOSING TO
MlcHUBIAL PATH o u t f i t:t T v
The terms 'pathogenicity * and Virulence* refer to
Copyrighted materia
 * Infection 67

the ability of a microbe to produce disease or tissue
injury bur it is c&nvenient to make a fine
distinction between them . " Pathogenicity ' is
, event but a specific reaction between surface
generally employed to refer to the ability o\ a
nicrobinl species rn produce disease, while the
lent ] Viralem* is applied to tile same property
in a attain of nnicrchorganisiia . Thus the species
\i. tuberculosis or the polio virus is referred to
as being pathogenic. The pathogenic species
AJ . rubcrLt/ loirs and the polio viruscontain strains
of varviog degrees of virulence including those
which arc avirulent, such as the vaccine strains .
The virulence of a strain is not constant and may
undergo spontaneous or induced variation .
Enhancement of virulence is known as exaltation
and can be demonstrated experimentally by serial
passage in susceptible hosts . Reduction ot
virulence is known as jttemudofl and can be
achieved hv passage through unfavourable hosts,
of many infections is the attachment of the bittern
to body surfaces. This attachment is not a dunce
receptors on host cells and adhesive structures
f /rgandsjon the surface of bacteria. These adhesive
structures are called .idhesuis. Adhesins may occur
as organised structures, such as fimbriae or
fibrillar and pill, or is colonisation factors. This
specific adhesin may account for rhe tissue
tropisms and host specificity exhibited by many
pathogen 5- Adhtsins serve as virulence factors,
and loss of adhesins often renders the strain
jviruleni. Adhesins are usually made of protein
and ate antigenic in nature. Specific immunisation
with adhesins has been attempted as a method of
prophylaxis in some infections, as for instance
against E. ctyli diarrhea in calves and piglets, and
gonorrhea in human beings.
Lnvasiveiiess: This refers fo the Ability of a r

repeated cultures in artificial media, growth under
high temperature or in the presence of weak
utiseptie, desiccation, or prolonged storage in
culture.
Virulence is the sum total of several
determinants, as detailed below.
pathogen to spread in the host tissues after
establishing uifetbon, Highly invasive pathogens
characteristically produce spreadi ng or generalised
-
lesions (fcg p streptococcal septicemia following
wound infection ) , while less invasive pathogens
cause more localised lesions ( erg p staphylococcal
«

Adhesion; The initial event in the pathogenesis abscess ). Some pathogens* though capable of

Table 9.1 Dlstin.guishir>g features Of eioioitins and endotoiins

fCxctoxins Endotoxins

1. PTI & EELEIIS I ,ifMpoLysaeckar ides

2 . Heat labile Heat stable
3. Actively SCOT red! by cells; diffuse Into
. Fonn part of cell wait; do not diffuse into
,

surrounding medium surrounding medium

4 Readily separable from cultures by physical
P Obtained only by cell lysis
means such a® filtration
5 Action often enzymic
, No enzymic action
6, Specific pharmacological effect for each Effect nonspecific; action common to all
cxoloxin endmtdai
7 Specific tissue affinities
, No specific tissue aifinitv
8, Active in very minute doses Active only in very large doses
9 Highly antigenic
, Weakly antigenic
.
10 Action specifically neutralised by antibody Neutralisation by antibody ineffective

Copyrighted material
hi * Te*ttwok a1 Micnotnology *
causing serious or even fatal diseases , lack
invusiveflW allugetlijer (e.g, , [he LeLunUS buillut
which remains confined r< > the site of entry and
produces the disease by elaborating a potent
tutiji) -
Toxijerucity: Huc- retix produce two types of
—
toxins exotmt i ns and endotoxi ns,
Ejtotoxins ATC heat labile prutrirts which are
secreted by certain species of bacteria and diffuse
readily into die surrounding medium. They are
.
highly potent in minute imounts and constitute
some of the most poisonous substances known.
One mgrst tetanus or botulinum toxin is Sufficient
to kill more than one million guinea pigs and it
has been estimated rhit 3 kg of botulinum toxin
cad kill .LL ] the inhahi runts of the world - Treatment
of cjiotoxins with formaldehyde yields toxoids
which are notonc hut retain the ability to induce
.a
Antibodies ( antitoxins}. They exhibit Specific tissue
Affinities and pharmacological Activities, each
toxin producing a typical effect which can he
made out by characteristic clinical manifestations
or autopsy appearances . EJKUKKUU aie generally
formed hv Gram positive bacteria hut may also
be produced by some Gram negative organisms
such as Shiga’s dysentery bacillus, vibrio cholera
and enterotoxigenic E. coti .
Endotoxins arc heat stable lipopoEysaccharidcs
{ EPS ) which torm jn integral pjrt of the cell
wall of Gram negative bacteria. Their toxicity
depends on the lipid component (lipid A ) . They
are not secreted outside the bacterial cell and are
released only by the disintegration of the cell wall .
They cannot he coxoided . They an? poor antigens
and their toxicity is not completely neutralised by
[ lie homolijguui Antibodies. They are active oulr
in relatively targe doses. They do not exhibit
specific pharmacological activities. All endotoxins,
whether isolated from pathogenic or
nonpathogen ic bacteria , produce similar effects.
Administration of email quantities of endotoxin
in susceptible animals causes an elevation ofbody
temperature manifested within \ 5 minutes and
luting tor several hour*. The pyrogenic effect of
fluids used for intravenous administration is
usually due to the presence of endotoxins from
contaminant bacteria . Intravenous injections of
large doses ot endotoxin uni massive Gram
negative septicemias cause cnUntmcic shock
marked by fever, leucnpenia, thrombocytopenia,
significant tall in blood pressure, circuiaCcry
collapse and bloody diarrhea leading to death
( Tables 9.1, 9.2 ) .
PtasniiJic Genes coding for some virulence
characteristics may be plasmid borne. Lixamplcs
of pilimid-barnc virulence factors are surface
antigens responsible for the colonisatiem of
intestinal mucosa by F . coli and enteroioxin
production by E, aoSand .Staph. aureus. Multiple
dmg resistance (R) plasmids increase the severity
of clinical disease by their resistance to antibiotic
therapy.
Llacteriophagei: The classical example of
phage directed virulence is seen in diphtheria. In
diphtheria bacilli, the gene for toxin production
LV present in beta or richer tux ' ooiyncphages.
Communicability: The ability' of A parasite
to spread from one host ro another is known as
communicability. This property does not influence
the production of disease in an individual host
but determines the survival and distribution of a
parasite in a commumcy. A CWIcUtlOXl need not
exist between virulence and communicability. In
t .LCt. a highly virulent parasite may not exhibit a
high degree of communicability due to its rapidly
lethal effect on the host . In general , infections m
which the pathogen is shed in secretions, as in
respiratory or in test in si diseases , are highly
communicable . In sortie instances, as in
hydrophobia, human infection represents a dead
end, there being an interruption in the spread of
the pathogen to other hosts.
Development oi epidemic and pandemic
diseases requires the strain of pathogen to possess
high degrees of virulence and communicability.
.
Other bacterial prciducttt Sr: me bacterial
Copyrighted materia
 * Inlection » 69

products other than toxins, though devoid of
intrinsic tonicity, nwy contribute to virulence by
inhibiting the mechanisms of host resistance.
Pathogenic staphylococci produce a ihrombin -
like enzyme conge!use which prevents
phagocytosis by forming a fibrin barrier around
the bacteria and willing off the lesion
lnhrinnlyritls promote the spread nf infect inos hv
breaking down the fibrin barrier in Einhues.
HvaJuronidascs split hyaluronic acid which is a
component of intercellular connective iissue and
thus facilitate the spread ot injection along tissue
or death , rcapectively, in a sustcprihlc animal
under
standard conditions . As animals exhibit
considerable individual variation in susceptibility,
these doses are more correctly estimated as
statistical expressions, ID SO and I ,D 50, as the
dose required to infect or kill 50 per cent of the
, animals tested under standard conditions .
Route of infection: Some bacteria, j^ h ns
EtTCptOcocci , Can initiate infection whatever he the
mode of entry. Others can survive and multiply
only when introduced by the optimal routes .
CbdLen vibrio* arc infective orallvhut arc unable
r

spaces. Leucocidini damage polymorphonuclear to cause infection when introduced subcutaneously. Jr

leucocytes. Many pathogens produee hemolysins
capable of destroying erythrocytes but their
significance in pathogenicity is not clearly
understood ,
Bacterial appentiniest Capsulitcd bacteria
such pneumococci , /C, ponuDcunt and / /
influenzae in nor readily phagocyiosed. Some
bacterial Hiirtace antigens such as the Vi antigen
of 5, tvphi , K antigen* of Er anti also help rhe
bacteria to wirhstind phagocytosis and the lytic
activity of complement
Infecting dose; Successful infections require
that an adequate number of bacteria should gain
entry into the host. The dosage may be estimated
as the minimum infecting dose ( MID ) nr
minimum lethal dose ( MLD ) which are ,
respectively, the minimum number of bacteria
required ro produce clinical evidence of infection
This difference is probably related to mode? by
which different hacterij are able to initiate tissue
damage and establish themselves. Bacteria also
differ in their rites of election in the host body
after introduction into tissues. They also differ in
'
the ability' to produce damage of different organs
in different species of luiimaJs. Tubercle bacilli
injected into rabbits cause lesions mainly in the
kidney and infrequently in the liver and spleen,
but in guinea pigs the lesions are mainly in ibe
liver mil spleen , the kidnew being spared. The
reasons for such selective multiplication in tissues
are largelv obscure, though they may he related
to the presence in rissucs of substances that may
selectively hinder or favour their multiplication,
-
TVIM I M OP INPKCTIOU DISKASKS
*
Infections diseases maybe localised or generalised.

Table 9.2 Biological activities of endotoxins

IVrogenidty Lethal action DrpreGBion of blood pressure
Afflvttsm of complement IntravMoiilsir coagulation Leueopeoia
LeUCOCYtOlk Inhibition of glucose and SEI mutation of B lymphocytes
glycogen gynrhcsifl Ln the Liver
M aerojvhjage inhibition Interferon release Induction of jWi ^ ratrkrLLlin
synthesis
IClotting of limuius Ivsate ( lysate of amcbocytes from horsc-ahra crab, Limuius pulyphcmus, used as a
VlW fur detection of endotoxins)

Copyrighted material
70 * Texibook of Microbiology *

Localised infections may hr: superficial deep or
seated Generalised infccrinn involves the spread
. infectious diseases may be classified into different
of the infecting agent from the sice of entry by
contiguity, through tissue spaces or channels,
along the lymphatics or through the bloodstream.
Circulation of bacteria in the blood is known as
bacteremia . Transient bacteremia is a frequent
event even in healthy iruiividud; and may occur
during chewing, brushing of teeth or straining at
Stools, The bacteria are immediately mapped up
by phagocytic cells and arc unable to initiate
infection, JJactemrus ofgreaterseverity and longer
duration in seen during generalised infections as
in typhoid fever, Sepricerma is the condition
where baden .1 circulate and mill [ i ply i n the blood,
form toot ic products and cause n igh, swi nging type
of fever. Pyemia is a condition where pvogenic
bactena produce septicemia with multiple
abscesses 1 n the 1 niernal organs such as the; spinn,
liver and kidney.
depending or their spread in the community,
-
types . Enticmi : diseases arc those which are
consrandy present in a particular area. Typhoid
fever is endemic in most parts of India.An tpicfemte
disease is one that spreads rapidly, involving many
persons in an area at the same time . Influenza
causes annual winter epidemics in the cold
countries. A pandemic Ls an epidemic that spreads
thmugh many areas of the world involving very
large numbers of people within a short period.
Influenza , cholera, plague and cntcroviral
conjunctivitis are pandemic diseases. Epidemics
vary in the rapidity of spread. Waterborne diseases
such us cholera and hepatite may cans? explosive
outbreaks, while diseases which spread by person
to- person contact evolve more slowly. Such
-
creeping or smouldering epidemics, as that of
cerebrospinal fever, are termed pnosudemiL-
diseases*

Further HonJinjl
Mint* CA- 1?S7. The /1ithqprne.sjs of Infectious Disease. 3*1 edn. London: Academic PTTJ*.
Rati ick S and MJ Larkr: 1995 . /FflJiWFKiAqpraJ and Molecular aspects of Bacterial K'imSence. Chichester: Wiley;
]

VrfyhijQ' tn< j Immunity, S" edn. VQLI , Lundon ; Edward Arnold.

1
--
IVwtun I li and Jl Aibuthnot 1990. Deter mi Hints- of bacterial vi rulenct In: Tripiey u.. i IVfJmn s Brittcipie-s of Bacteriology,
Roth JA et al, rda 1989. VrrWcncr jVfcWuj.'.:oni of Baetiriaf Ptr-hngeo*. Washington DCr American Society far
Microtia Lqgy.

I j opy righted materia

 Immunity

The term Hmmur- ityh traditional]) refers to the

1
general, or specific where resistance to a particular
resistance exhibited by the host towards injury pathogen is concerned.
caused by microorganisms and their products. Innate immunity may he considered at the level
However protection against infectious diseases is of the species, nice or individual. Species immunity
only one of the consequences of the immune refers to the total or relative refractoriness to a
response, which in its entirety is concerned with pathogen, shown by all members of a species. For
the reaction of the body against any foreign antigen. instance, all human beings are totally unsusceptible
Immunity against infectious diseases is of to plant pathogens and to many animal pathogens
different types: such as rinderpest or distemper. This immunity is
something a person obtains by virtue of being a
I Innate (Native) Immunity part of the human species. The mechanisms of
Species species immunity arc not clearly understood but
(a ) Nonspecific Racial may be due to physiological and biochemical
Individual differences between the tissues of the different Host
species, which determine whether or not a pathogen
Species can multiply in them. An early insight into the basis
( b) Specific Ra.ci .il of species immunity was gamed by Pasteur 's
J n dividual experiments on anthrax in Hogs , which are naturally
resistant Bo the- disease but become susceptible when
IT Acquired {Adaptive } Immunity thrir body temperature i ^ raised from 25 "C to
Natural 35 *C,
(a ) Active Within a species, different races may show
Artificial differences m susceptibility to infections. Trie is
known as mcraJ immunity, the classic example of
Natural which is the high reactance of Algerian sheep to
( li i
'
Passive anthrax. Such racial differences are known to be
Artificial genetic in origin, and by selection and inbreeding,
it is possible to develop, at will, races that possess
Injure or native immunity is the resistance
to hii'h degrees of resistance or susceptibility to
infections which an individual possesses by v irtue various pathogens. It h difficult to demonstrate
of His generic and constiniTianal make- up. It is not marked differences in immunity in human races, as
affected by prior contact with microorganisms or controlled breeding is not possible in the human
immunisation . It may be nonspecific ^ when it species. It has been reported that the people of
indicates a degree of resistance to infections in Negroid origin m the USA are more susceptible

Copyrighted material
72 4
-
Te Jtibaok of MjcrobioJogiy
than the Caucasians- to tuberculosis. But such
comparisons are vitiated by menial influences such
as differences in socioeconomic levels . An
interesting instance ot genetic resistance to
Plasmodium fakipanjm malaria is seen in some
parts of Africa and die Mediterranean coast. A
hereditary abnormality of ted cells ( sickling ),
prevalent in the area , confer immunity to infection
by the maJari .d parasite and may have evolved from
the survival advantage conferred by it in a malari . ii
environment .
The differences in innate immunity exhibited
bv. different individuals in a race is known as
indjvidudimfn iiyThc genetic basis of individual
'
^
immunity is evident ffnm studies on the incidence
of infectious diseases in twins. It is well documented
that homozygous twins exhibit similar degrees of
resistance or susceptibility to lepromatous leprosy
and tuberculosis. Such correlation is rtOT seen in
heterozygous twins .
Several factors influence the level of innate
immunity in an inLlividuaJ:
A.fce: The two extremes of life carry higher
susceptibility to infectious diseases as compared
with adults. The fetus in uttro is normally protected
from maternal infection by the placental barrier.
But some pathogens cross tins harrier causing
overwhelming infections resulting in fetal death -
Smnc , such as rubella , herpes cytomegaloviruses
and ,r . : injJ .^ .' ii . i IFONTJJJ , le .Lcl to congenital
malformations . 1"he hv ightencd susceptibility of the
fetus to infection -s related to the immaturity of its
immune apparatus . Newborn animals are more
susceptible to experimental infections than older
ones . Coxsackie viruses cause fatal infection in
melding mice but not in adults.
Increased susceptibility in the young nuty, in some
instances , be due to hormonal influence . Tinea capitis
caused by Microsportim audouiitii frequently
undergoes spontaneous cure with the onset of puberty
The susceptibility of the vaginal epithelium in
prepubertal girts to gonococcal infection is another
instance of the effect of sex hormones on resistance .
Some infections l i k e poliOntyel 11 i s and
chickenpox tend to be more severe in adults than
in young children, due to hypersensitivity that
causes greater tissue damage. Conversely hepatitis
B virus infcctionl in the newborn are usually
asymptomatic because clinical disease requites
adequate immune response wliuch. is lacking at that
age. Hovrever, the virus multiplies unrestrained and
such neonates end up as chronic viral carriers, often
developing late hepatic complications. Old persons
arc highly susceptible to infections due to the
waning of their immune responses and other
infirmities like enlarged prostate leading to urinary
stasis.
Hormonal Influence );: Endocrine disorders
such as diabetes mellirus , hypothyroidism and
adrenal dysfunction are associated with an enhanced
susceptibility to infections. The high incidence of
staphylococcal sepsis in. diabetes maybe related to
the increased level of carbohydrates in tissues.
Cort icostcroi Js exert an i mportant i influence on the
response to infection. Thev depress the host’s
resistance by their anr I inflammatory and
antiphagocytic effects and by the suppression of
antibody formation and hypersensitivity. They also
haw a beneficial effect m that they neutralise the
harmful effect of bacterial products such as
endotoxins The elevated steroid level during
pregnancy may be related to the heightened
susceptibility ol pregnant women to many
i nfections. The reported effect of stress in iitcreasing
susceptibility to infections, may in some measure
be due to the release of steroids.
Nutrit «m: The infraction between malnutrition
and immunity is complex but 1 in general , both
humoral and cell mediated immune processes are
reduced when there is malnutrition. Cell mediated
immune responses such as the Mantoux test become
negative in severe protein deficiency, as in
kwashiorkor. Because of its wide prevalence,
malnutrition may well be the commonest cause of
i n mu ii i identic in:v.
iff
Paradoxically [here is some evidence that certain
Copyrighted materia
 * Immunity »
infections may nor became clinically apparent in
the severely malnourished . Malarial infection in
the ranine stricken may nut induce fever but octet
chcir nutrition is improved , clinical malaria
develop, fr has also been reported that some viruses
may not multiply in the tissues of severely
malnourished individuals.
MECHANISMS O F INNATE IMMUNITY
ftpithelin! surfaces; The intact skin and
mucous membrane covering the body protect if
considerably against invasion by microorganisms.
They provide much marc rhan u mechanical
barrier. Healthy skin possesses bactericidal activity
In which the presence of high concentration of sail
in the drying sweat, the sebaceous secretions and
the long chain fatty acids and soaps contribute.
When cultures of typhoid bacilli placed on healthy
skin and on a glass surface are sampled at intervals,
the bacteria on the skin are seen to be killed within
minutes, while those on glass survive for several
hours. The bactericidal activity of skin secretions
JF
is illustrated by the frequent mycotic and pyogenic
infections seen in persons who immerse their hands
ip soapy water for l -: mg pnods OCCUpatLLipallv.
Though the skin frees itself readily of bacteria
deposited or it ( transients ), its reactions are different
to the bacterial flora normally resident on it .
Resident flora arc not easily removed evert hv
washing and application of disinfcctant*-
'
I hc mucosa of the respiratory tract has several
'
innate mechanisms of defence. The very architecture
of the nose prevents entry of microorganisms to a
Urge extent, the inhaled particles being arrested at
or near the nasal orifices. Those that pass beyond
flic held by the mi LOLLS lining the epithelium, and
arc swept hack to the pharvmt where they tend to
be swallowed or coughed out. The cough reflex LS
an important defence mechanism of the respiratory'
tract. The cilia on the respiratory epithelial cells
propel particles upwards. Nasal and respiratory
secretions contain mucopolysaccharides capable of
combining with influenza ailJ certain other viruses.
1$
Pianides. that manage to reach the pulmonary alveoli
are iogcsrcd bv the phapjotytic cells present there.
Tire mouth is constantlv bathed in saliva which
hn.s an inhibitory effect cm many miLTacsTgam ^ iTis.
Particles deposited in the mouth arc swallowed and
subjected to the action of the digestive juices. The
high acidity of the stomach destroys most
microorganisms. The pH becomes progressively
alkaline from the duodenum to the ileum. The ifcum
contains a rich and varied flora and in. the large
intestine,. the bulk of the contents is composed of
bacteria .
The intestinal mucosa is covered bv a bcelike
network of mucus. Particles get enmeshed in the
mucus and form small masses which ire propelled
by peristalsis.
The conjunctiva is freed of foreign particle* hy
the flushing action of lachrymal secretion*. 'Hie
eyes become susceptible 10 infection when
Lachrymal secretions are absent. Tears contain the
antibacterial substance lysozyme, first described hy
Fleming (1922), This is a thermolabllc low
molecular weight basic protein which acts as a
inuramiiudasc - Lysozyme is present in tissue fluids
and in nearly all secretions eveepe cerebrospinal
tluid , sweat and LLriue. It acts by splitting certain
polysaccharide components r ? t the cell walls of
susceptible bacteria. In the concentrations seen in
tears and other secretions, lysozyme is active onlv
against & nmc nnnpsthngenic fltani positive
bacteria. However, it occurs in phagocytic ceils in
concentrations high enough to be lethal to mint
pathogens .
The flushing action of urine eliminates bacteria
from the urethra. Spermine and zinc present in tbc
nemen carry out antibacterial activity. The acidity
of the adult vagina, due to the fermenration ul
glycogen in the epithelial cell* by the resident
aciduric bacilli , makes it inhospitable io many
pathogens .
Antibacterial substances in hi nod and
tisSUCSl The complement system possesses
bactericidal activity and plays ,in important role in
Copyrighted material
74 * Textbook ai Microcnology >
the destruction of pathogen / bacteria that ; nvade
the blood and Tissues (see chapter 14) ,
Several substances possessing antibacterial
properties have been described in blood and tissues.
These include ( 1 } beta lysi nh a relatively
thermostable suhstance active against anrhricf and
related bacilli ; ( 2 ) basic polypeptides such as
Lukins extracted from leucocytes and pkkins from
platelets; i -i I acidic substances, such as lactic acid
found in muscle tissue and in the inflammatory.
Tores; And ( 4) lactnperoxldase in milk. While these
substances possess antibacterial properties
demonstrable experimentally, their relevance in the
natural context is not clearly understood .
A method of defence ag . iinst viral infections is
the production or : utcHeror by cells sri - uulated by
live or killed viruses and certain other inducers.
Interferon has been shown ro be more important
than specific antibodies in procecdon against and
recovery from certain acute \ iral infections/! :" sues
'
and bodv secretions contain other antiviral
r
substances.
Microbial antagonism : The skin and
*
mucous surfaces have resident bacterial flora which
prevent colonisation by pathogens - Altera: Lon of
normal resident flora mav lead to invasion bv.
cxtnncoua microbes, causing serious diseases such
, lymphocytes called natural killer fNK ) cells are
as staphylococcal or clostridial enterocolitis
following oral inti bio tics. The importance of
normal hacterisil flora in native irnmunitv is
&
exemplified by the extreme susceptibility of germ
free animals to all types of infections .
Cellular factor* in innate immunity;
Natural defence against the invasion oiblouJ and
Tissues by microorganisms and other foreign
particles Is mediated to a large extent by phagocytic
cells which ingest and destroy them. Phagocytic
cells, originally d / eovered by Mctchnikoff ( 1883) ,
were classified by him into microphages and
macrophages. Micro phage a arc polymorpho
nuclear leucocytes . Macrophages consist of
hi * riocytcs which ate the wandering ameboid cells
seen in issues, the fixed ft- iu- ubenduthelLaJ cells
and the monocytes in the blood. A major fund ion
of the reticuloendothelial system is the removal of
foreign particles that enter the body. Phagocytic
cells reach the -. ites of inflammation in large
numbers, attracted by chemocactic substances, and
mgest particulate materials . Capsulated bacteria ,
such AS pneumococci are not readily phagocytoscd
,
except in the presence of opsonins. They are more
readily phagocytosed when trapped against a firm
surface such as the alveolar wall than when they
are free in [ issue fluids. Bacteria are phagocytosed
into a vacuole (phagosome ), which fuses wirh the
lysosomes round in the cell to form the
phagolysosome . The bacteria are subjected to the
action of the Ly 1 :c enzymes in the phagolysosome
and ate destroyed. Some bacteria, such as brucella
and lepra bacilli resist intracellular digestion and
,
may actively multiply inside the phagocytic cells.
Phagocytosis in such instances may actually help
to disseminate infection to different parts of the
hody. The importance nl phagocytosis in protection
against infection is evidenced by the enhanced
susceptibility to infection seen either when the
phagocy11 cells are depleted, as in agranulocytosis ,
or when they are func; innalIy deficient , as in
if
chronic granulomatous disease . A class of
important in nonspecific defence against viral
infections and tumours. They selectively kill virus
i ] i fee ted cells and tumour cells. NK cells are
activated bv interferons.
jf
Jnllammntinn: Tissue ir- iury or irritation ,
initiated by the entry of pathogens or other irritants ,
leads to iuflanimation, which is an important ,
nonspecific defence mechanism. The arterioles at
the site constrict initi . dly and then dilate leading to
ai L i ncieased h ]ood flow. There i s a slowing of blood
flow and margination of the leucocytes , which
escape into the [ i ssuc > by diaped*:sis and accumulate
- in large numbers , attracted by the chrmntACfii;
substances released at the site of injury.
Microotgamsms arc phagocytosed and destroyed .
[ here is an outpouring of plasma, which helps to
opyrighted materia
 i Immunity
.
dilute die roxic produces present A fibrin barrier is
bid, serving ro wall off the site of infection.
I ' ever A rise of temperature following infection
L:- .1 natural defence mechanism. it not merelv helps
ro iccekntt the physiological pcDcene but m^v,
in somf eases , actually destroy the infecting
* immune response occurs more quickly and
pathogens . Therapeutic induction of fever was
employed for the destruction of Treponema
pallidum in the tissues of syphilitic patients before
penicillin became available . Fever stimulates the
pnxductiun ofinterferon and aids recovery trum viral
infcctions-
Acule phase proteins: Infection or Injury
lead? to a sudden increase in plasma concentrations
of ccTfflin proteins, collectively called acute phase
proteins. These include C reactive pmtcil ( CRP),
mannose binding protein, alpha -1'me id
glycoprotein, strain amyloid P component rod many
otheir. CRP and some other acute phase proteins
activate the alternative pathway of complement.
rrhcy arc believed to enhance host resistance, prevent
tissue injury and promote repair of infiam maton
lesiions.
A t :y L I R H D i M M l N I T\
The resistance that an individual acquires during
life is known as acquired Immunity as distinct from
inborn inottt immunity. Acquired immunity is of
two types, active and passive. Affine rmruunirv is
the resistance developed hy an individual as a result
of an antigenic stimulus. It is also known as adapm?
immunity' ass it represents an adaptive response of
rhe host to a specific pathogen or other antigen
This involves the active functioning of the hour’s
immune apparatus leading to the synthesis of
antibodies and the production [if LmmunologicaUy
active cells. Active immunity sets ill only after a
tatenr period which is required for the
immunologic ! machinery to be scr in motion.
*
During the development of active immunity, there
is often a negative phase during which the level of
measurable immunity may actually he lower than
it was before t be an tigenic stimulus . Tbis is because
75
cite antigen combines with any pie outing antibody
and lowers im level in circulation. Once developed,
the active immunity is long- lasting. If in individual
who hi? been actively immunised against an antigen
experience? the same antigen subsequently, the
abundantly than during the first encounter. This is
known ;LH .tfeondarv respond. Besides the
development of humoral and cellular immunitv,
active immunity is associated with Immunological
memory. I '’
his implies that the immune system is
able to retain tor long periods the memory of a
prior antigenic eKposure and ro produce a secondary
type of response when it encounters the same
antigen again , Active immunisation is more
effective and confers better protection than passive
immunisation .
The resistance that is transmitted passively to a
recipient in a ’readymade' form is known as pltnfe
murium'ty. Here the recipient 's immune system plays
no active role. There is no antigenic stimulus;
1
instead, preformed antibodies are administered.
There is ro latent period in passive immunity,
protection being effective immediately after pssrive
immunisation. There is no negative phase. The
immunity ii transient, usually lasting for days «
weeks, only till the passively transmitted antibodies
are metabolised and eliminated. No secondary tvpe
ruspinse OCCUIS in passive immunity.In fact, passive
immunirv diminishes, in effect with repetition.
When i foreign antibody it administered a second
time, it it eliminated more rapidly than initially
. Following the first injection of (in antibody such as
immune hor*c scrum, the elimination is only hy
metabolic breakdown but during subsequenr
injections of horse serum, elimination is much
quicker as it combines with antibodies to horse
serum that would have hern produced following
its initial injection. This factor of immune
chmimTion ]im i rs the iLscti.il ness nt’repeated passive
immunisation. Passive immunisation is less effective
and provides an immunity inferior to that provided
by active immuniseion. The main advantage of
Copyrighted material
76 * Textbook ol Microbiology *
.
Ti 1 - -
i : immunisation i that H . kL immediately
and , therefore, can be employed w'hen ' instant '
-
immunity is de ired I. I .ible 10.1).
Active immunity may be natural or arrifieiaL
Natural active immunity results ITUKI either a
clinical or Jin inapparent infection by a microbe. A
--
pn r T I who lias recovered from an. attack of measles
develops natural active immunity. The huge majority
of adults in the developing countries possess natural
active immunity to poliomyelitis due to repeated
, c. Subunit { Hepatitis 13 vaccine)
inappsrent infections wi. rh polioviruses during
childhood . Such immunity is usually long-lasting
but the duration varies with the type of pathogen.
The immunity is lifelong following many ' iral
diseases such as chicken pox or measles. In some
viral diseases* such as influenza or common cold,
the immunity appears to be shortlived. Influenza
can recur in an individual after a few months or a
year hul this in not SD much due to lack of the
i menu n :ising capact ty of the virus as to : ts abil 1 ry to
undergo lAti mr variation so that immuniiy
^
following the first infection is not effective against
the second infection caused by an antigcmcally novel
virus . In common cold , the apparent lack of
immunity is because the same clinical picture can
be caused by infection with a large Humber of
diflfcrcn t viruses.The i mniulity follow 11 ig bactci i al
infection is generally less permanent than that
following viral infections. Some, such as typhoid
fever , induce durable protecti ^ n. In syphilis , a speci .d
type of immunity known ss ' premunition’ i seen .
I lere, the immunity to reinfection lasts only as long
- Natural passive immunity is the resistance
JV the original infcc’ Jon ncmnins active. Once the
disease is cured, the patient becomes susceptible to
the spirochete again. In chancroid, another venereal
disease, caused by Haemophilus ducreyi, there does
not appear to be any effective i mrnuii Lev as the pat i mi
may develop lesions following reinfection even
while the original infection in active.
Artificial active immunity is rhe resistance
induced by vno lines . Vaccines arc preparations of
I ivc or k i I led n i icrootgan i sms or thei r products used
for immunisation .
Examples of vaccines are
1 . Haute:ial vaccines
as follows:
a. Live ( BCG vaccine for tuberculosis)
b. Killed {Cholera vaccine )
c. Subunit i ryphuid Vt antigen I
d. Bacterial products (Tetanus taw ; id )
2. Viral vaccines
a. Live ( Oral polio vaocinc^Sabin )
b Killed {Injectable polio vaccinc-Salk)
r ive vaccines initiate an infection without
causing any injury or disease. The immunity
Following live vaccine administration therefore
parallels that following natural infection though it
may be of a lower order. The immunity lasts for
several years but booster [loses may be necessary'.
Lice vaccines may be admi -listened orally (as with
the Sabin vaccine for poliomyelitis) or paicntcrally
(as with the measles vaccine ). Kilted I' sccines are
generally less immunogenic than live vaccines* and
prnteclion lasts only 1 or a short period. They have,
therefore * to be administered repeatedly' generally
at least two doses being required for the production
of immunity'. The first is known as the primary
dose and the subsequent doses as booster doses.
Killed vaccines may' be given orally but this route
is generally not effective. Parenteral administration
provides humoral antibody response, which may
be improved by the addinon of ‘adjuvants’ (for
example* aluminium phosphate ).
passively transferred from mother to baby. In human
infants , maternal antibodies arc transmitted
predominantly through the placenta, while in
animals such as pigs* transfer of antibodies occurs
mainly orally through the colostrum - The human
colostrum, which i? also rich in IgA antibodies
resistant to intestinal digestion , gives protection to
the neonate.The human fetus acquires some ability
to synthesise anybodies ( IgM ) from about the
twentieth Week of life but LCE- immunological
capacity is still inadequate at birth. It is only by
about the age nl three months that the Infant
Copyrighted materia
 » Immunity
Table 10.1 Comparison of active and passive Immunity
Active immunity
Produced actively by hasts immune system
Induced by infection or by immunogens
Durable cffi&eiive protection
Immunity effective only alter lay period
-
Irntmann lrtgical memory present
Booster effect on ubtequent dene
'Negative phase may occur
1
Not applicable in rhe immunjodefieienr
acquires SUIUE measure qf immnnnhig Lea I
independence. Until then, maternal antibodies giivt
passive prelection against iriJbctknis dlKUc4 to the
infant - Transport of antibodies across the placenta
is an active process and therefore the concentration
of antibody in the fetal blood may sometimes be
hiirhui tllin that seen in the mother Profcr rion so
afforded Will ordinarily lie adequate against all the
.
common infectious diseases in the locality It is for
this reason that most pediatric infections arc DKHE
common after the age of three months than in
younger infants. By active immunisation of mothers
during pregnancy, it is possible to improve the
quality of passive immunity in the infants.
Immunisation oJ pregnant women with tetanus
toxoid is recommended for this purpose in
communities in which neonatal tetanus is common.
Artificial pawivt immunity is the resistance
passively transferred to a recipient by the
administration of antibodies . The agents used for
this purpose are hyperimmune sera of animal or
human origin, convalescent sera ami pooled human
gamma globulin. T hese are used for prophylaxis
and therapy, Equine hyperimmune sen such as
antitetanus scrum and ATS prepared from
hyperiimmunised horses used to be extensively
cm ployed. They gave Temporary protection but
carried the disadvantages of hypersensitivity anti
-
immune elimination Human hyperimmune
globulin ( for example , tetanus immune globulin,
TIG) is free from those complications and also
gives more lasting protection. Antisera of animal
* 77
Fmnv immunity
Received passively. No active host participation
Readymade antibody transferred
Transient! less effective
Immediate immunity
No memory
Subsequent dose less effective
No negative phase
Applicable in immunodeficient
origin arc now recommended only where human
] ! reparations are not available ( and gas gangrene
and anti botulimim scrat antivenoms ) .
CoautlciCClK sera (sera of patients recovering
from infectious diseases) contain high levels of
Specific aittihudy. Pturletl human gmutiggiobuUa
(gammaglobulin from pooled sera of healthy adults)
contains antibodies against Jill common pathogens
prevalent in the region, Convalescent sera and
pooled human gammaglobulin were used tor
passive immunisation against some vims infections
(like viral Hepatitis A). Human gammaglobulin is
also used in the treatment of patients with some
-
immunodeficiendcS Gammaglobulin tends to
aggregate and when injected intravenously may
cause anaphylactic reaction due to complement
activation. They are therefore to be given only
intramuscularly. It has to be ensured that all
preparations from human sera are free from the
risk of infection with hepatitis Bn hepatitis C „111V
nd other infective agents.
PSSHVC immunisation is indicated for Immediate
and temporary protection in a nonimmune host
faced with the threat of an Infection, when there L 5
insufficient time for active 1minimisation to take
effect, It is also indicated for the treatment of Home
infections , Passive LTD mu nidation may also he
employed for rhe suppression of active immunity,
when the latter may be injurious. An example is
the IISC of RJl immune globulin during delivery to
prevent Immune response to the khesus fectnr in
Rib negative women with Kh positive bablCS-
'opy righted materia
7a * Te*itiook
Sometimes a combination il active and passive
methods of immunisation is employed. This is
known ns combined immunisation. Ideally ,
whenever passive immunisation is employed for
IITIMO- Liiatc protection, combined immunisation is
- -
to IT- L p re hr rred , a.- i n the prafeefi i > n of a non ' mrnune
1 |Schick testi methods . Where protection is
individual with a tetanus - prone wound . The
method is to inject TIG in one arm and the fust
dose ill tetanus toxoid in the other. This is fallowed
by the full course of phased tetanus toxoid Injections.
TIG provides the protection necessary till the active
immunity is able to take effect.
A special type oJ immunisation is the injection
of immunoloj-ricaJlv cumpereiit lymphocytes. Tics
is known as .u /oprnc immunity and does nut have
general application. Instead of whole lymphocytes,
an extract of immunologically competent
1yr lip h my let , known a& the ' transler factor", can be
used. Tin -- has been attempted in the treatment of
,
certain types of diseases (for example lepmmatnus
leprosy ).
MEASUREMENT OF IMMIMTI
'Hie tnily valid measurement of immunity is to test
-
the re .btance of an individual to a challenge by
-
the pathogen.Thi IH, however, nor applicable since
the challenge itself altera the state of imitiunily. it
is, therefore, rot possible to measure accurately the
level of mmunity in an individual. Estimates of
immunity are generally made by statistical methods
using large numbers n ) individuals.
A simple method of testing immunity is to relate
its level to some convenient indicator, such as
demonstration of the specific antibody. This i > not
always reliable as the immune response to a
pathogen consists of the formation of antibodies to
several antL ens present m it , as also to the
^
production of cellular immunity. The antibodies
maybe demonstrated by a variety of techniques such
as agglutination, precipitation, complement fixation,
hemagglutiItalian inhibition, neutralisation , Kl . iS.A
and others. In the absence of exact information as
to which antigen of the pathogen constitutes die
of Microbiology *
protective annijert, serological attempts to measure
i mmunity arc at best only approxi mati i ms. In some
instances, as m diphtheria where pathogenc^ is is
due to a well-defined antigen ( the toxin ) , the level
of immunity can he assayed by in vitro or in vivo
associated with cell-mediated immunity, skin tests
for delayed hypersensitivity and in vitro tests for
CM! ariord an indication ot i mmun i ty.
LOCAL I MUI NITlf
fhe concept of local immunity, proposed by
Besredka (1919-74), has gained importance in the
.
treatment of infections wiii - h arc localised or where
il is operative combating infection at the site of
lit
primaiy entry of the pathogen . In poliomyelitis, for
'
instance, systemic immunity provided by active
immunisation with the killed vaccine neutralises
the virus when it enters the bloodstream, but it
docs not prevent multiplication of the virus at the
sire of entry ( the gut mucosa ) and its fecal sheddi ng.
-
Thi - is achieved by the local intestinal immuniry
acquired either as a result of natural infection or
immunisation wirh the ILVC oral vaccine. In
influenza, immunisation with the killed vaccine
elicits a humoral antibodv response. But the
antibody litre in respiratory secretion? is often not
h igh enough to prevent infection, N atural infection
or the Lve virus vaccine administered intranasally
provides local immunity. A special class of
immunoglobulin ? ( IgA ) forms the major
component of local immunity.
One type nf IgA antibody called secretory IgA
is produced locally by plasma cells present on
mucosal surfaces or in secretory glands. There
appears to be a selective transport of such antilxhlics
between the various mucosal surfaces and secretory
glands . Thus, following intestinal exposure to an
antigen, the Specific IgA anubody and the plasma
cells (ormiogsuch an anribody can bedemonstrated
in breast milk - This indicates the existence of a
common mucosal or sccmroij immune system .
Besides providing local defence against
Copyrighted materia
 n I nmunhty 79

miaoorifjiiisiios the mucosal lmmuiw system may

! community (had ) ire immune to i pathogen , the
also be involved iri handling vnrinus ant n cns rhat
^
mav come L:ito contact wirh mucosal suriaces from
m
the external environment M through food.
HERD [ HMUNFTI
Th ^ refers to rhe overaU ]evel of immunity J
i :i a
community rind is relevant in the control of epidemic
diseases. Wbtt) a Large proportion of individudl in a
herd immunity m the pathogen L$ satisfactory. When
herd immunity is tow, epidemics sire likely to occur
on the introduction of a suicable pathogen, due TO rhe
procrux of large number* of sunpribk individuals
in the community. Eradication of communicable
diseases depends on the development of a high kind
of herd immunity rather than on the development of
a high level of immunity in mdivktuak .

-
l uirth gr itctfcliiiij
.

Janeway CA and P Travers 1S94. J.namunaJ3w>A3gy. London: Current Biology

Gaboy C and I Kuthner lW9 ALUCC phase proreins. N EfiglJ Med 34th44S.
f

Kwairkowsky D. 2000. Suscepribiliry to iofbctwa* flrM / 311;10ilr

^
Roitt IM. 1W4. EssHiriaJf Immmobgy, Kk edn. London; Blackwell Scientific.
-
Weir DM and ] Srewarr lW?. ZjninuHiJqgy S* cdn. Edinburgh: Churchill Livingitune.

- 1
—
Copyrighted material
1
 Antigens
An antigen has been defined as any substance
which . when introduced parentcralU into the body,
st i mutates the product in of an an t Lbody with w] i i eh
^
u reacts specifically and in an observable manner ,
-
Thi ; traditional description of an antigen is no
longer comprehensive enough in the light of current
nancepts about the immune response. Some antigens
may rot induce ant i bodi LLS but may sens i r i sc spec ific
lymphocytes lead nig to «11 mediated immunity or
may .. uisc i mmunologi cal tolerance.
The word ' parenteral' ( meaning, outside the
intestinal tract ) is used in the definition because
orally administered antigens arc usually denatured
by digestive enzymes and their antigenicity
destroyed, so that no sntiiwdv formation takes place.
When given parenwrally, antigens do nor undergo
any such inactivation and can induce antibody
production. However, there are exceptions and Home
antigens can be immunogenic when given orally,
such as oral vaccines .
The word 'specifically’ in the definition is
important as specificity is the hallmark of all
immunological reactions. An antigen introduced
into the hndy reacts only with those particular
iinmunocytcs ( B or T lymphocytes) which cany
the specific marker for that antigen and which
produce an antibody or Cells complementary to that
antigen only. The antibody so produced will react
cully with that particular antigen and with no other,
though immunological cross reaction may occur
between closely related antigens.
The two attributes of inti genicity are (1)
induction of an immune response ( immuno
genicity) , and ( 2) specific reaction with antibodies
- [sequen t ial or linear epitope ) or formed by bringing
or sensitised cells (immunological reactivity ). Based
on the ability to carry out these two functions,
antigens may be classified into different types. A
complete antigen is able to induce anlibudy
formation and produce a specific and observable
reaction with the antibody so produced. Haptens
are substances which are incapable of inducing
antibody formation by themselves but can react
specifically with ami bodies. (The term hapten is
derived (TOITI the Greek h&ptcin which means ' to
fasten’. ) Haptens become immunogenic (capable of
inducing antibodies ) on combining with a larger
molecule carrier. Haptens may be complex or
simple; while compkis; haptens can precipitate with
specific antibodies , sim /ife haptens are
nonprecipitalirtg. Fhey can inhibit precipitation of
specific antibodies by the corresponding antigen
or complex hapten. Complex and simple haptens
have been described as polyvalent and univalent,
respectively, since it is assumed that precipitation
requires the antigen to have twn or more antiiiody
combining sites,
[ iic smallest unit of antigenicity is known as
the anf jgenic determinant or epitope . The epitope
^
is that small area on the antigen, usually consisting
of tour or five aminoacid or monosaccharide
residues, possessing a specilie chemical structure,
elect ] ical charge and stone [spatiid ) configuration,
capable of s e n s i t i s i n g a n immunocyte and of
reacting with its complementary site on the specific
antibody or T-ceU receptor. Epitopes may be present
as a single linear segment of the primary sequence
together on the surface residues from different sites
V- opyrighted materi3
M
 * Anlfcg & ns SI

of RLIL:peptide :II:L n during :LH folding into [ Lie .Susceptibility to tissue enzymes: Only
L ]

tcrtiair ttniCtVR (conformational epitope ) . I cellf substances which arc metabolised and arc
recognise sequential epitope* while B cells identity susceptible to the action of tissue enzymes Liehave
*
the tertiary Configuration of the conformational as antigens . Antigens introduced into the body are
epitopes . The combining area on the antibody degraded by die host .it Lto fragments of appropriate
molecule, corresponding to the epitope , it; called size containing the antigenic determinants .
the pnnur>iK - llpitopcs and paratopes detemninc tire Phagocytosis and intracellular enzymes. appear to
,

specificity of immunologies! reactions . Antigens such play an essential role in breaking down antigens
at bacteria or viruses carry many different types of
cpitofies,presenting an antigenK mosaic.!he presence unsusceptible to tissue enzymes such as polystyrene
nf the ^mc or siniilar epitopes on different antigens
accounts for one ty|>e of antigenic cross- reaction.
DETERMINANTS A NTIUENUJIT Y
OF
A number of properties have been identified which
make a substance antigenic but the exact basis of
antigenicity is; still not clear.
Siv.arr Antigenicity is related to molecular size. Very
large molecules, such as hemocyaruns {MW h . 7 s
millioo), are highly antigenic and particles \i i rh
lots molecular weight ( less than SOUO) arc
nonantigcnic or feebLv HO . LOW molecular weight
substances maybe rendered antigenic by adsorbing
them on Large inert particles such as bentonite or
kaolin . Some Low molecular weight substances
(such as picrvl chloride , formaldehyde and
penicillin) may he antigenic when applied on the
skin, prohablv hv combining with tissue proteins.
'lTev are haptens of Low inimnnogenicitv, effective
in some persons only and related to hypersensitivity,
Chunriunl nature: Most naturally occurring
antigens are proteins and polysaccharides, Lipidi
and nucleic acids are Less antigenic . Their
antigenicity is enhanced by combination with
proteins. A certain degree of structural diversity is
required for antigenicity. That probably explains
why proreins which are composed if about 20
different jmiiiuAetds better antigens than
ire
polysaccharides which have Only four or five
monosaccharide units. However, not all proteins
arc antigenic , A wed- known exception is gelatin,
into immunogenic fragments , Substances
Latex are rot antigenic. Substances very rapidly
broken down hy tissue enzymes arc also not
antigenic. Synthetic polypeptides , composed ofD-
aminom-icls which are not metabolised in the body,
are not antigenic , while polypeptides consisting of
[ ,- ainmoacida are antigenic ,
1
hordfintiests: Only antigens \ v ]iich arc “ foreign
to the individual ( nonself ) induce an iulmuile
response . The animal body contains numerous
antigens which induce an immune response when
introduced into another individual or species. An
individual tides not normally mount an immune
response against Liis (iwn normal constituent
antigen'- "Hiis was first recognised hy Ehrlich who
proposed rite concept oThorror autotoxieus ' ( which
means fear of self 'poisoning) . ToLeraTLce of Self
antigens is auditioned by contact with them during
the development of the immune apparatus .
Ifeeakdown of this homeostatic mechanism result ^
in autoimmuniiaiion and autoimmune disease .
Io general , the antigenicity of a substance is
related to the degree of its foreignness. Antigens
from other individuals of the same species ;irc less
antigenic than chose front other species. Antigens
from related species are less antigenic than those
from distant species.
Antigenic specificity': The basis of antigenic
specificity is stereochemical , as was fim
demonstrated by Obermajer and Pick and
confirmed by Landsteitier. Using haptens such as
:Lt ( > xvi coupled with protein , it was shown that

which is nonircimunogcnic because of its structural antigenic specificity is determined by single

unstabilitv. chemical groupings Mid even by a single acid

Copyrighted material
82 * Textbook oF Microbiology
radical. The importance ot the position of the
antigenic determinant group in the antigen
molecule was evidenced by the differences in
specificity in ootopfliods with the group attached
at the ortho, mefa fir /wa positions . The infinciue
of spatial configuration of rhe determinant group
was shown by differences in antigenic specificity
of the efejftro, leva and meso comers of substances
such as tartaric add.
Antigenic specificity is not absolute . Cross
reactions can occur between antigens which hear
stereochemical similarities - In some instances,
apparent cross reactions nuy actually be due to the
sharing of identical antigenic dctcrminants hy
different antigens .
The specific] tv of natural tissue antigens of
animals may be considered under different categories
as species, iso-, auto- and organ specificities.
Specie *peuificit > i Tissues of all individuals
in a species contain iperia-specific antigens. There
-
exists some degree of cross reaction between
antigens from related species. This immunological
relationship parallels phylogenetic relationships. It
has been used in tracing evolutionary relationships
between species. If also has. forensic applications
in the identification of the species of blood and of
seminal stains. Phylogenetic relationships are
reflected in the extent of cross reaction between
antigens from different species that cause
hypersensitivity. An individual sensitised to horse
scrum will react with serum from other equities
bur may not do so with bovine serum.
Jr
I &oapecificilv: lsoantigcm arc antigens found
in some but not all members of a species. A species
may be grouped depending on the presence of
different isoantigens in its mem hern . The hest
examples oJ isumltigens are the human erythrocyte
antigens based on which individuals can be
classified into different blood groups. 'These are
genetically determined . They are of clinical
importance in blood transfusion and in
isoimmunisation during pregnancy. They were of
help in determining disputed paternity cases, hut
have been supplanted by the more discrimiuatoiy
DNA fingerprinting tests . Blood groups find
application in anthropology.
Histocompatibility antigens arc those cellular
determinants specific to each individual of 4 species.
Thcv arc recognised by genetically d Life rent
individuals of the same species when attempts arc
made to transfer or transplant cellular material from
one individual to another [described in Chapter
15 ) .
Autoapecifioity; Autologous or self antigens
are ordinarily nunantigenii: but there are exceptions.
Sequestrated antigens that are not normally found
free in circulation or tissue fluids [ such as eye lens
protein normally confined within its capsule ) arc
not recognised as self antigens. Similarly, antigens
that are absent during embryonic life and develop
Later {such as sperm ) arc also not recognised as self
antigens .
Organ specificity : Some organs, (inch as the
brain, kidney and lens protein of different species-,
share the same antigen. Such antigens, characteristic
of in organ or tissue and found in different species-,
are called organ specific antigens. The
neuroparalytic complications following jLHtirabic
vaccination using sheep brain vaccines arc a
cumequence of brain specific antigens shared by
sheep and human heiogs. The sheep brain antigens
induct immunological response in the vaccinees,
damaging their oemus tissue.
11tie rotfe no He ( heterophile) specificity:
'
Hie same or closely related antigens may sometimes
occur in different biological species , classes and
kingdoms. These are known as heterogenetic or
heterophile arttigenfiT best exemplified bv the
Forssman antigen which is a lipid carhuhydrate
complex widely distributed in many animals , birds,
plants and bacteria. It is ahsent in rabbits, so anti -
Forssman antibody can be prepared in these
animals. Other heterophile antigens are responsible
for some diagnostic serological reactions in which
antigens unrelated to etiological agents are employed
[ heterophile reaction ) . The Weil — Felix reaction in
Copyrighted materia
 4 Anttgena *
typhus fever, the Pau ] Bunncl test in infectious
mononucleosis and the cold agglutinin test in
primary atypical pneumonia are examples.
liLOLOGicAL CLASHES OF ANTIGENS
Depending on their ability to induce antiihody
formation, antigens are clarified asT cell dependent
(TD) and T cell independent (TI ) antigens .
Antibody production is the property of B
lymphocytes. For the full express ion of rh i --. function,
however, the coopcrarion of T lymphocytes is
necessary. Some antigens can directly snmulate
antibody production by B cells, without the apparent
participation of T cells. Such antigens are called TI
antigens. Others that require T cell participation to
generate an immune response are called TD antigens.
Several important differences exist between
TI and TD antigens. TT antigens arc structurally
simple , being composed of a limited number of
repeating epi ' opcs , as- in the cane of the
I urthrT Rndiii
1
Wi. ir DM . - 1 I Stewart 1997. lmmunvhyp , 9 th edit. Edinburgh ; Churchill Livingstone .
'
83
pneumococcal capsular polysaccharide , bacterial
lipopolya ace ha rides and the flagellar protein
flagd I in . Their immune response is critically dose
dependent . Too little is non iinmunogtnkp while too
much results in immunological tolerance rather
than immunity. Their antibody tesponse is usually
limited to IgM and IgG 3. They do not show
immunological memory. I [ antigen:- dn not appear
to require preliminaiy processing by macrophages.
They are metabolized very slowly and remain in
the body for long peril ids.
TD antigens, on the other hand, are structurally
more complex, such as erythrocytes, scrum proteins
and a variety' of protein-hapten complexes. They
are immunogenic over a wide dose range and do
not cause tolerance read illy. They induce the full
— IgM. IgG, IgA
gamut of immunoglobulin isotypes
and IgE They show immunological memory,
, '
require preliminary processing, and are rapidly
metabolised in the body.
^
Copyrighted material
im Qe
not
n
A <
J
ble
 * Antibodies
immunoglobulins, but :ill immunoglobulins may not
be antibodies.
Immunoglobulins constitute 30-2S pcx cent
the rord serum proteins . Based on phyticcchemid
and antigenic difKilcnc4s , five classes fit
bnmtmoftEobulj ns hive been recognised— IyG , I gA*
IgM, [ LfO and. IgE . ( Both I;LT and y are accepted
abbreviatione for immunoglobulins!.
S rklJ ^ TtJKE
Studies involving rite cleavage of tluL
immunoglobulin molecule, pointered by ftirter,
Edtdmim, NinttoS and their colleagues, have led
to ,i detailed picture of its structure . Rabbit IgG
antibody to egg iThumin, digested by papain in the
pRKQCe of cysteine , W; LH split into two frmtionji- -—
an insoluble fraction which crystallised in the cold
(called JjC for cn-ntalhx.ihlr ] , .ind . I soluhle fragment
which, while unable to precipitate w i t h egg
1
album in, could still bind w i t h it . This fragment in
LCHAiN
M
I AmmolorrTwiLjs j
\ 1
+1
!
fab
Fig . \22 Basic structure ol
Fc
an immunoglobulin molecule end the Iragmenla obtained
papain and pepsin
— immunogioOins » S5
called the Fab (ar? riitrcr? binding ) fragment. Each
molecule n1 immunoglobulin is split 1» papain into
'
three partu, one Fc and two F.ih pieces, having a
sedimentation coefficient of 3.5 S. When treated
with pepsin , a 5 S fragment in obtained, which i*
composed essentially of two Fab fragments held
together in position . It in bivalent and preripiliLteS
^ vith the jiutgcn . This fragment is tailed Ffab72-
rrhe Fc portion it digested by |>cpsin into smaller
fragments ( Fig. 12.2).
Immunoglobulins arc glycoproteins, each
molecule consisting of two pairs of polypeptide
chains ot Jiflereul sizet. The smaller chains art
called ' light' ( L ) chains and the larger ones 'heavy 1
( H ) chains. The I. chain has a molecular weight
«1 apprnximitdy 25/100 and the H chain ( lt 50,000.
The L chain is attached to the H chain by a
disulphide bond. The two H Plains arc joined
together by 1-5 b- S bonds, depending on the class
of immunoglobulins ( Fig. 123).
3 H -CHAIlV
a * iK C
( Gairboxyte rnnmus )
2
2 ^-
a.
LL
U
///
) Flab ),
^ by the cleavage by
Copyrighted materia
86 * Tesihook of M b

The H LEI . LI n arc Structurally Lind iniigertically

S

distinct for each class and arc designated by the

Greet letter corresponding to the Lrnmuuojriobulm Afftlft 'QVCIJIlfti flu.5
dass a * shown below:
J -
£
J*H mttn&giohuim ctofi H chnin
I-
IgG y i mnu )
IgA ^
a (alpha ) 31 Hi ngu H chain
IgM p (mu ) L
srj

isn 6 {delta ) - v.::

kE E ( epsilon )
C
u'
L"
5S
.
_
T
The I . chains arc similar in ail classes of a- &
LmmuDoglobuifos. They occur in two varieties * ci
: '
CartoxYternninua
kappa ( t ) and lambda iXh A molecule of
&
immunoglobulin may have either kappa or lamhda
chains, but never both toother. Kappa and lambda
are named after Komgold and Lapari who originally
described them. The kappa and lambda chains. occur
in a ratio of about 2 :1 in human sera.
The antigen combining site of [ lie TNOlecule is
at hi ununorermimis . Lc is composed of both L and Fig .12.3 The ioi^peplids chain sl? Lft(un& of She lg &
H chains. Studies on the primary structure of J „
and H chaini show that they consist of rwo portions
.
rtioIncuts compound «4 IWD identical heavy (H) and
: Vo Idenliul ligtil jLf chains linked by Interchains
disulphide hands. Leons formed by inhjiehBfn
each . Of the 214 jminojcid residues that mike up disulphide bonds art pomamt ishown Bhppied ) .
E ,>ch chain has one bo main in (he variable region
the |, chain , about 107 that constitute the .
carboxytcrminal half * occur only in a constant < VH gnd VL| Each light chain has me domain in
1he constsni region < CLj iwhiie eiich heavy chain
sequence . This part of the chain is therefore called has Ifcrw domains in the con slum region ; CH' CK - '

the constant region '. Only two sequence patterns
3 re seen in the constant region - those determining
the Js/ jpi and lambda specificities. On the other
hand, the aminoacid Sequence in lltC ammo!union at
half of the chair is highly variable , the variability
determining the immunological specificity of the
antihody molecule. It is therefore called the variable
region * "Hue H chain also has 'constant ' and Variable'
ivgiuns. Wlule in the L chain the rwo regions are of
equal length, in the H chains the variable region
constitutes, approximately onEy a fifth of the chain and
is located at its aninotcrmunis. The infinite range of
the antibody sjjeeificUv of immunoglobulins dejiends
on the variability gf the aminoacitl sequences at the
Variable legions' of the H and L chains which form
the antigen combining sites.
and CHI- Beiween CH' and CH: is the hinges region
'
.
I he LuiiinoiLfid sequences of die variable rejpons
of the J.. and H chilin > arc not uniformly variable
along their length , but consist of relatively invariable
and si mat highly variable zones. The highly \ ariable
zones numbering three m the L and four in die H
chains .uv known .is hypervuriable regions {or hot
-.puts l and arc involved with the formation of the
antigen hinding sites.The sites on the hypervarinble
regions that make actual contact with the epitope
arc called ‘complementarity determining regions'
or CDKs .
rite Fc fragment Is composed of the
cathox terminal portion of the H chain .. If does
^
not possess antigen combining activity hui
-
determines the biological properties of the

I
M
 -
* Antibodies -immurmgleibifls 87

iitimunuglubulin molecule «sdi u complement IMMLNOGLOBI I IN Cl ASSES

fixation , placental transfer , skin fisatian and
catabolic rate The portion of the H chain present
,
In the Fab fragment is called the Fd piece - The 11
chain carries a carbohydrate moiety which is distinct
for each class of immunoglobulins,
Each immunoglobulin peptide chain lias
internal disulphide links in addition to intervhain
disulphide hi. 111 ds which bridge the H and L chains.
These interchain disulphide bonds form loops in
the peptide chain, and each of the loop* is compacdy
folded to form a globular domain , each domain
having a separate Junction. The variable region
domains, VL and VHr arc responsible for the
formation of a specific antigen binding site , The
CHJ region in JgG binds Clq in the classical
complement sequence , and rhe CM ’ domain
mediates adherence ro the monocyte surface. The
areas of rhe H chain in the C region between the
fiftt and second C region domains (CH : and CH - )
is rhe hinge region . It is more flexible and is more
exposed ro enzymes and chemicals. Papain acts here
to produce one Fc and two Fab fragments ( Fig.
12 , 3),
Table 12.1 Same properties ol Immunoglobulin classes
Human sera contain IgG , IgA , IgMn Igl ) and IgE
in rhe descending order of concentration. Table
12.1 shows their characfcristics.
IgG: This is the major serum immunoglobulin ,
constituting about SC per cent of die total . It has a
molecular weight of 1150 ,000 ( 7b ) . IgG may
occasionally exist in a polymerined form . It is
distributed approximately equally between the
intravascular and cxtravaseular compartments. It
contains leun carbohydrate thin other
immunoglobulins . It has a half -life of
approximately 23 Javs . The catabolism at IgG is
unique in that it varies with its serum concentration .
When its level is raised , as in chronic malaria , kala
azar Or myeloma , the IgG -svnthes ised against a
pamcular antigen will be cirabolised rapidly and
may result in the particular antibody deficiency.
Conversely, in hypogammaglobulinemia the IgG
given tor treatment will be cataboliscd only slowly.
The normal serum concentration of IgG is about
-
S lb mg per ml .
IgG i -H the only maternal immunoglobulin that

IgG IgA' IgM IgD IgE

Sedimentation coefficient ( S ) 7 7 7 $
Molecular weight 150 , 000 160,000
1 ^
00,000 ISO ,000 190,000
Serum. concentration ( mg / ml ) 12 2
^
1.2 0.03 0 - 00004
Half life (days) 23 6 5 2-51 1 -5
1 >i!ily production (mg/ kg ) 34 24 3.3 0.4 0.0023
Infftwcuk ditfriburioA {per ticni) 45 42 B0 75 50
Carbohydrate { per cent) 3 fi 12 13 12
Cnmplemenc fixation
- - -
Clastifd
Alaniint
Placental transport
Present in milk
—
++

+
+
—
*++
-
— — ——
+ + " " "

Selective secretion by
seromucus glands
Heat stability ( 56 °C)
>

—
+ +
t-
— — ——
IRA may occur m 7S, 9S and. 11 S forms .

Copyrighted material
89
is normally transported a-cioss- the placenta and
provide ^ natural passive immunity in the newborn „
It is not synthesised by the ictus in any significant
amount. lgG binds to microorganisms and enhances
their phagocytosis Eitmcdhdar killing of r .irgct
cells coated with IgC antibody is. mediated through
recognition of the surface Tc fragment by K cells
lieming the appropriate reCfptOfit Interaction oi lgG
ootripIcKes with platelet be receptors probably leads
to aggregation and vasoactive amine release. IgG
.1 le i -.e, .Li Lie 11 L” hu m*i L I I nmunoglohljl] i LK , can : i itse 11
to guinea pig skin , but the significance ot this
property Is not known . IgG participates in most
immunological reactions such as complement
fixation , precipitation, am! neutralisation of toxins
and viruses . It maybe considered a general purpose
antibody, protective against tltnse infectious agents
which are active in the blood and tissues. Passively
administered IjG suppresses rhe homologous
antibody synthesis by a Feedback process. This
property is utilised in the isoumminissiiun ofwnmen
hy the administration of aatijKh ( D} IgG during
delivery. With most antigeot, IgG is a late antibody
and makes its appearance after the initial immune
response which is IgM in nature.
Fatu subclasses of IgG have been recognised
( JgGU IgG 2 , lgG3, IgG 4) , each possessing ,i
distinct tvpc of gamma chain , identiliable with
specific antisera. The four IgG subclasses are
diitributcd in human scram in the approximate
proportions oft 5 per cent , 23 percent , 8 pci cent
and 4 per cent, respectively
IgA: IgA is the second most abundant class,
constituting about 10- Id per cent ot serum
inrununoglobulins. The normal scrum level is 0.6-
4.2 mg per ml. It has a half lite of 6-B days. It in
the major immunoglobulin in the colostrum , saliva
and tears.
IgA occurs in two forms. Serum IgA is
principally a monomeric 7S molecule ( MW about
160,000}. IgA found on mucosal surfaces and in
secretions is a dimer formed by two monomer units
joined Together at theii carboxylennirials bv a
i T& itjGOOK n ( Microbiology »
glycopepfide termed the J chain ( J For joining ).
This is colled the secretory IgA ( SJgA ). Dimeric
SIgA is svnthrsised by plasma cells situated near
the mucosal or glandular epithelium , llie j chain
is also produced in the same cells. J chains are
present also III other polymeric miinunogLobulms
such ns IgM ( Fig. 12.4 ) .
SlgA OtJUftains aiiothct give int rich polypeptide
called the secretory component u secretory piece.
This LS not produced by lymphoid cells but bv
mucosal or g .mdnlar epithelial cells Dimeric IgA
binds to a receptor on the surface of the epithelial
-
Ctlls laid ii cndOLytostd and ran porlrvl across the
cells to rhe luomul surface . Durin this process, a
part of the receptor remains attached to the IgA
dimer . This part is kuem - n as the secretory
component. The secretory piece is believed to
protect IgA from denaturatiem by bacterial
proteases in sites such as the intestinal mucosa
which have a rich and varied bacteria flora , SlgA
is a much larger molecule than scrum IgA ( 1 IS;
MW about 400,000).
SlgA is selectively concert trilled in secretions
2
i
ill
"
3
Fig . 12.4 SecrvlOiy IgA. 1. - pavy Chain. 2. Light chain,
. .
i . J chain 4 Secretory componanl . 5 . Disulphide
bond
Copyrighted material
 • Antibodies— Immunoglobins *
anti on mucus surfaces forming an Antibody pastr '
and is believed ro play an important role in local
immunity against respiratory and intestinal
pathogens . Secretory IgA is relativeLv resistant to
the digestive enzymes and reducing agents. IgA
mirihudies may tune [ tent by inhibiting the adherence
ot microorganisms to [he Surface of mucosal cells
by covering the organisms and thereby preventing
their entry into body tissues. IgA does [ ]Ol fix
complement hut can activate the alternative
complement pathway. It promotes phagocytosis and
intracellular killing ot microorganisms.
Two IgA subclasses have been described , IgA .
and IgAj. IgA lacks interchain disulphide bonds
^
between the heavy and light chains. Though IgAj
is J minor component of serum IgA, L L LS the
dominant form in the secretionS-
IgM: IgM constitutes 5-8 per cent of serum
immunoglobulin with a normal level of Q - 5-2 mg
^
per ml. It has a half-life of about five days . It is a
heavy molecule (19S' mol, m 900,000 to 1 ,000,000,
hence called ‘the millionaire molecule!, IgM
molecules are polymers of five tbur -pcplidc subunits,
each bearing -an extra CH domain (Fig. 12.5). As
with IgA, polymerisation ot the subunits iiepends
upon the presence of the J chain . Though the
theoretical valency is ten , this is observed only with
small haptens. With larger antigens , the effective
valence bills to five, pruhably due Do stone hindrance.
Most of IgM ( 80 per cmr ) is intravascular in
distribution. Phyloge n erica!IY, IgM is the oldest
immunoglobulin class . Ir is also the earliest
immunoglobulin to be synthesized by [ he fetus,
beginning by about 20 weeks of age , As it is not
transported across the placenta, the presence oflgM
in the fetus or newborn indicates intrauterine
infection and its detection is useful in the diagnosis
of congenital infections such as syphilis , rubella,
HIV infection and tOXOpfrvMYlOJU IgM antibodies
are relatively short lived, disappearing earlier than
IgG, Hence, tlitirdemonstration in serum indicates
recent infection. Treatment of serum with 0.12M
2 - nt cre a p toet h a n t> ] sel c c t i vel y d c s r roy s IgM
69
.
t
Fig. 12,5, IgM molecule. 1. Heavy chain. 2. Light .
1J chain .
without affecting IgG antibodies. This is a simple
method for rhe differential estimation of IgG and
IgM antibodies .
The isoheitiagglutinius (anti- A , antii- li ) and
many other natural antibodies to microorgaTri&ms '
are usually IgM, as also antibodies to typhoid 'O'
antigen ( endotoxin) and re agin antibodies in
syphilis .
The unique structural features of IgM appear
particularly suited to the biological role of
providing protection against microorganisms and
other large antigens that have repeating antigenic
determinants on their surface, A single molecule
of IgM can bring ahnut immune hemolysis ,
whereas 1000 IgG molecules arc required for the
same effect. IgM is also 500- 1000 rimes more
effective diau IgG in opsonisation, 100 times more
effective in bactericidal action and about 20 times
in bacterial agglutination. In the neutralisation of
rmins and viruses, however it is less active than
,
IgG . Being largely confined to the intravascular
space , IgM is believed to be responsible tor
protection against blood invasion by-
microorganisms. IgM deficiency is often associated
with septicemias .
Monomeric IgM is the major antibody receptor
on the surface of H lymphocytes for antigen
recognition .
Copyrighted material
W * Textbook of MicrotiiDlogy
IgD resembles IgG structurally Lt 15 present
m a concentration of about 3 mg per 100 nnl of
Krvm and is mostly incE' av^.gctjl^r. Ir bis a half-life
of about three days . IgD and IgM occur on the
surface of uustnnulared B lymphocytes and serve
as recognition receptors for antigens. Combination
oi cell membrane bound IgD or IgM Wi I h the
corresponding anrfeen leads to spec irk stimulation
of the R cell — CLther aul 1 vUIion and cloning to
produce anclbtidv, or Rupprefii- inr -
IgK - This iimnnnoglobulin was discovered in 1966
by Ishizaka duri ng the invest 1gallon of atopic rt agin
an n bodies . It SR an SS molecule (MW about
190,000), with a half life of about two days , it
resembles IgG structurally. Jr exhibits unique
properties such as hear lability (inactivated at 56 °C
in one hour) and affinity lor the surface of r issue
cells (particularly mast cell5 ) of the same species
(homocytotropism ), ft mediates the Erausnirz-
Kustner reaction . It is susceptible to
rttencaptoethanol. It does ncit pans the placental
bat 1 KT or tbf complement. lt is mosdy exiravascukr
in distribution. Normal scram contains onlv . traces
SI
{ a tew nanograms per ml) bur greatly elevated levels
are sect-, in atopic ( type 1 allergic) conditions such
ay asthma, hay fever and eczema. Children living
Lit insanitary conditions , with a high load of
intestinal punishes, have high serum levels ol lg£ .
Jg£ is chiefly produced in the linings of the
respiratory and intestinal tracts. IgE deficiency bus
been associated with IgA deficiency in individuals
^ ilh impaired immunity who present undue
susceptibility to infection.
Ig£ is responsible for the anaphylactic type of
hypersensitivity. The physiological role of IgE
appears to be protection against pathogens by mast
cell dcgranulation and release of inflammatory'
mediators. Ec is also believed to have a speciat role
in defence agonist helminthic infections.
In general, IgG protects the bodv duiils, IgA the
body -mifeceH anti LgM the bloodstream, while ] g£
mediates rcaginic hypeniensiti'.-lly JgD is a too ignition
molecufe on the surface of B lymphocytes.
ABNORMAL IMMUNOGLOBULINS
Apart from antibodies, other structurally similar
proteins arc seen : n scrum in many pathological
processes, and sometimes even in healthy persons.
I he earliest description of an abnormal
1 mmunoglobulin was the discovery by Bence Jones
(1847) of the protein that bears his name. Bence
Jones protein is found typically in multiple
myeloma . It can be - deimfled in urine by its
eharacicrisdc property of coagulation when heated
Co SO °C but rcdissolving at 70 ?C- B-CIICC Jones
proteins arc the light chains of rmmunoglohiilins
and so may occur as the hyp or .urnhe /a forms.
But in any one patient, the chain is either L ; ji < a - nr
y.ur . hJif cully, and never both, being uniform in . L 1
'
other respects also, This is because myeloma is a
plasma cell dyscrasia in which there is unchecked
proliferation of one clone ufplaRitia cells, resulting
in the excessive production of the particular
immunoglobulin synthesized by the done. Such
immunoglobulins are, therefore , called monoclonal.
Multiple myeloma may affect plasma ceils
synthesizing IgG , IgA, IgD or IgE. Similar
involvement of IgM producing cells is known as
Hk .MfrtSrj'ujn V rti dcroglohulincmis , In this
.
condition, there occurs acesj : vc production of the
respective myeloma proteins (M proteins} and of
the 1 r light cha 1 ns (Bence Jones pn Nrcins }. A different
disorder i -. found in "heavy chain disease", which is
a lymphoid neoplasia characterised by the
overproduction of the Fc parts of the immuno-
globulin heavy chains.
CiYogtnhulincrnk is a condition in which there
isthe formation of a gel or a precipitate on cooling
the serum, winch reclissolvts on warming. It may
not always be associated with disease but is often
found in myelomas , macroglobulinemias and
autoimmune condition? such as systemic lupus
crythcmab ^us. Most cryoglobulins consist of either
IgG, IgM or their mt.vcd preL -iiutates-
Becausc of the monoclonal nature of Bence
Jones and other M proteins , they have been
Copyrighted materia
 * Annbadios
valuable models For the understanding of
immunoglobulin smicrure and function.
lMMLNOGLOBl/1 EN SfBClPIClYlEa
The immunoglobulin specificity of the greatest
biological importance is idiotypic specificity
pertui r.ing to the nature of the antigen h i tiding sites
( paratopes).The specific antigenic determinants on
the paratope are called idiotopcs . The sum total of
idiotopcs on an lg molecule constitutes its idiotype,
By immunisation with Fab fragments *
anti idiotypic antibodies can be produced. These
resemble the epitopes of the original antigen. Used
as a vaccine, these show prcnecnon against the
original antigen ( pathogen or tumour ) in
experimental animals. Sequential antiidiotypic
antibody formation is the basis of Jernc’s network
hypothesis of immune regulation .
Immunoglobulins exhibit other genetically
determined specificities based on their antigenic
structure . The antigenic specificities which
distinguish between the different classes and
subclasses of lohuliris present in all normal
iitimum
^
individuals of a gjvcr species ate termed isotypic
(
JW . ] W?1 . I m muno globulin sirwiurc
Rodrt IM and O DeJvei cd . 1992 . ErHyrlopn' -' j
— ImmijnoglQtrin* * 91
specificities . Antigenic specificities which
distinguish immunoglobulins of the name class,
between different groups of individuals in the same
species, are called allotypic specificities.
Immunoglobulin allotypes have been studied in
detail in the rabbit and guinea pig by using type-
sjDCtific 11 ni i Lime Sera . Such deliberate immun isatiem
is not possible in human beings, but ami allotype-
spec i tic antibodies may develop following blood
tranfusion or passage of maternal IgG into the fetus.
Antiallotype antibodies arc atso found in sera
containing 'rheumatoid arthritis factor’.
Two allotypic systems are known in humans
the Gm system (for gamma marker) and the InV
-
system (abbreviation of patient’s name ). 'Fhe Gm
is associated with the Fc portion of the IgG heavy
chain. More than 25 Gm types have been identified
so far. The InV system is associated with the kappa
light chain and so has been renamed Km. Three
Km allotypes have been identified.
Gcnctic markers associated with IgA arc called
LAm\ To date, in the human system no allotypic
markers have been found for lambda light chains
or 5 ore heavy chains -
Further Reading
and 1 . n r: i . V" c d n. In fljjji and Clin ..* } Immunology.
1
London: Academic Prew ,
opy righted material
 CO Antigen— Antibody Reactions
Antigens u) jj antibodies, by definition, Combine
with each other specifically and in ar observable
manner [ l i e nuiiuHi between antigens and
, of motile organisms ami enhancement ot
intibodici serve several purposes. In the body, they
fomi [ JLL basis of antibody mediated immunity in
inlKtUUI diseases, or of tissue injury in wine type*
of hypersensitivity and autoimmune diseases. In
die laboratory, they help in the diagnosis of infections ,
in epidemiological surveys , in the identification of
infecrious agents and of ntminfectnuE anrigens such
as enzymes. ITL general tliese reactions c.m be used
, so on, and the corresponding antigen, agglutinngcn ,
lor the detection undtjuu nfitutjo n of either antigens
or antibodies. Antigen — antibody reactions in vitro
are laHWD Ss scstiiogicd DCScdtUs.
"
J HLL reactions hetwrer antigens and antibodies
occur in rh ree The primary stage is rhe in i ri al
interaction between rhe two, without anv visiblt
effects. [’[ us reaction IS rapid , occurs ever at ttjw
temperatures .uni obeys rhe general laws of physical
chemistry and thermodynamics. The reaction is
reversible , the combination between antigen and
antibody molecules being effected by the weaker
intermoJeculsr forces such as Van der Waal 's forces,
ionic bonds and hydrogen bonding, ruber ih ; m by
the firmer covakm bonding. The primary reaction
can bedet ecced tnv estm iati riLi; free and boimd antigen
nr antibody separately in rhe reaction nurture by a
number o 1 physical and chemical methods ,
-
including die use oi markers inch as radioactive
isotopes , fluorescent dyes ^ r ferritin.
In mutt instances, but not all , the primary stage
LS followed bv cite secondary stage , leading to
demonstrable events s u c h a s precipitation ,
agglutination, lysis tit cells, killing of live antigens,
neutralisation of toxins and other biologically active
antigens, fixation of complement, immobilisation
phagocytosis . When such reactions were discovered
one by one, cl was believed that a different lyjie of
anti hotly was responsible for each type of reaction
and the antibodies came to be designated by tire
reactions they were thought tu produce . Thus, the
antibody causing agglutination wits called
agglutinin, chat causing precipitation precipitin* and
precipitinogen, and so on . By the 1920s , this view
was replaced by Zinsser's Unitarian hypothesis
which held that an antigen gave rise to only one
antilWy, which wat- capable uf producing ill the
different reactions depending on the nature of the
antigen and [ he conditions id the reaction . Both
these extreme views are fallacious. While it is true
rhdT a single antibody can cause precipitation,
agglutination and must of the other serological
reaction *, it it also true that an antigen can stimulate
the production (it d i f f e r e n t closes id’
immunoglobulins which differ in their reaction
capacities as well as in o th L- r properties [Table 13.1 ) .
^ -
Some anUj ei i .U Ltilxidy reactions occurring in vivo
initiate chain reactions that lead to neutralisation or
DESMICIM > n of injur LOUS tu i ligens, i IT to tissue damage.
These an; tertiary' reactions and include bumonil
i mm 111 Li ry against infectiousdiseases* as well as. clinical
.dleigy id other immunological diseases.
GSNEHAI FflATI liKS OF
ANTLGBM-ANTHBODY REACTIONS
Antigen -antibody reactions have the following
general characteristics:
Copyrighted material
 * /Wigen
Table 13.1 Comparative efficiency ol the immunoglcbulin classes in dilterent serological reactions
Reaction IgG
Precipitation Strong
Agglutination Weak
Complement fixation Swing
1. The reaction is specific, an antigen combining
only with its tromoiogous antibody and vice
.
versa The specificity, however, is not absolute
and 'cross-reactions' may occur due TO antigenic
simiLiritv or rdatedness .
r
2. Er ri re molecules react and not fragments. When
an antigenic determinant present in a Large
molecule <> r on .i ‘carrier ' partible reacts with
its antihndv. whole molecules or particles arc
agglutinated .
3. I herc is ms den am ration of the antigen or the
"'
antibody during the reaction.
4. The combination occurs it the surface. nierefene,
it ]& the Surface antigens that arc
immunolngieally relevant , Antibodies to the
surface antigens of infectious agents arc
generally protective ,
5. The combination is firm but reversible. The
firmness of the union is influenced bvthc affinity
and avidity of the reaction . Affinity refers to the
intensity of attraction between the antigen and
antibody molecules. It is a function of the
Jr
closeness of fit between an epitope and the
antigen combining region of its antibody
Avidity is the strength of the bond after the
formation of the antigen-antibody complexes.
It reflects the overall combining property of the
various antibody molecules in an antiserum ,
possessing different affinity constants with the
multiple epitopes of the antigen.
6 . Both antigens and antibodies participate in the
formation of agglutinates or precipitates.
7. Antigens and antibodies can combine i n varying
proportions, unlike chemicals with fixed
valencies. Roth antigen and antibodies are
multivalent. Antibodies arc generally bivalent,
Afilibody readions. 93
IgM IgA
Weak Variable
Strong Mode rate
Weak Negative
though IgM molecules may have five or ten
combining sites. Antigens may have valencies
UpTO hundreds.
MEASUREMENT nr AVTKIEX AND
ANTIbCJLll
Many methods arc available for the measurement
of antigens and antibodies participating in the
primary, secondary or tertiary reactions .
Measurement maybe in terms of mass ( for example,
mg nitrogen ) or more commonly as units or BtBft.
The antibody here of a serum is the highest dilution
of the serum which shows an observable reaction
with the antigen in the particular Test . The title ot
a serum is influenced by the nature and quantity of
the anrigen and the type and conditions of the test.
Antigens may also be ri [ rated against sera.
Two important parameters of serological tests
are sensitivity and specificity. Sensitivity refers Id
the ability of the test to denser ever very minute
quantifies of antigen nr antibody When a test is
highly sensitive, false negative results will be absent
or minimal. Specificity refers to the ability of the
test to detect reac tions between homologous
antigens and antibixhcs onlv, and with m > oilier . In
n highly specific rest, (ahe positive reactions arc
absent or minimal . In general , sensitivity and
specificity of a lest arc in inverse proportion.
Origiruillv, reagents for serological tests were
prepared by individual laboratories, leading to batch
variation, ami lack of reproducibility and
comparability. The commercial availability of
ready - made standardised test kits has simplified test
procedures, improved quality and greatly enlarged
their scope and use.
Copyrighted material
94 * Texttwok 01 Microbiology

SEROLOGICAL REACTIONS For a given antigen-antibodv system, the optimal

or equivalent ratio will be comtuit, irrespective of
PlMiCIPITATION REACTION
the quantity of the reactants . If rhe amounts of
When a soluble antigen combines with Its antibody precipitate in the different tubes arc plotted ort a
in [ be presence of electrolytes ( Nad ) at a suitable
"

graph , the resulting curve will h .iv < - three phases:

temperature and pH, the antigen-antibody complex
form* an insoluble precipitate. When, instead of
sedimenting, the precipitate remains suspended as
floccules, the reaction is known as floccutzticNi .
Predpitstuoii can take place in liquid media or in
gels such as agar, agarose or polyacrylamide
Tlic amount of precipitate formed is greatly
influenced by the relative proportions of antigens
and antibodies. 1f Lncreatirtg quantities of antigens
are added to the same amount of antiserum m
different tubes, precipitation will be found iu occur
most rapidly and abundantly in one of the middle
tubes in which the antigen and antiliudv are present
in optimal or equivalent proportions. In the
preceding tubes in which the antibody it ill excess ,
and LII the later tubes in which the antigen is in
CVCCRS the precipitation will he weak or even jbsciir.
an ascending part [ piozone or zone of antibody
CKCCSS ) , a peak ( zone of equivalence ) and a
descending part ( postzoneor zone it antigen excess)
( Fig, 1.1.1). This is culled the zone phenomenon.
Zoning occurs in agglutination and Rome other
scroll jgiical reactions.The prazone is of importance
in clinical serology, as sera rich in antibody may
sometimes give s false negative precipitation or
agglutlnation result , unless seven! dilutions are
tested .
MECHANISM OF PRECIPITATION
Marrack (1 ^14) proposed the lattice .hypothesis to
explain the mcchanicm of precipitation. According
to this concept, which is supportedbj considerable
experimental evidence and is now widely accepted ,
multivalent antigens combine with bi.vs.lent

+ i
-a- ,-
*
--.
V
*r
- -- nraJii *
- II

B
b. j

-
r *

-
«i J*
* P * * *
- m m ,r

l
l
s r

* + +
* ::fef r

fN "«
.- -*- XV
^e
i

A
.
Fig. 13 1 A quantitative precipilalion teal snowing : A prozone ( zone of antibody excess] , B . zone o1
equivalence , C - zone of antigen excess

opyrighted material
 * Arligen- -Antibody reactions * 95

antibodies in vnyuig proportions, depending on the The following types of precipitation and
-
antigen antibody ratio in the reacting mixture.
Precipitation result * when a large lattice is formed
flocculation tests arc in. common use:
"

King titsfc Tins, the simplest type of precipitation

consisting of n ] rcmating antigen xnd antibody
molecules. This is possible only in the zone of
equivalence . In the zones of antigen or antibody
Otoes-s, the lattice does Iior enlarge, as the valencies;
of tbe . mtibodv and the antigen, respeclively, are
fully satisfied ( I jg. 13.2) The lattice hypothesis
1
holds good for aggju rinadon also.
APPLICATIONS OF PRECIPITATION
REAt ; T!tJt\
The precipitation tint mav tie earned out as e]ther a
qualitative or quantitative test. It is very sensitive in
tile detection of antigens and as little as 1 fig of protein
can he detected ha precipitation tests. It thercton: finds
'
forensic application in the identification of blood JIIKI
seminal stain*, and in testing fur fluid adulterants.
Precipitation is relatively lew sensitive fbt the detection
of antibodies.
TtXl BOOK OF MICROBKH CX 5 Y
test , consisrs nflayeriiig the antigen solution over a
column of an t isenj m in a narrow tube. A preci pitate
Forms at the junction of tin.- two liquids. Ring rests
have only A few clinical applications DOW. Examples
are AfCOliV thermoprecipitin tent artd the grouping
of streptococci by the Lancefield technique.
Slide lesli When a drop each of the antigen and
antiserum ate placed on a slide and mixed by
shaking, flocculus appear. The VDRL test for
syphilis is an example of slide flocculation.
Tube test’ The K a h n tost for syphilis is an
example of a tube flocculation test. A quantitative
tube flocculation r e n t IN employed lor the
standardisation of toxins and toxoids. Serial dilution*
of the toxin/tcooid arc added to the tubes containing
a fued quantity of the antitoxin. The amount of
toxin or toxoid that flocculate * optimally with ore
u n i t of the antitoxin is defined as an IJ done .

Fig, 13.2 Mechanism 0i preclpitallon by lattice formation. In K (antigen excess) and C lantibody excess),
lattice formation does not occur. In B ( zone of aquivafonea), lattice formation and precipitation occur optimally.
The dark spheres indicate antigen and ihe spindles bivalent antibody molecules.

Copyrighted material
9
* * Tejdtjoo
* Q{ Wicrooiology

,’1

aggg
n% © o
i 2
4
©

o o o o o o
A B c
Fig. 13.3 Dillerent types ol imiminodltruslon ; t . Single drHusmn in one dimension t Double 11 uSian
In one dimension jQakley-Fulihorpti 3. Radial immunoctiHusion 4 . Double dirJu & mn In two dimensions
( Quchlffrior ) , ghpwing ruction p-f mill | ui lelflluLfm l M: :^, [ i r
ICf , The ^
wells marked $ contain n id n ? J conlaln ar > :
inirsa

[imrnmodiffiistnn { Precipitation in gcfte precipitation formed at the advancing front of tbe

Thcrc arc several advantages in allowing
prcci pita tion to occur m a jpd rtther than in a Itqu id
medium . Trie reaction is visible its a distinct band
of precipitation, which is stable and tin be stained
for preservation , if necessary. As each an tiger -
Antibody reaction, gives rise to a line of precipitation*
the number of different antigens i:i the reacting
mixture can be readily observed- timmunodiffusion
also indicates identity, emss-rcaction and nonidentity
between different antigens.,
Immunodiffusion is usually performed in a soft
( 1% ) agar or agarose gel. Different modifications
of tbe test are available;
1. Single rff /ftisron CD one dpmejrsion (Oudin
procedure ): 'Hie antibody is incorporated in agar
gel in a test nibfi and the antigen solution is layered
over ir . The anrigcn diffuses downward rhruugb
the agar gel, forming a line of precipitation that
appears to move downwards [’his is due to the
, bands of precipitation 1
antigen , and is dissolved as the concentration of
antigen ir the >ne in-cfL' c-.^c :- JILI id Jifhision, Hie
number of bands indicates the number of different
antigens present .
2. Double ilhiusiuir in one dimension (( X i k l f \ —
Fulthnrpe procedure ); Here, die antibody is
incorporated in gel, above which is plated a
column if plain _ fhc antigen is
top of this , i : irigi . lid niribodj movt
towards each other through the intervening
column ; ii'
dad foul .ibfcnd oi precipltal
where t Iter meet al opt mum propon . .
J, Single difivsiotl in hvo dintentioni ( Rnclizl
immunoditYiision ): Here the antiserum is
incorporated in agar gel used n a flat surface
(slide Or Pfctri dish ) , The . 1 : HI|>< . is added 10 rhe
wells cut on the surface of i " gel. It diffuses
radially front the well and form ring- shaped
' concentrically

Copyrighted material
 * Antigen— Anribody reactions *
around the well. The diameter of the halo gives
(in estimate of the concentration of the antigen .
Thr- method has been employed for the estimation
of the i : 111111. . i > glubuii. n classes in sera and fur
screening sera lor antibodies to influenza viruses,
, by this method in human serum. This is useful
among others.
4 . Double diffusion in t wo dimensions
{Oochterlonv procedure ): This is the
. inmunodiffusion method most widely employed
and helps To compare different antigens and
antisera directly. Agar gci. is poured on a slide and
wells are cut using a template. The an i scrum is
placed in the central well and different antigens
in the surrounding wells. If Two adiaccnt antigens
are identical , the lines of precipitate formed hy
them will fuse. If they are unrelated, the lines
will cross each other- Cross- reaci ion or partuE
identity is indicated by spur formation ( Fig . 13.3 ) .
A special variety of double diffusion in two
dimensions is the Elek test for toudgeniciTy in
diphtheria bacilli . When diphtheria bacilli arc
streaked at ri . ht angles to a filter paper strip
^
carrying the antitoxin implanted on a plate of
suitable tncdijm, arrowhead shaped lines of
precipitation appear on incubaiion, il the bacillus
'
is toxigenic.
5. fmnrunoetcctrophoresis: The resolving power
of immunodiffusion was greatly enhanced when
Grabar and Williams devised the technique of
imnuinoeleetrephoresift. This involves the
clectiophoretic separation of a composite antigen
( such as strum ) into its constituent proteins ,
followed by immunodiffusion against its antiserum,
resulting in separate precipitin lints, iiuinmating
reaction between each individual protein with its
antibody . ! In - enables identification and
approximate quantitation of the various proteins
present in the scrum. The technique is performed
on agar or agarose gel oo a slide , with an antigen
L . l and ait ambody trough cut on it . The test
serum is placed in the antigen well and
elcvtmphorcsed for about an hour. Antibody'
against human serum is then placed in the trough
97
and diffusion allowed to proceed for 13— 2-4 hours .
The result! nu precipitin Lines can be photographed
and the slides dried, stained and preserved for
record. Over 30 different proteins can be identified
for testing for normal and abnormal proteins in
serum and urine ( Fig . 13.4).
Hkclroimmunodi ffus t > n: The development
of precipitin lines can be speeded up by electrically
driving the ,miLgen ami juilibody. Various methods
have been dewrihed combining electrophoresis
with diffusion . Of these, otic-dimensional double
electroimmunodiffusion ( counterimmuno -
clcctrnphnrc Ls ) and one - dimensional single
-
electroimmunodiffusion ( rocket electrophoresis )
are used frequent!v lit the clinical laboratory.
J. CoHnferimmunw/MtTopfroreNis (C/E. counter-
cmreiif i mmu si oelec rropVi arts is h This involves
simultaneous electrophoresis of the antigen and
antibody in gel in appetite directions resulting in
predpitatiiin at a ptint between them ( Fig. 13.5).
This method produces visible precipitation Writes
within thirty minutes and is ten times more
sensitive than the standard double diffusion
techniques . The clinical applications are for
detecting various antigens such as alphaferoprorein
in scrum and specific anrigens ofcrypttrcooeus and
meningococcus in the cerebrospinal fluid,
2. OnetfirmensK ^ria/ sijyffc dft.'flrt ' / mmtirtotirfluMOji
(rocket eIccftophorc.ds ); The main application of
this technique is for quantitative estimation of
antigens. The anti serum to the antigen to be
quantitated is incorporated in agarose and gelled
on the glass -slide . The antigen, in increasing
concentrations, is placed in wells punched in the
set gel. The antigen is then eleccrophoresed inro
the antibody containing agarose ( Fig. 13.6 ) . The
pattern of immunopreL ipitation resembles a tucket
and hence the name .
A variant of this is Laurdls two-dimensional
electrophoresis . In this technique , the aniigen
mixture is first electTopharcrically separated in a
direction perpendicular to that of the final rocket
Copyrighted materia
 9S 4 Tsstiijagk gl Microbiology
*

stage. Ry this method one can quantitate each of

P!
K
m V

VJV mw several antigens in a mixture (Fig. 13- 7) .

-
'I

t
> -x- irajs
AGGLUTINATION REACTION
m
; When a particulate antigen is mised with its antibody
in fhc presence ofelectrolytes ai a suitable temperature
and pi lh the panicles are dummied nr agglutinated.
- . .
"i? f

-
Agglutination is more sensitive than prpt ij iration for
' '

2 ! . the dctectu tn of antibodies. J “he same priIKlples gtfVtin

sgghitiiwtior and precipitation. Agglutination occurs
vvl
xx - X
^ i

optimally when antigens and antibodies react in

equivalent proportions. The zone phenomenon mav
he seen when either an antibody or .an antigen is in
1

excess - "Incomplete’or 'monovalent ' antibodies do not

2 C
3 > MSR V cause agglutination, though they combine with the
antigen. They may act as ‘ blocking antibodies,
1

13
inhibiting agglutination by the complete antibody
added subsequently.

H .
'r j
m APPLICATIONS AGGLUTINATION
OF
. Y
* ’T REACTION
4 v

Slide IJI RIL 31

1 i 11 ! When 3 drop of tire
x
* V
appropriate antiserum is added ro ;L smooth, uniform
suspension of a piimculate anrigeo in a drop of saline
7TTT v - on a slide or tile , agglutination takes place, A positive
:: : --
HKE
result ts indicated by the clumping tugether of rbc
»

. j#.? particles and the clearing of the drop . The reaction

f
a'

5 v
i*
is facilitated by mixing the antigen and the
- — antise rum wirh a loop or by gen tly HK hi ng t he slidtr
Depending cm the titrenfthr scrum, agglutination
. may oeeur instantly or wkbin seconds, CLuitiping
Lv-
: v- xi
x
V

“-“ — occurring after a minute may be due to drying ol

j

“ x-x > < the fluid and should be disregarded. It j essential

; *i
! vi
»

Fig . 13.4 Immunoelectrophoresis 1 Samisolld agar
layered on 1+ie glass siide A well lor aniigen and
a trough For anliserum cul out oF agar.I. Antigen
well Filled wllh human swumj Serum separaled
Ay electrophoresis 4. Annse rmx trough filled wllh
antiserum to whole human serum . 5, Serum and
antiserum allowed to diffuse into agar 6, Precipitin
lines rcrm Tor individual serum proleing .
0 i
. - .
*
to have on the gumc slide a control consisting of
S . - . -.
in
"' ' 'ri'i' the antigen suspension in salineh without the
antiserum, to ensure that the antigen is not
autoagglutinable. AggluitiLatum is usujiElv visible
to the naked eye but may sometimes require
confirmation under the microscope , Slide
. agglutination is a routine procedure for the
idcntificatinn nf many bacterial isolates from
. .
clmictl specimens It is also the method used for
blood grouping and cross- matching.
 * Ap1 *gcn— Antibody raaclions » 99

antigens are used. The Tl ' or the flagellar antigen

on Loirthming ich its antibody forms large, 1OO.HC1
^
flufly clumps iHliEij wisps ot eotrorrT vooL The
EtectfopfiorslHHnfUnt ^ '
O or >m i r ic antige 11 f< imit tight, ear IIPACL dcj>i NSI t*
iesemi Ling eh .dk powder . Agglutinated hat illi
spread our in a djscliltt ]>.i £ Cei]i at the hi strum of the

©HI=©
tuhtt -
n Le tube agglutination COST tor brucellosis may
"

IK Complicated EH lliLr promrsc phenomenon .ind

the presence of 'blocking' antibodies . Several
dilutions of the scrum should be tested to prevent
fake negative revolts due to jwoMnc. Incomplete
or blocking antibodies mav he detected hv doing
the test in hypertonic ( 5 saline or aEbumin saline,
Fig . 13.§ Oounterimimin-oeleclrophofwii (CIE) Anllfltfn
and antibody an? driven. Together by an sleelric
^
or more reliably b \ the anriglobulin (Cwmhs) test ,
turrenl fihd a predpJlHi line forms. Jfht VVcil - l- cliif reaction for semdiigntHiih of
typhus fevers is a Iwtnrophile agglutination test and
is based on [ EK- SIIJRING of a common antigen
i
_ between typhus tickettsiar and some straini nJ
protem bacilli . Another example of the heteicphile
agglutination n- st is the Strc dfcus Mfj
^
agglutination test lor the diagnosis of primary
atypical pneumonia.
n n i Examples ni agglutination tents Lining red eells
i • i as antigens are the Paul Runnel test and the cold
agglottnattcm tert. The former is based on rhe
prOntCt of shtep cell agglutinin-- in the sera ul
Infectious mononucleosis patient, which are
* adsorbed by M red cells but not by guinea pig ktdnev
.
Fig, 1 3 .6 flocks ? elfrc I rep " a
S AnliHi' n is driven inlrj
q- c-i conuinmq antibody i Aniibody in agarow gel .
CJitf .i . r . J i | i- cold agglutination lest : positive in
mycoplasmal {primary atypical! pneumonia. The
.
2 . PrecipItLnsrea 3 Antigen wells 4 . increasing
patients sera agglutinate human O group
antigen concentraiion ,
erythrocytes at 4° C , the agglutination being
Tube lntinntinn: This is a standard reversible at 3" °C.
^ fl
quantitative merited for the measurement of The pntiglohulin ( Coombs) t e s l i "1 he
antibodies. When feted volume ot a particulate
a antiglobuli ti rest was devised by Coombs, Mourani
antigen suspension ]s added To an equal volume of and Race { 194 S ) for the detection of anri - Rh
serial dilutions of an antiserum in test tubes, the antibodies that do rnt agglutinate Rh positive
agglutination litre of the serum can be estimated- erythrocytes in saline . When sera containing
Tubc agglutination is routinely cm ployed for the incomplete anti-Rh antibodies are meted with Kh
acroEogical diagnosis of typhoid, brucellosis and positive red cells, the anti bud v globulin coats the
typhus fever . surface of the erythrocytes, though thev arc not
1 n the Widal hint used in typhoid,* TWO types of agglutinated. When such erythrocytes coated with

opyrigniea mstGf
 100 H Tonttwok al Micros - ology *
Antibody
in gel
+
Staring waH
Fig .13.7
tockal daclraphotesls jtwo dlmenBlonal nimun&elcctrophoresl ? |. FIr run: antigens
Lagrpll' j variant ol
are separated on Ihe basis or el«ErophofeUc mobility. The second run ai dghi angles to " first drives the
antigens into the antiserum confining gel to form precipitation pej](s. The ares of me
to Ihe concentration 01 the antigen.
the antibody globulin arc washed tree of all
unattached protein and treated wirh a rabbit
antiserum against human gammaglobulin
( antiglobulin or Coombs serum ) , the cells are
agsdul iniled . 1 in > is the pfiiunplt: of the intiglobolm
test ( Fig. 13.8) .
The Cfjombs te:» t muv be direct nr indirect. In
the direct Coombs test , rhe sensitisation of the
erythrocytes wid |mcuiTtplele antibodies fatuii place
in vivo, as in the hemolytic disease or [ be newborn
due to Rh incompnidbilitj, When the red cells af
-
tty thru bias Lot ic EEiiuntH an washed free of
unattached protein and then mixed with :L drop of
Coombs serum, agglutination results. The direct
Coombs [esl is alien negative in Siemulvtie disease
due to ABO incmnpiribilitv.
In the indirect Onmibs test, sensitisation of ted
cells with the anrihndy globulin is performed in
vitro . Originally employed for detection of anti -
Rh Antibodies, the Coombs test ES useful tnr
demonstrating any rype of incomplete or
nonagglutinating antibody, as, for ex Ample , ir
brucellosis.
PA $S[V« :iggltiilin:iliim lest : I he only
difference between rhe requirements for the
precipitation and agglutination tests is rhe physical
nature ot die antigen. Bv ACL aching Soluble antigens
Precipitin
arc
t
r o
-
is proportional
to the surface of carrier panicles, it is possible to
convert precipitation rests into . luuiiaiiu ] LCSLS,
which are more ronveiiient anti n sensitive lor
the detection ot Antibodies. > LU . tents are known
as Aauiir Ugfutuation re.srs.
-
The Lonmionly u ed carrier t > .i r t . . - are red
cells, lutes particles or bentonite . II . or sheep
erythrocytes adsorb a variety of antigens
Polysaccharide antigens may be , . -
Ibv l . npi .
mixing with the cells, tor adsorption of protein
antigens, tanned red cells are
A special type of passive hei lagghnJiiimori test
is tile Ruse Waaler test. In rheumati EI arthritic,
-
art auluanribcidy ( RA lacttsr ) ,ip . . in the SC rum,
which aers as an antibody m cUnUm . . 11 > . TTle
RA factor LS able to m red Cetls Coated
-
with globulins. Th -e antigen ised for the test is a
suspension uf sheep erythrocytes s c n s i t j-s cd with a
subagglutimating dose ot rabbit antishccp
erythrocyte antibody (amboceptor) .
Rllyslyiene latex, which can be manufactured
.
as uniform spherical pArtictcs O -S-l m in diameter,
can adsorb several types of antigens . Latex
agglutination tent - ( latex fixation tests) are widely
employed in the clinical laboratory tor the detection
of ASO, CRP, RA factor, HCG and many other
antigens.
' iQhfl
 * Antigen- Antibody tactions 101

J 3 HSH ivt ugglu t i nat ion tests are very sensitive and
yield iiigl ] tltres,. but mayjtb' C false positive results.
When instead of the antigen , the antibody is
adsorbed to currier particles in tests for estimation
of antigens, the technique is known as reversed
passive agglutination.
COMPLEMENT! FIXATION TEST (CFT]
Complement takes part in many immunological
reactions and is absorbed during the combination of
antigens with ihrir :intihK >dieH. In the pfCKUOC of the
appropriate antibodies, complement lyses eiythnxytes,
kills and, in some oases, lyses hactcria, immobilises
motile organisms, promotes phagocytosis and immune
adherence and contributes to tissue damage in certain
types of hvpersensitivity.
The ability' of antigen antibody [ ompieKes to
'fix ' complement is made use of in the complement
fixation test (CFT") . This is ; L very versatile and
sensitive test , applicable with various types of
antigens and antibodies and capable of detecting »
little as (1.04 mg erf antibody nitrogen and 0.1 mg
of antigen - CFT is a complex procedure consisting
—
of two steps and five reagents antigen, antibody',
complement , sheep erythrocytes and amboceptor
{ rabbit antibody to sheep red cells). Each of these
reagents has to be separately standardised.
The antigen may be soluble or particulate . The
antiserum should be healed at 5b "C ( inactivated )
for half an hour before the test to destroy any
complement activity tile serum may have and also
to remove some nonspecific inhibitors of
complement present in some sera
(anticomplementary activity ). The source of

#
2
’ O. I I
+
4!i
1
4 <2
5
ojo
Fig . 13.0 Anilgtabulln ' Coombs ; teat . Rh positive erythrocytes ( 1 ) are mixed with Incomplete mlibody ( 2 ) The
antibody coats the cells ( 3) but , being incomplete , cannot produce acjgtuiination . On addition of
anllglobullri « rum ( 4 ) which Is complete antibody to immunoglobulin, agglutination takes place .

Copyrighted material
102 * Textbook of Microbiology
complement is guinea pip scrum. AH- complement
activity is heat labile , rhe serum should be freshly
drawn , nr preserved either in the b'ophDised or
frozen stare or with special preservatives , as in
Richardson's method. The guinea pig serum should
he titrated fur complement activity. One unit or
minimum hemolytic dose ( MHD) of complement
is defined as the highest dilution of the guinea pig
serum that lyses one unit volume of washed sheep
erythrocytes in the presence of excess hemolysin
(amboceptor) within a fixed time ( usually 30 or 60
minutes ) at a fixed temperature ( 37 " C ) . The
amboceptor should be titrated for hemolytic activity.
One MHD of amboceptor is defined as the least
amount (or highest dilution ) of the inactivated
amboceptor that lyses one unit volume of washed
sheep erythrocytes in rhe presence of excess
complement within a fixed time ( usually 30 or 60
minutes) at a fixed temperature (3? ::C ).The diluent
used for the titrations and for CFT is physiological
saline with added calcium and magnesium ions .
The classical example of CFT is the
-
Wagscrmann reaction , formerly the rout i in method
for the senodiagnosis of syphilis , The test consists
of two steps. In the first , the inactivated scrum of
the patient is incubated at 37 fur one hour with
the Wasscrmann antigen and a fixed amount ( two
Units ) of guinea pig complement . If the Serum
contains syphilitic antibody the complement will
be Utilised during the antigen antibody interaction .
If the serum docs not contain the antibody, no
antigen - antibody reaction occurs and the
complement will therefore be left intact. Testing
for complement in the postincubation mixture will
thus indicate whether the scrum had antibodies or
not. This constitutes the second Step irt the test and
consists of adding sensitised cells ( sheep
erythrocytes coated with 4 MHD hemolysin ), and
incubating at 37 °C for 30 minutes. Lysis ot rhe
erythrocytes indicates that complement WJS not
fixed in the first step and , therefore, the scrum did
not have the antibody ( negative CFT). Absence of
erythrocyte lysis Indicates that the complement was
used up in the first step and, therefore , the serum
contained the antibody ( positive CFT) ( Fig. 13-9).
Appnopriate controls should be used, Including
the following: antigen and serum controls to ensure
that they arc not anticomplemertary, complement
control ro ensure that the desired amount of
complement is added, ami cell control to see that
sensitised ervthrocvtes do not undergo Ivsls in the
absence of complement.
ln direct complement fixalinn lent:
Certain avian (for example, duck, turkey, parrot ) and
mammalian ( for example, horse, cat ) sera do not fix
guinea pig complement. When such sera are to be
tested, the indirect complement fixation rest may he
employed. Here the test is set up in duplicate and
alter rhe first step, the standard antiserum known to
fix complement is added to one set. If the test serum
contained antibody, rhe antigen would have been used
up in the first step and there lore the standard antiserum
added subsequently would not he able to fix
complement, Therefort, in the indirect test, hemolysis
indicates a positive result.
( lon lutinatirtg complement nhsorption
^
luMt : For systems which do not fix guinea pig
complement , an alternative method is the
corglulinating complement absorption test. This
uses horse complement which is nonhemolytic. The
indicator system is sensitised sheep erythrocytes
mixed with bovine serum. Ravine serum contains a
beta globulin component called conglutinin, which
aers as antibody to complement . Therefore,
conghitinm causes agglutination of sensitised sheep
erythrocytes ( conglutination ) if they have
combined with complement . If the horse
complement had been used up by the intigen-
,
antjjoody interaction in the fiisl step, agglutination
of sensitised cells will not occur.
Other complement dependent
serological tests: When some bacteria ( for
example , Vibrio WioJerse, Treponema pallidum )
react with the specific antibody in the presence of
complement and particulate materials such as
erythrocytes or platelets , rhebacterii arc aggregated
Copyrighted material
 1 Anligen
and adhere to the cells, This is known as humans
adherence. The 'rtirnobjJrj- ation tejfr is another
j good antigens and induce the formation oi
turn filcmc nt dependent inKtUOi In the Treponema
pallidum inuoohiliatioa rest , A highly specific test
formerly considered the 'gold standard' for the
uerodiagnosis of syphilis * the tMt serum i > mixed
with a live motile suspension of T. palbdum in the
presence of complement . On incubation, the specific
antibody inhibits the imutility of treponemei.
Cytolytic Of cytotidoJ Jests are also complement
dependent. When a suitable live bacterium, such as
the cholera vibrio, is mixed with its antibody in ihe
presence of complement , tlie bacterium Ls killed and
lysed. This forms the basis of thevjhriocidal antibody
test lor the measurement of anticholera antibodies.
NEUTRALISATION TESTS
Virus neulralilstion tests: Neutralisation
of viruses by their antibodies can be demonstrated
in various systems. Neutralisation of bacteriophages
can he demonstrated by the plaque inhibition test .
When bacteriophages are seeded in appropriate
dilution on tawn cultures of susceptible bacteria,
plaques of lysis are produced . Specific anti phage
serum inhibits plaque formation. Neutralisation of
—
animal viruses can be demonstrated in three s ^ -stems
animals, eggs and tissue culture .
W
I ANTIGEN + TEST SERUM
{CONTAINS ANYBODY) l - C< >
+ COMPLEMENT J
+ HEMOLYTIC SYSTEM
l|, ANTIGEN + TEST SERUM
{ CONTAINS NO ANTIBODY )
+ COMPLEMENT
+ HEMOLYTIC SYSTEM
.
Fig 119 Complement fi Kalian test
— Arlib ay reactions
& 103
Toxin neUtraliiiHLinn: Bacterial QQtO)d(U are
neutralising antibodies ( antitoxins) which arc
important clinically, in protection against anal
recovery from diseases such an diphtheria and
tetanus. The toxicity of endotoxins is not neutralised
by antisera.
Train neutralisation can be tested in vivo or ifl
vitro. Neutralisation tests in animals consist of
-
injecting toxin antitoxin mixtures and estimating
the least amount of antitoxin that prevents death or
disease in the animals. With the diphtheria toxin,
which in small doses Causes a cutaneous react ion,
neutralisation Tests can he done on rabbit skin . Tlie
Schick test is based on the ability of circulating
antitoxin to neutralise rhe diphtheria toxin gimi
iniridem:ally and indicates immunity or
susceptibility to the disease. Toxin neutralisation
in vitro depends on the inhibition of some
demonstrable toxic effect . An example is the
antistreptolysin O test, in which antitoxin present
in palienl sera neutralises the hemolytic activity of
the streptococcal O hemolysin,
OPSGNISATION
The name 'openin' was originally given by Wright
(1903 ) to a heat labile substance present in fresh
M PI EMENT FIXED
RESULT - NO HEMOLYSIS
POSITIVE CF TEST
COMPLEMENT NOT FIXED
RESULT-HEMOL Y SIS
NEGATIVE CF TEST
Copyrighted material
104 * Textbook flf Microbiology
normal sera, which fat i Litated phagocytosis, This
'
factor was subsequently identified as complement.
A heatstahle scrum factor with similar activity wa
harttnutripin'. TliLt appears to be a specific
called ‘
.
antibody The term opsonin is nqw generally used
to refer both these factors. Wright used the
to
'opsonic index' to study the progress of resistance
during the course of diseases. The opsonic index
was defined as the ratio of the phagocytic activity 1
of the patient s blood for a given bacterium, to the
phagocytic activity of blood from a normal
individual- It was measured by incubating fresh
citrated blood wirh the bicteriiL] suspension at
37 “C for 15 minutes and estimating the avenge
number of phagoLytosed bacteria per polymorpho
nuckar leucocyte [phagocytic index) from stained
blood films.
IMMUNOFLUORESCENCE
Fluorescence is the property of absorbing li ht rays
^
of one particular wavelength and emitting rays with
a different wavelength. Fluorescent dyes show up
brightly under ultraviolet light as they convert
ultraviolet into visible light . Coons and his
colleagues (1943) showed that fluorescent dyes can
be conjugated to antibodies and that such labelled'
antibodies can be used to locate and identity
antigens in tissues. This ‘
fluorescent antibody ' or
immunofluorescence technique has several
diagnostic and research applicates.
n its simplest forms ( direct immuno -
fluorescence ,
it can be used for the
test)
idcntificarion of bacteria, viruses or other antigens,
using the specific antiserum Labelled with a
fluorescent dye . For example, direct immuno-
fluorescence is routinely used as a sensitive method
of diagnosing rabirs,by detection of the rabies virus
antigens in brain smears . A disadvantage of this
method is that separate fluorescent conjugates have
to be prepared against each antigen to be tested.
The " indirect immunofluorescence wf overcomes
this difficulty by using an antiglobulin fluorescent
conjugate. An example is the fluorescent treponemal
*
antibody test for the diagnosis of syphilis, i [ere, a
drop of the rest serum is placed on a smear of T
paJirddm on a slide and after incubation, the slide
b washed well to remove all free serum, leaving
behind only amibody globulin, if present, coated
on the surface of the rnrponemei ' The smear ii then
treated with a fluorescent labelled antiserum to
human gammaglobulin. The fluorescent conjugate
reacts with antibody globulin bound to the
[reponemes. After washing away all the unbound
fluorescent conjugate, when the slide is examined
under ultraviolet illumination, if the tesr is positive
Mid trepnemp will be s«n ,LS bright objects .L .Linst
a dark background. If the serum does not have
^
ami treponemal antibody, there will be no globulin
coariiig (in the Ireponemes and therefore they will
not take on the fluorescent conjugates, A single
anti human globulin fluorescent conjugate can be
employed for detecting human antibody to any
antigen ! Rg_ 13.10).
Fluorescent dyes may also be conjugated with
.
complement Labelled complement is a versatile
tool and can be employed for the detection of
.
antigen or antibody Antigens also take fluorescent
labelling bur not as well as antibodies do. For
detection of anti bodies by immunofluorescence, the
sartdwfcA technique can be rfrtpli > ved. Thc autiboJv
IN first allowed to react with unlabelled antigen,
which is then treated with fluorescent labelled
antibody A sandwich is thus formed the antigen
being In the middle and libelled and unlabeUed
antibodies on either side .
The fluorescent dyes commonly used are
fluorescein isochiocynate and lissamine rhodamine,
-
exhibiting blue green and orange-red fluorescence,
.
respectively By combining the specificity of
serology with the localising capacity of histology,
immunofluorescence helps in the visualisation of
antigen-antiihody reactions :.n situ. It thus an
mmunohistochemical technique The major .
disadvantage of the technique is the frequent
occurrence of nonspecific fluorescence in tissues
and other materials.
Copyrighted materia
 * Ariiigen- — Anlibody reactions * 105

RADIOIMMUNOASSAY (R|A )
Besides tluorvscenl JvesH many i>t t ] t;r distinctive
'labels’ nlsn can he conjugated to antigens and
antibodies . 3 he most commonly used Libels arc
' '
[bt hinder. The first reaction rjI this; Ivjir wan
radioimmunoassay ; 1UA) described hi- Rerson and
YalloW ! [ ] 1 59. RIA jjennits thfi measureme nl of
^
analytes upto piengmm (10 11 g] quantities . RIA

radioisotope ami enzymes. A variirlv of tests have
*
been devised for the measurement of antigens and
.
antibodies using such labelled reactants The term
hindcr -ligtfld -assay lias been used for the HI:
reiactLoniH. The: substance ( antigen ) whose
and its modifications have versatile applications in
various areas of biology and medicine, including
the quantitation of hormone $ „ drugs, tumour
markers, IgE and viral antigens . The importance
ol RIA was acknowledged when the Nohcl Rrizv

-
L- om-mt ration i-s ro be derL rmiued ]S termed the
awarded ro Yallnw for ITS discovery in 1977 .
ftFUt/vre Or ligdiul . d lie binding protein (ordinarily, RIAcompetitive binding assay in which
LS a
the antibody) which bunds to the ligand in called fixed amounts of antibody and radlolahelled antigen

Hfe& -r f f
A

1 + - *"
J
J. I
.
-
s«a»

2
A

+ is \ \} s

1
!

11
'x /
*i
Fig 1 J . 1 C ImmunoHuoneacMiee. 1. Direel lest: Am - cjtn ( A ] Is mixed with fluorescem- conjugaled anlibody (B),
The anHgen- antlfccdy complex is fluarvscenl. : Cj. 2 Indirect lesi : Antigen ( A ) IB mixed with antibody
iBi .The antigen - anti body complex (C ] is treated with fluorescent conjugated enllglobulin serum (D), The
final jjrodud is fluorescent (E|.
106 i Texlhaok of Microbiology
react in the presence of H labelled antigen - "fhc
labelled .L' I -LI unlabelled antu ens compete fur the
^
limited binding sires on the ant . body This '
. homogeneous El A is enzyme multiplied
competition it determined by the level of tlte
unlabelled ( test ) antigen present in the reacting
system. After the reaction * the antiijen is separated
i uto Tree* and ‘bound ' frac r i i > ns and their radioar rive
counts measured. The concentration of the test
antigen can be calculated from the ralio nf the
bound and total antigen labels, using a standard
dose response curve.
For anv reacting system , the standard dose
response or calibrating curve has to be prepared
first - This is done by running the reaction with
fixed amounts uf antilxody and labelled antigen * and
varying known amounts of unlabelled antigen. The
fating [ > j bomul : total labels i B : 1' mfio ) plotted
agaiost the analyte concentrations give the standard
calibration curve. The concentration of antigen
in the test sample is computed from the R : T ratio
of the test by interpolation from the calibration
curve.
ENZYME IMMUNOASSAYS (ELA )
Enzyme labelled conjugates were first introduced
in 19*6 for local iwr ion of antigens in tissues, assn
alternative to fluorescent conjugates . In 1971,
enzyme labelled antigens and antibodies were
developed as serological reagents for the assay of
antibodies and an Ligens. Thei r versati : m , sensitivity*
si m pi icity, economy and ahsence of radiation h azard
have made ElAs the most widely used procedure
in clinical serology. The availability of test kits
and facility for automation have added to their
popularity.
rhe term enzyme immunoassay { El A) includes
all assays ha d on the measurement of enzyme
^
labelled antigen * hapten or antibody. EIAs are of
two basic types - homogeneous and heterogeneous.
In homogeneous E1A, there is no need to separate
the bound and free fractions so that the test can be
completed in one step , with all reagents added
simultaneously, This type of El A can be used only
Y
for assay of haptens such as drugs and not for
microbial antigens and antibodies. An example of
'
i mmurtuaraay rec/imgue ( EMIT ), which is a simple
assay method Tor small molecule drugs such as
opiates* cocaine, barbiturates or amphetamine in
serum.
Heitj eriet - i-r!- * El A requites the separation of
^
the free and hound fraction* eithe r by centri fugation
or by absorption on solid surfaces. and washing. It
is therefore a muliisrep procedure * with reagents
added sequentially. l*hc major type ofhcterogcncous
El A is Enzyme Linked Immunosorbent Assay
( ELISA ) .
Enzyme Linked lmmunosorbenf Assays
( ELISA ) .
E1TSA is so named because the technique
involves the use of an immunosorbent an
absorbing mareiul specific lor one of the
-
.
components of the reaction the antigen or antibody.
This may he particulate, for example cellulose or
-
agarose Or i solid phase such as polystyrene,
polyvinyl or polycarbonate tubes or microwells
or membranes or discs of polyacrylamide , paper or
-
plastic. ELISA is usually done using 96- well
TTiic- roritre plates miii table for automation . The
principle of the test can be illustrated by outlining
i is applies do ci for the detection of rotavirus andgen
in feces.
The wells of a microti me plate arc coated with
goat anti rotavirus antibody. After thorough wii -thing*
the fecal samples to be tested are added and
incubated overnight at 4 Dil- or for two hours at
37 aC . Suitable positive and negative controls are
also set Up. The wells are washed and guiltca pig
antirotavi rus annserum , labelled with alkaline
phosphatase, added and incubated at 37 °C for one
hour . After washing, a suitable substrate
; paraniTrophcnyi phosphate) is added and held at
room temperature till the posidve controls show
the development of a yellow colour. The
phosphatase en me splits the suhstrate to vivid a
yellow compound. ^
.
*“ 4
opyrighted materia
j
 < Anliger> -Antibody reactions 1& 7

If the t« r
t« die antibody
^ mplc crsn ruins
H rniavinj ,
i r i s Fured
* -
coatinf; the weLls , When the enzynvc
Labelled antibody is added subsequently, it is in turn
fixed. The presence of residual enzyme activity,
indicated by the development of vellow eolour,
i here tore denotes i positive test ( Fig . 13.11), If the
sample is negative , then: is no significant colour
change. An ELISA reader provides quantirativc
colour recordings.
'IllC detection of antibody by EJISA can be
iLLu & rrated bv the anti - 111 V antibody tesr . Purified
i
inactivated HIV antigen is adsorbed onto microassay
plate wells. Test serum diluted in buffer is added to
the well and intubated aC: 37 JC for 10 minutes .
The well is then thoroughly washed. If the scrum
contains anti HIV antibody it will form stable
,
complex with the HIV antigen the plate . A
a
on goat
antilmmafi immunoglobulin antibody conjugated
with horse radish peroxidase enzyme is added and
Lncubated for 30 minutes. After thorough washing,
-
the substrate O phcnylene diamine dahydrochloride
is added and after 30 minutes , the Colour that
develops is read using a m i L roansay plate reader,
Positive and negative controls should invariably be
used with test sera ( Fag. 13.12),
The examples described above are of simple
noncompetitive sandwich ELISA. The test can be
made more specific by making scram antibody and
enzyme labelled antibody compete for the binding
sites on the antigen (compefidw ELISA ). Tests

U 1 2
*

..
©

Fig. 1111 ELISA far dalecticn of rotavirus in leces 1 MicrMssay plate coaled with goal antibody to rotavirus.
1When local suspension is added and incubated , rotavirus if present, will absorb to Ihe coaled anttbedy.
1 After thorough washings add guinea pig antirotavirus antibody conjugated wllh alkaline phosphatase
. .
enzyme If relaviruj is present the conjugate will compter with It 4 - Alter washing add Ihe substrate,
.
paranitrophenyl phosphate, S II Ihe enzyme conjugate is present, Itie SUbitrate Will be $p1h 10 term yellow
.
coloured product This indicates a positive test In a negative lesl liters will be no significant cotour formation .
s

w
JQ 'ft

..
Fig. 13.12 ELISA for HIV anlibody in serum. 1. Micruaasay plate coated with HIV antigen 2 Tesl serum 1$ added
. .
and incubaled Anli-HIV antibody 11 present In the serum will Mach to Hlv aniigen. 5. After washing, add goat
amihurnin immunoglobulin antibody conjugated with horse serum peroxidase enzyme. The conjugate will
- .
attach lo anti HIV antibody in a positive test. 4. Alter washing, add substrate OPD. 5 A colour develops in Ihe
.
positive tesl white teere will be no colour in A negative lesl.

Copyrighted material
108 * Textbritk oh MiCrcbioioqy
tor specific immunoglobulin classes {for example,
IgM spct’ jt ic ELISA) arc also available Capture
'
ELISA and imnninometric tests am even more
specific.
Several VJ nations oftbf ELJSA technique have
been developed to provide unojile diagnostic texts,
Including the card and dipstick methods fuitable
for clinical laboratory and bedside applications,
A pimple modification of ELISA which has
found wide application far testing OTIC or a few
samples of sera at a rime Is the cylinder or otaeflr
ELISA . Here each specimen is tested in JI separate
disposable cassette . The test is rapid, taking onlv
about It ) mi notes as compared with the 2-4 hours
taken for rnicropiatc ELISA. There is no need for
miCfOplate washes or readers. Tlic result is H&d
visually. Inbuilt positive and negative controls are
usually provided foe validation of the test procedure.
An example of cassette ELISA is the one used
for the detection of HIV type 1 and 2 antibodics-
Speci fir type 1 and 2 antigens are immobilised at
separate freed dtes on the nitrocellulose membrane
ML the cassette . Test serum is added on the
membrane and allowed to tiller into absorbent
material placed befow it in the cassette basc-
Antibody, if present in the sc runt will bind to the
appropriate antigen . After washing to remove
unbound antibody, enzyme labelled antihuman
immunoglobulin antibody :is added . After additional
washing to remove unbound conjugate, u substrate
yielding a coloured product is added . A positive
result is indicated hy a coloured spot developing at
the site of the antigen against winch antibody is
present in the serum . Human immunoglobulin
immobilised at a spot on the membrane acts as a
control for the test procedure , as shown by the
development of colour at the site.
CHEMILUMINESCENCE IMMUNOASSAY ( CLIAj
Chemiluminescence refers (o a chemical reaction
emitting energy in the form ( if light just as
, contains HbsAg, due to the formation of a HbaAg-
radioactive conjugate $ are employed in RJA
fluorescent cojugares inIFA and enzymes in ELISA.
f
chcm idittincsccnt compounds (such as luminpl nr
'
acridinium esters) arc used in CLIA ns the label to
provide the signal during the tntigcnantibody
reaction . The signal (light) can be amplified *
measured and the concentration nf the ana Lite
calculated. The method has been Killy automated
and is being increasingly used in laboratories where
rhe volume of work is large
IMMUNOELECTHO& LOT TECHNIQUES
lmmunoclcctrobLot or { electrojmmunnhlot )
techniques combine: the sensitivity of enzyme
immuno?ssjy with much greater specificity. The
technique is a combination of three separate
procedures: ( a ) separation of ligand - antigen
components hy polyacrylamide gd electrophoresis '
lb ) blotting nl the elcctrophorcscd Ligand fraction
on nitrocellulose memliraric strips; and (c) enzyme
immunoassay (or radioimmunoassay) ro ( 1) detect
antibody in [ r*.c seta against the various ligand
fraction bands; or ( 2) probe with known antisera
against specific antigen bands . The Western Blot
rest , considered die definitive test for the
sercwliaj nosis of HIV infection, is an example of
^
the immuDQcIntroblot technique (see chapter 62
for details).
IMMUNOCHHOMATOGRAPHIC TESTS
A one- step qualitative iinmunochroiTurtographic
technique h ^ s found wide application in
'.crodiagrosis due to its simplicity, economy and
reliability. A description of its use for HBsAg
detection illustrates the method. The test system is
, a Km .ill cassette containing a membrane
impregnated with ami - HbsAg umiljody-cuLLoidaJ
gp]d dye conjugate. The membrane is exposed it
three windows on the cassette. The test serum is
dropped into the first window. As- the serum travels
upstream by capillary action , a coloured band
appears ar the second window ( test site ) if the serum
antibody conjugate complo/Thii is the positive
reaction . Absence of a coloured band at the test
Copyrighted materia
 + Amtig & r} — Anubody reactions » H)9

sire indicates A
-
negative reaction Simultaneously A
coloured band should appear tn every cue at rhe
third window, which form* an inbuilt control , in
the absence of which the te^C is invalid . The Belt LH
claimed to be ready as sensitive and specific as
EIA tests.
IMMUNOELECTRONMlCflOSCOPlC TESTS
[ rnmur ] m:lectri}niiiicr[) \copy: When viral
particles mixed with specific antisera arc observed
under the electron migrowope, they are seen tn he
clumped . This finds application in the study of some
viruses Such as the hepatitis A virus and the viruses
causing diarrhea .
Itnmunnferritin lesl: Ferritin (an electron -
dense substance from horse spleen ) can be
conjugated, with antibody , and such labelled
antibody reacting with an antigen caul be visualised
under tire electron microscope.
Immunnenzyme lost: Some stable enzymes ,
SDCfa as pSOOCidasc, can La: coningateJ with re i Moodies.
Tissue sections carrying the cunesfiording antigens
are treated with peroxidase labelled antisera - The
perraddStsC bnifld to the arti Ti can he visualised under
^
the election mkTOKOpC, by rnicrohistochemical
mcthock Some o&cr enzymes, suchasghicose oxidase,
phosphatases and tyrosinase, may also he included in
imrmLnncnzyTTie tc^ts -

Furlher Heading
Bairns A ct al. 1991 . AJanmJ . / Opnjfff / . Witrn fcrn
' , gy-Wp.^>iLn tcni DC; Amakin Swiety of Mkrtbaologji
f

Hudson L And FC Hay 19 H 9. JVJLTI . .J hnnauulogy

"
^ ^
^ cdn . Oxford: hlaclcwell .
Kennedy DM and S|Ch.ilia combe 19 B9. ELISA sad Other Solid Phase Imiimno-tsays. New Tforfc John Wiley.
Kore NR ec al. AJanuz/ of Clinical Jmjnuiioic y, 3ri edn , Washington DC: Afnerlcin Society for Microbiology,
^
Stircif l ) P LLI ILL AI Terr 1991. HITTi, c inti Clinical lirijnurttiltjgy, 7* i'( ln . NHWWIJIC Applptun-l -anije.

Copyrighted material
 The Complement System
The term Lcumplement ' (C ) r L-: L C to a system of
factors which occur in normal scrum and arc
-
activated characteristically by antigen antibody
interaction and subsequently mediate a number of
biologically significant consequences,
Towards the end of the nineteenth century; it
was noticed that bactericidal , bacteriolytic and
hemolytic actions nf the appropriate anti bodies
requi ned the part ic i pari on of a heat labile component
present in the normal sera of human beings and
animals. Buchner (1889) was the first to observe
that the bactericidal effect of scrum was destroyed
by heating at 55 aC ( br one liour . Pfielfer (1B94)
discovered that cholera vibrios were lysed when
injected intraperi toneallv into speci Really
immunised guinea pigs ( bacteriolysis in vivo or
PFitfiet 's yihenonienort ). Bordet ( 1895) extended
these observations and established that immune
,
bacteriolysis and hemolysis required two factors
the heat stable antibody and a heat labile factor,
which was called alexine . This term has been
— serological reagent in that complement from unc
replaced by the present name rampleme/ir which
was coined by Ehrlich , because this factor
complemented the action of anfibodv.
Bordet and trengou (1901) described the
complement fixation test, using the hemolytic
indicator system , as a sensitive serological reaction .
Thin found wide application, and the Wasscrmann
complement fixation test for syphilis became one
of the most popular serological tests. For the next
half century, interest in complement remained
confined to its use as a tool in serological reactions.
-
Since t liL ti the structural and functional complexities
of the complement system have been defined and
its rule LIS a mediator and an i pii lie r of many i mmunc
and inflammatory reactions recognised ,. The
complement system belongs to the group of
biological effector mechanisms (called triggered
enzyme cascades) which also includes coagulation,
fibrinolytic and It in in systems. Such biological
cascades havedistinct advantages. For example, each
enxyrrie in the cascade i * able to activate many
molecules of the succeeding component providing
for amplification of the response at each step. Every
step has its own control mechanisms so that the
cascade can be regulated with prevision.
GEN Eft AI PhopHftTiEa
Complement is present in the seta of all mammals
and also in that of most lower animals, including
birds, amphibians and fishes . It is a nonspecific
Species can rear t with an t i bodie s from other species,
though the efficiency of ncacrion is influenced by
the taxonomic distance between the species.
Complement constitutes about five per cent of
normal serum proteins and is not increased as a
result of immunisation .
Though some of its components are heat stable,
complement as a whole is heat labile, its cytolytic
activity undergoing spontaneous denaturation
slowly at room temperature and being destroyed
in JO minutes at 56 nC. A serum, deprived of
its complement activity by heating at 56 °C for
30 minutes, is then said to be 'inactivated'.
Complement (C ) ordinarily docs not hind to
free antigen orannbody but only to antibody which
has combined with its antigen. Various terms such
Copyrighted materia
 * Ths Complement System *
as fixation, £ij r?dj ngor consumption have been used
' '
fa refer U> the cOrtibinJtrOil of C WLTH bolicld
immunoglobulin , leading to the activation of the
classical C pathway, All classes of Ig do not fix
Only lgMh IjyGd , 1 and 2 (in that order ) fix (.’ , but
not IgC4, IgA, Igl ) or IgE . The site ofC binding
is located on the Fe piece of the Ig molecule ( CHJ
domain nn IgG . CHJ on JgM ), and is expressed
only when lg is combined with its antigen . The
fixation of C is nut influenced by the nature of
, biological effects which contribute to defence
antigens, hut Orly by the dass of immunoglobulins.
COMPONENTS
The complement system consists of at least twenty
chemically and immunologically distinct scrum
proteins comprising the complement components,
the properdin system and the control protein !.
Complement is a Complex of nine different
fractions called Cl to C9. The fraction Cl occurs
in scrum as a calcium ion dependent complex , which
on chelation with EDTA yields [ luce protein
subunits called Clq, r , and s. Thus C is made up of
A total of 11 different protc i IH . C tractions arc named
Cl to Cy in the sequence of the cascading reaction,
except that C4 comes after Cl , before C 2 -
The model trodiliunaUv used ro explain C activity
in immune cytolysis is the lysis of erythrocyte
sensitised by its antibody. The erythrocyte ( L )
antibody ( A) complex is called EA, and when C
components are attached to FA, the product is
called EAC, followed by tlie components that have
reacted ( for example, EAC 14235 or EAC 1 5).
When a C component acquires enzymatic ui ocher
demonstrable hiologlcil activity, it fc indicated by
a bar over the component number, for example, the
enzymatically activated Cl is shown as Cl .
Fragments cleaved from C component * during rhe,
cascade are indicated by small letters (C3a , C’3b) .
Inactivated forms of C components are indicated
by the prefix T ( iC3b ) .
ACTIVATION
Complement is normally present in the body in an
111
inactive form but when its activity is induced
-
by antigen antibody combination or other tumuli*
C components react in a specific sequence as a
cascade , Basically, the C cascade is a series of
reactions in which the preceding components act
as enzymes on the succeeding components, cleaving
them into dissimilar tragments-. The larger
fragments usually join the cascade. The smaller
fragments which are released often pusses*
mechanisms hv amplifying rhe inflammatory
process , increasing vascular permeability, inducing
smooth muscle contraction , causing chcmotaxis of
leucocytes , promoting virus neutralisation ,
detoxifying endotoxins and effecting the release of
histamine trom matt cells .
1
The C cascade can be triggered off by two
parallel Hut independent mechanisms nr pathway*
which differ only in the initial steps. Once C3
activation OCCUR , the subsequent stops ant common
in both pathways, which have been called the
classical C pathway and the alternative or properdin
pathway.
The classical pathway is ho called because it
was the Unit one identified . But actually it is a more
recently evolved mechanism of ^ pceiTiL active
immunity, while the alternative pathway represents
a more primitive system of nonspecific innate
immunity.
CLASSICAL, PATHWAY
- The chain of events in which C components react
m a specific sequence loll owing activation of Cl
and typically culminate in immune cytcdysls it Itnown
as the classical pathway ( Fig . 14.1), It consists of
the following steps:
1. The first Step in the binding ( if Cl to list
antigen-antibody complex ( traditionally
represented as EA ), The recognition unit of Cl
is Clq. which reacts wi th the Fc piece of bound
IgM or JgG , Clq has six combining sites.
Effective activation occurs only when Clq ia
attached ro immunoglobulins by at least two of
Copyrighted material
112 * Toxlbook of Microbiology
its l > i riding sires. One molecule of IgM or two
molecules of IgG can therefore initijirc rhe
process. Clq binding In the presence of calcium
ions leads to sequential ICtiviuioilot Clf mid S.
2. Activated t ’ I s is an esterase (CI a esterase ) , one
molecule at which can cleave several molecules
ofC4 - an instance ot amplification. C4 is split
into C 4 an which is an anaphylatoKin , and C 4b
which binds to cell membranes along with Cl .
3. C 14 b in the prese ncc of magnevhun Lonl delves
C2 into C3a , which remains linked to cell-
bound C 4b, and (.! 2 b which is rclcasd! intofluid
phase. C 4 bia has enzymatic acriv Ltry and is
referred to as the classical pathway Cl
4 Cl convcrtase splits C3 into two fragments :
r system was zymosan , a polysaccharide from the
C3i which is an anaphylattmn, bud C3b which
remain* celt - bound along with C4 b2i to fom
a trimokcular complex C *lb2a 3 b which has
entymatic activity and is called C5 conwrtasc .
5. The membaae arrack phase of complement
activity begins at this stage with C.5 ecatvotue
cleaving C 5 into C5 a, :in aruphytatovin which
is released into the medium, and CSb which
continues with the cascade. C6 and C 7 then
join together. A heat stable IT i molecular complex
C5b 7 3S formed, part at which binds to the cell
membrane and prepares it for lysis by C 6 and
C9 which join the reaction subsequently Most
of C ?b7 escape and serve to amplify the
react inn by adsorbing onto un -sensitised
'bystander cells' and rendering them susceptible
to lysis by C8 and C9, The unbound C567 has
daemotac t ic activity, though the effect is
transient doe to its rapid inacrivaritm. The
mechanism of complement mediated cytoh'sis
is the production of Lhoics', approximately 100
A in diameter on rite cell membrane. This
disrupts the osmotic integrity at the membrane
leading to the release of the cell contents.
Although the classical pathway is gen era fly
activated by the antigen-antibody Com pieces at
Aggregated immunoglobulin, activation may also
t
be due to other stimuli, such as DNA, C-reactive
protein, trypsin - like enzymes or some letrovi ruses.
ALTERNATIVE C PATHWAY
The Central process in the complement cascade is
the activationofG , which is the major component
of Cr In the classical pathway, activation of C3 is
achieved by {J 42 (classical C3 convertase). The
activation ufC3 without prior participation ofCl 42
is known as the Haitemitive pathway'.
The first example of the alternative pathway was
the demonstration by EJilLemer (1954) of the
,
‘properdin system ' aii a group of serum proteins
Contributing to antimicrobial defence without
requiring specific antibodies. The activator in this
yeast cell wall, but many other substances can also
acrivatc the pathway. These activators include
bacterid endotoxins, IgA and I ), the cobra venom
factor and the nephritic factor ( a protein present in
the serum of glomerulonephritis patients) .
The liftl step in llie alternative pathway is the
binding at C3b to an activator. C3b is continuously
generated in small quantities in the circulation but
in the free state it LS rapidly inactivated by the serum
protein factors II and I. Bound C3b which is
protected from such inactivation interacts with a
srnmi protein called Fact nr R ( also known as ' C3
proactivator1} to form a magnesium-dependent
complex 'C 3 b, B \ This complex is cleaved by
another serum protein Factor D (also called 'C3
proactivator CKwertue1) into two fragments - Ba
and 13b. Fragment Ba is released into the medium.
Fragment Bb remains bound to C3h* forming the
esterase C 3 bfBb complex, which 15 the alternative
pathway f ’3 connrbec. This enzyme C 3b, Bb is
extremely labile. The function of properdin (also
called Factor P) is to stabilise the C3 convcrtRSC,
which hydrolyses C3, leading to further steps in
the cascade, ay in the classical pathway ( Fig. 14.2).
RTIFJULATION ni - CJ ACTIVATION
Unchecked complement activity can cause not only
Copyrighted material
 « The Complement System * 113

nflj a MH AONT SYSTEM

_ CI M. ri .
FA
' -
LA
EACH

HACMih I J FAn +Fli C*b

Liiiim
*
CA* c

C5 ti. 1 LAS lin; , toflw ’

FATL 4 h2a.lfr
FAf

/
S

^ '

*
CIi Di. ^

EAC w

,
CTTtl YSlS -

exhaustion of the
—
Fig. 14.1 Complement cascade the classical pathway
complement system but also activating factor or KAF), provides homeostatic
strioua damage to tissues. Several inbuilt control control of Co activation , particularlv bv the
mechanisms reflate the complement cascade ,n alternative pathway.
different steps. These are mainly of two kinds: 2. Another beta globulin Factor H acts in concert
inhibitors which bind to complement components and with Factor I modulating C3 activation ,
halt their further function,. and inactivators wluch are j. Anaphvlatoxin iomctivMtor is an alphaglobulim
emmtiEH that destroy complement proteinh. that en / vmari tally degrades Co .i . C4a and C5a
,

which nre anapbylatoxins released during the

A . 1 XHUIITDHK
1. Normal serum contains an inhibitor of Cl
esterase [CIHTNH ). This heat labile alpha
nruruminaglyooprotein also inhiblEs many other
esterases found in blood , such as plasmin ,
kininOgen and the Hg eman fktH. Thit no(
^
prevent the normal progress of the complement
cascade hut checks- its autocatalytic prolongation .
2. The S protein present in normal serum binds to
C 567 and modulates the cytolytic action of the
membrane attack complex .
Ti . INACTIVATORS
1 , A scrum bctaglobulin , called Factor l {formerly
known a* C3h . (_’ 4h 1 NAC , conglutinogeti
C cascade.
4. C4 binding protein controls the activity of cell-
hound C4 b.
Many other regulators of C activity have also
been repotted .
BIOLOGICAL EfFBcTs OF C
Complement mediates* immunological membrane
damage (cytolysis , bacteriolysis}, amplifies the
inflammatory response and participates in the
pathogenesis of certain hypersensitivity reactions.
It exhibits antiviral activity and promotes
phagocytosis and immune adherence. It also
interacts with the coagulation , fibrinolytic add
kiuinogcnie systems of blood. i

Copyrighted material
114 * ToutLnck cl Microbiology *

An important function of C is to facilitate rhc immune complex ( Type III ) hypersensitivity

uptake and destruction ofpuThgugcns In phjguCytiC 1
reactions. The destruction ofervr hmeytes, 1i wing
cells. This opsonic effect is- based on the presence
.
on the surface of phagocytic cells ( macrophages,
incompatible transfusion and t
sedormid purpura , art'
iTnp
mpk > of Type 1J
in -
monocytes* neutrophils and others) of complement reactions . C contributes to thu p .ir - i igennis of
receptors or CRs. Mane such receptors have been nephrotoxic nephritis , irurtmTioIugical
identified, such as CR 1 , 3 , 3 , 4 and Clqr which Icidnev damage muy also occur in its absence of C-
stimulate phagocytosis and removal of immune C is required for the produrti n of imuiune
complexes. The CR 2 receptor O i l B cells also acts complex diseases sucb as serum ckm and Artiius.
—
as a receptor for the Epstein Ban- virus ( EBV ),
the causative agent nfinfecrinus mononucleosis, and
reaction . C, however, appear* prevent immune
complex disease by solubilising ir t gi n anrihod)
so has a rtllfi in the pathogenesis ot tim condition. complexes and preventing : r precipitation in
The classical C pathway results In bacteriolysis vessels and tissues . Serum C vnmporirr rs are
and cyrolysis , Cells vary m their susceptibility to decreased in many i to! nuuum - diseases such as
complement mediated lysis . 11 ram negative bacteria systemic lupus erythematosus and rheumatoid
are generally sensitive to lysis, while firam. positive arthritis , C also ptays a n .- : r role in the
cells are killed without lysis. The neutralisation of pathogenesis of autoimmune ; anemia,
'
viruses under some conditions requires the paroxysmal nocturnal lobii u r i s a n d
participation of C. hereditary angioneurotic edema .
C fragments- released during the cascade reaction Findoloxin is an cfficienr .ictivsn i of rhe
help In amplifying the inflammatory response. C2 alternative C pathway. In : : fi » shock there is
k ini ns arc vasoactive amines and increase capillary massive C3 fixation and platelet adheteno l u e
permeability C 3a and C5 a are anuphvluroxic scale platelet knis and release ^
( histamine re I easing ) and cite mot act ic. C 5fi 7 is platelet ta Lead 1
- : i un .ri I i t . - il .-
'
L
1 r

vhcmotactiv and also brings about reactive lysis. coagulation and t b mm btnylope ri In. Li ram negative
L participates in the cytotoxic (Type II) and
"

septicemias and rrhugii sViidrunir

'
:

true E" 5h
by
/' TbN
" * FjICiDfN Jl jrid f
l in cirrulubmi I

- hcfn nifnhined
^“ HHUMHH
' nil ivuiiH t.fl.
i flC[
Wi

, cynuimn

CJhs B FatliW II *
ri C 3h

Fflfwr D

Bd- » C3b nh . + PtapcnJin > j C3b. fib , P

C3

Gusudc
/
Fig. 14,2 Complement cascade Ihr alternative pathway

Copyrighted material
 * lira Complement
Table 14.1 Clinical syndromes associated with ganelic deliciencies ol complement components
Group Deficiency
1 Cl inhibitor
11 Laxly components; of classical
pathway Cl , C2* C4
III C :\ and in regulatory protein
C3b inactivator
IV C5 to CTS
vr C
* No particular disease
may haw a similar pathogenesis. Depletion of C
protects against the Schwartzman reaction.
C hound To antigen-antibody complexes adheres
to erythrocytes or to nonprimate platelets. This
reaction, called, UHfllllH adherence* contributes to
defence against pathogenic micnoorganiiiimH since
such adherent particles arc rapidly phagocytosed .
C3 and C 4 are necessary lor immune adherence.
Rovi nc sera m contai ns an unusual prate i n called
conglutimn ( K) which causes clumping of pare ides
or cells coated with C , a process known as
conglutination . Conglutinin reacts exclusively with
hound Cl . Though conglutinin behaves an an
antibody to C, ir is not an immunoglobulin and
requires CaH + for its activity. Antibodies with
corgluririn -like activity' (immunoconghrinin, IK)
car be produced by immunisation with complement
coated materials . They may also occur frequently
in human. beings and other rn .mmuh an
autuanlilmdies to fixed C.. The times of tenim IK
rise in conditions such as infections and
autoimmune diseases associated with mere used
fixation of C in v[vo, High IK levels have been
noticed in the saliva and jejunal secretions. They
are IgA antibodies whose significance is not
krurwn .
QUANTITATION OF C AND ITS
COMPONENTS
Complement activity of serum is measured by
estimating the highest dilution of scram lysing
System * 115
Syndrome
Hereditary angioneurotic edema
SLE and other collagen
vascular diseases
Severe recurrent pyogenic
infections
Bacteremia, mainly with Gram
negarive dipbeoed, toxoplasmosis
sheep erythrocytes sensitised by smderythrocytic
Ulinbody. Estimation of individual complement
components also uses hemolytic activity ill a system
containing an excess of all complement components
except the one to he measured . C components can
be quantitated also by radial immunodiffusion in
agar bur this method does nor differentiate between
active and inactive fractions.
BlOfl VNTllfi &IS OF C
Complement components are synthesised in various
Mit s m the body such as the intestinal epithelium
'
*
( Cl ) , macrophages (C 2, C4), spleen (C5» CS) and
liver (C3, CO, C9 ). C is, to some extent , an ‘acute
phase substance' and rise in C levels ( particularly
C 4, C3, C5 and Cb ) is observed during the acute
phase of inflammation .
DEFICIENCIES O F THE COMPLEMENT
SYSTEM
Complete or partial deficiencies of all the classical
complement components and several of the C
inhibitors have been described in humans or
animals. Some are associated with severe diseases,
while in others clinical manifestations arc sporadic .
C deficiencies result in the host being unable to
efficiently eliminate the microbial antigens or
circulating immune complexes. Recurrent bacterial
and Jungal infections and collagen diseases also occur
( Table 14.1) .
Deficiency of the Cl inhibitor is associated with
Copyrighted material
116 H TcKlb& oii 01 Microtoology

hereditary anginneurnfif edtmah a condition breakdown of C 4 and C 2 . 71ie train mediator of

characterised hy episodic apgincdrma of the
suboui . un diis tiaauei or of the mucosa of [ he
'
respiratory or alimentary tracts. It may be fatal when
the larynx and trachea are affected. The attack L-s
preL imitated by the local exhaustion of the reduced
amount of the Cl inhibitor present , leading to the
autocatalytic activation of Cl and the unrestrained
the edema appears tn he the 12 kirin released. The
attack may be created by infusion of fresh plasma
as a source of the inhibitor . Prophylactic
administration of epsilon aminocaproic acid (or its
analogues) is useful. They are believed to inhibit
the activation of plasma en mes^ thus sparing the
^
small amounts of the Cl inhibitor present.

Further Reading
l.tncw y CA - ind PTTPVFT* I* edn - London: CTirrmt Biikij y-
^
Trim-lin^on 5 199
- - J , CrHpphmtpt ( Ip/ pfl
'e i7rrohanJ -
1"

-
*m *, C NIT Op hnmvnnf , 5 , 33
^
DM and J Snewirr 19V 7. Immunoiapy . If* edn . Edinburgh : Churchill Lim ingstune.

Copyrighted material
 Structure and Functions
of the Immune System
Hie lymphnrcHcular system is a complex
organisarion of cells of divert morphology
distributed widely ill different organs and tissues of
-
the body i es}Hi[isibk for immunity Lyrnpharediuilar
cells consist: of lymphoid and reticuloendothelial
components, with clearly dems reared functions. The
lymphoid cells - lymphocytes and plasms cells
arc primarily concerned with the specific immune
- being responsible tor the cellular and humoral
response. The phagocytic cells, forming part of the
rcticukjCndothriiall system , arc primarily concerned
With the 'scavenger' functions of eliminating etfete
cells and foreign particles. They contribute to
noi ispcdtic immun Ley by removing microcupui i s i ns
frurrt blood and ti-smies. They ilsn * play a role in
Specific immunity, both in The afferent arid efferent
limliK of rhe immune response.
The functional anatomy ufthe lymphoid system
can be appreciated only against the background of
1
llic ' Lvro component concept of immunity. The
inti [ tune response to art antigen, wli ateveT irs nature,
—
can he ot twn broad types the humoral or antibody
mediated immunity ( AMI ) and the cellular- or cell
mediated immunity [CMI ) . Humoral immunity is
mediated by antibodies produced by plasma cells
and present in blood and other body fluids ( hence
the nunc luimoal' from fun nor ' the old term for
body fluids). Cellular immunity is media ted directly
by sensitised lymphocytes. Cells for each of these
enmponen ts develop th rough separate du nine Is and
remain independent , though they may also interact
in some instances ( Fig. 15.1 ).
The lymphoid system consists cjf the lymphoid
cells (lymphocytes and plasma celle) and lymphoid
organs. Based on the different roles they perform,
lymphoid organs cut be classified into the cental
( primary) and the peripheral (secondary ) lymphoid
organs . The central lymphoid organs arc
lymphoepithelia ] structures in which the precursor
lymphocytes proliferate , develop and acquire
immunological capability, The thymus and the bursa
of Kabricius itl birds ait primary lymphoi J organs,
immune responses, respectively. The equivalent of
the avian bursa in mammals is bone marrow. After
acquiring imnumncompe fence , the lymphocytes
migrate along blood and lymph streams, accumulate
ill ihc peripheral lymphoid organs and , following
antigenic stimulus, effect the appropriate immune
response. The spleen, lymph nodes and mucosa-
associatcd lymphoid tissue ( MALT ) ccmsiitute the
mayor peripheral lymphoid organs. Lymphoid tissue
in the gut , lungs , liver and bone marrow and
lymphoid collections in the adventitious tissue of "
all organs also form part of rhe peripheral lymphoid
system .
GB ^ TRAI . ( PRIMARY ) LYMPHOID
Oftt:
Thymust rite thymus milage develops from the
epithelium til the third and fourth pharyngeal
pouches at about rhe sixth week of gesrarion . By
the eighth week , mesenchymal stem cells
[ precursors of lymphocytes ) from the yolk sac., fetal
liver and born.- marrow reach the thymus and
differentiate into the thymic lymphoid cells
( thymocytes ) . It is. thus the tirer < h|-gan in all animal
species ro become predominantly lymphoid. In
human bemgi, the Thymus reaches ire maximal
relative size just before birth . It continues to grow
till about [ lie twelfth year. After puberty, it undergoes
Copyrighted materia
118 * Textbook o1 Microbiology *

Ir
-- .
j h i . . V i, t 1 \W . H.i i
"Ii
'

ACTIVATED T mi
&
Jrd.1 T CELL EYIMPHCKWES
* u
Pouches

mm

£ R * THflOC f

?WGWCAliMCrTi
f MYELOO i
suw 3
LVMPHQIG’ ¥
(
X
!s-® f .
* s. r -
BMW
Q CtLL
flL,fl5 A Df P
-_ a$M
* oil
Flgr 15.1 Development of T and E cell systems

spontaneous progressive involution, irudicJltiog that only about one per cent leave thq thymus . The rest
ii functions best in early life. are destroyed locally. T h e ' . t ^ - I f " 1 pparent!}
i

I he thymus in hunted behind the upper part of wasteful process is not known . In 1 Sit thvi - . I lie
the Ettraum. Aberrant thvmie tissues are often found lymphocytes acquire new Surface citigen: Thy'
in neighbouring sires , The thymus has. two loben antigens ) , Lymphocytes >ndidoited in the thy -
surrounded by a fibrous capsule. Septa arising from are called ^ thymus (T) dependent Ivniphocvio or
the eapsulc divide the gland it ] CO lubiiks which HT cells'. Unlike in rhe ; pheral . r. .. - . lymphiK-ytc .
are differentiated into an outer cortex and an inner proliferation in the thymus is - i- n dependent on
medulla . The cort -eit is crowded with actively antigenic stimulation . In tact, jteriphersd untigeclic
proliferating small Ivmphncvtes-. The medulla Stimuli do not lead to any in m m* response in the
consists mainly of epithelial cells and mature thymus. Antigen introduced (End tly intot n- : : .
lymphocytes in the middle of which arc the .LV lead
[ TI local Immune i r
to a
Hawaii ' s corpuscles , which .ire whorl - like The rbvmus confers MI ii '. . : i - u - . ter
aggregations of epithelial cells. or tbc lymphocytes during theii stay in the organ .
Till the l%Os, the thymus was an organ without Fret hymic lymphocytes are not mpermit
.LI LV (ecqgmsfid Iunction.The for tui coos obscr virions In the thymus they arc educated
' chaL th •
bv Good ( 1954) of thymoma and impaired become capable ot mounting cell media' d irm tuj c
:

immunity in a patient, and by Miller ( 1961) of response against appropriate antigens . The
mi memo della envy in neona tally rhymectomi&ed importance of thymus in km pi i< . vr . proliferation
mice, paved tlie wav for the understanding of the and development of CMI is evident from the
pivotal role of rhe organ in the development of cell lymphopenia, Jelk-lenr graft rejection ami [ he so
mediated immunity. TEie primary function <it the called ‘runt disease * seen in neonatally
thymus i -, the production of thymic Lymphocytes. thymccromised mice, Deficient t Mi is also seen
It is the major site tor lymphocyte proliferation in f
in congenital aplasia thymus ithumanbeings.
rhe body. Howc^r, nf rhe lymphocytes produced , (DiGeorge syndrome ), and in mice) (' nude mice') .

!opyrEoht&d material
 * Structure and Functions ol the Immune System *
T lymphocyte jure selecris e!y seeded into certain
aite^ in the peripheral lymphatic tissues, being found
in the white pulp of the spleen, around the central
arterioles, and in the pararorticnl areas of lymph
nodes. These regions have been termed 'thymus
dependent as they are found grossly depleted after
neonatal thymectomy. While thymectomy affects
CM!primarily, it also diminishes antibody response
to many types of antigens { thymus dependent
antigens ) such as sheep eryrhrocyrcs and bovine
serum albti m in, FI uni oral response to or her antigens
is unaffected.
LiurMJ of FabrirauK This is a Lvmphocpithdiil
organ arising as a pouch from the dorsal part of
lliu cloaca ill birds . It becomes J lymphoid organ
hy about the day 15 of cmbiyonatian, develops full
functional ability near hatching and starts involuting
by 7-13 weeks of age, corresponding to the age of
puberty. The buna is also a site of lymphocytic
proliferation and differentia(ion. Stem cells from
the yolk sac , fetal liver and bone marrow enter the
bursa , proliferate and develop into immuno-
competenr 'bursal lymphocytes' or B cells ( II for
bursa or bone marrow ). These migrate and seed
( elective are an in the peripheral lymphoid
organs— the mantle, germinal follicles and
peritidlicular regions of [ be spleen, and the far
COrtlCal ureas and medull arv turds of Ivmph nodes.
These are known as ' hursa dependent or thymus
1
independent areas' , Following appropriate antigenic
stimulation , B lymphocytes transform into plasma
cells and secrete antibodies .
The vital role of the bursa in humoral immunity
was discovered accidentally by Click and Chang
( 195b) who found that chickens bursectomised i[
batching failed to form antibodies wlien challenged
with a bacterial antigen . IrnnmmHrojnpetence is
conferred on the lymphocytes by the bursa in
stages , Competence for IgM production is
acquired early ( about day 14 of embryonation )
and for IgG late ( about day 21 ) . Birds
burscctomised On IS to 2U days synthesise IgMh
but not fgG-
119
In humans and other mammals , the bone
marrow acts as the bursa equivalent. All
lymphocytes originate in the hone marrow; While
T lymphocyte develop in the thymus , B
*
lymphocytes develop in the bone marrow itself. In
rhe human fetus , Pejer's patches develop and
himphnid cells appear in the spleen and lymph nodes
bv the 20th week of gestation. From then on the
fetus is able to produce IgM and IgJJ . It receives
maternal IgC , but IgA and Igt arc nor present. At
birth IgM production is enhanced , hut IgC level
tldis steadily, [o reach minimum levels by the 3 rd
mo nth . IgCi production then picks up and becomes
adequate by 2-5 years . Full immunocompctcnce is
attained only at ter tin first decade of life .
m
PBRLFHBRA ] . ( SECONDARY ) LYMPHOID
OltCANS
Lymph nodes: Lymph nodes are placed along
die course ot Icmpliatic vessels . They ate surrounded
by a fibrous capsule from which trabeculae
penetrate into the nodes . The node cm be
differentiated into an outer cotta and an inner
medulla . In the cortex are accumulations of
lymphocytes (primary lymphoid follicles ) within
which germinal centres ( secondary follicles)
develop during antigenic stimulation. The Iodides
contain, besides proliferating lymphocytes, dendritic
macrophages which capture and process the
antigen . In the medulla , the lymphocytes, plasma
cells and macrophages arc arranged as elongated
branching hands (medullary cords). The cortical
follicles and medullary cords contain B lymphocytes
and constitute the bursa dependent ureas. Between
the conical follicles and medullary cords, there is
a broad, ill- defined intermediate zone (paracortical
urea ) which contains T lymphocytes and
inrcrdigiraririg cells, "Hiis constitutes the thymus
dependent area (Fig, 15 , 2),
Lymph nodes act as a Biter for lymph, each
group of nodes draining a spec i lie part of the body,
They phagocytose foreign materials including
microorganisms. They help in the proliferation and
Copyrighted material
120 « Textbook of Microbiology 1
circulation ofT and R cells. They enlarge following
local antigenic stimulation .
Spleen; The spleen is ilie larges L oi the Lymphoid
organs tr has a capsule from which trabeculae
, in the respiratory tract, the bronchus associated
descend , dividing die organ into seven!
in te rco mienl -ed compartments. The brandies ol the
splenic juxery travel along the trabeculae, and on
Leaving iheui branch again to form the central
arterioles, which are surrounded by a sheath of
-
Lymphoid tissues. This part is known as LILL white
pttlp of the spleen and may uonstirure about bait to
three quarters of the organ, hi [lowing antigenic
stimulation.The central arterioles- proceed onto [1111
red pulp , so called because at the abundance ot red
blmid cells in it .
Tbe periarterial Lymphoid collections in the
white pulp n ( the spleen are called the Malpighian
corpuscles or follicle*. Germ in til centres develop
following antigenic stimulation. Surrounding the
germinal centre is a ' mantle layer ot lymphocytes.
Immediately outside the periarterial lymphatic
sheath and separating it from the red pulp lies the
marginal wnc . The lymphatic sheath immediately
surrounding the centra ) arteriole is the thymus
dependent area of tbe spleen. The perifollicular
region , germinal centre and munrte layer form the
bursa dependent ( thymus independent} areas ( Fig.
15.3}.
The spleen serves as the graveyard for effete
blood cells, as a reserve tank and settling bed for
blood and as a systemic filter for trapping circulating
hlondluirne foreign particles . The immunological
function of the spleen is primarily directed against
btoodbCHK antigens.
Mucosa associated lymphoid tissue
( M VLTf The mucosa lining the alimentary,
respiratory, genitourinary and other lumina and
surfaces arc constantly exposed to numerous
antigens. These areas are endowed with a rich
collection of lymphoid cells , either speculated
aggregates like the PeverV pate be s or sea tte reel
—
isolated lymphoid follicles collectively called the
mucosa associated lymphoid tissue (MALT). Such
»
lymphoid tissues in the gut, from the adenoids and
tunsils to the follicles in the colon , are called the
gut associated lymphoid tissue (GALT} smd those
lymphoid tissue ( HALT ).
MALT contains lymphoid as well a± phagocytic
cells liorb and T cells arc present. While the
,
predominant immunoglobulin produced in rite
mucosa is secretory IgA , other immunoglobulin
.
classes , IgG IgM and lgE are also formed locally.
There appears In he a free traffic of antigen -
specific effector lymphocytes between die various
mucosal and secretory areas , so that an antigenic
exposure at one site may cause production of the
specific antibody ai the other nuicosal and secretory
sites also. I'bis indicates the existence ol a common
mucoeal or weretory immune system anti explains
the superiority of oral or nasal immunisation over
the parenteral nuUCt for many enteric and respiratory
tnfoctions-
Cei - l -3 O F THIi t. V M F H O R H T I C i : I AH
SYSTEM
Lymphocytes: Lymphocytes arc small, round
cells found in peripheral blood , hi up Is, Lvinphoid
organs and in many other [issues . In peripheral
-
blood , they constitute 20 45 pet cent of rhe
leucocyte population, while in lymph and Lymphoid
organs they form the predominant cell type. The
human body contains about 10l! lymphocytes,
^
approximately lC of them being renewed daily.Only
about one per cent of the total body lymphocytes
aie present in flic blood . Rliriieh ( 1J17 ) who
inrroduted a staining technique for blood cells
^
described lymphocytes AS nonmotile end cells with
no recugnis- abic function ! Lymphocytes- ate now
recognised as the major cellular elements
responsible for immunological responses.
According to their size , lymphocytes cm be
classified into small (5^S pm ), medium (S-I2 pm )
arid lafgc (12 -15 pm ) lymphocyteKr StUiill
lymphocytes arc the most numerous. They consist
of a Spherical nucleus with prominent nuclear
Copyrighted materia
 4 Structure and Functions ol llie Immune System w 121

chromatin and a thin ricu of cytoplasm, containing
scattered ribosomes but virtually devoid of
endoplasmic reticulum or other organelles. They
arc capable of slow motility and during itHVHtlttlt
assume a 'hand minor1 form, with the nucleus in
Front and the cytoplasm as a tail behind.
Depending on their lifespan, they can he
classified as short-lived aid long-lived lymphocytes-
ll human beings, the short lived. LymphOrytes Liave
a lifespan of about two weeks , while the Long lived
cells miy last for three years or more, or even lor
life. Short lived lymphocytes are the cflTeuL-Oc cells
in immune response, while the long lived cells act
as the storehouse for immunological memory Long
lived cells arc mainly thymus derived .
Lymphopoiesis Hikes place mainly in die central
lvrnphold organs where they differentiate and
mature before entering the circulation and then the
in a process known as ‘lymphocyte recirculation' .
There is a constant traffic of lymphocyte through
rbc blood, lymph , lymphatic organs and tissues .
*
This recirculation ensures that following
introduction of antigen into any pari of the body,
lymphocytes of appropriate specificity would reach
the site during their ceascles-s wandering and mount
an immune response . A Lymphocyte compiles one
cycle of recirculation in about one or two days .
Recirculating lymphocytes can be recruited by ( Lie
lymphoid tissues whenever necessary. Recirculating
lymphocytes are mainlyT cells , U cells tend to be
more sessile. Chronic thoracic dnet drainage will
therefore result in selective T ceil depletion.
A lymphocyte that has been ' educated' by the
central lymphoid organs becomes an
immttnoJcjgically competent cell' (ICC), Mature
L and B celts , before they encounter antigens are
"

peripheral lymphoid organs Rnd tissues Like a called naive cells. Such cells, though not actually
polLcemait on bent patrol. These populations of engaged in an immupnLogical response , are
lympfuKTTes do not remum distincthue mis: together nevertEieless fully qualified to undertake such a

AFFERENT
LYMPHATIC
CAPSULE

TRABECULA
GERMINAL
- l CENTRE
CORTICAL
FOLLICLE
MEDULLARY
vv V SIN JS
I
!

o ft 4
1

* IV
t
9 a 1
ESB *E
^ARAgOflTlCAL
AfltA
rv
P&) WJ *
/1
- 1
.

ms* v
r

- i
MEDULLARY
CORDS

- f EFFERENT
LYMPHATIC

.
Fig 15.2 Diagrammatic section of lymph node (arrows indicate the path ol lymph flow) .

opyrighted material
122 ^ Texttoo ol Microtvclogy
*
mpansibiliiy when appropriately stimulated bv an
antigen . LTicy subserve the fcllining timedons—
1
recognition of antigens , storage of immunological
memory and immune response to specific antigen ^
Lj!rnplKKytCS have antigen recognition mechanisms
"
on their surface, enabling each eel! to recognise
only one antigen , I ’ hr reaction of an
immunocompetent cell ro its specific antigen may
>
be induction of either ltoleniHt or the immune
response. The nature of immune response depends
on whether the lymphocyte is a 11 or T cell .
Stimulated T cell * produce certain activation
prepared the antibodies, the same marker came to
hr known hy different names (T 4r Leu3 , and so
on ) , Order was introduced at the 'International
Workshops for Leucocyte Differentiation
Antigens' bycomparing the tpeofitities ofdifferent
antisera.. When a duster of monoclonal antibodies
was found to react with a particular antigen, it was
defined as a separate marker and given a CD (cluster
of differentiation) n umber. Over 15D CD markers
have been identified so far. Tabic 15,1 lists a few
CD markers with their eel! association and
*
previous designations fin spite of the CD
,

products (lymphoktnes ) and induce CM1, while nomenclature, some popular old names continue
Stimulated B cells divide and [rsQifbqn ill Co plasma to be iti use, tor example, T*l and TS are still in use
cells which synthesise immunoglobulins . for Cl >4 ( helpcr/ induccr) and CDS ( suppressor/
A number of surface antigen or markers have cytotoxic) cells) .
*
been identified on lymphocytes and other Leucocyte* The irust e!ea r\ut dilfercnti atinn between' \ and
by means of monoclonal antibodies These , B cells is by fheir surface markers, for example, by
marker * re fleet 1li c :--1JLTLU ot ditterenfiatiun ami demonstration of CD!) tin T cells and Ig on R OCILH,
,

functional properties of the cells. As they were given Many other tests help in their differentiation ( Tabic
different designations bv rhe investigators who 15.2). These include:

sTRLirn UF wit u ' vn m^ ui- mi IMMUKR stvn M

TRABECULAR-
VESSEL
-
£77*
TP 4BEOAA
RE & PULP
Q $IMJ$Q|D
WHITE PULP
MALPIGHIAN CORPUSCLE
GERMINAL CENTRE
MANTLE LAYER:
MARGINAL ZONE
Vi .
m.--
HFP 1*1LF
i

i+
%

CENTRAL ARTER JU
^

SWJE« ARTERV

Fig . 15.3 Schematic diagram oi splenic archllKturi

riqhted
 4 Structure and Functions of the Immune Syslem >
1. T cells bind ro sheep erythrocytes forming
rosettes (SRRC or E rosette ) by CD2 antigen.
El cells do not.
2. B cells bind to sheep emhrocyres coated with
antibody and complement forming EAC
rosettes. due to the presence of a C3 receptor
{ CR2 ) on the B cell surface . This receptor
(CR2) also acts as A receptor for the Epstein - ^
Earr virus. T celb do not possess this.
3. B cells have immunoglobulin on their surfacc-
Each B cell carries about 10 s Identical Ig
molecules on its surface. The first Ig class to
appear On the Reel] surface is monomeric IgM.
Subsequently other classes {either lgG, IgA or
IgE ) may be present , along with IgD. The
surface lg on a B cell will have only a single
anrigen specificity. It therefore serves as the
antigen recognition unit. T cells do not have
surface Ig. Instead they have T cell receptors
{TC R} CQCnpttiCd tif two chai ns of polypeptides,
linked to CD3.
4. T cells have thymus- specific antigens , which
are absent on B cells.
i . T cells undeigo blast transformation on treatment
with mitogens such Uphytohcmagglutinin ( PH A )
or Concanavalin A (Con A ) , while B cells
undergo similar transformation with bacterial
endotoxins, Staphylococcus aureus ( Cowan 1
strain} or EB virus.
6. Viewed under the scanning microscope , T cells
are generally free of cytoplasmic surface
projections while B cells have an extensively
filamentous surface, with numerous microvilli .
T CELL M VTURATION
T cell precursors From the yolk sac, fetal liver and
bone marrow migrate to the thymus during the
embryonic and postnatal stages. The earliest
identifiable cells of T lineage arc the CD7+ pro-T
cells, which acquire CD2 on entering the thymus.
They synthesise CD3 in the cytoplasm and become
prc-T cells, T cell receptor (TCR ) synthesis also
takes placc.
123
TCK is A hetenodimer of glycoprotein chains
expressed on theT Cell surface, which in association
with CD3 acts as the antigen recognition unit ,
analogous to rhe Ig on the surface of B cells. TCR
occurs as two pairs of glycoprotein chains, either
-
tip or K& EVc 'l ‘cells ditforen ti ate in to two 1 i rcages,
expressing either «3 or TCR chains . The laigc
majority of T cells carry ftp TCR. TCR chains
contain four separately encoded regions— V or
Variable, P or diversity, ] or joining and C or
constant, as in the case of immunoglobulins and
hence belong to the iiamunoglobuHn gene
superiamily. Ry rtassortmeot of these regions a very
wide repertoire of antigen spedficities can be
formed on theT cell surface ( F g. 15.4).
^
Contact with self antigens within the thymus
lead s to the destruction of i inmatune T cells carryi ng
the corresponding TCR . Thus, self tolerance or
eliitimaiion oi T cells capable of reacting with -self
antigens takes place EH. the tbvmuS. But cells Capable
of reacting with iiutoantigens continue to arise
throughout life . These potentially harmful
' forbidden clones' are deleted
by antigen specific
suppresor CCIIR . I mmunneompetr nee jgai tut fore ign
antigen* is also developed in the thymus.
T cells also develop MHC restriction so that
CDS' cells respond Cully To foreign antigens
presented along with HLA Class I, and CD 4 ' cell*
to those presented with HI . A Class II molecules .
Immature '1' cells in the thymus exhibit CD7,
2 , J , 1 , 4 and S , besides TCR . On functional
they lose CDl and differentiate into the
maturity,
two major subsets CDS 4* or CD4 S' . Mature
COS 4 ' TCR ftp cells are hclper/inducer cells,
inducing R cell differentiation , stimulating
pro]] forarion of CDS' cytotoxic cells, producing
lymphokines and regulating certain stages of
erythropoiesls . CD4 8 ' TCR Ctfi cells ane
"
suppresHjr/cytoToxic cells, inhibiting B cell antibody
synthesi s and acting as cytotoxic effector cells . Minor
subsets ofCD4 + cells and CDS' cells also exist.
Small numbers of CW 8* and CD4 8 cells arc " “
also present in circulation.
Copyrighted material
124 * Te*lbook of MfcrobioKjgy
Thi“ func cir of TCR "/fi cell? in not well
^
understood, but they art believed to be immune
surveillance cells on epithelial surfaces and a form
of defense against intracellular bacteria .
Sequential antigenic changes characterising T cell
inanar-jcnm enable their enr identification . Tins has
P
, kill and lyse target celk earthing new or foreign
application ir defining T cell malignancies. Acute
cell malignancies such as Lymphoblastic leukemia and
KTnphrKmjs involve carle f cells, pro -T cells anil other
immature forms - Chronic T cell malignancies like
mycosis Eimguides, peripheral T cell lymphomas and
HTIV - 1 associated . i . Lu. Lt 1 ted leukemias involve
4
mature T odlst mainly C134' relfc .
T cells arc broadly classified as n ilaton and
^
efieeturcellL Based on their surface marhes, target
cells and functions the folluTwing 1' cell category
have been identified:
1 . Helper/ inducer cell ( TH ), with CE>4 surface
marker, MHC class U restriction; generally
stimulating and promoting the growth ofT cell*
and macrophages , Based 0:1 the different profiles
of cytokines produced , two subsets art identified .
TH1 and TH2.
THl celts produce mainly the cytokines
interferon gamma ( IFN - y) and interleukin 2
(11.2 } which activate inatrophages arid 1 celk
- IgM , IgG , IgA or IgE . By mswnnicnt oflg genes,
promoting < ’ Ml. destruction of target cells and
4 which can met with all the possible epitopes. By
killing of intracellular microbes , such as
tubercle and lepra bacilli.
TH 2 cells produce mainly the cytokine* 11 A , 5
and 6 which stimulate B cells to form antibodies.
2 . Suppressor T Cell ( TsJ: these have Cl surface
Table 15-1 Leucocyte differefrtlalion antigens (a few examples)
CD liujfiibej
’
Cell typc iHocfiQoii
CD 1 Thymocytes, Lingertuuii alls
CD 2 T evil SRBC receptor
CD 3 T cell antigen receptor complex
CD I
CD 8
- . Helper T tell ( receptor fbr HJV )
Supprotor/cytotmic T cells
CD B cells
marker and MHC class I r*LNtrk- ti on They down
.
regulate immune responses and check over
.
stimulation .
3, Cytotoxic/cytoIvTiL- T cell (T ) with CDU surface
(
marker and MHC class I restriction. The* can
imtigetis- r including tumour, allograft and virus
infected cells .
4 . Memory cells Tin 1mth CI 4 and CT)S tells
provide memory and anamnestic immune
^
responses.
B GEI . I. MATURATION
B lymphocyte precursors, prcrli cells, develop in
the letai Liver during cnibivotjic life and in the hone
marrow afterwards continuously throughout life .
Rearrangement of immunoglobulin genes takes
place O I L their he coming prc - U cells , which
synthesise cytoplasmic IgM , In the next stage -
immature 11 cells - IgM is expressed on the cell
surface . These Crib migrate t t h e periphery and
undergo immunoglobulin inotype swi telling HO that
instead of IgM alone , the ctH expresses on its
surface IgD, as well as one of the other Ig classes—
B cells- denkp the capac i tv to produce I g nrolecu 1*5
i process of alk- tic exclusion , each B cell becomes
programmed to form only one class oflg, with cither
Aap/wand lambda light chat n , possessing specificity
to a single epitope alone , and to express it on the
cell surface . By contact with self antigens during
Former designations
T6, Leu 6
Til , Uu 5
T3, Leu J
T 4 Leu 3
f
Tfi. Leu 2
fU , Leu 12
Copyrighted materia
 * Slnuoture and Functions ot the immune Syelern * 125

Table 15 , 2 Seme distinguishing characteristics of T cells , B cells and macrophages

Property T cdl B cdl Mmcrophugc

CDS receptor
Surface i mmunoglobulins
Reccpror for Fc piece of JJJJG
EAC rcMtte
(CL! recepror; CR2; EBV receptor}
——
* =
*
+
=
--
SRBG rosette ( CD2; measles receptor}
Thymus - specific antigens
* -
-
—
—
* *

Numerous microvilli, on surface 4 *

Blast transformation with:

i, anti -CD 3
b- Koti - Ig
c. PHA
+ - —
d Conanaviditi! A
f . Endotoxins
Phagocytic action
Adherence to glass surface

development, self toiuancc is developed by clonal
deletion or anergy.
O n contact with its appropriate antigen , the
mutunt B L L ' 1 I undergoes donal proliferation , Home
'
activated B cells become long-lived memory cells
responsible for the recall phenomenon seen on
producing cell, lymphocytes , lymphoblasts and
transitional cells may also synthesise Ig to home
extent,
A separate lineage of B cells , which arc
predominant in fetal and early neonatal life, express
tbe 1 cdl marker CDS on their surface and have
'

subsequent contact with the same antigen. The been named as 111 ndh . Their progenitor cells move
majority of activated R cells ire transformed into from the fcr.il liver to rhe peritoneal caviry where
they multiply. rl hcy secrete Low affinity palyrcaciivc
"

plasma cells,
Plasma cell is the antibody accreting cell. It is IgM antibodies many ot them autoanti bodies." ITiey
,

oval, about twice the size of ,t small lymphocyte,

w i t h an eccentrically placed oval nucleus containing
large blocks of chromatin located peripherally
( Lurtwhccl appearance}. The: cytoplasm is targe urtd
CONTAINH abundant endoplasmic reticulum and a
well - developed Golgi apparatus;. It is strut tu rally
designed to be an immunoglobulin producing
factory E ^ tasma cel 1st are eml cell* ami haw a short
lifespan of two nr three days. A plasma cell makes
an antibody of a single specificity, of a single
immunoglobulin class and allotype, and of a single
light chain type only. An exception is seen in the
primary antibody response, when a plasma cell
producing IgM initially; may later be switched to
IgO production . While plasm? dl is the b«t antibf >djr
-
arc responsible for the T independent Vtutur ^ l ' IgM
antibacterial antibodies which appear in neonates
seemingly without antigenic stimulus . CD5 ^ B
CcDl nnybe relevant in the ranfftfipfi ofqtftoiiwmill*
Conditions.
NULL CELLS
When circulating lymphocytes IK classified by
their surface markers into T and R ce!k, about
-
5 1(1 per cent of the cells an: found to lack features
of either type. They were called ntlJJ ce/k Because
of their morphology, they arc also known as large
granular lymphocytes ( LGL), They arc nearly
double the size of the small lymphocytes, with
indented nuclei and ahundant L V1 I .L ^ ETI Containing

Copyrighted material
126 i T& xiOOQk of Microbiology *
several azuroph i I ic granules , Composed of
milpfhoiuiii .L , ribosomes, endoplasmic reticulum
and Golgi apparatus , LGL arc a heterogeneous
group of cells with differences in rheir functional
and surface marker features. TILC [] iosl important
member of This group is the natural killer ( NK )
cell , Others arc the antigen dependent eytatnwc
cells. (ADCC) and the tymphalanc activated killer
( LAK ) cells, '[‘he term NK cell is sometimes used
as a common name tor all uu.ll tells.
Natural killer cells possess spontaneous
cytotoxicity towards various target cells mainly
malignant acid vims infected cells. Their cytotoxicity
is not antibody dependent or MHC restricted - NK
ICtfritv is ‘natural or 'rtOfii runjune' as it does not
require sensitisation by prior antigenic contact. NK
cells therefore form part of the innate immune set -
up. Tile v belong ro a different lineage from T and
B cdls and are therefore normailv active in 'severe
1*
combined immunodeficiencr diseases', in which
rnuture T and l? cells arc absent . They hive CD16
and CD56 on rheir surface . They bind to fhe
glycoprotein receptors on the surface of autologous
as well as allogeneic target cells and release several
Cytolytic factors. One of these, perform, which
resembles complement component CN, causes trans
membrane ports tlirough which cytotoxic 1 actors ,
such as the tumour necrosis (actor beta, enter the
cell and destroy it by apoptosis ( programmed cell
death ). NK cell activity is augmented by interferon .
They arc considered to be important in immune
surveillance and natural defence against virus
injected and malignant mutant cells.
A subpopulation of LGI . H possWCS surface
receptors for the Ft part ( if Ig. They are capable of
lysing or killing target cells sensitised with ) gt!
antibodies . This antibody dependent cellular
CytOTOXicity is distinct from the action of cytotoxic
T cells, which is independent of antibody. ADCC
Cells were formerly called killer ( K ) cells but are
row rlaMifiwI with N K cells.
ki iler ( LAK ) cells a re N K
] ,ymphokine activated
lymphocytes treated with interleukin - 2 ( IL2 ) ,
which are cytotoxic to a wide range of tumour cells
without affecting normal cells . LAK cells have
shown promise in the treatment of some tumours
such as renal cell carcinoma . 1 L 2 also acts as a
growth factor fur NK cells.
PtiRgemylic evils: Phagocytosis is pliylo -
gtinctically the oldest defence mechanism in
Animals . Originating in protozoa as a combined
mechanism tor nutrition and defence, along the
course of evolution, the phagocyte lost its trophic
functions with the development of digestive
enzymes - In higher organisms it specialised in the
removal of foreign and autochthonous particles.
Phagocytic cells are the mononuclear macrophages
(of blood ami tissues ) and the polvmurjihnnuclear
microphages.
The blood macrophages ( monocytes ) are the
largest of the lymphoid cells (bund in peripheral
blood (12 - 15 pm ). The tissue macrophages
( histkbytes ) arc larger (15-20 pm ) . Mononuclear
macrophage cells originate m the bone marrow from
pVKtmor cells and become monocytes in about SI
day ^ Monocytes in circulation haw an approximate
half- life of three days. They leave the circulation
and teach various tissues to become transformed
- into macrophages , with morphological and
-
functional feature - characteristic of the tissues, such
as alvtokr macrophages in the lungs and Kupffer
cells ill the liver. Tissue macrophages survive for
months . Multinuclcaied celts and epithelioid cells
seen in granulomatous inflammatory lesions such
as tuberculosis originate from mononuclear
macrophage cells.
The primary function ot macrophages is
phagocytosis. These cells move slowly in a
ponderous and purposeful manner , their abundant
cytoplasm thrusting our restless pseudopod i . L that
glide harmlessly past normal body ceils but engulf
effete cells And foreign particles. They accumulate
in areas ot inflammation or tissue damage by
chemotajiis- Particles sensitised by antibodies arc
phagocytosed more readily. The phagocytosed
particle is held inside i vacuole ( phagosome ), the
Copyrighted material
I
« Structure and Functions of the Immune System
membrane of which inses W ich :L lysosome, forming
a ' phagolysosome*, Lysosomal enzymes digest the
particle , the rtituuntibtm | extruded from the cells .
While phagocytosis is an effective defence against
most microorganisms, some {such as the bacilli of
typh < i id , bracictlo}. i * and tubcrculo-.: s) res: st digesti on
and may multiply in the cells and he transported in
them to other locations.
Macrophages express many surface receptors
including la proteins, those for the Fc part of IgG ,
activated complement components and various
lymphokines. Mac 1 is a protein antigen found on
mouse macrophages. A similar protein on human
macrophages has been named Afl marker. This
appears closely related to CR3, a cell receptor for
C3 components.
Macrophages may participate in several ways
in the induction and execution of the specific
immune response.They trap the antigen and provide
it , in optimal concentration to the lymphocytes,
Too high a concentralion of antigen may he
tolerogenic, and too low a concentration may not
be immunogen . It has also been shown that with
^
SOrtie ancigensj prior processing by macrophages is
an essential prerequisite for indue - ion of antibodies.
The processing and presentation of antigen by
the macrophage to T cells reeju. i re that both the
cells possess surface determinants coded for by the
same major histocompatibility complex ( MHC )
genes. The T cell can accept the processed antigen
only if IT i presented by a macrophage carrying on
^
its surface the self-MHC antigens. When the
macrophage bears a different MHC antigen, it
cannot cooperate with the 1 jells. This is A/f /C
r&trictii m.
The functional efficiency of macrophages can
be increased in many ways. They may be 'activated’
hy lymphokmes , complement components nr
interferon. Activated macrophages are not antigen-
specific. For example, activated macrophages from
animals infected with one microorganism are
cytotoxic to Tumour cells as well as to many other
microorganisms. Activated macrophagL - show - and atnpic allergy.
127
morphological and functional changes as compared
with un &ti undated quiescent macrophage5- They are
larger adhere better, spread faster on glass and are
.
more phagocytic They secrere a number of
biologically active substances, including hydrolytic
.
enzymes, b:. ndl ng protci n s (fibronect i n transferri n ),
tumour necrosis factor {cachectinl , colony
stimulating factor ( CSF) and interleukin 1
(formerly called the leukocyte activating factor ).
Interleukin 1 acts as an endogenous pyrogen and
also induces svnthcMS of interleukin 2 bv T cells.
CB nB
Interleukin 2 facilitates the activation of 1’ cells.
When stimulated by cytoplnLic antibodies and
1
certain lymphokmes, macrophages become ‘aimed .
Such armed macrophages are capable of antigen -
specific cytotoxicity, which is important in
antitumour activity and graft rejection.
Microphages are the polymorphonuclear
—
leucocytes of the blood neutrophils, eosinophils
and basophlk Neutrophils arc actively phagocytic
and form the predominant cell tvpe in acute
inflammation . The phagocytic property of
neutrophils is nonspecific , except for its
augr.imlaliim by npsonns. They do not appear Co
have any role in specific immune processes.
Eosinophilic leucocytes arc found in large numbers
in allergic inflammation , para- itic infections and
around z\.‘igen-antibody completes. They primar il y
inhabit tissues rather than the bloodstream. 'I'heir
distinctive feature is the presence of two types of
—
granules the small, round , homogeneous ones and
the large ovvid one*. The granules contain a variety
of hydrolytic enzymes which bring about extracellular
killing af large parasites . Eosinophils possess
phagocytic activity but only In a limited degree.
Basophil leucocytes are found in the blond and
Tissues ( mast cells ). Their cytoplasm has large
numbers of prominent basophilic granules
containing heparin , histamine, serotonin and other
hydrolytic enzymes. Degranulation of mast cells,
with release of pharmacologically active agents,
constitutes the effector mechaniim in anaphylactic
uu py righted
/“
material
i
128 * Tanthe&k l Micr & tHalm y >
^
ffiwini ' Hi R vm iMMUfJi : itrsTHM

BONE | STEM 1
MARROW
v!v
SURFACE
CD MARKERS

7 PPG - T

THYMUS 7, - 2 fPLASMIC
OTO \ PRE T

kV'
7, 3, 3 IMMATURE - f
1.4. K
TCP -p
EX I Si A THYMIC -
MATURE 1

7.2. A 7.2. 3 . K 7. X ?
TCR « P
HELFER/INDUCER
TCR |i
CYTOTOXIC/
- TCRTS

SUFPRLSiiOR

Fig. 15.4 T it It inuiuralion

Copyrighted material
 4 Structure and Functors <A Ito Immune System
Dendritic ccl ] r< : While ma crophagcs are the
mi: i ir
anti gtit present!ng cells another type of cell
, muliiallelic cluster of genes, which was called the
known a & rhe dendritic cell also performs this
fui]triu[ ]. Dendritic cells are bone marrow derived
cells of a lineage different from the macrophages
and T or R lymphocytes. I hey possess MHC class
II antigens bur not Fc or sheep RUC receptors or
surface immunoglobulins. They have little or no
phagocytic activity. They are highly pleomorphic,
with a small central body and many long needte-
libc processes, and are present in the peripheral
blood and in the peripheral lymphoid organs,
particularly in the germinal areas of the spleen and
lymph nodes. Dendritic cells are specially involved
in the presentation of antigens tod' cells during
the primary immune response.
The B cell is another antigen presenting cell,
pan iculaHy du ri i ig the secondary i mmune response.
Langcrhans cells in the skin possess features of
macrophages and dendritic cells. They process :md
present arvigens that reach the dermis.
M ^ ioa HISTOCOMPATIBILITY
COMPLEX ( MHC )
The primary function of the immune system is the
recognition and elimination of foreign cells and
antigens that enter the body. Tissues and organs
grafted from one individual to another member of
the mini; species {'allogral K .l are recognised as
foreign and rejected. Jt was the early workofGorer
in rhe 1930s on the antigens respond iblc for allograft
rejection in : n.bred mice that led to the discovery of
the major histocompatibility complex ( MJ 1C).
GofCI i JtritiJied tWO blood group antigen
systems in mice „ one of which (antigen 1) was
common ro all the strains. Antijjen 2 was found
only in some strains and appeared to he responsible
for allograft rtiection , This was called the H-2
anligen ( H for histocompatibility; Chapter 20}.
The hiitocompatibilitv antigens are cell surface
antigens that induce an immune response leading
to rejection of allografts. ( H -2 amigen system was
found to be the major histocompatibility antigen
129
{dr mice and to becoded for by a closely linked
major histocompatibility complex.
The development of congcnic and recombinant
strains of mice by Snell enabled the detailed analysis
of the various loci of this complex. {The term
L ongenich means animals which differ only at a
:
single genrlic ItHius) . Dausset pioneered Studies Oil
human leucocyte antigens, which were later found
ro be the ma : or histocompatibility antigens in
human beings.
The genetic basis of immune response, which
had been suggested by many early observa:; o ns, was
proved by Qenacerraf and colleagues, who
established that rhe ability to respond
immunologically to an antigen was conditioned by
specific genes called the Immune response (Ir)
genes . For their work un MHL and the genetic
control of immune response, Snell, Dau &set and
bcnaccrraf were awarded the Nobel Prize for
Medicine in I 9fl0.
Earlv- studies on MHC were carried uut in mice.
However, all species of animals ( including human
brings) examined subsequently were found to
,
possess a -.iiuilar complex of genes on a segment of
one eliromosome pair, coding for three different
classes of proteins:
L Class I protL - ins that determine histo -
compatibility, and the acceptance or reiocrion
of allografts { tissues or organs from different
individuals within the same species);
2 . Class II proteins that regulate the immune
response; and,
3, Cl ASS [ ][ proteins that include some components
of the complement system and a few others.
I’hc name 'histocompatibility complex' arose
because its discovery was based on transplantation
experiments, and only Later were the Other two
components of the complex identified - The major
antigens determining histocompatibility in human
beings are alloantigens, characteristically found on
rhe surface of leucocytes. Human MHC anugens
are therefore synonymous with human leucocyte
Copyrighted
^8 1
material
130 i T& xlhOok ol Microbiology *
antigens ( HLA ), and the MHC complex of genes
with rhe HLA complex .
HLA complex: The HLA complex of genes is
located On the short arm of chromosome 6 {Fig.
15 ,5), Ti consists of three separate clusters of genes:
1 ) HLA class I comprising A , E and C loci ;
2) Class II or rhe D region consisting of DR , DQ _ distribution, being found only on cells of the
and DP loci , and
'
3) Class [ II or the complement region containing
-
gene t for complement components C2 and C4
of the classical pathway, as well as properdin
factor B of the alternative pathway heat shock
proteins and tumour necrosis factors a and |1.
HLA loci are multiallelic , chat ish the gene
occupying the locus can be any one of several
alternative forms (alleles ). As each allele determines
a distinct product (antigen ), the HLA tyiitem is
very pleomorphic . For example , at least 24 distinct
alleles have been identified at HLA locus A and
50 at S.
HI , A molecules HLA antigens arc rwcnchain
glycoprotein molecules anchored on the surface
membrane of cells {Fig . 15 -6 )-
Ckss I molecules consist of a heavy pep ride
chain (alpha chum ) noncovalendy linked to a much
smaller peptvlc called beta 2- muroglobulin ( beta
chain ) . The beta chain has a constant aminoacid
sequence and is coded for by a gene on chromosome
15 . The alpha chain consists of three globoid
domains (alpha ] , alpha 2 , alpha .? ! which protrude
from the cell membrane and a small length of
transmembrane C terminus reaching into the
cytoplasm . The distal domains {alpha 1 and alpha
2) have highly van able aminoacid setjuences and
are folded to form a cuvi tv or groove between the m.
Protein antigens. processed by macrophages or
dendritic cells to form small peptides ait bound to
this groove for presentation to CDS T cells - The T
cell will recognise the and gen only when presented
as a complex with the MHC das& 1 molecule and
not otherwise ( MHC restriction ) . When so
presented the CDS cytotoxic killer cell destroys
, polymorphism of the MHC helps maximise
the target cell [for example , a viios infected cell ).
HLA class I antigens ( A , B and C) are found
nn the surface of virtually all nucleated cells. They
are [he principal antigens involved in graft rejection
and cell mediated evtotysis. Class I molecules may
function as components of hormone receptors.
HLA class II antigens are more restricted in
immune system— macrophages, dendritic cells ,
activated 1’ cells, and particularly on B cells.
Class II antigens are hecerodimers, consisting
of an alpha and a beta chsi i n ( Fig. 1 $.7] . Each chain
has two domains, the proximal domain being the
constant region and the distal the variable. The two
distal domains (alpha 1 , beta 1 ) constitute the
antigen- binding site, for recognition by CD4
lymphocytes , in a fash ion si rm I nr to the recogn i fi on
of the Class 1 antigen peptide complex by CDS T
cells . HLA class 11 molecules are primarily
responsible for the graft - versus -host response and
the mixed leucocyte reaction ( MLR) .
Both class 1 and II molecules ate members of
the immunogicihi] I i n gene supetfain iiy. The i mmune
response ( Jr) genes which control immunological
responses to specific antigens arc believed to be
situated in the HLA Class II region , probably
associated viLth rhe DR locus. It genes have been
studied cxten -uvelv. in mice and located in the I
region of mouse MHC. They code far la ( 1 region
Mociated ) antigens consisting of IA and IE
proteins . However, the relevance of Jr genes in
humans is not dear.
HIA class III molecules are heterogeneous.
They include complement components linked lo
the formation ofC3 convertases, heat shock proteins
and tumour necrosis factors . They also display
polymorphism.
The MHC system was originally identified in
the context of transplantation, which is an artificial
event. In the natural state* besides serving as cell
surface markers that help infected cells to signal
cytotoxic and helper T cells , the enormous
protection against microbial infections . Bv increasing
Copyrighted materia
 * Structure and Functions ol ttie Immure System * 131

STRUCTURE *NlJf \ r
^Ofr-SOFTllL IMML NtSTiTtiM
O H-1W I
CantmrnwTi QBM il O
'll p

A
n
nr &
*p
S'
ft *-1
'
InVj :V2
:,
0 1 *
LI
DO DP CiB 0 F C? THF H ft

Fig , 15.5 HLA compleK loci On Chromosome

the specificity of self antigens, the Ml IC prevents they tend TO have antibodies co the HLA antigens
microbes with related antigenic make up sneaking of their husbands, due to sensitisation during
past host immune defences by molecular mimicry. pregnancy. Monoclonal antibodies to HLA antigens
The primary aim o! the MHC may be defence have been developed- Typing is done serologically
against microbes and not against the graft . by mkrocytotorarify, which tests for complement
MHC has been implicated an a number of , mediated lysis of peripheral blood lymphocytes with
TiDniimminoIogkil phenomena such as individual a standard set c]f typing sera. However* -serological
odour, body weight in mice and egg laying in typing is not possible for HLA -jDR antigens, which
chickens. are detected by the mixed leucocyte reaction ( MLR )
HLA typing* Antisera for HLA typing were and primed lymphocyte typing ( PLT)5 respectively.
obtained principally from multiparous women as
, Genetic methods are being used increasingly far

13

P-
at
HHi
m2
a Chain [i Chain

i
)
Pi
|ij Mtcroglobulin J

-
r.1?1!

-
U 0

GOQH COOH
cytopiasjri C iopi asm
^
Fig . 15.& HLA Class I mulacult Fig. 15 ,7 HLA Class II moleeuls
132 * Textbook ol Microbiology »

HLA-typing m advanced centres, 'I'hcsc employ
restriction fragment lctigrl’i polymorphism ( RFLP)
and gene sequence specific oligonucleotide probe
*YP“ g
The HIA antigens coded for by the
combination of alleles at each locus on one stfud
of a chromosome pair represent the haplotype. I'hc
complete FI [ .A type ol ml individual comprises [ tie
antigen* represented on both strand *, of the diploid
chromosome and so will consist of TWO haplotvpvS
-
(for example, HLA Al , -A2; - R 7, - Bi2i -Cw3,
-Dwfit L>w 4; -Dw7t -DRl ; -DR7; DQyrl ; Qw3-
-DPw 4; -DPwti ) ,
-
lihn. to the extreme pleomotphism ol the HLA
system, delineation of the I1LA type provides a
method of typing of individuals, that is far more
discri mi nating than blood grouping. 11LA typing
i $, used primarily for testing computihilitv between
recipients and potential donors before tissue
tnuriHjilantation. Tt also has applications in disputed
paternity. As the prevalence of HLA types varies
widely between different human races and ethnic
groups, HI . A typing i n used in anllirof J O logical
exhibit a he reditu rv tendency. Kor example, strong
association has been found between antqrlosing
Spot idyl ids and .HLA- H 2 7, rheumatoid jrtli ri t is an LI
f 11 ^ A - DR 4, and m any autoi m mu nc cond i tions and
HLA- DR 1 .
MHC RESTRICTION
The importance of MHC antigens in immune
reaction is indicated by the finding that '1' cells
respond to processed antigens on the macrophages
and other accessory cells only when they are
presented along with the self - MHC antigen , This
is known as MHC restriction. Both class I and class
11 antigens o]K.Lrale in this phenomenon. CytOtOXic
T lymphocytes from immunised mice arc able to
kill and Ivsc virus infected target cells only when
the T cells and target cells ate of the tame MHC
lvpeNso that theT cells can recognise clans I MHC
antigens on the target cells. I JclperT cells can accept
antigen presented by macrophages unlv when the
macrophages bear the same class II Ml 1C
molecules on the surface. For T cells participating
in delayed type hypersensitivity the antigen has to ,

studies, I puliation studies of HLA polymorphism

^ he presented along with class II MHC
suggest the origin of fbe human tpedn in. Africa determinants.
and emigration as different subtypes to other In view ol the great importance of MHC
continents.. An association has been observed restriction in immunological control, the Nobel
between HI .A types and certain diseases. Such Prcic far Medicine lor the year 1996 was awarded
diseases arc generally ol uncertain etiology, to Peter I >oherty and Rolf Zinkernagd for their
associated with immunological abnormalities and seminal contributions in this area.

Further liunding
Ahhds AX cr (L iy 4. CeJJuIirand MflArcrjAtrliFhinLino/cgT. 2*: edn. Philadelphia : Saunders.
^
Chapel H and M Haeney 1993. Essentials ofOinic tirimtino/t y. fllLedn. [.(melon: Htnckwell Jkwirce.
^
/mrnujKiiNoJoijv, 2 lI cdn . London : Currant Biology.
' ^
JanewayGA and P Travers 19%,
. -
'
.
IVakrTun M and [) VerRani 1997 Basic and Clini al fn? mimcJc>£p Edinburgh, Churchill - Livingstone -
Iflfeir DM a n d j Stewart 1997 . laUBUJKrlvgf' S* ndrt . t Lli ntwicgl:: CtlUIXhall-I . Lvidj -tniu:.
"

^
Klein JK and A 2000. The HLA system - NewEngf Nfeti 34.L702

Copyrighted materia
 Immune Response
The specific reactivity induced in a host by an
antigenic stimulus is known as the immune
response . In infectious disease it is generally equated
with protection against invading microorganisms .
I5 ut the immune response has a much wider scope
and includes reactions against any antigen , living
OT nonliving. It may lead to consequence ^ that are
beneficial, indifferent or injurious to the host. It
also includes die state of specific oonreictivity
( rule ran ue 1 induced by certaini types of antigenic
—
Stimuli , The immune response rim be of two types
the humoral ( antibody n led i ated ) and the cellular
(cell mediated ) types. The two ate usually
developed together, though at times one or the
other may be predominant or exclusive. They
usually act in conjunction but may sometimes act
in opposition.
Antibody mediated immunity' ( AMI ) provides
primarv defence against most L'xiraciellliLLr bacterial
pathogens, helps in defence against viruses rbiit infect
through lilt respiratory or intestinal tracts , prevents
recurrence of virus intections and participates in the
pathogenesis of immediate ( types l , 2 and > )
"
hypersensitivity and certain autoimmune diseases. Cell
mediated Immunity ( CM I ) proteurs against fungi,
viruses and facultative intracellular bacterial pathogens,
participates in the rejection of hotnograftn and graft-
, the response to subsequent stimuli with the same
versus - host reaction , provides immunological
surveillance and immti uity against cancer, and mediates
the }twtliogenesis of delayed ( type 4) hypersensitivity
and certain autoimmune diseases.
HUMORAL IMMUNE RESPONSE
The production nf antibodies consist; of three stops:
1 . The cntTy of the antigen, its dis-Triburiun and fete
in the tissues and its contact wirh appropriate
immunocompetent cells ( rhe afferenr /djiab).
2. The processing of antigen by cells and the
control of the antibody forming process [cenfruf
functions).
3. The secretion of antibody, its dislributicm in
tissues and body fluids and the manifestations
.
of its effects { efferent limb )
Antibody production follows a characteristic pit tern
consisting of
a. A lag phase, the immediate stage following
antigenic stimulus during which no and body is
detectable in circulation.
b. A log phase in which there is steady rise in the
titre of antibodies.
c. A plateau or steady state when there is an
equilibrium between antibody synthesis and
catabolism.
d. The phase of decline during which the
catabolism exceeds the production and the titre
falls {Fig. 16.1).
PRIMARY AND SECONDARY RESPONSES
The antibody response to an initial antigenic
stimulusdiffers qualitatively and quantitatively from
antigen.The former is called the primary response
and the latter the secondary response ( Tig. 16.2).
The primary response is slow', sluggish and short-
lived , with a long lag phase and low ricre of
antibodies that does not persist for long. In contrast,
the secondary response is prompt powerful and
prolonged , with a short or negligible lag phase and
Copyrighted materia
134 * TGnttOOk Of Microbiology

a much higher level of embodies that lasts for long

periods. The antibody formed in the primary response
la predominantly IgM and in the secondary response
[g|G.The early antibody Is mote specific but less avid
than the late antibody The duration of the lug phase
and the persistence ofthc antibody vary with the nature 4
of the antigen. With liome antigens such as diphtlieria
toctoifl, the lag phase in the primaiy response may he
-
-
as Jong as 2 3 weeks, while with pneumococcal
- 2
polysaccharide, antibodies Can be detected as early as 3
within a fcw hnurs.
A single injection of an antigen helps more in 1
sensitising or priming the immunocompetent cells i T T I 1

producing the particular antibody than in the actual

elaboration of high levels of antibody. Effective
levels of antibody are usually induced nnly by
subsequent injections of the antigen. It is for this
reason that nonliving vaccines arc given in multiple
defies for active immunisation. The first injection
is known is the priming dose and subsequent
injections as hastier doses. With live vaccines, a
single dose is sufficient as multiplication of the
organism in the body provides a continuing
antigenic stimulus that acts as both the priming
and booster dose.
When an antigen is injected into an animal
already carrying the Specific antibody in circulation,
3 is primary or secondary. Antigens introduced
2 intravenously are rapidly localised in the spleen ,
1 fiver, bone marrow, kidneys and lungs. They are
r
i f T f
Fig. 1fi.1 Primary Immune response. An antigenic
stimulus 1. latenl period. 3, rite In titrs of tnuin
antibody. 3. Steady slate of anlibody litre. 4. decline
of antibody litre .
A
t
B C
.
Fig 16.2 Effect erf repeatec ant titnit stimulus. A 8, .
.
C antigenic stimuli . 1 primary immum response.
1. secondary immune response.. 3 negative phase. .
A . high level of antibody following booster injection.
a temporary tail in the level of tin ulating antibody
occurs due to the combination of the antigen with
the antibody. This has been called the negative
phase. It is followed by an increase in the titre of
the antibody acceding the initial Level.
FATE OF ANTIGEN IN TISSUES
The manner in which an antigen is dealt with in
the body depends on factors such as the physical
and chemical nature of the antigen , its dose and
route of entry, and whether the antigenic stimulus
broken down by the reticuloendothelial cells and
excreted in the urine, about 70 80 per cent being-
thus eliminated within one or twr> days. In contrast ,
antigens introduced subcutaneously are mainly
localised in the drairing lymph m >ilesr only small
amounts being found in the spleen.
Particulate antigens are removed from
circulation in two phases- The first is the

Copyrighted material
 4 immune Response
noniinrrunc phase during which the antigen is
cn Eled by the phagocytic cells broken down and
^ ^
eliminated . With the appearance of the specific
antibody, rhe phase of immune elimiLiar ion begins,
during which antigen — antibody complexes are
formed and arc rapidly phagocytosed, resulting in
an accelerated disappearance of the antigen from
circulation. With soluble antigens , three phases can
be recognised — equilibration, metabolism and
immune elimination. The phase of equilibration
cnn5 i -.ts of diffusion of the antigen to the extruvascular
spam. During the metabolic phase, the Level of the
antigen falls due to catabolic decay. Dun ng the phase
of immune elimination, them is rapid elimination of
the antigen with the formation of antigen— antibody
complexes. Snell complexes can cause i issue damage
and may be responsible for ' immune complex
diseases' such as scrum sickncss -
Thc speed of elimination of an andgen is related
to the speed . u which it i & metabolised. Protein
antigens arc generally eliminated within days or
weeks , whereas polysaccharides which arc
metabolised slowly, persist for months or years.
Pneumococcal polysaccharide, for instance , may
persist upto 20 yean in human beings, following a
- ingle injection .
PRODUCTION OF ANTIBODIES
Immune response to an antigen is brought about
hy three types nf cells : antigen processing cells
( APC— pri ncipally maemphages and dendritic cells ),
T cells and E cells. The first step is the capture and
processing of the antigens by APC and their
presentation, In association with the appropriate MHC
molecule, to T cells. While this step is essential for
most antigens (T cell dependent antigens such as
proteins and erythrocytes ), in the case of T cell
independent antigens, such as polysLreharidcs and
other structurally simple molecules with repeating
epitopes, antibody production does not requmeT cell
participation.
Only when the processed antigen i - presented
on the surface of APC, in association with Ml IC
135
molecules , to the 1 cell carrying the receptor
(TCR) for the epitope is the T cell able to recognise
i :. In the case of CD4 ( helper T/TK ) cells, the
antigen has tn he presented completed with MHC
Class II and for CDS (cytotoxic T/T . ) cells with
^ possess
MHC class I molecules . B cells, which
surface Ig and MHC class II molecules, can also
present Antigens to '1 cells, particularly during the
secondary' response .
The TH cell requires two signals for activation.
The first signal is a combination of theT «11 receptor
-
[TCR) with the MHC class II completed andgen .
Hu.- second signal is iuterleukin-1 (1L1 ) which is
"
produced by the APC . The activated TH cell forms
i ritcrlcukm-2 and other cytokines required for B cell
stimulation. These include 1L4, IL5 and JLfr which
act as E cell growth (actor ( ROGF) and the B cell
differentiation factor ( BCDF) that activate B cells
which have combined with their respective antigens
to donaliy prohleratc and differentiate iinto antibody -
sccre ring plasma cdls- A small proportion of activated
H cells , instead of biLing transformed into plasma
cells , become long lived memory cells producing a
secondary type of response to subsequent contact with
the amigen.
Cyrormtic T ( CDfl /TC) cdls are activated when
they contact amigens presented along with MHC
class T molecules. They also need a second signal
Th
IL2, which is secreted by activated 3 cells. On
contact with a target cell carrying the antigen or
Its surface, the activated Tc cdls release cytotox ms
'
that destroy the target, which may be virus infected
or tumour cells. SomeTc cells also become memory
cells [ Fig . 16.3).
MONOI :I ONAI ANTIBODIES
A single antibody forming cell or done produces
antibodies specifically directed against a single
antigen or antigenic determinant only. However,
antibodies produced ordinarily by infection or
anti gens have multiple epitopes or antigenic
determinants, each of which generates separate
clones of lymphocytes. This results in antisera
Copyrighted material
136 + Textbook or Microbiology

rviML nr RESPOND

w
c

* / ®
§k TCH
cw

Wtwkmi
I •"!

B.- ZHI

0 Hi.ItiF

IL - ?
©

CytaloMia

MHC - I
tO
^
.
CTrt
_wafh
'r |
Tc
M
4
- ©r
/ \
HC
®

f ‘\
Fig.16 3 : Outline stbameaNmimjneresponse a ainstTceltcfep^nddnl antigen
^
1. Anl ige n praso ntl ng ee lls ( AFC) proees & phagccytosed antigen { A ] in thair cytoplasm I present oi thscefl
*
s u rf ace imm u nog eni c c p hope s (E f com piexcd wilti M HC Class II molecules .
.
2 Signals forlhe activation of CP 4 ( THJ cells are a combination of the T Cell antigen -
rht MH(
Classilantigen complexes APC . and stimulation by I L-l released by A PC .
1. The activated TH celt spteutslL- ZteCBptgrssndi secretes cytgfcines hich act on g cells i’ lgfor
IheepHope , causing cellgrowlh gnrl dilterenlielmn ) ,
*
^
5. Some of the activated 0 c Ns become memory cells (6U1 , wh He outers ate transformed c :: i i r: .
*
S 0C teti ng irttiba dy against ( he anti gen ( 6 f .
i

7 Some TH cellsremain as memory cells ( THM)

r

4. Cytotoxic T cells are stimulated by IL- 2 released by the activated Th cell. On pitepa
complexed with WHC Class t mdeCutee On tire Surface ol target Cells , (he Tc cell Ins which
destroy the target cells ( 9 }.
id . Some Fc cells become memory colls tT:u|.

Copyrighted material
 * Immune Response *
containing immunoglobulins of different da--- with
^
specialities against different epitopes of the antigen.
On the other hand, when a done of lymphocytes or
plasma cells undergoes. selective prul iteration, LLH in
multiple myeloma, sinribodieG with a single antigenic
.
specificity accumulate Such antibodies produced by
a single done and directed ; LJ HJU >[ a single antigenic
^
determinant are Called monoclonal antibodies.
Monoclonal antibodies ire very useful tools for
diagnostic and research techniques .
An ingenious method for rhe luge scale
production of monoclonal antibodies against any
des- i red antigen was developed by Kr>h I < -T and MUsed n
in 1975. In recognition of the great importance of
this hybfidoma technology, the Nobel Prj c for
^ beings . Various modifications were introduced to
Medicine was awarded to them in 19fl 4.1 Iybridonuts
arc somatic cell hybrids produced by tiising anlilwdy
fanning spleen cells with myeloma cells.TIK resultant
hybrid retains the antibody producing capacity of the
spleen cell and the ability of the myeloma cells to
multiply indefinitely ( fig. 16.4).
Lymphocytes from the spleen of mice
immunised with the desired antigen are fused with
mouse myeloma «Dl grown in culture which di >
not form immunoglobulins and are deficient m the
enzyme hypOKanthinc phosphorihnsyl transferase
( HPRT ). The fused cells are placed in basal
culture medium ( HAT medium containing
hypoonthinc, ammopterin and thymidine ) which
does not permit the growth of the enEyme
deficient myeloma cells. Av normal lymphocytes
cannot replicate indefinitely, only hybrid cells
possessing properties of both the splenic
lymphooytei anti myeloma Cells can grow in
culture . These hybrid cells, called liybridomas* are
cloned and examined for the production of
antibodies. Clones producing antibodies jigainst the
desired antigen arc selected for continuous
cultivation. Such hyhridomas can be maint .lined
P
indefinitely in culture and will continue to form
monoclonal antibodies. They may be also injected
intrapcriioireally in. mice and monoclonal antibodies
may be obtained bv harvesting the ascitic fluid
137
produced - Hybridomas may he frozen far prolonged
Storage . The discovery of tlie hyhndoma technology
for the production of unlimited quantities of identical
[noiuxJnnal antibodies of die same lg das*, possessing
uniform spedlieky, affinity and other properties ,
created a revolution in immiLnology by opening up
numerous diagnostic , therapeutic and research
application*. Monoclonal antibodies against scvcrel
anrigens are now available orwnmervLlly.
Murine monoclonnl antibodies, however, proved
unsuitable for human therapeutic use hecause they
induced strong anti mouse immune response .
Moreover* the Pc piece of mouse [g could not
initiate effector defence mechanisms in human
improve efficiency. Cleaved Fab fragments could
be coupled TO various active substances like toxins,
enzymes, radionuclides or cytotoxic drugs. Mouse
monoclonal* have been humanised by genetic
manipulation ro inalte chimeric antibodies
consisting of murine variable region# and human
constant region*. Grafting of murine monoclonal
Gl > R loops cm a human Ig framework provides a
virtually human molecule . ( The antigen binding
surface of an antibody is composed of six
hypervuiible loops of ami noadds. These are called
oaaplementanty determining regions or CDRs).
I Jumati monoclonal antibodies have subsequently
been developed. Genes for particular antibody
fragments have been fused to hacrcriophagC genes.
Whole libraries of such antibodies have been built
using bacteriophages. I Jige quantises of the desired
antibody can be prepared by infecting bacteria with
the appropriate bacteriophage. Such antibody
engineering holds great promise for immunotherapy
FACTORS INFLUENCING ANTIBODY
PELODI CTION
Genetic factors: The immune response is
under genetic control The differences in immune
response to ( lie same antigen shown by different
individuals in a species is determined by genetic
differences . The t e r m s ' responder 1 and
Copyrighted material
m 4 Textbook n\ MiCf &biOtogy *

IMMUNE RESPONSE

Wrrt «DJ*ip !lwnur'U.^ion

Mfl CxJlur
*

MPBI »g
I^RT lg 1-« H«
i
^ Hn

,V> :
r © t fW 1
l
\
'
- '

SftilbEC -on nr Hybrid m\m

rnhAT iTiMiUpn /
Aun- ! _
niibodv

r_ - .-
r i' C zdv
.
"

Jjn&dliE^g [rDi-!
Hp-binh
^

lurnwa liidurliuii

Mm -
Cw liir
*

. .. .
W.:rT r r: -mi;

fliiliEudy
y 4^
SMcmri.mnBi
niibodv
Freer

| .-
*
> 4i.ILHIH ilH

. .
Fig 1 & 4 : Monoclonal antibody flr &ductlon by bybridoma

Copyrighted marorial
 lmmun & Response
Hnnn responder arc used to describe the individual's
capacity to respond to a particular antigen. The Ir
( immune response ) genes control this property.
Aj ,“ The embryo is immunologlcally immature.
^
The opacity to produce antibodies start only with
the development and JrfieienTiatLon. of lymphoid
organs. The age at which embryos acquire
immunological competence varies with different
specie?. When the potential immunocompetent cell
comes into contact with its specific antigen during
-
embryonic hie, the response i elimination of the
cell or induction of tolerance, This is believed to
be the basis for the nonaTiligenicity of self antigens.
During embryonic life , the developing lymphoid
cells come into contact with all the tissue antigens
of the body released by cellular breakdown , so that
all the clones of cells that have specificity towards
seif antigens arc eliminated.
Immunocompetent is not complete at birth,
but continues To develop U the infant grows . The
in font has to depend on itself for antibody produce ion
-
from 3 6 months of age, by which time the maternal
antibodies- disappear Hiiwever, lull competence is
-
acquired only by about 5 7 years for lgG and 10
15 years for IgA . The ontogeny of antibody response
- hypersensitivity} rather than antibody formation.
also depends on rhe antigens concerned . B cell
responses to most proteins and other T cell
dependent antigens develop earlv, while responses
to polysaccharides find other 3’ cell independent
antigen ? develop only later, usually by two years.
Most IgG antibodies to polysaccharides arc of the
lgC 2 type, and lgG 2 producing 11 cells art the last
to mahm: during infancy . is effective only above a minimum critical dote
N LI Li ' iE irpilul S to Los; Malnutrition
affects the
immune response adversely, though serum
components necessary for immunity are conserved
selectively till the nutritional deficiency becomes
marked . Protein calorie malnutrition suppresses
both humoral and cellular immune responses, the
latter more severely, Deficiences of ami noacids
{ tryptophan, phenyl alaninet methionine, glycine,
i sole neine ) and vitamins [vitamin A, and B group
factors rilujfljvine, pyridoxine, pantothenic acid
t 139
folic acid) have beer shown to cause a decrease in
antibody synthesis.
Route of administration: The humoral
immune response is better following parenteral
administration ot antigen than through oral or nasal
routes . Large particulate antigens, such as bacteria
or erythrocytes, are more effective when injected
into tissues. The route of administration may also
influence the type of antibody produced. For
production of IgA antibodies, the oral or nasal route
is must -suitable . Inhalation ot pollen antigens
induces IgE synthesis, whereas rhe same antigens
given parenterally lead to IgG antibodies . With
some antigens the route of administration
determines wherher tolerance or antibody response
results. Injection of protein antigens into the
mesenteric vein or intrarhymically usually induces
Tolerance , Sulzberger ( 1929 } and Chase ( 1959 }
showed that guinea pigs can be rendered upecifu - ally
tolerant if certain antigens arc ted before a parenteral
-
challenge (Suliberger Cbase phenomenon ).
Application of simple chemicals to the skm usually
leads to cellular immune response ( delayed
With some antigens tile site Ot injection aeeins
relevant. 1 Icparitis B vaccine is less immunogenic
following gluteal injection than following Injection
Into the deltoid . This mav be due to the paucitv of
antigen presenting cells iit the gluteal fat, delaying
presentation of antigen to T and B cells.
'
Size find number of doses: Artihndy
response is., roan extent , dose dependent. An antigen
.
Further increase in dose enhances the intensity of
the anTih«ty response hut beyond a certain level,
,
increase in the dose df antigen docs not improve
rhe antibody response but may even inhibit it and
induce tolerance. Mice injected with 0.5 pg of
pneumococcal capsular polysaccharide produce
specific antibodies but those injected with 5Q pg of
the antigen not only fall to form antibodies but may
not respond even to subsequent doses of the same
. antigen. The massive antigenic stimulus appears
Copyrighted material
140 * Tgxibook
swamp [ III annlxhl/ producingsystem and paralyse
1
it. This phenomenon wasdesigniLtecTirninLinolLiLdcai
paralysis' by Felton ( 1949 ) .
The increased antibody response ro a secondary
antigenic tdfflulur lias already been noticed. With
repeated antigen injtctioru, the .11 .i;':KK!V response
' Adjuvants: The term adjuvant refers to any
increases progressively hot after a certain stage, no
further increase occurs.
The term "anamnestic re actio n' wan originally
applied to the production, in response to an
antigenic stimulus, of a heterologous but related
untiHody that the ho*r had earlier produced hbr , lead to the production of 'udjLtvant diseases" such as
instance, A person who had been immunised earlier
against typhoid bacilli may sornetinio: produce
ant Ltypho id antilhodic > in response to i1 Lfeetion wiih
some other bacterium. This may cause L- onfusiort
in the serological diagnosis ot typhoid tcver but
urcu-m nestle reaction can he differentiated from a
true secondary response as it is transient. The term
'anamnestic reaction’ has been employed by some
ro refer to the secondary response as well, so there
is some confusion in the use of this term.
Multiple antigens: When two or more antigens
art administered simultaneously, [he effects may
wary. Antibodies may he produced against the
different antigens just as though they hardbeen given
separately or antibody response to one or the other
of rhe an tigers may he enhanced* or the response
to one or more of them may he diminished
(antigenic competition). When two bacterial
vaccines ( for example, tvph- uU and cholera vaccines)
are given in a mixed form, the antibody response to
.
each is nor influenced by the other When toxoids
Art giycn ALONGWILIL h.1,rkr - iI ¥
| CQnes ( for esample,
triple vaccine containing diphtheria and tetanus
toxoids along with Bord pertussis vaccine) the
response to the toxoid is potentiated. When
diphtheria unci tetanus toxoids Aft given together,
with one in excess, the response to the other is
.
inhibited When triple antigen is givtn to a person
who had earlier received a primary dose of
diphtheria teweid, the response to the tetanus and
pertussis antigens will be diminished.Such antigenic
of Microbiology *
competition is important, from a practical point of
"
view, in immunisation wuh polyvalent antigens For
, .
optimal effect, the nature and relative proportions
of the different antigens in a mixture should be
carefully adjusted.
substance that enhances the immunogcnicity of an
antigc n. Adjuvants may confer 1m munogcn icily on
[icmantigcnic substances, increase the concentration
and persistence of the circulating antibody, induce
or enhance the degree of cellular immunity and
.
allergic disseminated encephalomyelitis A variety
of substances exhibit adjuvant acLivhv.
Repository adjuvants such HE aluminium
hydroxide or phosphate and incorporation of
protein antigens in the water phase of a water - in
oil emulsion (Freunds incomplete adjuvant ), delay
-
the release of antigen from the sire of injection and
prolong the antigenic stimulus . Others Euch as -silica
particles , beryllium sulphate and endotoxins activate
macrophages, The most potent adjuvant is Freund’s
complete adjuvant, which is the incomplete adjuvant
along with a suspension of killed tubercle hue ill'..
Besides increasing the humoral immune response,
it induces a high degree of cellular immuni tv
.
{ delayed hypeftcnwtivitj) as well As It produces a
'
local granuloma, it is unsuitable for human use .
The adjuvant effect of tubercle bacilli is due to a water
soluble peptide MDP (muramyi dipepiide) which
induces good antihc -ilv response without causing
,
granuloma . Given in mineral oil or as tposomes, it
also stimulates cell mediated immunity. Derivatives
of MDP are hc-int; developed for human use. Gram
negative bacilli show an adjuvant effect due to their
lipif Htlvvtfccharide fraction.Bordrldla pertussis, which
-
has, in atkiirion, a Eyinphocytosis pnomoting factor
anting on both T and K cells, act? as a gtxxl adjuvant
for diphtlieri-a and tetanus tmuids in triple vaccine
Ollier adjuvants otnumunlv uaed with human vaccines
are alnrYiinium hydroxide or phosphatC -
L mmunoguppro strive agents: These inhibit
the immune response lltey are useful in certain
,
Copyrighted material
 4 Immune-
situation;; Like transplantation, when it becomes
necessary to prevent graft rejection . Kxamples of
immunosuppressive agents are X- irradiation,
ndiwninKtic drugs, corriooRfernidR, anrimctaboliteK
and other cytotoxic chemicals, and antilymphocytic
semm .
fiuhlethui whole body irradiation suppresses
antibody response. When antigen if stimulus folkmi
24 hours after irradiation, antibody Jinn 3 ucn -. ni does
not occuii whereas if the antigen is administered
2-!l davs before irradiation, the antibody response
is acto , LE 3 V enhanced. The primary response is more
radiosensitive than the secondary response .
Radiomimetic drugs are agents with an action
resembling that of X rays. They belong in genera ]
to the class of alky La ring agents { fur example ,
cyclophosphamide , nitrogen mustard ). In human
beings, cyclophosphamidegiven for three days after
the antigen , completely suppresses the antibody
resjJOrtse. 1! is: much less effective when given before
the antigen.
Corticosteroids cause depletion of JjJTjnphocytCfi
from the blood and lymphoid organs - They also
stabilise the membranes of cells and Lysosomes ,
inhibiting histamine release , md the iTiflummarory
response, They suppress antibody formation in the
rat, mouse and rabbit but are much less effective in
guinea pigs , monkeys and human beings.
Therapeutic doses have little effect on the antibody
formation in human beings . They inhibit the
induction and manifestations of delayed
hypersensitivity in human beings .
Anrimcrabolitcs arc substances that interfere with
the synthesis of UNA, KNA or both and thus inhibit
«11 division and differentiation necessary for humoral
and cellular immune responses. They inchide folic
acid antagonises { methotrexate), alkylating agents
( cyclophosphamide ) and analogues of purme {6-
mcnzaptopurinc, oxathioprine), cytosine {cytosine
arabi noside ) and uracil ( 5-fluorouracil ) . Many
am ! metabolites find clinical application in the
preveniiun of graft rejection.
The drug most widely used now for
Response 141
immunosuppression is cyclosporine , a cyclic
polypeptide derived from the soil fungus TbJppo-
eJjtiinm jj-ttfarum. It la not cytotoxic for lymphocyte
*
and has no antimitotic activity. It selectively inhibits
helper T cell activity. A related drug is rapamycin .
Antilymphocyte scrum ( ALb) is a heterologous
antiserum raised against lymphocytes. Arnihodv
prepared againsr thymus CHL 11S is called
intithymocyte serum ( ATS) . The corresponding
globulin preparations ,irc called A LG and ATG.
Tfeey Were used to prevent graft rejection. While
all other immunosuppressive agents have
unde? i rahle side effects, ALS is devoid of a ny action
other than that on lymphocytes. ALS acts primarily
against T lymphocytes and therefore specifically
on cell mediated immunity , Humoral antibody
response to thymus independent antigens is
unaffected and may even be enhanced. ALS acts
only against Ivmphocvtes in circulation and not cells
in lymphoid Organs . As ALS is a foreign protein,
it ? effect is decreased on repeated administration,
which may also lead Co serum sickness ami other
hypersensitivity reactions. Monoclonal antibodies
against specific lymphocyte membrane antigens
have hern prepared.
Effect of antibody: The humoral immune
response to an antigen can be suppressed specifically
by passive administration of the homologous
antibody. The action appears TO be by i feedback
INEEHLL [] ism . The primary response is mure
Susceptible to inhibition than the secondary
response. The antibody may also combine with the
antigen and prevent its availability for the
immunocompetent cells "["he Inhibitory effect of a
,
passively administered antibody on the humoral
i mmu n c response has been applied i n the prevention
of Kh sensitisation in Rh negative women carrying
Rb positive fetuses . This L S achieved by the
administration of anti - Kh globulin immedinrclv
following delivery ( within 72 hours).
This effect is also relevant in the practice of
combined immunisation as in diphtheria and
tetanus. In such eu«, the toxoid and Antitoxin
Copyrighted material
142 * Texl & ook
should he giveo at separate sites, Adsorbed toxoid
'. bould be used as the inhibitory effect is much Less
r
than with fluid toxoid -
Inmu - m / js adm nihtiatnm cC immune gkAulin I:. L -
been shown to haw: immtmninrsdulatmy effete It has
been used in the treatment of many diseases of presumed
^
TiiriuiHjpadi£ilpg etioli /jji such as thiwnbocyKjpeni- is
i[
and smtranunune honolytie anemiaa.
S i J F R R A N T1G B N S
Superarrigcns ate certain protein molecules, such
as staphylococcal enterotoxins that activate very
large numbers of T cells irrespective of tlieir
ailtigcnic specificities. While conventional antigen
fragments bind to the a (3 hetemdlmer groove of
the MHL’ molecules through the V regions ol
TCR a and [1 chains, ? uperantigens bind directly
to the lateral aspect of the TCR |1 chain. Updo 50
per cent of the circulating T cells may be so
activated , compared to conventional antigenic
stimuli which involve only about 0 -001 per cent of
them. This exaggerated T cell activation leads to
massive outpouring of T cell cytokines , causing
multisystem dysfunctions, such as seen lit
staphylococcal toxic shock syndrome.
MITOGENS
Mitogens are certain > ubscance & chat induce division
of lymphocytes and other cells. Some of these, like
the lectin glycoproteins hind to sugars DO the surface
of responsive cells and activate them, causing a
polyclonal reaction. At low concentrations , they
stimulate B cells without polyclonal activation .
Lipopolysaccharide is such a B cell mitogen.
Some Large molecules with repeating epitopes,
such as pneumococcal polysaccha ride directly i nteract
with B cell surface imrmmogjobLilins, leading to an
TgM immune response. Such T- i ndependent immune
response with IgTvI antibody; is not associated cither
with Ig class switching or memory.
CELLULAR IMMUNE fl ESPONSE
The term 'tell mediated immunity (CM1) refers
1
to the specific immure responses that do not
o Microbiology r
'
involve antibodies - for long the only demonstrable
facet of CM I was the phenomenon of delayed
hypersensitivity ( DH ) which resulted in injury
-
lather titan protection The first description of a
CM! response was the observation by Jennet
( ] ?V8) that inoculation of vaccinia virus in an
immune individual led to a local erythematous
-
papule in 24^75 hours 1 le called this the 'reaction
of immunity ' - Koch US 90 ) described the
exaggerated cutaneous reaction of tuberculous
guinea pigs to the intradcimal injection of tubercle
bacillus or a protein extract of the bacillus
( tuberculin ). Thereafter, the tuberculin test became
the paradigm for [JH . The term 'delayed
hypersensitivity refers to the appearance of a skin
1
lesion 48-75 hours after administration of the
antigen . The lesion is an indurated nodule with
infiltration hy mononuclear cells. DH was found
to be immunologically specific but IT did not have
any relation to antibodies and could not be
r
Transferred passively by serum. The cellular basis
ofDH was shown by Landsteiner and Chase (1942)
bv its passive transfer in guinea pigs through the
injection of leucocytes ftom sensitised donors, With
the recognition of the two component concept of
immunity, DH and other types ofCMI were found
to be medi .ited by T lymphocytes, A variety of
techniques are now available for the detection of
CMI, though they lack the sensitivity and precision
i h antibody assays fbr humoral inmmnil y.
SCOPE OF ( 1 M 1
CMT participates in the following immunological
functions:
1. Delayed hypersensitivity.
2. Immunity in infectious diseases caused by
obligate and facultative intracellular para -.ites -
These include infections with bacteria ( for
example , tuberculosis, leprosy, listeriosis,
brucellosis), fungi ( for example, histoplasmosis,
coccidioidomycosis, blastomycosis ) , protozoa
( for example , leishmaniasis, trypanosomiasis)
and viruses (for example, measles, mumps).
xJ opyrighted materia
[
 * hvnu'ie
3. Transplantation immunity and graft -versus-host
reaction .
.
4 Immunological surveillance and immunity
against cancer.
.
5 PathogeneMS ofeercu n aucn i immune diseases (lor
example , thymiiiiti -;, encephalonryelifiis) .
INDUCTION DI GM1
The nature of the anri genic stimulus is important
m the induction of CMI, It is best developed
following infectious with intracellular parasites.
Killed ViCC i nes and Other noi Jivi ug antigens do not
induce CMI unless administered with the Freund
type of adjuvants. Only T cell dependent antijfent
lead to CMI. The application of certain cherriiL als'
on the skin induces DH.
Each T cell bears on its surface a specific
receptor (TCfe) (br one epitope and combines only
with antigens carding that epitope . On contact
with the appropriate antigen, T cells undergo blast
transformation, clonal proliferation and
differentiation into memory cells and effector cells
providing CMI - T cells recognise antigens only
when presented with MHC molecules . Helper T
cells react with antigens presented on the surface
of macrophages or other cells cumplexed with
, been grouped under the term cytokines.
MHC class II molecules. They then release
biological mediators (lymphokines ) which activate
macrophages, enabling them to kill intracellular
parasites. Cytotoxic T cells recognise antigen on
the surface of cells (such as virus; infected, turn urn:
or allograft cells), in association with MCH class
1 molecules, secrete lymphokines and destroy the
target cells.
C VTOJilNKS
Biologically active substances released by activated
T lymphocytes were called lymphokines. Similar
substances produced by monocytes or macrophages
were called monokines. Initially they were given
names based on rhe i r demonstrated biologi cal effects
(Table 16,1 ) . As most lyirphoki ncs exhibit multiple
biological effects and rhe same effect may be caused
R HSPO - i HE? * 143
Table 16-1 Examples of lympluldnM
I. Affecting macrophages
a . Migration inhibiting facmr ( MIF )
b. Macrophage activation/ aggregation factor
c.
( MAFJ
Macrophage chemotaeric factor ( MCF)
II. Affecting lymphocytes
I- Blastogenic/ micogenic factor ( BbYMF )
b T cell growth factor ( TGE )
u B teQ growth factor ( BGF )
III. Affecting granulocytes
,L Chemotactic factor {CF}
b. Colony stimulating factor ( CSF)
IV. Affecting cultured cell*
. i Lymphotoxiii {LT )
b Interferon (1FN )
C Tumour necrosis factor ( TNF )
V. Others
a . Skin reactive factor (SRF)
b . Transfer factor fi t')
by different lymphakLues, their names lack
prccisiDr. The term interleukin was therefore
introduced for those products of leucocytes which
exert 9. regulatory influence on other cells .
Interferon ^, growth factors and others were found
to have similar effects. Therefore all of them have
Cytokines are peptide mediators or intercellular
messengers which regukre immunological, inflam-
matory and reparative host responses. They are highly
potent hormone like substance active even at
^
femtomolar (10 ]*M ) coiicencniL^ns. They differ from
"
endocrine hormones in being produced not by
specialised glands but by widely distributed cells (such
as lymphocytes, macrophages, platelets and fibrobtets),
and acting not systemically but locally near the producing
cells (paracrine effect ) or directly on the produdi cells
^
themsdves (autocrine effect]. They arc, in general,
pleintmpic, hati ng multiple effects on the growth and
differentia* ion of various cell types . There is
considerable overlap in the effects produced by
different cytokines . Cloning of cytokines and the
availability of monoclonal antibodies against them
have helped to characterise them better {Tabic 16.2),
Copyrighted materia
144 * Textbook of Microtuologv

Table 16.2: C okmct

^
C
^ fptj' nf

A. INTERLEUKINS:
Mufl WWro« Major function*

1L- I ( H and p.) Macrophages and other cell types f Liferation and differentiation of T, B and other
^
Ctl ]a; pyrwprnio; induce acute phase jwntdflp; bone

IL 2
- T *
cell
-
mArrow < cU prolifentiun ,
PmiAOU i ywdi ; iid tiiffHHitiirimi ofT acu. l B
. ^
cells Cytotodrity ot T and N 6L. cells, secrethm of
other lymphokines .
1L-3 T cells Multi CSF
IJ . -4 T„« U* FroLiter at ion of F and MUDDCT cells: increase
IgCI and igE production; enhance MHC class II
SiElrd IgE rcMptOtfS.
1 L- S TM celt* Proliferation of eosinophils, stimulate IgA and IgM
,

production.
11 . fn TH, macrophages, fibroblasts Promote B cell difftraitkboa; IgG pnxiuctiun ,
acute phase proteins.
1L-7 Spleen , bone marrow stroma] cells B andT cell growth factor.
iL - e
IL 9 -
.
IL - 10
Macrophages, others
Ted )
T. n odls, macrophages
Neutrophil chemoractic factor.
T cell growth and! proliferation
Isiliibrt IFN production and mononuclear cdl functions.
iL n
-
IL 12
- Bone marrow ttroinil cells
T cells
Induce acute phase proteins,
Activate NK cdk
IL 13 T cells Inhibit mononuclear cdl fiinctikH]*.
B. COLONY ST1MU LATIN Cl FACTORS:

--
GM C5F T cells, macrophages, fibroblasts T cell and macrophage growth stimulation
CSF Fibroblasts, endothelium Granulocyte growth stimulation .
M CSF- Fibroblasts, endothelium
C . TUMOUR NECROSIS FACTORS:
Macrophage growrh stimulation.

TNF- K Microphlgct, monocytes Tumour cytotoxicity, lipolysis, wasting, acute phase

proteins,. phagocytic cell activation, antiviral and
antiparasitic effects, endntoodc shock.
TNF- b T cells IFUIUCV Other cytokines.
D INTERFERONS:
IFN * Leucocytes
IFNb Fibroblasts Antiviral activity
[FNS T cells Antiviral, macrophage activation.! MHC Class I and.
11 -expression on cells.
K OTHERS:
TCF b T and B cell* Inhibit T and! B cell proliferation and hemaroposcEU ;
promote wound healing;.
UF T cdla Ptaiftnlkfll of stem cells; eosinophil chemotaxis.
The features nt some important c \ rkJn -us are cytokine was renamed interleukin-1 (IL!) in 1979..
presented below: ILl is a stable polypeptide retaining its activity up
-
Interleukin 1: Originally detcribed an the
leueotvte activating factor ( LAF) in 1972 and an
—
to 56 :C and! between pH .1 11.1L.1 occurs in two
molecular fbrmst ILl alpha and beta ILl is -
rhc 15 cell tKtilting factor { BAk ) in 1974, this principally secreted by macrophages and monocytes

Copyrighted materia
 < Immune Response >
but cin be produced by most ocher nucleated trc Lls
also. Trs production if - NTimulATedby antigen*;,, toxins,
injury and infkmmaioty processes and LEI} hi ted by
cyclosporin iY, coitimwidt and prostaglandins,
Hit immunological effects or IL 1 include
stimulation of T cells for the production of 11.2
and other lympliokj nes, U cell proliferation and
,Lilt ] body synthesis , neutrophil rhcijiotaxb and
phagocytosis. h mediates a wide range of metabolic,
physiological, inflammatory and hematological
effects. I : v nclirlg on bone marrow, epithelial and
synovial cells, fibroblasts, osteoclasts, hepatocyica,
vascular trulothelium ami other targets li . l is an
, has a Stimulator,’ effect on lueputocytes, nerve cells
important endogenous pyrogen . Together with the
tumour necrosis factor (TNF), it is responsible for
many of the liematologicul changes in septic shodk
ami also enhances the initial meningeal
inflammation in bacterial meningitis. Cytokine
inhibitors such an Jesimcrbasone have been found
In protect against the sequelae of such CWUSMVL -
meningeal inflammation . On the Other hand, ILl
Has a beneficial effect in severe infections in
immunocompromised hosts.
-
Interleukin 2: The discovery in 1976 of a T
cell growth factor (TCG F) produced by Activated
T cells., which induced 1’ cell proliferation and
enabled their maintenance in continuous culture,
contributed greatly to the understanding t>fT Cell
functions. This cytokine, renamed 1L2, is a powerful
muduLlor of the immune response. It LF the major
activator of T and R cdls und stimulates cytotnic
T cells and NK cells. It converts some null cells
(LGL ) intotympbakioe activated killer ( J .AK ) cells
which can destroy NK rcsLstanr tumour Octli- TIrfo
property has been used in the treatment of certain
types of cancer.
lnlerkuikiti-1 1L3 is a growth factor for bone
marrow stem cells . It stimulates multilincagc
hematopoiesis and is therefore known also as the
multicolony stimulating factor (multi- C 5 F ).
Interleukin- 4: Formerly known as the 11 cell
growth factor -1 (BCGF-1), IL4 activates resting
B cells and acts as a H cell differentiating factor. Ir
,
145
-
also acts as a growth factor for ! cells and mast
cells. It enhances the Action of cytotoxic T eelU. It
may have a role in atopic hypersensitivity as it
augments IgE synthesis.
[ merle Likin -5: Formerly known as the H cell
-
E
growth factor II , 11 0 causes proliferation of
activated B cells . Ir also induces maturation of
eosinophils.
-
Inlcrlcukin ti; 1L6 is produced by stimulated
T and H cells , macrophages and fibroblasts . It
induces immunoglobulin synthesis by activated 15
cells and formation of 1L 2 receptors on T cells. It
and hematopoietic cells . It acts as an .inflammatory
response mediator in host det enre aguinst infections.
'
Colony stimulating factors (GSFk These
cytokines stimulate the growth and differentiation
f pluripotent item cells in the bone marrow. They
have been named after tike types of ceil colonies
-
they induce in soft agar culture for example ,
granulocyte (G ) t nr mononuclear (M) C5F ] L3
which induces growth of all types of hematfjpoictic
cells is known as multi -CSF. In the bntlv they cause
nther effects also, presum .ibly by inducing cascades
of other cytokines. They arc responsible for adjusting
the rate of production ot blood cells according to
requirements, for example , the massive granulocyte
response seen in pyogenic infections . Colony
stimulating factors have clinical applications for
treating hematopoietic dysfunctions in infections
and malignancies.
Tumnur necrosis factors (TNF ): The
rumour necrosis factor occurs as two types, alpha and
beta . A scrum factor found to induce hemorrhagic
necrosis in certain rumours Wt& mured the tumour
neciOtiLii factor. Tlte tame substance was independently
_
described as ( itchcctin , a serum factor causing the
wasting syndrome (cachexia ) during chronic
infections.This has been reranvedTNFtx . It is formed
principally hy activated macrophages; and
monocytes . It resemble ^ 11.1 in possessing a very
wide spectrum of biological activities* such as
participation in the manifestations of endotoxic
Copyrighted material
146 ^ Textbook pf
Other cytokines , TNFP+ formerly known JX
iymphotcndrL, is produced principally by T helper
cells . Its effects are similar to chose of TNFa.
Interferons UFN ); Originally identified JH
antiviral agents (see chapter 49), interferons are now
clarified as cytdkinCE. There are three etsisHCi. of
IFNs, alpha produced by leucocyte*, beta produced
by fibroblasts and gamma by T tells activated by
antigens, mitogens or expusoire Co IL2. IFNy causes
many immunological effects, such as macrophage
activation , augmentation of neutmphi and
monocyte functions, and antitumour activity.
Othflr cyttikintHT The transforming growth
factor beta (TGFp) was RO named because of its
ability to transform flbroblasts. He sides acting as a
growth factor for fibroblasts and promoting wound
lieal ing* it also acts as a down regulator of some
immunological and hematological processes .
ITic leukemia inhibitors factor ( LI F), produced
by T cells, helps stem cell proliferation and
eosinophil chemotnxis-
Cytokine production is regulated by exogenous
stimuli such us antigens and mitogens, as well as
by endogenous factors such as neuroendocrine
hormonal peptides (corticosteroids, endorphins )
ami piKjducts of lipoxygenase and cyclooxygenase
pathway! .They i&o regulate each other by positive
Fig. 16.5 Inhibition sT migration of macrophage cells lift Exposure to antigen or cells Irom Rensilfc &ed guinea
,
pigs Migration inhibited Right Control Exposure tt mtlgs r of cells iron- normal guinea pigs Show i
. . .
foam — like migration of macrophage*.
Microbiology
and negative feedbacks. A number of cytokines (for
example, ILl , 2 , 3, colony stimulating factors*
interferons) have already found therapeutic
application, With better understanding of their
properties, it is possible that many cytokines, their
igofiuits and antagonists could eventually be used
in the m an age men r of inflammatory, infectious ,
autoimmune and neoplastic condition.
DETECTION OF CM I
The original method fur delecting CM1 Wfll the
5 kin rest delayed hypersensitivity ( for example,
for
the tuberculin test ). A number of In vitro correlates
ofCMI have now become available . These include
tlie lymphocyte transformation test ( transformation
ot cultured sensifiwd T lymphocytes on contact
with the antigen ), target cell destruction ( killing
trf cultured alls by T lymphocytes sensitised against
them ), and the migration inhibiting factor test
which is commonly cm ployed. As uriginally
d t ihed, r i . i . iiisi 'icki oJ incubating in .i culture
chambrt, packed peritoneal macrophages in a
cap ! Uy tube. Hue maUuphageB migrate 1 form a
-
bey, fun like pattern. If the macrophages are from
iguinea pig en ti cd to rubcrouloprotrin, addition
[ | tubcr-culin in
j ;
culture handle: will inhibit
the migration ( Fig 1 m5) , Hi is h . i - been adapted
? materia
 4 Immune Response
lor clinical use by IIIV. JI v. l i:ig human peri ] rtic- ral
leucocytes in cap:. Hjiry tune - in culture chambers.
When an antigen to which the individual has CMI
is introduced into the culture medium, the leucocytes
am prevented from migrating , By comparison with
the control , it is possible to make a semiijiianritHtiw?
assessment of the migration inhibition.
TEIANS^ BR FACTOR
Passi ve transfer of CMI was first achieved by the
injection of viable leucocytes from sensitised donors.
T -awrcncc ( 1954) reported transferof CMJ in human
beings by the injection of extracts from leucocytes.
Thn extract is known as the 'transfer facror' (TF).
The transferred immunity is specific in that CMI
can be transferred only to those antigens to which
the donor is sensitive.
TF is a dialysable , low molecular weight
substance ( MW 2000 to 4000), repi -itant to trypsin,
DNAase , RNAase and freeze thawing. Tt LH stable
for several years at — 20 C and in the lyophilised
form at 4 ^C . It is i nacf i rated at 56 5C in 30 mi mites.
It is not anCii cniL . Chemically* it appears to be a
-^
polypeptide polynucleotide .
TF is highly potent , an extract from 0.1 ml of
packed leucocytes lx Ing sufficient for transfer. The
transferred CMI is SYSTEMLL: and. not local at the
:
iu ecte (i site alone . FollowingTF injection , DH and
various in vitro correlates of CMI can be
demonstrated in the recipient . Hu morn I immunity
is not transmitted by rF; TF transfers CMI to all
the antiL enn to whi .: b the donor is sensitive, en
^
htne . It is possible to transfer CMJ from the
recipient to another in a serial fashion.
The mechan i sm of action of the TF i s not known.
TF could be an informational molecule or a specific
gene dcrepressor capable of inducing antigenically
uncommitted lymphocytes to produce artigen -
specitic receptors . TF activity was till recently
demonstrable only I n human beings but it has now
been reported in monkeys , guinea pigs and mice.
TF has several applications. It has been used to
restore immune capacity in patients with T cell
147
deficiency ( Wiskott-Aldrich syndrome). It has also
been used in the treatment of disseminated infections
associated with deficient CMI {lepromatous leprosy,
tuberculosis, imicocutaiieoLis candidiasis). IT has been
employed 111 the treatment of malignant melanoma
and may be be nefivial i n other types of cancer as well.
Its use has been suggested in some autoimmune
diseases ( systemic lupus erythematosus, rheumatoid
arthritis ) and diseases of unknown etiology ( sarcoidosis,
multiple sderusis).
IMMUM0 LOGICAL TOLER AM CE
Immunological tolerance or immunological
unresponsiveness is tlie condition in which contact
with an antigen specifically abolishes the capacity to
mount ar. immune response agionat that partkiilar
artCij rt when it ns administered subsequently- Hns
^
nonreactiVLty is specific to the particular antigen,
immune reactivity to other an - igens being unaffected .
The first example of immunological tolerance
was the nhservatinn by Owen ( 1945 ) of erythrocyte
chimcrism in dizygotic cattle twins, each of the
twins having erythrocytes of its own and the other’s
blood groups. As dizygotic twins are genetically
dissimilar, they do not ordinarily accept transplants
from each other but such transplants survive in
cattle twins. The reason for this tolerance was shown
to be the sharing of the same placental blood supply
by the twins during intrauterine life . Based on this
observation, Burnet and Fenner (1949) suggested
that the unresponsiveiLess of individuals to self
antigens was due to the contact of the i rpmature
immunological system with self antigens during
embryonic life. Any anriLcn that comes . nro contact
wi:h the immunological system during embryonic
life would be recognised as a self antigen and would
not induce any immune response. They postulated
that tolerance could he induced against foreign
ant i .L ens i f they were adm ini *tcred during embtyon ic
^
life. This was proved experimentally by Medawar
and his colleagues ( 1953) using two strains of
syngeneic mice . When a skin graft from one inbred
strain of mice ( CBA ) is applied on a mouse of
Copyrighted material
148 A Textbook of Microbiology
another strain (A ), it is rejected. If GHA cells arc
injected into frtll nr newborn Strain A mite ,
however, the lamer when they grow up will freely
accept skir- grafts from CRA mice. The content of
the self antigen appears to have been enlarged hy
contact with ti foreign antigen during embryonic
life. This phenomenon is called 'specific
immunological tolerance'.
Development of tolerance is not confined to rhe
embiyo or newborn but can occur in adults also.
Tolerance may he total or partial* short-lived of
long -lasting. The induction, degree and duration
of tolerance depend on cite species and
imlEUnocoinpctence of the host , nature and dose
of the antigen and the route of administration .
Rabbits and mire cun be rendered tolerant mure
rapidly than guinea pigs and chickens . Strain
differences in tolerance induction arc seen within
species . The higher the degree of immunO -
yompctcncc of the host, the more difficult it is to
induce tolerance. It is fm this reason that embryos
and newborns arc particularly susceptible to
induction of tolerance . Tolerance can he induced
in adults in whom immunncompplence is
temporarily interrupted by immunosuppressive
agents. Induction of tolerance is very difficult in
adults already immunised Against the antigen.
The physical ‘rate of the antigen is important.
Soluble antigens and haptens are more tolerogenic
than particulate antigens. The tolerngen reify of an
antigen can be modified by certain procedures.
When liun i ; m g - i i nmiglohaiLirt is heit aggregated,
iris highly immunogenic in mice hut is tolerogenic
when deaggregated. Solutions of scrum proteins
-
centrifuged at high s}M. cd separate into tolerogenic
supernatant and immunogenic sediment fractions
the induction of tolerance is dose dependent There
is JI threshold dose, below which tolerance is not
induced. Further increase in dose increases the
duration of tolerance. With certain antigen*,
tolerance can be induced by two types > f doses, one
high and the other low, with intermediate doses
producing immunity instead of tolerance. These arc
known ns 'high none’ and low mne tolerance
1
lesfJCLtively. A special type of high lOtle tolerance
is Felton's immunological paralysis. The duration
of tolerance iti variable.Tolerance can be prolonged
k... J ..
1 L: TL _e
administration th .tr best induces tolerance is that
whereby rhe antigen equilibrate* throughout the
extra - and intravascular compartments. With
antigen* tha t do lint LCJU i I ihra I e readi ly or ire rapidly
eliminated , the route of choice is intravenous.
Certain haptens that are immunogenic in guinea
pigs hy the intraderm& l route are tolerogenic orally "
or intravenously.
Tolerance ear he overcome spontaneously or hy
Ati injection of cross-reacting immunogens. For
example, U iterance ro bovine serum album in in
rabbit* can be abolished by immunisation with
cross reacting human scrum albumin . In general,
tolerance to living agents is more lasting than that
to nonliving substances. Naturally occurring
tolerance is found in certain viral infections such
as congenital rubella and cytocncgilovinii infections
m which there is persistent viremjl with adeeieased
ability lor the production ot neutralising antibodies
( persistent tolerant infection ). In lymphocytic
choriomeningitis infection in carrier nice, the vims
may persist in virtually all the cells and tissues and
be transmitted vertically to the offspring without
any demonstrable immune response or pathogenic
effect. When the tolerance is interrupted hy an
induction of antibody or an injection of sensitised
lymphocytes, disease results . The mechanism of
tolerance is not dear, Jr specific immunological
tolerance in embryonic life , the clones of cells
,
responding to tile particular antigen were believed
to he anniliiluted hy contact with the antigen . This
may not be entirely tong as self - reactive R cells can
he found in adults . The more likely mechanism may-
be elimination ofTlf cells, effectively preventing
b cell activation , This j* the 'central mechanism’ of
tolerance induction . In Other instances , the
mechanism may be an 'afferent block’ in which
access of the antigen to immunocompetent cell* is
Copyrighted materia
 Irnmuns Response
*
interfered with or an 'efferent block ' in which the
*
antibody Evnthesihcd LR Hdltn£jxd or destroyed. T
and B lymphocytes appear to possess differing
sensitivity to tolerance induction, the former being
more susceptible. In general, high doses of antigen
induce H cell tolerance and negated minute doses
of antigen induce T cell tolerance.
Tolerance to humoral and cellular types of
immunity LHusually induceif simultaneously. S-plit '
tolerance can also occur where unrespon&ivenesa
is established for one paiumeter' of the immune
, explain'd .i JugfulLv the specificity of the antibody
response and not to the other. In guinea pigs, i)H
tu tuberculin can. be inhibited, without affecting
the production of a circulating antibody, by the
injection oftuberculoprotcin prior to immunisation
with BCG.
THEORIES OF IMMUNE RESPONSE
A succession of theories h vc been put forward
*
from time to time in order to explain the versatility,
specificity, memory and other features oi the
immune response. Theories of immunity' fall into
two categories : instructive and selective ['lie
,
'
instructive theories p o s t u l a t e that a n
immunocompetent cell is capable of SytttilCtibng
antibodies of any peeifi city. Tlae antigen encounters
*
an immunocompetent cell and instruct? lr to produce
the complementary antibody. Instructive theories
were proposed by chemists who were more
concerned with explaining the physicochemical
aspects of specificity than with the biological
principles of immune processus . Stltttivt theories ,
on the contrary,shift the emphasis from the antigen
to the immune competent cell. 'Hicy postulate that
immunocompetent cells have only a rust rifted
immunological range. The antigen exerts only a
selective influence by srtrrmUring the appropriate
-
immuno competent «11 to synthesise JIL antibody,
Side chain theory: The first plausible theory
of immune response was the 'side chain' theory
proposed by Ehrlich (1900) , Cells were considered
to have surface 'receptors' capable of reacting with
substances having complementary 'side chains’. The
L 49
physiological significance of such receptors was in
anchoring nutrients to cells before assimilation.
When foreign anrigens arc introduced info the body,
they combine with those cell receptors which have
a complementary fit. This inactivates the receptors
and interferes with the absorption of nutrients, As
a compensatory mecbariitm, there Is an
overproduction of the same type of receptors, which
spill over into the blood and circulate as antibodies.
This was the first of the selection theories , ii
response. However, when 3 ,anJ.slcincrdemonstrated
that aitihiJies could be formed not only against
natural antigens but also against various synthetic
chemicals, this theory was abandoned, Ii was
believed that an Impossibly large number of
receptors would be needed to account for the
seemingly endless scope of antibody specificity. It
is, however , remarkable bpw closely Ehrlich
anticipated modern views on the immune response .
Direct template ihenries: Instructive
theories were proposed by BrcinJ and I laurowita
(1930), Alexander (1931) and Mudd ( 1932),
According td these, the antigen (or the antigenic
determinant! enters antibody to ruling cells and
serves as a 'template ' against which antibody
molecules arc synthesised so that they have
combining sites complementary to the antigenic
determinant. 1’hese are lliuretore known as 'direct
template' th iriw, J’sud i ng ( 1940) presented a more
detailed model suggesting that specificity was
determined by the folding of ihe antibody
jicrlypcptrilc chains to form a tertiary structure fitting
the antigenic determinant.
Indirect template theory, Burnet and
Tenner ( 1949) proposed this instructive theory " to
explain rhe synthesis of antibody as an adaptive
.
protein They postulated that the entry of the
antigenic determinant into the antibody producing
cell induced in it i heritable change . A 'genocopy7
of the antigenic determinant was thus incorporated
in Lrs genome and transmitted to the progeny cells
fiudinecr template) . This theory explained specifidry
Copyrighted material
150 i Texlbqgk ol MipmbioJQgy *

find fhc -iccnndary response l^oit became untenable ( Ihm . i

selection Burnet { 1917 }
with advances in the molecular binlngy ot protein proposed this theory which liiftcd imm urological
*ynthesis. Burnet and Fenner were; the first to explain specificity the cellular level .
to
the iKma -nNgenictry of self anrigeiu by postulating the According Eo the clonal i: hypothesis,
embryonic recognition of ' self mailers'. during immunological L udupn ent cells capable
Natural selection theory: roc { 1955 } J
^
reintroduced the concept of the selc-erivt: fimetion
qf antigens in his natural selection theory. This
of reacting with different at gen were formed by
a process of somatic mutation. Cl<
hid imrtiunofogitil reaeliviry ll self antigens
of cells that

pustulated that about £ million globulin (antibodv) Were eliminated during : niu l Such clones
molecules were fonned in embryonic life , which are called forbidden dcutcj. persistence or
covered: the full range of antigenic specificities . development in later ifchi mutation could
These globulins were the 'ualura] antibodies". When lead to autoimmune processes Each
ar. antigen was introduced, il combined selectively iniTnuiuHiompEtenC cell was capable of reacting with
with the globulin that bud the nearest tme antigen (ora small numlier antigens} which
complementary ' fit ' . The globulin , with the could recognise and > with antigens
combined antigen, homed in on the antibody introduced into [he body. The : - H : of the contact
'

forming cells 3 rul Stimulated rhem to synthesise the with the specific antigen was ; i r proliferation
'
same kind of antibody. Here; selection was to form clones synthesising the tntibods
postulated at the level of the antibodv molecule, It Tbe clonal Selection li is more widely
did not explain the fact that immunological memory accepted than the other theories, t ! n i - unable
resides in the cells, and not in serum. to account for all the features of the immune i&pi nue.

CONSTANT
* VARIABLE HEOlOH REGION
T
Genmhrce
tjflriaa

VM 1 1 DO Ol - J J l -6 C it TN yj if , . . 7*
.
f a,, ns
+ TRANSLOCATION /

^
B cell genes V5 D3 Jt 1

Fig. 1 S.6 ' llustration of gone shuffling to form a 5 cell encoding for lyGM heavy chain with Dy , J variable
region sequence (lidlRtr shuffling also occurs for light chains — nol shown)

iopy righted material

 * Ifnrrure Response 151

jl -
A vari ntv of modificationsand altcrmri v? theoti L S have
been proposed in recent timesblit rvnnr has succeeded
M L explaining all that is known of immunity.
As an explanation for the mechanism of
regulation of antibody response, Jeme has pi ^ tulatcd
the network hypothesis. The variable region of an
immunoglobulin : molecule currying the .ouL'cn -
combining site Is tliflfrrenl in tliflfrrenl antibudicH.
''
J hc distinct ami noacid sequences at the antigen
combining site and the adjacent parts of the variable
region are termed idiorypes. The idiotype can , in
turn , act as an antigenic determinant and induce
antiidiotypic antibodies, These in turn can induce
antibodies to them and so on, forming an idiotype
network which 1s postulated to regulate the amount
of am i bodies produced and the number oLuitih ' idv -
forming cells in action . For hi * theoretical
contribution to antibody formation and regulation
of the immune system, Niels It Jeme was awarded
the Nobel Prize- for Medicine in 19S4.
of the aJidhtidv molecule. As the constant regions
.Lre identical for immunoglobulins of anyone type,
there need be only one gene or a few genes for
each constant region, as ag .ii. nst a very large number
of genes for the variable regions. For example , the
kappa L chain genes are composed of three separate
segments; V, J and C . There are about a hundred
ditfefrnt types of V ( variable ) domain sequences
- and only one C (constant) segment, with some five
J ( joining) segments in-between . By combining
different V and J sequences with the C domain, it
is possible to provide for antibodies with at least
500 different specificities . By p a l i n d r o m i c
arrangement (sequences that can be attached by
cither end ), it is possible to generate many times
more different specificities. The lambda chain has
additional C sequences. The H chain gene has also
a C (diversity ) segment. By the shuffling of these
different segments of the C and EE chains, it is
possible to have antibodies with far mure than 10 '

The genetic basis of antibody diversity has been
clarified recently. An individual has the capacity to
produce an estimated 1(P different antibody
molecules. Io have each such antlhodv molecule
coded for by a separate gene would requi re millh ms
of genes to be set apart for antibody production
alone- This would obviously be impossible. The
phenomenon of splir genes explains this . The
genetic informal Lon for the synthesis of an
immunoglobulin molecule is not present in a
continuous array of codons. Instead, this information
occurs in several discontinuous stretches of DNA
('gene segments') , each coding for separate regions
types of specificity, a total repertoire that can react
with any conceivable antigen. The split gene
shuffling takes place during cell development and
a mature B cell DNA w i l l have Only one
combination of the different segments of the
unmunoglobLilin gene and cati therefore produce
only one type of antibody ( Fig. 16. &),
The discovery of split genes for
immunoglobulins demolished the long standing
dogma of one jztnc-onc protein and has impunant
implications in biology beyund immunology. For
this discovery, Susumu Toncgawia was awarded the
Nobel Pp7e for Medicine in 19fl 7.

V u r L h e r Remit inti
Enjjfclhjpd VH . 1994 , Hcwoclh proms antipenE. Scimt American 44,
Jancway CA 1993.1 low the immune system recognises invaders. Scieni American 41 .
.

Jeme NK. 1985. The generative grammar of the Immune aysrem- Sriena 29,1057.
^
Llewellyn MB et al. 1992. Monoclonal antibodies, BMJ 305: 1269, 1348, 1424.
Remkk DG arid JS Friodfcunri 1997 . OytAfnn JJI hcaith zred fibnur . 2nd cdn. Base ]; MttCdFDefcker
Tonegarwa S. 1983. Somatic generation of antibody diversity, Nnuir 302 = 575 .,
Weir PM find J S(cw?Lrt 1 W7 finUuMJiqgpr. 8 , b cdn . EdmbiUKh: Chnrrhill - Iiringihane.
S

Sdvwvfz RS 2003. Dimity of i ht mmiH New Eng J M±J 348:1017-

opy righted material

0 j w
 1^ Immunodeficiency Diseases
InimuntHlctuziencv. diseases are uundilmn -H wlicre
‘
JF
the iletencE mechanisms of the IMHIV aie impaired ,
leading to repeated microbial infections of varying
severity ;* Tui srjnuciriTncs enhanced susceptibility to
malignancies . Deficiencies of defence mechanisms
may involve Specific immune functions - humoral
immunity, cell mediated immunity or both - or
nonspecific mechanisms such as phagocytosis and
complement , which augment and act in conjunction
with specific immune processes . I mm u no
deficiencies may be classified as primary or
AtconcUry. ftinuij'inimiijBdeiiciendetnsuit from
abnormalities in the development of the immune
mechanisms . Secondary inununudchcicncics- are
consequences ( it disease , drugs , nutritional
inadequacies and other processes il . it interfere
with the pfOpd functioning dl the (nature immune
system,
PRIMARY IMMUNODEFICIENCIES
The established types of primary imitiuiudtfiden
syndromes are listed in Tabic 17.1 . Though primary ^
deficiencies of specific immunity can be
conveniently ciassifled as those a Meeting M cell
responses, T cell responses, of both , it must be
realised that there is considerable overlapping due
co the intimate interaction between the H cell and
cheT cell systems. For instance, ] ceil deficiencies
' '
involving helper or suppressor T cells will haw: a
profound effect on antibody response .
r
H L : M O K A I . IMMUNODEFICIENCIES
.
a
X linked agnmniJiglohiilinemia : fids
'
syndrome described by iStutor ( 1957 ) is the first
immumwleficicucy disease to have been reCOglUKtf
It is seen only In male in but H. Mini I ^stations are
not apparent till about six months of age due to the
passive protection afforded by maternal antibodies.
! ho disease present ? as recurrent serious infections
with pyogenic bacteria , particularly with
pneumococci , streptococci , meningococci ,
scLfJomojiit,? anJ / /. infhrcnzaf. Patients respond
^
normally to viral infcctioni such as measles and
chkkcnpm, though there have been reports of
paralytic poliomyelitis and progressive encephalitis
following immunisation with live vims victim# or
exposure to wild virus. As a general rule , live
microbial va.co.ncs should not 1re given To children
with any type of primary imrtnmcdrfdency.
AH classes of immunoglobulins are grossly
depleted in the serum, the IgG level being less- than
tenth , and IgA and IgM less than a hundredth of
the normalkvei . Tonsils and adenoids arc atrophic.
Lymph m >de biopsy reveals a depletion uf cells uf
the bursa-dependent areas . Plasma cells and
germinal centres are absent even after antigenic
stimulation . There is a marked decrease in the
proportion of B cells in circulation . Antibody
formation dot ^ not occur even after injections of
antigens.
Celt mediated immunity is rot affected ,. I Delayed
hypci sensitivity of tuberculin ai i J contact detnuiii is
types can be demonstrated . Allograft rejection is
normal. Arthritis, hemolytic anemia and atopic
manifestations are- frequently observed - However,
the wheal and - flare response of atopic
hypersensitivity cannot be dcinonsnared.
The incidence of this condition has been
Copyrighted materia
 * immunodeficiency Diseases * 153
Table 17 1 Classification of primary Immunodeficiency syndromes

A. Disorders ol sp« ific immunity

1 Humoral immunjodcfidnidn ( B cell deHtn:)
i , X - linkcd agimma lobuiincmia
^
b. Transient >iypojamm* kibulii>ciTiij of infancy
^
c . Comjricin variable immunodeficiency date onset hypogammaglobiilinjemiii )
JL Selective otimuno lobulin defii icndt? [IgA , IgM or lgC subdwws )
^
e , Immunodeficiencies with Kyper - lgM
£ Transect Akmin II deficiency
II. Cellular immunodefitienties (T cell defects)
a. Thymic hypoplasia (DipOCge! 1
iyadrame)
b. Chronic mucocutaneous candidiasis
Ci Purine nuclwsidc pho horyla* (PN1P) deficiency.
^ *
III . Combined immuoexidveiencies (B and T odl detects.)
a. Cellular immunodldMeftCV with. fltoOfffltl ammunogj]oboJkh wnfait
( Nntlof syndrome )
h.. Ataxia. tdajigiectsLi&ia.
—
c. W Lsfco tt Aid rich sjudioms
d. Immunodeficiency with. dqfDUffll
e. Immunodeficiency with short-limbed dwarfism.
£ Episodic lymphopenia with lymphocytotoxin .
$ Severe camhsncd ommunadcficicndes
1. Swiss type agammaglobulinemh
L

2. Reticular dysgenesis of de Va*l

3. Adenosine deaminase ( ADA) deficiency
B.. Disorders of complement
a. Complement component defidencies
b, Complement inhibitor ddidcntics
G« Disorders of phagocytosis
a . Chronic granulomatous disease
b- Myeloperoxidase deficiency
c. Chediak-Higashi syndrome
d . Leucixyte G6PD deficient
Jobs syndrom*
£ Tuftsin deficiency
g. Lazy leucocyte syndrome
h. Hyper- IgE syndrome
_ -
i Ac tin. binding protean deficiency
j . Shwachmans disease
reported as one in a hundred thousand population fractional catabolic rate of IgC in this condition
In the United Kingdom. Its manage mmc consists enables the maintenance of effective levels with this
of the maintenance of an adequate level of dosage. Commercial preparations of
immunoglobulins. This can be achieved with an gammaglobulm contain only Traces of IgA and IgM .
Initial administration! of 300 mg of gammagtobulirii To provide these, whole plasma infusions have been ,

per kg of hody weight in three doses followed by employed, the donors being tested for hepatitis and
monthly injections of 100 mg per kg. The slow other transmissible infections.

Copyrighted material
154 « Textbocm ot Microbiology *
TH r M \ I IH. .
AM.U AC I.OBI LINI : .MLA
Oh i \l ANCl
Tliis is due toan abnormal delay in the initiation
of IgG synthesis in Home infants . Maternal IgG is
SIQWIV catabnlised in the newborn and reaches ;L
level of 200 mg per 100 tnl by the Second month .
Ordinarily, the infant begins synthesismg its own
IgG by this age . When there is a delay,
immunodefkicnev occurs . Recurrent otitis media
Jr
and respiratory infections arc the. common diseases
found ill thin condition . Spontaneous recovery occurs
between 18 und 30 months of age. It maybe found
in iofaurs of both sexes . Treatment with
gammaglobulin mij be required in some case; but
it is not recommended [ imphylactically, as it may
contribute to prolongation ul immunodeficienCY bv
i negative feedback inhibition of IgG synthesis..
( lOIIHtiN V ART ADI V elevated. IgM . " Hu.- Ig.\ l molecules appear to have
IMMLNODGKICIENCY
This wmmvr form of inoutriodefidcnt}' is also
known as late onset hypogammaglobulinemia
because it usually manifests only by 15-35 years of '
age. It is characterised by recurrent pyogenic
infections and an increased incidence of
autoimmune disease . MflllbtOrpfiori and giardiasis
arc common . The tots I immunoglobulin level is
usually Less than 300 mg j'er 100 mb with IgG less
than 250 mg per 100 ml, U cdb may be present in
circulation in normal numbers , but they appear
defective in their inahiliry to dittenentiate into plasma
cells and secrete iinmunoglorutlinK . Increased
suppressor T cell ml diminished helper T cell activity
hsvc been proposed as a cause cif tKi -s disorder.
Treatment is by administration ot" gammaglobulin
preparations inlrajuLLScularlv or intravenously.
StiLliCTi v i; h i m JNot : i n u t it . tN
DrarrciRVLii's
In these conditions, there is selective deficiency of
one or more immunoglobulin cl asset, while ihe
others remain normal or elevated . These
dysgammaglobulirimias’ are common and haw
‘
been reported in about one per Cecil of all patients
with recurrent infections , Isolated IgA deficiency
is [ fie most common condition in this grotlp, with
a repaired incidence of ab< mt 0.2 percent in normal
populations. These pa dents exhibit increased
susceptibility to respiratory infection and
steatorrhea - IgA deficiency is often accompanied
1 > y atopic disordc rs. Anti - IgA antibodies arc present
in many of these patients .
Selective IgM deficit; my has been found ro be
associated with septicemia . Deficiencies of IgG
subclasses hive been observed in relation with
chronic progressive bronchiectasis ,
IramunodtiliciencieB with hypwp IjM: In -
this group of immunodefidcTtcics, some of which
arc X-linked and some inherited as autosomal
recessive, tow IgA and IgG levels are seen with
normal structure and possess antibody activity.
Patients show enhanced susceptibility to infections
and ILL toil untune processes such as
[ hrumbocytopeuix, neutropenia , hemolytic anemia
and renal lesions . Some patient ? develop malignant
infiltration with igM-producing cells. Elevated
IgM level with immunodeficiency is sometimes seen
LU congenital rubella.
TrqnsaohnluniLn II deficiency : In this
disorder, inherited as autosomal recessive, patients
show metabolic effects of vitamin B12 deficiency
including megaloblastic anemia and intestinal
villous atrophy. The associated immunological
defects arc depleted plasma cells , diminished
Immunoglobulin levels and impaired phagocytosis.
Treatment with vitamin R12 hm been reported lo
restore hematopoietic, gastrointestinal and B cell
functions but not phagocytic activity.
CKJ I . L I . All iMMUNOUEPICIEiNCIES
Thymic hypoplusia ( Digeorgc **
syndrom? }. This is i develop mental defect
involving the endtidermil derivatives of the third
and fourth pharyngeal pouches, It leads ro aplasia
Copyrighted material
 - Immunodeficiency
or hyjoiplasia of ilie thymus and parathyroidglands
It docs not appear to be hereditary and does not
show a TIMIII . LI jjyijflKt It in probably due to Home
intrauterine infection or other complications, Jr is
usually associated with Fallot's necrology and other
anomalies of the heart and the great vessels, and a
characteristic facial a ] i] >earj] icc . Neunatal tetany is
present. ftitients who survive the neonatal period show
.
enhanced susceptibility to viral fungal and bacterial
infections, which ultimately prove fatal.
The immunodeficiency primarily involves cell
mediated immunity. The thymus dependent areas
of lymph nodes and spleen are deplored of
lymphocytes. Circulating T cells are reduced in
number delayed hypersensitivity :md graft rejection
, are susceptible to recurrent fungal, bacterial , viral
.
are depressed The humoral immune mechanism
is largely unaffected. Antibody response ts> primary
antigenic stimuli is normal hut secondary response
to many antigens is impaired. Transplantation of
fetal thymus tissue has been reported l-o restore the
immunological function.
Chronic mucocutaneous candidiasis:
This constitutes an ahnurmal Lmmunological
response to Candida albicans. Patients develop severe
chronic candidiasis of the mucosa, skin and nails.
rfhcy do not show increased susceptibility' to other
infections but often have endoCrinupatlries. Cell
mediated immunity to Candida is deficient . In some
cases there is a total failure of T cell response to
any test antigen. Delayed hypersensitivity to Candida
antigens is absent hut circulating imLibudics to them
arc found in high litres, Intracellular killing of
Candida is defective. Transfer factor tbcrapv, along
with amphotericin B, has been reported effective.
Purine nuuduoside phusphnrylase
(PNP ) deficiency; The enzyme purine
nucleoside pho-sphoryliiHe is involved in the
sequential degradation of purines Co hypo.tonlbine
and finally to uric acid. Patients who have PNP
deficiency as on autosomal recessive inherited trait
show decreased cel ] mediated imum II I [ y and
recurrent or chronic infections. They usually
present with hypoplastic anemia and recurrent
.
*
pneumonia , diarrhea and candidiasis. A low serum
155
uric acid may point to the diagnosis .
<. ^< iM IUrS ED lM MUNODEPJCIE NCI FS
i T- iJu hi r irtrniuriodefiuiency with
abnormal immunoglobulin synthesis
{ Nezelof syndrome): The term Nezelof
syndrome has been rather loosely applied to a group
of disorders, probably of varied etiology, where
depressed cell mediated immunity' is associated with
•immunoglobulin
datively elevated, decreased normal levels of
of
. The consistent features are a
marked deficiency of T cell immunity and varying
degrees of deficiency of B cell immunitv. rodents
.
and protozoal diseases Abundant plasma cells are
seen in the spleen, lymph nodes, intestines and
elsewhere in the body, Thymic dysplasia occurs
with lymphoid depletion. Autoimmune processes
such is hemolytic anemia are common. In spite of
normal levels of immunoglobulins, antigenic stimuli
do not induce anlibudy formation.
Histocumparible home marrow transplantation,
transfer factor and thymus transplantation have been
used for treatment, with success in some cases.
Adequate antimicrobial therapy is essential.
Ataxia 11Inngiectasia: This is a hereditary
condition transmitted in the autosomal recessive
mnde , where combined immunodeficiency is
associated with cerebellar ataxia, telangiectasia,
ovarian dysgenesis andchromosomal abnormalities.
The earliest signs are ataxia and chorioathctold
movemean which are usually noticed in infancy.
Telangiectasia involving the conjunctiva, face and
other puns of the body usually appears at Five or
. -
six years of age Death occurs due to i uupu.t ] i ion nr v
infection uarlv in life, or malignancy in the second
o: third decade . The majority of patients lack serum
and secretory IgA and. some possess antihody to
IgA . IgE deficiency is also frequent. Cell mediated
immunity is also defective, resulting in an
impairment of delayed hypersensitivity and graft
rejection. The disease is progressive, with both
Copyrighted material
156 i TgxtbQOk L'l M Lrobiu C- qy
neurological defects ..Liu: immunodeficiency
becoming more severe with time.. Transfer farter
therapy and fetal thymus transplants have been tried
with some benefit.
Wiskutl-Aldrieh syndrome: This is an X -
linked disease characterised by eczema ,
thrombocytopenic purpura and recurrent infections.
Affected boys rarely survive the first decade of life ,
death being due to infection , hemorrhage or
lympboreticufar malignancy. Cell mediated
immunity undergoes- progressive deterioration
associated with cellular depletion oi the thymus
and the paracortkd areas of lymph nodes, berum
IgM level is low tun IgG and IgA levels are normal
-
or elevated Isuhcniagglutinins ate absent in the
serum. The humoral detect appears rn he a specific
inability tu respond to polysaccharide antigens. Bone
marrow transplantation and transfer factor therapy
have been found beneficial .
Immunodeficiency w i t h thymoma: This
syndrome, occurring usually In adults, consists of a
benign thymic tumour, impaired cell mediated
immunity and agammaglobulinemia. It is frequently
accompanied by aplastic anemia.This is of historical
importance as one of the experiments ot nature
which Suggested tlie immunological function of the
thymus .
Immunodeficiency with short ' limbed
dwarfism: The features of this condition arc a
distinctive form of short - limbed dwarfism ,
ectodermal dvsplasta , thvmic defects and enhanced
susceptibility to infection . These defects arc
appatendy inherited as autosomal recessivcs.
Epiioditi l y m p h o p e n i a with l y m p h o -
oytptoxin: In this syndrome there occurs ait
episodic but profound depression ofT cell (unction
by the action of a circulating cample ment dependent
Ivmphocy lotos in . The toxin appears to he an
antilymphocyte antibody . The p a t i e n t s lack
'
immunological memory so the secondary antibody
1
response is abolished . The disease is familial.
Severe c o m b i n e d immunodeficiencies:
T h e s e include many syndromes w i t h severe
deficiency of hoth
E
humoral and cell mediated
i m m u r e responses . They arc inherited in the
autosomal recessive mt >de and the primary defects
are at the l e v e l o f the early precursors of
immunocompetent cells in [ he fetal liver and bone
miirifw. Many distinct patterns of severe combined
immunodeficiency have been described.
X
In 1 5 H , Swiss workers reported
^
agammaglobulinemia with lymphocytopenia and
severe defect in cell mediated immunity This has
b e e r referred to as Swiss type aga /nma -
ghhulincmig . The basic defect is presumed to be
at the level of the lymphoid stem cell.
The most serious form nf combined
immunodeficiency is the reticular dysgenesis of de
Vagi , Here the defect is at the level of the
mnltipotenr hemopoietic stem cell, as a result of
which there is a total failure of myelopoic&LS leading
tolymphopenia, neutropenia, thrombocytopenia,
anemia and bone marrow aplasia. The condition is
invariably fatal in the first week of Life.
AcfencMEJte deaminase (ADA ) deficiency is the
first immunodeficiency disease associated with an
enzyme deficiency. ADA catalyses the conversion
of adenosine to irosine , an important step in the
purine metabolic pathway: How this deficiency
causes L:11 oui no logical impairment is not clear, I "he
range <> t immunodeficiency' varies from complete
absence to mild abnormalities of B and T cell
functions . The condition is associated w i t h
chondrocyte abnormalities which can be discerned
radio logically.
DISORDERS OF COMPLEMENT
Complement component deficiencies:
Genetic deficiencies have been detected for almost
all the complement components in human beings.
The defeers ate transmitted as autosomal recessive
Traits. Hemolytic and other functional activities are
completely restored by supplying the deficient
factor. Complement component deficiencies have
been frequently associated with systemic lupus
erythematosus . Recurrent pyogcnk infections WCft
CoDvriahted
rJ * material
 * immunodeficiency Diseases »
found associated with C3 deficiency and neisserial
in it.LL fi )]i H with deficiency of Cb, C ? und CS.
"
Complement inhibitor deficiencies:
I leredirary angioneurotic edema is due to a genetic
deficient of Cl inhibitor. This relatively common
^
defect is transmitted as an autosomal dominant.
Androgen H , aminocuproie acid and its analogue
IranejtamiL- acid haw been found useful in the
management of this condition. Plasma infusions,
once recommended for treatment , haw been given
up as they were found to worsen the condition in
some eases.
Tiie rare deficienci' of Cdb in activator has been
associated with chronic recurrent pyogenic lesions.
DlSQRDEflS OF PHAGOCYTOSIS
Phagocytosis may be impaired by either intrinsic or
extrinsic defects, Intrinsic disorders may be due to
defects within [he phagocytic Cell, such as enzyme
deficiencies. Extrinsic disorders may he due to a
deficiency' of opsonic antibody, complement or other
factors promoting phagocytosis, or to the effects of
drujp or antineutrophil autoantibodies. Phagocytic
dysfunction leads lo increased susceptibility to
infection, rjmgntg from ntild recurrent skin infections
to owrwhdming systemic infection.
Chronic granulomatous disease: 'Ibis
familial disease manifests itself as rccumcnt infection
with low grade pthegens, starring early in life, The
progress is chronic and the outcome fatal . Chronic
suppurative granulomatous lesions develop in the shin
itod lymph noties, along with hepahKplennmegaly,
progressive infiltration of Lungs and granulomatous
septic osteomyelitis. Humoral and cellular Immune
responses arc normal
The bacteria involved in the recurrent infections
arc catalase positive pyogenic pathogens such as
staphylococci and coliforms . Catalase negative
pathogens such as streptococci and pneumococci
are handled normally. Leucocytes from the patients
are unable to kill catalase positive bacteria following
phagocytosis.The bacteria multiply in the cells and,
being protected from antihcidies and antibiotics bv
157
their intracellular position , sec up chronic
suppurative infection - The: diminished bactericidal
capacity' of the phagocytic cells is associated with
a decrease of some metabolic processes like oxygen
consumption , hexose monophosphate pathway
activity and production of hvdrogen peroxide . The
diminished HjOa production appears to be the
major reason for the bactericidal defect . The
leucocytes do not undergo degnmulation following
phagocytosis . The delayed granule rupture and
defective release of myeloperoxidase also contribute
to inefficient bactericidal activity. TreuctMtytes from
the patients fail to reduce nitnablue tetrazolium
(NBT) during phagocytosis. Thia property has been
used as a screening method { NET test ) for the
diagnosis of chronic granulomatous diseasC-
Thc disease shows two types of inheritance-^
the more common X-linked type seen in boys and
the rare autosomal recessive lype seen in girls.
Myeloperoxidase deficiency : In this rare
disease, leucocytes have reduced myeloperoxidase .
Patients are particularly liable to Candida albicans
infectinn -
Chediak -Higaahi syndrome: This is a
genetic disorder characterised by decreased
pigmentation of the skin , eycn and hair,
photophobia, nystagmus and giant peroxidase
positive inclusions in the cytoplasm of leucocytes.
The inclusions may be the result of autophagocytic
activity. The leucocytes possess diminished
phagocytic activity. Patients suffer from frequent
and severe pyogenic infections,
I ciicocytu G6PO deficiency: Ju this rare
disease , leucocytes arc deficient in glucose 6
phosphate dehydrogenase and show diminished
bactericidal activity after phagocytosis . The
condition resembles chronic granulomatous disease
in reduced myeloperoxidase activity and .
susceptibility to micnjbi J agents, but the NBT test
ii Lav be normal.
f
Job s syndrome: This is characterised by
,
multiple large "cold' staphylococcal abscesses
containing large quantities of pus , occurring
1
Copyrighted material
158 * loxltxia ^ 01 \tiurOh uloQy *
various organs, vvirh
repeatedly on the: Kkin :md in
little inflammatory response. Atopic tacmj, *.
chronic nasal discharge and otitis media are
common features . The serum immunoglobulins are
normal except for elevated igE /J’hc pathogenesis
of the syndrome is nut clear but it is probably a
primary defect in phagocytic tuncrion .
Tuftsin deficitncy: A Icucnkinin capable ot
stimulating phagocytosis , discovered at Tufts
University, Boston , has been designated ' tuftsin *.
Chemically, it is A email teirapeptide (Thr - Lys-
-
Pro Arg ), Patients with tuftsin deficiency have
been reported to be prone to local and systemic
bacterial infections.
Lazy leucocyte syndrome: The basic defect
here is in chemntaxis and neutrophil mobility. The
bone marrow has a normal number of neutrophils
but there is a peripheral neutropenia , with poor
leucocyte response to chemical and iiiflammaroiy
stimulation . Patients show an increased
susceptibility to bacterial infection , with recurrent
stomatitis, gingivitis and otitis.
Hyper- [ gK syndrome These patients, nf both
sexes, have an earty onset of eczema and recurrent
bacterid infections such as abscesses, pneumonia and
secondary infection of eciema . The organisms
responsible include Sruptov/ocaceus anreu? and
Streph^Majs yt enes. Cellular and humoral immune
^^
mechanisms are normal but serum IgE levels are
usually mote than ten rimes the normal level.
Actitvhirulmg protein deficiency ;
Frequent infection and slow mobility of leucocytes
result from the defective act!n - hi riding protein in
these patients.
Slmaehmanki disease; In this condition .
Further Rending
Rosen FS CT ai. 1984. The primary Lmmunodeflcreiicits. New Engl J Med 311L23SN 300.
-
Fischer A and A Amtix Villcna 1995. Immunodeficiencies of genetic origin ,
Sriita DP and AJ Ten 199 L BASIC and Clinical Immunology. 7* cdn. Connccricur: Appleton - Lange.
Wizgcl H . 1993. Immunodeficiency. CLI /T Qpin Tmimanoi 5 :567 .
frequent infections am found together with decreased
neutrophil mobility, pancreatic malfunction and
bone abnormalities,
SECONDARY IM Mil NO DEFICIENCIES
A variety of factors such as malnutrition, malignancy,
i nfecrinriH, metabolicdisorders and cytotoxic drugs may
lead to deficits in specific and nonspecific immunity.
AIDS is a secondary immunodeficiency. Secondary
Lm munodefLeiendei are therefore very much more
common than primary deficiencies.
Deficiencies of humoral and cellular immune
response may occur secondarily during the course
of many disease processes . Humoral deficiency
results when 13 cells are depleted as in lymphoid
malignancy , particularly in cEirOnic lymphatic
leukemia - when immunoglobulin catabolism is
increased as in the nephrotic syndrome, when
excessive loss of scrtiin protein occurs as in
exfoliative skin disease and in protein 'losing
enteropathies, and when excessive production of
abnormal immunoglobulins occurs as in multiple
myeloma. Cell mediated immunity is depressed in
lymphorcticular malignancies , as in Hodgkin 's
disease ; obstruction to lymph circulation or
lymph orrhea when the thymus dependent areas
^
of lymph nodes are infiltrated with ronlymphold
cells as in lepromatous leprosy; and , transiently,
following certain viral infections such as measles.
Nutritional deprivutinn afJbcts both types of
immune responses adversely. Ageing also causes
waning in the efficiency of acquired immunity,
Immunodeficiency follow? the intentional or
unintentional administration of immunosuppressive
agents.
Today 16:510,
Copyrighted materia
 Hypersensitivity
Immunity was originally considered a protective
process, helping the body to overcame infectious
agents and their tmira. This however is only one
aspect of thebroad phenomenon of tmmu nity wh ieh
includes all manner of specific responses TO antigens.
Immune response may some rimes be injurious to
the host. Sensitised individuals respond to
subsequent antigenic stimuli in an inappropriate nr
exagger.ited manner, leading to tissue damage,
disease or ever death. The term hypersensitivity
refers to the injurious consequences in the sensitised
host, following contact with speedier antigens. In
the protective processes of immunity', tta focus of
attention is the antigen and what happens ro it —
for example, hilling of a bacterium or neutralisation
of a tojcin. In hypersensitivity, on the other hand,
antigens arc of little concern and often * they are
innocuous or bland substances such as scrum
.
proteins or pollen Elyperscnsitivicy is concerned
with what happens ro the host as a result of the
immune reaction.
Considerable confusion is attached to the use
of the term 'allergy . As originally used hv WOT
1
Pirquct, allergy meant an altered state of reactivity
to an antigen, and included both types of immune
responses, protective as well as injurious ft is still
, Serum sickness
used in this broad sense hv some . Others use: the
dr
allergy' to mean all immune processes harmful
term ‘
to the host , such as hypersensitivity and
autoimmunity. Allergy is probably most com muniv
used as a synonym for hypersensitivity. It is
sometimes employed in a narrow sense to refer to
only one type of hypersensitivity, namely ‘ atopy’.
For induction of hypexsensitiviry reactions,the host
should have had contact with the antigjcn (alktgen).
The initial contact sensitises the immune system *
Leading to the priming of the appropriate R or T
.
lymphocytes This is known as the 'sensitising' or
'priming dose. Subsequent contact with the allergen
1
.
causes manifestations of hypersensiriviry This is
known as the ’shocking' dose.
CLASSIFICATION OF
HYFFRSKNSITI v m RK ACTIONS
Hypersensitivity reactions have been classified
traditionally into 'immediate' and 'delayed' Types,
based or the time required for p. sensitised host to
develop clinical reactions on rc -exposurc to the
antigen. The major differences between the
immediate and delayed types of bvpereensitivitv
reactions am shown in 'I’abk 1@J .
The immediate and dclaved reactions are
subdivided into several distinct clinical types:
1. Immediate hypersensitivity ( E cell or
anfibodv mediated)
Anaphylaxis
Atopy
Antibody mediated cell damage
Artbus phenomenon
[ ]. Delayed hypersensitivity (T cell
mediated)
Infection ( tuberculin) type
Contact dermatitis, [ ype
Coombs and Cell [ l96 d !< classified
hypersensitivity reactions into feur types bused on
the different mechanisms of pathogenesis. Their
classification, now widely used, is outlined below:
Copyrighted material
160 * Texttiook ol Microbiology
Fable 16.1 Distinguishing features ol immediate and delayed types ol hypersensilivily
I mm edizte hypenensirivity
1. Appears and recedes rapidly.
2. Induced by antigens nr haptens by any route .
.1. Circulating antibodies present and respnnsibEe
for reactions 'antibody mediated reaction.
'
4, Pwaivt rrartsfor possible with serum.
5 . Dcsentidution ewv, but short - lived.
1 . Typfl I ( Anaphylactic , IgE -nr reagin
dependent ): Antibodies ( ' cytotropic' IgE
antibodies) arc fixed on the surface of tissue cells
(mist cells and basophils ) in sensitised
individuals. The antigen combines with thecell-
fixed antibody* leading to release of
nharmacnlngicallv active substances ( vasoactive
amines) which produce the clinical reaction.
2 . Type IT ( Cytotoxic or cell stimulating): This
, mechanism* described are
type of reaction is initiated by lgG ( or tardy
lgM ) antibodies that react either with cell
surface DC tissue antigens. Cell or tissue damage
occurs in the presence of complement or
mononuclear cells . Type II reactions are
intermediate between hypersensitivity and
autoimmunity. Combination with antibody may
in some instances, cause stimulation instead of
damage. An example is the 'long acting thyroid
stimulator ( LATS), an antibody against some
1
determinant on thyroid cells, which stimulates
excessive secretion of thyroid hormone. ( Such
antibddv mediated cell stimulation has also been
called type V hypersensitivity. )
3. Type IN (Immune complex or toxic complex
disease ): Here the damage is caused by antigen
antibody complexes. These may precipitate in
and around small blood vessels, causing damage
to cells secondarily, or on membranes, interfering
with their function .
4 .Typc 1 Y ( Delayed or cel ] mediated
Delayed hypersensitivity
1 . Appears slowly, lasts longer.
2 . Antigen nr hapten intradetmallv or with
Freunds adjuvant or by shin contact
3. Circulating antibodies may be absent and
nut responsible for reaction: 'cell mediated'
reaction .
4. Cannnot be transferred wjth scrum; but
possible with T cells or rrtnsftr factor.
5 . Difficult, but long- UsTitvgr
hypersensitivity ) : This is a cell mediated
response . The antigen activates specifically
sensitised CD4 and CDfi T octls , leading to the
secretion of lymphokincs, with fluid and
phagocyte accumulation.
The classification and wire of the features of
KypefSeflsitivity reactions are shown in Table
1 &,2 . The four types of immunopathogetiic
mutually
not
exclusive. Any given hypersensitive reaction may
comprise the components of more than one , or
all nf these mechanisms. The pathology and
clinical features of such immunological diseases
would also bo influenced by the contributions
of many mmimmune body mechanisms such as
inflammation , complement , coagulation ,
fibrinolytic and kinioogenic systems, collectively
called the humoral amplifiataon systems.
TYPE I REACTIONS ( IgE DEPENDENT)
These occur in two forms - the acute, potentially
fatal , systemic form called anaiphy]axis and the
chronic or recurrent, nonfatal , typically localised
form called atopy.
ANAPHYLAXIS
This is the classical immediate hypersensitivity
reaction. The term anaphylaxis (ana = without ,
-
phyiaxis protection ) was coined by Richer (1902)
to describe his observation that dogs which had
Copyrighted material
 1 Hyper s- en sin vi1\
Table 18.3 Types P( hyyrmiiHfrtty rwdkH
Type of reaction Ciinicml syndrome
Type h IgE type L Anaphylaxis
2. Atopy
Type II: Cytolytic Antibody mediated damage- Variable: hours
and CTTOCOXLC
P
thfomboejtopcnia-ajfranulo to days
eyiflBLs, hemolytic anemia., etc.
Type III : Immune I . Arthni reaction
complex 2. Serum sickness
Type IV: Delayed 1 . Tuberculin
hypcFsensjlrjVLiry 2. Contact dermatitis
survived a sublcthfll injection of a toxic extract of
sea anemones wen; rendered highly susorptitile to
minute doses of the toxin given days or weeks laici,
instead of becoming immune to it - Theobald Smith
(1905) had noticed H similar phenomenon in guinea
pigs, following widely spaced injections of toxin
antitoxin mixtures . Ehrlich named this the
— Icucopcnia and thrombocytojvenia.
Theobald Smith phenomenon ' and showed that it
wan independent of tile toxin and. unliLoxm used,
since the phenomenon canid he induced with
normal serum also.
Sensitisation is most effective when the antigen
is introduced parenteraliy but may occur by any
route , including ingestion or inhalation. In
susceptible s-pecies, very minute doses Can sensitise
the host . Antigens as well as haptens can induce
anaphylaxis. There should be art Interval of at 1e?st
2- 3 weeks between the sensitising dose and the
shocking dose . Once sensitised, the individual
remains so for lung periods- TliC shocking d(3K is
mast effective- when injected intravenously, less
effective intrupcritoneully or subcutaneously and
least effective mcradcrmaMy- 'I'hc shocking antigen
must he identical or immunologic a Ely closely
related to the sensitising antigen , The clinical
features of anaphylaxis are the same with aHV antigen
but vary between species. The clinical effects are-
due to smooth muscle contraction and increased
mta 11 AT permeability. The organs affected vary with
t 161
sno their features
Time required for MtdlttOn
mnnifestutim
Minute* IgE: histamine and
other phannacologkal
agents
I*C; IgM, C
-
Variable; hours lgfklgM, Cf
to days IcUCOLTttS
Hours to Javs T cellsi lynlphokiiiie
w
macrophages ^
the species. Tissues or organs predominantly
involved in die anaphylactic reaction are known as
Target tissues.' or 'shockorgans’. Other changes seen
in anaphylaxis arc edema, decreased coagulability
of blood, lull in blood pressure and temperature,
There is considerable species variation in
susceptibil i ty to anaphylaxis. Go i nta pigs are h ighly
susceptible and rats very resistant. Rabbits, dogs
and human beings are of intermediate susceptibility.
Anaphylaxis can he readily induced in guinea pigs.
If a small dose of egg albumin is injected
intraperitoreally, fallowed 2 - 3 weeks later by a
slightly larger dose of the same antigen
intravenously, the guinea pig will exhibit a dramatic
sequence of events. Wilkin minutes the an [ mil
becomes- irritable, sneezes , coughs, experiences
respiratory distress , develops convulsions and dies.
The heart continues to beat for some time after the
respiration has stopped. At autopsy, the lungs are
marked ly emphysematous and do not collapse when
the thorax is opened or ever when they arc cut
into pieces. The shuck organ is tile lung. Death LS
due to the constriction of the smooth muscles of
the bronchioles causing respiratory standstill.
ID rabbits, death in anaphylactic shock is due
to constriction of the pulmonary' artery and its
brunches, leading to extreme dilatation of the right
side of the heart - Respiratory movements continue
Copyrighted material
162 H Textbook ad Microbiology
after the cessation of the heartbeat. In dugs, the
reaction is slower and takes 1 -2 hours. There is
constri cti jn of the hepa Me venous system with gross
engorgement oftlie liver and profound fall of blood
pressure. In human beings, fatal anaphylaxis is
fortunately Symptoms and si ^ ns of
rare.
anaphylactic shock begin with itching of the scalp
and tongue, flushing of the skin over the whole
body and difficulty in breatlung due TO bronchial
spawn. There may he nausea, vomiting, abdominal
pain and diarrhea , sometimes with blood in the
Stool. Acute hypotension, Loss. dfnonScinusness and
death follow. Human anaphylaxis , once commonly
associated with heterologous scrum therapy, is now
seen mostly following injections of antibiotics or
other drugs. Insect stings can also cause anaphylaxis
m human beings, Prompt treatment with adrenaline
tan be lifr -savi itg. Adrenaline is to be ad mi n istered ,
0.5 ml of a 1 in 1000 solutvm, subcutaneously or
intramuscularly, the dose bring repeated upto a total
of 2 ml Liver 15 minutes, if necessary.
Cutaneous anaphylaxis; When a small
shocking dose of an antigen is administered
intradermally to a scnsiti'ied host, there will be a
local whcal-and -fUie response (local anaphylaxis).
The wheal! i s a pale, central area of puffi ness due
to edcmah which is surrounded by a flare caused by
hyperenu .i and subsequent erythema. Cutaneous
anaphylaxis (skin test for Type I hypersensitivity')
is useful in testing fur hypersensitivity and in
identifying the allergen responsible in atopic
diseases. In highly sensitised individuals, even
the skin test may lead to serious and even fatal
reactions. Hence a syringe loaded with adrenaline
should always be kept ready whenever a skin
test is performed to detecr anaphylactic
hypersensitivity.
Ebisaive cutaneous aimphylavis ( 13 ( ’ \ > e
Th is test developed by Ovaiy (1952) is an extremely
sensitive in vivo method for detection of antibodies.
A small volume of the antibody is injected
intradermaJly into a normal animal. If the antigen,
along with a dye such as Evans blue, is injected
»
intravenously 4-24 hours afterwards, there will be
an immediate blueing at the site of iutradermal
injection due to vasodilaia : iun and increased
- -
capillary permeability ( wheal and flare reaction ).
PCA can be used to detect human Ig£J antibody
which is heterocytotropic (capable of fixing to cells
of other species ) but not IgE which is
homocytotropic (capable of Fixing to cells of
homologous species only) .
-
Anaphyta \ i : i in vitroi Isolated tissues, such
as intestinal or uterine muscle scrips from sensitised
guinea pigs, held in a bath of Ringers solution will
contract vigorously on addition of the specific
antigen to the bath. This is known as the Schultz
Dale phenomenon. The reaction is specific and will
—
be elicited only by the antigen to which the aiiiimal
is sensitive. Tissues from normal animals can be
passively sensitised by treatment with scrum from
sensitised animals.
Mechanism of anaphylaxis: The
i immunologic basis for hypersensitivity is cytotropic
IgE antibody Free IgE antibody in circulation is
not relevant in anaphylaxis Thus, an animal
with a high litre of Liiculating antibody may be
refractory to shock, while anaphylaxis may be
caused by cell fixed antibody, even in the absence
of detectable circulating antibody. While in human
beings, IgE is the cytophilic antibody, in the guinea
pig and mouse the analogous mop hi I ir antibody
is IgGl .
IgE molecules arc bound to surface receptor*
on mast cells and basophils. These cells cany large
number* of such receptors called Fc ER receptors,
analogous to TCR receptors on T cell surface. IgE
molecules attach to these receptors by their Fc end.
Following exposure to the shocking dose, the
antigen molecules combine with the cell bound
.
IgE bridging the gap between adjacent antibody
molecules. This truss - linking increases the
permeability of the cells to calcium ions and leads
to ckgranukrion , with release of biologically active
substances contained in the granules. The
manifestations of anaphylaxis are due to
LJ opyrighted materia
 * Hyper^fisilivity
pharmacological mediators, which arc of two kinds
'
— primary mediators which are the preformed
contents of mast fell and basophil granules
( histamine, notHlin* eosinophil chemuCactic factur
nf anaphylaxis, neutrophil v he mot aerie factor,
heparin and various proteolytic enzymes ) and
secondary mediators which are newly formed upon
stimulation by mast cells, basophils and other
leucocyte* {slow reacting suhstance nf anaphylaxis,
prostaglandins and platelet activating factor, and
cytokines such as JL3, IL4, 1L5, IL6: GM-CSF) .
Primary mediators of anaphylaxis:
Histamine. This is the most important vasoactive
amine in human anaphylaxis. Histamine is formed
by the decarboxvlation of histidine found in the
granules of mast cells, basophils and in platelets.
Released into the skin, histamine stimulates sensory
nerves, producing burning and itching sensation ^.
It causes vasodilatation and hyperemia hv an axon
reflex [ Hare effect ) and edema by increasing capillary
permeability ( whenl effect ) . Histamine induces
smooth muscle contraction In diverse [issues and
organs, including vasculature, intestines, uterus and
especially the bronchioles. It also stimulates
secretions (secretogogue effect ).
1 . Serotonin ( 5 - hydroxy try ptamine):
This is a base derived by decarboxylation of
tryptophan. It LS found in the intestinal mucosa,
brain tissue and platelets. It Causes smooth
muscle contraction , increased capillary
permeability und vasoconstriction , It 1 s
important in anaphylaxis in rats and mice but
its mle in human beings is uncertain.
2. llhcmotndic factors: The eosinophil
cbemohictit factors of anaphylaxis f F.CF-A ) are
acidic tctmpepridcs released from mast cell
granules which arc strongly chemotactic for
eosinophils . These probably contribute to the
eosinophilic accompanying many hyper
sensitivity states . A high molecular weight
diemoiactic factor has been identified, which
attracts [leutrophils ( NCI7) .
Heparin is an acidic mucopolvsacchjride . It
163
contributes to anaphylaxis in dogs, but apparently
not in human beings.
Enzymatic mediators such as proteases and
hvdrotates are ftlfQ released from mast cell
granules.
Second ary mediatan of tntphykxii
1. Prostaglandins and leukutrienes: They
are derived by two different pathways from
a rich i thin ic acid , which is formed from
disrupted cell inembninesof mastcdls and other
leucocytes . The lipoxygenase pathway leads to
the formation ol leukotrieres, while the cvclo-
oxygenase pathway leads to prostaglaodins and
thromboxane. A substance originally
demonstrated in lungs , producing slow,
sustained contraction of smooth muscles , and
therefore termed slow reacting substance of
anaphylaxis ( SRb -A ) has since been identified
a ? a family oflcukotricncs [ LTB4, <S 4, E 4).
Prostaglandin F2f and thromboxane A 2 are
(
powerful, but transient , bronchoconstrictors .
Prostaglandins also affect secretion by mucous
glands, platelet adhesion , permeability and
dilatation of capillaries and the pain tlireshold .
2 , Platelet activating factor ( PAF ) t PAF
is a low molecular weight lipid released from
basophils which causes aggregation of platelets
and release of their vasoactive amines.
Other mediators nf anaphylaxir Besides
the products Of mast cells and Other Leucocytes ,
several other biologically active substances have
been implicated in anaphylaxis. These include the
anaphylatoxins released by complement activation
and bradykmin and other kioins formed from plasma
kininogens .
An n p h y l u u t n i d react!cm: Inifivcfloas
injection of peptone, typ'd U and Certain Other
- substances provokes a clinical reaction resembling
anaphvlactic shock - TTiLs is termed 'anaphylactoid
reaction’- The clinical resemblance is due to the
same chemical mediators participating in both
reactions. The only difference is that anaphylactoid
Copyrighted materia
164 ^ Ts < rt>c 0i< of MiCrtSfcioloqy
shock has no immunological basis and is a
nonspecific mechanism involving the activation of
complement and the release of anaphylaiuodiiis.
Atopy; The term 'atopy (literally meaning nut
1
of place or strangeness ) was introduced by Coca
! 1* 23 ) to refer to naturally occurring familial
^
hypersensitivities of human brings* typified by hay
fever and asthma.The antigens commonly involved
in atopy are characteristically inhalants (for
example , pollen, house dust ; or ingest ants (for
example, eggs, milk ). Some of them are contact
allergens, to which the skin and conjunctiva may-
be exposed. These atopens are generally not good
antigens when injected parcntcrally but induce IgE
antibodies, formerly termed as ' reapin .mtihodies.
'
Atopic sensitisation is developed spontaneously-
following natural contact with atopens. It is difficult
-
to induce atopy ariiiicially.
Predisposition to atopy is gencncallydetennLncd,
-i n drably I i oked Do MHC j. jcnOtypc - . A [ Opy 11 LL rttfut
runs in families. What is inherited is not sensitivity
to a particular antigen , or a particular atopic
syndrome but the tendency to produce IgE
antibodies in unusually large quantifies. All
individuals arc capable of farming IgE antihodics
in small amounts but in atopies IgE response is
preponderant - About 10 per cert of persons have
this tendency to overproduce IgE. Jt has beer
reported that borlJefed infants tend to develop atopy
in later life more often than breastfed babies.
IgE differs from other immunoglobulins in the
following respects:.
1. It cannot be demonstrated by the conventional
serological rcact > > nfi such as precipitation or
complement fixation. 'l'he first in vitro method
for IgE detection was the mdioailergosorbent
test ( RAST ) . Simpler techniques such as
ELlbA and passive agglutination have since
lieen introduced.
2. While atopy occurs commonly in human beings,
it is not easy to induce it experimentally in
animals.
3. IgE is lmmi wmimpiv, that is, species specific.
Only human IgE can fix to the surface of human
UJK ; reaction, which was the original method
-
cells. This is the basis of the Prausnitz Kustner
for detecting atopic antibody. Prausnitz and
Kusiner ( 1921) reported that if serum collected
from Kustner, who had atopic hypersenativity to
certain species of cooked fish, was injected
iotracutaneously into Prausnitz, followed 24 hours
later by an iniracutaneous injection of a small
quantity of the cooked fish antigen into the same
- -
site, a wheal and flanp reaction occurred within a
-
few minutes. As rcagjnic IgE is homo- cytotropic>
the test has to be carried out on human skin. It
carries the risk of transmission o1 infection and so
is no longer used.
A . Unlike other antibodies, IgE is heat sensitive
and inacl ivated at 56 nC in 2-4 hours .
Hearing appears to damage the Fc part of the
IgE molecule, which is necessary for fixation
tocells.
5- Atopic antibody docs not pass through the
placenta.
Atopic sensitivity is due to an overproduction
of IgE antibodies - Tbi -. i -. otter associated with a
deficiency of IgA. This association has led to the
suggicsrinn that IgA deficiency may predispose to
atopy. The distribuiinn of lymphocytes capable of
synthesising IgA and IgE is closely parallel,
especially m the submucosa. In normal individuals,
the inhalant and ingestant antigens are dealt with
by JgA lini ng the respiratory and i ntestinal mucosa
and therefore they do nut come into contact with
the pntenti d JgE producing cells. When IgA is
deficient, the antigens cause massive stimulation of
IgE forming cells, leading to overproduction oflgE.
The symptoms nt atopy are caused by the release
of pharmacologically active substances following
the combination of the antigen and the cell fixed
IgE. l’he clinical expression of atopic reactions is
usually determined by tile portal of entry uf the
—
antigen conjunctivitis, rhinitis , gastrointestinal
.
J*“ 4
Lj opy righted materia
 4 Hyp«f sensitivity
symptoms and dermatitis following exposure
through the eyes, respiratory tract, intestine or skin,
respectively. Sometimes the effects m»V he at sites
remote from the portal of entry, for example,
tirtitdtia full owing ingestion of the allergen.
Specific deseftsirisatiuti (hyposensitization) is often
practised in the treatment of atopy.
TYPE II REACTION : CYTOLYTIC AND CYTOTOXIC
These reactions involve a combination of TgG (or
rarely IgM) antibodies with tire antigenic
-
determinants on tin surface of cells lending to
cytotoxic or cytolytic effects, Kxamples are lysis of
'
red cells caused by antierythrocvie j-ruilHidies in
autoimmune anemias and hemolytic disease of the
-
newborn Alternative!!', a free antigen or hapten mar
lie absorbed on cell surfaces . Subsequent reaction
of the combined antigen or hapten with its
corresponding antibody leads to cell damage. Many
-
drugs may act in I hi manner, leading to
complement mediated Lysis of red cells, leucocyte*
and platelets, causing hemolytic anemia,
agranulocytosis and thrombocytopenk; purpura.
In some Type II reactions, the antihodv
combines with cell surface receptors and disrupts
normal time I ion, either by uncontrolled activation
-
( agonist effect a caused by the antibody ‘
1
long- acting
thyroid stimulator in Grans' disease ) or hy
blocking (antagonist cftcer as in myasthenia gravis) .
TYPE III REACTIONS: IMMUNE COMPLEX
DISEASES
Arthus reaction: Arthus {1903 } observed that
when rabbin were repeatedly injected
subcutaneously with normal horse scrum, the initial
inject ions had no Local effect but with Later
injections, there occurred intense local reaction
consisting ot edema, induration ami hemorrhagic
necrosis, lliis is known as the Arthus reaction and
is a Local manifestation of generalised
hypersensitivity, The tissue damage is due to
-
formation ofantigenr Mitibody pretip'rtHes causing
complement activation and release of inflammatory
165
molecules. This leads to increased vascular
permeability and infiltration of the site with
neutrophils. Leucocyte— platelet thrombi arc formed
that reduce the blood supply and Lead to tissue
neefosis . The Arthus reaction can be passively
transferred with sera containing precipitating
antibodies (IgG, IgM) in high litres.
Arthus reaction forms a pathogenic Component
.
of many clinical syndromes For example,
intrapulmonary Arthus ‘like reaction to inhaled
antigen , such as thermophilic actinomyceces from
*
mouldy bay or grain causes Farmer;' ; Jung mid other
types of hvpcrsensi tivitv pneumonitis.
Serum sickness: This is a systemic form of
Type 111 hypersensitivity. As originally described
hy von Ehrquct and Schick ( 1905 ), this appeared
7-12 days following a single injection of a high
concentration, of foreign scrum such as the
diphtheria antitoxin. The clinical syndrome consists
of fever, lyniphadenopachy, splenomegaly, arthritis,
glomerulonephritis, endocarditis, vasculitis,
urticant] rashes, abdominal pit in, nausea and
vomiting, 'llie pathogenesis is the tbrmation of
immune complexes (consisting of the foreign serum
and antihody to it that reaches high enough litres
by 7-12 days), which get deposited on rhe
endothelial Lining of blood vessels in various parts
of the body, causing inflammatory infiltration .
I'hc plasma concentration of ’ complement falls
due to massive complement activation and fixation
.
hy the antigen antibody complexes The disease is
self limited. With continued rise in antibody
production, rhe immune complexes become larger
and mote susceptible to phagocytosis and immune
.
dimination When all foreign antigen is thus
eliminated, and free antibody appears, the symptoms
clear without any sequelae, The latent period of 7~
12 days is required only for serum sickness following
a single injection. With subsequent injections , the
disease manifests earlier, her uni sickness differs
from other types of hypersensitivity reaction in that
a single injection can serve both as the sensitising
dtjse amt the shocking dose. As heterologous serum
Copyrighted material
166 ^ Textbcon oJ Microbiology *
injections ire not used often now, the syndrome is
currently more commonly seen following injections
of penicillin or other antibiotics ,
Immune complexes occur in many diseasest
including bacteii .iL , viral and parasitic infections (for
example , poststreptococcal glomerulonephritis ,
hepatitis type li , malaria ) , disseminated
imEgnifieiH and autoimmune conditions . The
nephritis and arthritis seen in these conditions may
be caused by deposition of immune complexes .
TYPE IV REACTIONS: DELAYED
HYPEflSENSmvrTY
Type !. V hypersensitivity reset ions ( delayed
hypersensitivity) constitute one aspect ot cell mediated
immune response. These are typically provoked by
intracellular niLLTK dual infections or haptens like simple
chemicals applied on the skin, evolve slowly and
consist of a mixed cellular reaction involving
lymphocytes and macrophages in particular.. The
reaction is nut induced by circulating antibodies hut
by sensitisedJ 1' cells [Tdtb, "!hi „"I'hS ,1” c ) which, or
contact with the specific antigen, release cytokines
t h a t cause biological effects on leucocytes ,
macrophages and tissue cells, Delayed hypersemitivity
cannot he passively transferred by scrum but can be
transferred by lymphocytes uf die transfer factor Two
types of delavud hypersensitivity an: recognised — the
tuberculin (infection) type anti the contact dermatitis
type.
Tuberculin ( infection ) type; The archetype
of delayed hypenenaitivity is the tuberculin
reaction. WliL’a a small dose of tuberculin is injected
in tr a derm a lly in an individual sensitised to
tubcnculoprotein by prior i nfcction nr i mmun i sati nnn
an indurated inflammatory' reaction develops ar the
site within 40-72 hours, In unsensitised individuals,
the tuberculin injection finwokes no response. The
tuberculin test therefore provide!useful indication
of the stare of delayed hypersensitivity ( cell
cited!ated immunity) the bacilli. The tuberculin
to
test differs from the skin test for Type 1
hypersensitivity not only in the longer interval for
appearance hut alio in its morphology and
histology.
Tuberculin type hypersensitivity develops in
many infections with bacteria, fungi , viruses and
paruiles, especially when the infection is subacute
or chronic and the pathogen intracellular. A similar
hypersensitivity is developed in allograft reaction
and in many autoimmune diseases.
Cutancouf bnsophil hypenenaitivity; A
local reaction resembling the tuberculin response
may he produced by intradcrmal injection of some
protein antigens. This is not a delayed
hypersensitivity reaction as it Can be passively
transferred by serum. Its histology is different from
the tuberculin response, being characterised by
prominent basophil infiltration. This was formerly
known as the Jones-Motc reaction but is now
ter met ] cutaneous basophil hypersensitivity. Its
signiticance is not known .
Contact dermatitis typer Delayed
—
hypersensitivity sometimes results fiom skin contact
with a variety of chemicals metals such as nickel
and chromium , simple chemicals like dves, picryl
,
chloride , dinitruchlurnbenaenc , drugs such as
penicillin , and toiletries. Sensitisation is particularly
liable when contact is with an inflamed area of
skin and when rhe chemical is applied in an oily
base. Antibiotic ointments applied on patches of
dermatitis frequently provoke sensitisation . The
substances involved are in themselves not antigenic
but may acquire antigenicity on combination with
skin proteins-. Sensitisation requires percutaneous
absorption . As most of the substances involved arc
far soluble, passage along sebaceous glands may be
the method of entry of tlie allergen* .
Langerhans' cells of the skin capture locally
applied hapten , along with the modified tissue
proteins , and migrate to the draining lymph nodes
where they present the processed antigen along
with MHC molecules to T cells.The sensitised T
cells travel ro rht skin site , where cm contacting
Copyrighted material
 < HyptruntitMly
the antigen they realeasc vari ous lymphoki ncs. Thl
cell!-! secrete II - Ny and It ,2 which activate
macrophages and other lymphocytes Th 2 cells
release IL 4 h ILS, GM-CSF and other factors that
lead to an influx of eosinophils and tissue damage.
Acrivatcd Te cells mediate killing of target cells .
Contact with the allergen in a sensitised
individual leads to 'contact dermatitis', the lesions
varying from macules and papules to vesicles that
break down , leaving behind raw weeping areas
typical of acute eczematous dermatitis ,
Hyperser.sitis iry is- detected by the 'patch test’. The
allergen is applied to the skin under an adherent
dressing . Sensitivity is indicated by itching
appearing in 4-6 hour ?. , and local reaction which
may vary from erythema to vesicle or blister
-
B CP MR
formation , after 24 28 hours,
SHWARTZMAN REACTION
This is not an immune reaction bur rather a
perturbation in factors affecting intravascular
coagulation, lc is traditionally described along with
hypersensitivity reactions because of a superficial
resemblance .
Shwartzman (1928) observed that if a culture
filtrate of S. ryphi is injected intradermally in a
nhhit , followed 24 hours later hy the same filtrate
intravenously, a hemorrhagic necrotic lesion
develops at the site of the intradermal injection.
The Intradermal and intravenous injections need
not be of the same or even related endotoxins.
Culture suspensions or filtrates of a variety of
bacteria will Sensitise the skin to intravenous
injection by an equally wide variety of cultures or
filtrates. This absence of specificity and the short
interval between the two doses preclude any
immunological bai- is for the REACTLOII .
The initial ( preparatory ) dose is
characteristically an endotoxin. The intravenous
{provocal i ve) L:i ject i on can be a variety of substances
— bacteri al e n dn (ox i n s , a n I i gc n-an 11 b n dy
complexes, starch , serum, kaolm and others. The
preparatory injection causes accumulation of
167
leucocytes which condition the site by release of
[ ysosomat enzymes damaging capillary walls ,
Following the provocative dose, there occurs
intravascular clotting, the thrombi leading to
necOisK of vessel walls and hemorrhage .
If both the injections are given intravenously,
-
the animal dies 12 24 hours after the second dose.
Autopsy showy hiiateral corneal necrosis of the
kidneys and patchy hemorrhagic necrosis in the
Liver, spleen and othe r o rg.m > . An essent i .dly s im i [ j r
phenomenon was dcscriljcd by Sanarcl ! i (1924) in
experimental cholera. The reaction is therefore
-
called the Sanarelli Shwartzmati reaction or the
generalised Shwartzman reaction.
It has been suggested that mechani -- ms similar
to the Shwartzman reaction may operate in some
clinical conditions such as the purpuric rashes of
meningococcal Septicemia and the acute
hemorrhagi .; adrenal necrosis found in
overwhelming infections ( Waterhouse -
Friderichsen syndrome)-
Many infections, particularly Gram negative
septicemias can lead to a septic sliock syndrome
with profound hypotension, hypoxia and oliguria.
This may sometimes be accompanied by the adult
respiratory distress syndrome ( ARDS ) with
overwhelming neutrophil inva-vinn of lungs.
Massive adivalion of complement by the
alternative pathway, associated with release of
thromboxane A2 and prostaglandins from platelets
may lead to disseminated intravascular coagulation.
The mechanism may be the excessive release of
cytokines such as the tumour necrosis factor and
:utexlcukhis 1 and 6- by macrophages and endothelial
cells in response to contact with large cpi .ml Hies of
-
Tipopoly acchari'. k endotoxin. Some Gram positive
infections may also cause similar effects. Staphylfr-
coccus aureus can induce T ISF secretion hy
macrophages and peptidoglycan mediated platelet
aggregation , leading to disseminated intravascular
coagulation . Staphylococcal entcrutuxin can ac1 i:-
a super antigen, act i va Ling whole fan lilies t>l r -LieUs
irrespective of their antigen specificities and causing
Copyrighted material
I JF W
m HI Texlbook of Microbiology *

massive release TKF cytokines, IcJ-d-ing to the toxic origin but their pathogenic mechanisms jnEsemtle
shock syndrome. These tioadhionfl arc not of immune
jf
those in mumme inflammation.

K u r t her Keidini
Chapel HL and M Haemy 1993. Es&mtizls of CUnicil Immunology. 3rd edn. Oxford: Blackwell.
Even PW, 199®. AiuphyLaxis, iUWJ 316- 1442.
Frank M et ?il (eds ). 1995- louniJHafcgicif JDIWMJW- Boston: Little Emm.
.
Ho t ST mA MK Church 1993- Atksgy* Londoo:Gnu
^ *
Howanh PH. 1998. AltVjy ; Parbngtruc mechanism. New EqgJMtd 343:712,
LKIUDHII P et J1. 1993. OrUiVjJ ofJirtm-urkafr -. Oxfinrd: Blackwell.
Rmn IM. 1994. EsscrHeaJ Imatturnifc y. S*41 -edn. Oxford ^: Blackwell.
^

Copyrighted material
 Autoimmunity
Self -antigens arc not ordinarily immunogenic .
Ehrlich (1901) observed that goats produced
antibodies against erythrocytes from other goats but
rot against their own , and postulated the concept
of 'horror autotoxieus’ . Uur he did not regard
a utoLminumsat La n JH jn impossibility and even
envisaged its pathogenic possibility.
Autoimmuniiy is a condition in illicit
structural or functional damage is produced bv the
action of immunologic ally competent ecELs nr
antibodies against the normal components of the
body. Autoimmunity literally mcniH protection
against self but it actually implies 'injury to self
a nd therefor. it h as been c ri rictscd as a con tndicbo 11
1
in terms . Autoallergy ' has been suggested as an
acceptable alternative but the term autuunmunity
has the sanction of wide usage.
The earliest example of autoimmunity wa * the
duiervanon by MeTsilmkjofF ( l 90()l that guinea fujp.
injected with their own spermatozoa produced
sperm Immobilising antibodies, Donat h and
Lindstciner ( 1904 ) identified circulating
autoantibodies in paroxysmal cold hemoglobinuria
— a hemolysin which binds with the patients
erythrocytes low temperatures and produces
at
complement dependent hemolysis on warming.
This was the first description ot an autoimmune
disease in human beings, Dameshek aid Schwartz
{ 1938 ) established the autoimmune basis of acute
hemolytic anemia. With the discovery of Coombs
test for incomplete lantilwHiies it became possible
to demonstrate globulins bound to the surface ot
erythrocytes in this condition, Antoimmunisation
could be induced in experimental animals bv
injection of self -antigens along with the complete
Kreund’s adjuvant . The use of sensitive serological
techniques led to the demonstration of
autoantibodies in several diseases and even in. a
proportion of healthy individuals ,. Some
auroantihodies, such as the anti idiotypic antibody
may even be essential for the normal functioning
of the immune system.
When the concept of auloimmuritv came to he
accepted as a pathogenic mechanism, a Large number
of diseases were suggested to hive an autoimmune
etiology, based on the finding ot autoantlbodies in
rbc patients . Thill was soon recognised to be
tin ten able as nutuartibodies could be often
incidental or the result , and not the cause of disease.
Criteria were proposed For proving the
authenticity ot putative autoimmune diseases ,
similar to Koch's postulates in infectious diseases .
The« were found to lx- not applicable in the case
of such spontaneous and multi factorial conditions
as autoimmune diseases . It may be proper to restrict
the rerm 'autoimmune diseases' to those where
autoimmune processes , humoral or cellular, are
shown to be responsible for the pathogenesis , rather
than merely HMrirjptrrl Tfrii is not strictly adhered
to. Mrnuiiu, the border between autoimmunity and
hypersensitivity is Largely ill -defined or evert
nonexistent .
Diseases of autoimmune origin usually exhibit
the following tenures:
1 . An elevaCcd level ot immunoglobulins.
2 . Demonstrable autuantibodics.
3- Deposition of immunoglobulins or rheir
derivatives at sites of election , such as renal
Copyrighted material
170 * Textbook of Microbiology
glomeruli .
4 Accumulation of lymphocytes and plasma cells
, ,
at the Sites of lesion.
5, Benefit from corticosteroid or other
immunosuppressive therapy.
6- The occurrence of more than one type of
autoimmune lesion in an individual.
7. A genetic predisposition towards autoimmunity.
ftr Incidence higher among females.
9 , Chroniciry, Usually nonreversible.
MECHANISMS OF AUTOIMMUNISATIGN
Cells or tissues may undergo antigenic alteration
as a result of physical , chemiical or biological
influences. Such altered or 'neoantigens may elicit
1
an immune response. Ncoantigcns can arise in a
variety of ways. Physical agents such as irradiation
can cause antigenic alteration. Photosensitivity and
cold allergy may represent sensitisation to self-
antigens., altered by light and cold, respectively.
Sever .il chemicals, including drugs, can combine
with cells and tissues and alter their antigenic
nature. Contact dermatiLis, which is traditionally
considered a type o f delayed hypersensitivity can
also be taken to be an autoimmune response to skin
ant igens altered by the i r comb i nation wi r h chert ical
allergens. Drug induced anemias, leucopenias and
thrombocytopenias often have an autoimmune basis.
Infectious microorganisms, particularly viruses and
other intracellular pathogens, may induce alteration
of cell antigens. Viral infections, such as infectious
mononucleosis , arc known to often precede
autoimmune diseases. Bacterial enzymes also induce
alteration of cell antigens. Neuraminidases formed
by myxoviruses and many bacteria act on
erythrocytes releasing the 1’ antigen . The almost
universal occurence of T agglutinins in human
sera is believed to represent a harmless autoimmune
response following infecrions. NcoanTigens may also
arise by mutation. Such mutant cells may be
immunogenic.
Immunological damage may result from
immune responses induced by cross- reacting
foreign antigens. The fortuitous similarity between
some foreign and self-antigens is the basis of the
‘cross reacting antigen’ theory of autoimmunity.
Organ specific antigens are present in several
specif . Injection of heterologous organ specific
antigens may induce an immune response damaging
the particular organ or tissue in the host. An example
is the neurological injury that used to be a
complication of antirahic immunisation in human
beings with the neural vaccine of infected sheep
brain tissue partially denatured by treatment with
phenol. Its injection elicits an immune response
against sheep brain antigens. This may cause
damage to the individual's nerve tissue due to the
cross-reaction between human and sheep brain
antigens . Immunological injury due to cross-
reacting antigens can also follow infections.
Streptococcal M proteins and the heart muscle share
-
antLgemc characteri itics . The immune response
induced by repeated streptococcal infection can
therefore damage the heart. Nephritogenic strains
of streptococci possess antigens found in the renal
glomeruli . Infection with such strains may lead to
glomerulonephritis due to the antigenic sharing.
A related type of autoimmunisation is molecular
'
mimicry' which is due to the presence in some
infectuig microorganisms and self - antigenn , of
fij ' iwpes with identical peptide sequences ( instead
of Similarities in 'cross- reactions ]. Examples of
1
such homologous sequences arc seen in
arih Erogenic Shigella flerneri and HLA B27 ,
Afyvotactenirm fubemritMis and joint membranes,
-
Uoxsackie B and myocardium .
Another hypothesis is polyclonal B cell
activat i on. While an antigen generally activates only
i t s corresponding B cell, certain stimuli
nonspeafically turn on multiple B cell clones. Such
stimuli include chemicals ( for example , 2-
mercaptoethanol ) , bacterial products (PPD,
lipopolyweeharide], enzymes (trypsin), antibiotics
( nystatin ) and infections with some bacteria
( mycoplasma ) , viruses [ EM virus) and parasites
( malaria) . Multiple nonspecific antibodies form
Copyrighted materia
 « Autoimmunity »
during some infectious diseases, such ns anti human
erythrocyte cold antibodies in mycoplasma
pneumonia and anti sheep erythrocyte antibody in
infectious mononucleosis.These polyclonal
antibodies are IgM in nature, similar 10 the 'natural
antibodies’ produced by CCS * B cells.
-
Breakdown ot immunological borne stasis may
lead to cessation of tolerance and the emergence of
forbidden clones of immunocompetent cells capable
of mounting immune response against sel antigens.
^
Autoimmunisation may result when tolerance to a
self-antigen is abrogated, as for instance bv the
injection of the self- antigen with Freund’s adjuvant ,
Enhanced helper T cell and decreased
suppressorT cell functions have been suggested as
causes of autoimmunity Defects in the thymus* in
stem cell development and macrophage (unction
have also been postulated as causes.
Certain self - antigens :tre present in closed
systems and are not accessible to the immune
apparatus. These are known as sequestered antigens.
An example is the lens antigen of the eye. The lens
protein is enclosed in its capsule and Joes not ,
ei ten I ate in the blood . Ed e nee immunological
tolerance Against this antigen is not established
during fetal life . When rhe antigen leaks out,
following penetrating injury, ir mav induce an
immune response causing damage to the lens of
the other eye . An example of Hseque &tiari on in time'
is seen with sperm antigens . As spermatozoa
develop only with puberty, the antigen cannot
induce tolerance during fetal lifeThe sperm antigen
is therefore nut recognised as self And when ir enters
the circulation, Li is immunogenic. This is be lieved
to be the pathogenesis of orchitis following mumps.
The virus damages the basement membrane of
seminiferous tubules leading to the leakage of
sperms and initiation of an immune response
resulting in orchitis.
—
Defects in rbe idiotype antiidiotype network
have also been said to lead to autoimmunity.
Genetic factors such as defective It at
immunoglobulin genes have also been postulated.
171
Jl hum JO autoimmune diseases and in animal
models genetic factors appear to influence the
development and fate of autoimmune states . Inspire
of so many different possible mechanisms proposed ,
their actual role in autoimmunity, if any, has not
been established ,
Many animal models of spontaneous and induced
autoimmunity have contributed to an understanding
of this condition. Examples of spontaneous
autoimmune diseases in animals arc autoimmune
hemolytic anemia in the New Zealand Black
( NZR ) mouse strain, systemic lupus erythematosus
in NZB X NZW cross, insulin dependent diabetes
mellitus in the nonobese diabetic ( NOD) mouse,
and thyroiditis in the obese strain ( OS) chicken .
Experimentally, autoimmunity can he induced in
—
many animal species by injecting tissue extracts in
the complete Freund 's adjuvant for example ,
experimental allergic L' liL-ephaJomyeiitis with brain
or spina] cord extracts, and thyroiditis with thyroid
,
gland extract.
CLASSIFICATION OF AUTOIMMUNE DISEASES
Based on the site of involvement and nature ot
Lesion*, autoimmune diseases mav be classified as
hemocytolyiic , Localised (or organ specific}* systemic
( or nonorgan specific ), and transiton, diseases.
1
HEUOC 'I TOLYTIC AUTOIMMUNE
DISEASES
Autoimmune hemolytic anemias:
Auto antibodies against erythrocytes are
demonstrable in this condition . Serologically, two
groups of autoimmune anemias can he
distinguished, characterised by 'cold * and ' warm'
antibodies, nenpective ly.
The cold autnantibodies arc* generally, complete
agglutinating antibodies belonging to the IgM class
and agglutinate erythrocytes at 4 'C but nor at 37 " L\
Cold agglutinins were fifft detected by Dunaih and
Landsteincr in p .LTOxvsru .il cold hemoglobinuria.
This condition , which used to FICQLICULLV
accompany syphilitic infection , is seldom seen
Copyrighted material
172 r Textbook of Microbiology
nowadays. Cold agglutinins arc also seen in [ primary
atypical pneumonia trypanosomiasis and bkekwater
fever.
Warm smtoantibodies arc generally incomplete,
Ticinflggliiriniting antibodies usually belonging to
the IgG class. They can be shown coating the
erythrocytes in the direct Coombs test. Warm
jinticrythrocytc antibodies are frequently seen in
patients taking certain drugs sueli as gulphonamides,
, ,
antibiotics, and alpha methyl dopa.
In aiifnimmnrwi anemias , the red cells coated
with antibodies arc prematurely destroyed in the
spleen and liver. Complement dependent
intravascular hemolysis appears to be a rare event.
Auto immune thrombocytopenia;
Au roan LI bodies directed against platelets occur in
idiopathic thrombocytopenic purpura. Sedormid
purpura is an instance of immune response against
drug induced neoantigens on platelets. This
condition is traditionally considered an antibody
mediated hypersensitivity.
Autoimmune luucupcniat Nonagglutinating
antileucocyte antibodies can he demonstrated in the
serum of patients with systemic lupus
erythematosus and rheumatoid! arthritis.
LOCALISBP lUK ( i , \.N SPEtJFIt :) '
AUTOIMMUNE DISRASRIS
. Vutouiiiniiiiu ul the thytuid
gland
1. Mnshimoto's disease ( Lymph adenoid
gnltre ): This is the must typical ami best
-
studied of organ specific autoimmune diseases
hi 1956 P Roitt ind Doniacb in England
, condition some times follows mumps orchitis.
demonstrated andthyroglobiiltn. antibodies in
the sera of patientfl by prec ipitation in gyl , and
Witcbsky and Rose in the USA by the more
sensitive passive hemagglutination test . The
latter workers also reproduced the disease in
rabbits by immunisation with autologous thyroid
tissue obtained by hemichyroidectomy
Hashimoto's disease occurs more frequently in
females and is associated wirh an enlargement of
the thyroid gland and symptoms of hypothyroidism
nr frank mneedema. Histologically, the glandular
structure is replaced by lymphoid tissue consisting
of Lymphocytes , histiocytes and plasma cells.
Antibodies with different specificirics have been
found in this condition. They include antibodies
that react with thyroglobulin, a second acinar
Colloid, microsomal antigen arid a thyroid cell
surface component .
2. ThyrotoxicotiB { Graves ' disease)! The
majority of patients with thyrotoxicosis possess
antibody to thyroglohulin . Lymphocytic
infiltration is common in thyrotoxic glands. 'I’he
immunological basis ot thyrotoxicosis is
supported by the identification of the 'long
7
acting thyroid stimulator ( LATS) which is an
IgG antibody to the thyroid membrane antigen.
Combination of LATS with the surface
membrane of thyroid cells seems to stimulate
excessive hormone secretion.
Addison’g disease:: The immunological basis
of Addison * disease is suggested by lymphocytic
i nfiltration of the adrenal glands and the presence
of circulating antibodies directed against the cells
of the zona glomerulus .L. Similar lesions Can be
produced in experimental animals by immunisation
with adrenal tissue in Freund’s adjuvant .
Autoimmune ( it'd litis: Experimental allergic
orchitis with progressive damage to germinal
epithelium and aspermatogenesis can be induced
in guinea pigs by the injection of autogenous or
allogeneic testes with Freund's adjuvant. A similar
Lymphocytic infiltration of the testes and
circulating antibodies to the Sperms and germinal
cells can be demonstrated in this condition.
Myasthenia gravis: Jn this disease , there is
an abnormal fanguability of muscles due to
malfunction of the myoneural junction. An antibody
against acetyl choline receptor on myoneural
junctions of striated muscles is present in these
patients . This prevents acetyl choline from
combining with its receptor, and impairs muscular
Copyrighted material
 * AulOimmun -i ty
contraction . Tbti thymus shows lymphoid
hyperplasia and numerous germinal centres. Infants
born 10 affected mothers show symptoms of the
disease but [Boner spontaneously by the age of two
months, coinciding with the disappearance of
maternal antibodies. This suggests that the
pathogenic factor in neonatal myasthenia may be
the autoant ibody passively acquired from the
mothc r.
Autoimmune diseases of thu eye: Two
types of autoimmune diseases arc seen in the eye-
Cataract surgery sometimes leads to intraocular
inflammation caused by the autoimmune response
to the lens protein . This is known as
phico&n&phjinxit'
Perforating injuries of the eye, particularly tlutSC
involving the iris orciliaiyrbodies arc often followed
by i/iupadteftc op/iEhaiirfa in the opposite eye . The
disease can. he produced in experimental animals
by immunisation with uveal or retinal tissue in
Freund's adjuvant and can he passively transferred
with the spleen or lymph node cells but not wirh
serum.
Pemiciriu * an ami a: Two types ot
auloantibodtes arc present in this condition. The
first is directed against the parietal cells of the
gastric mucosa . This is believed to cause
achlorhydria and atrophic gastritis . The second type
of antibody is directed against the intrinsic factor
and prevents absorption of vitamin 11 , , either by
blocking its attachment to the gastric intrinsic facto:
or by binding to the intrinsic: factor complex
and interfering with its uptake hy the intestinal
dfluOOH .
Autoimmune disease* of the nervous
i ) stern: The ‘neuroparalytic accidents' following
rabies vaccination represent injury the nervous
to
system hy the immure response against the sheep
nervous tissue in the vaccine, which cross reacts
with human nerve tissue . An essentially similar
condition, experimental allergic encephalomyelitis
( F, AE ) . can be produced in animals by
immunisation with nervous tissue in Freund s
173
adjuvant . The cncephalogenic protein has been
identified sis the myelin basic protein ( MR?) which
shows no species specificity.
Idiopathic polyneuritis ( Guillfiin- Ilarre
syndrome) is considered an autoimmune response
against rhe peripheral nervous tissue. It tan be
reproduced in experimental .LiiimaU by
immunisation with peripheral nervous tissue in an
adjuvant
Autoimmune diseases nf ihe skin : Three
serious diseases of the skin art considered to
have an autoimmune basis . Pemphigus vulgaris
may he caused by an antibody to the inlcrceitular
cement substance. In bullous pemphigoid *
antilmdies directed against the dermal cpilhcli.il
junction have been demonstrated - Specific
antibodies in dermatitis herpetiformis have not been
identified .
SYSTEMIC ( NONOROAN SPECIFIC )
AI miMMtJNE DIHEAHEK
This group includes conditions characterised by
immune response against a variety of self-antigens
and damage to several organs and tissue systems.
Klemperer ( 1942) classified a number of diseases
of unknown origin wrirh the common feature of
connective tissue lesions as 'collagen diseases' .
Iiieluded in this category are systemic lupus
erythematosus ( SLR ) , rheumatoid arthritis ,
polyarteritis nodosa * Sjogren 's syndrome,
denoato myositis and scleroderma . All these
«r
conditions arc associated with generalised
autoimmune processes.
Systemic lupus cry them AtOAUK This is H
chronic, multisystem disease with remissions and
exacerbations, Terminating fatally. Patients have , L
variety of iutOantibodiev directed against Cell nuclei ,
intracytoplasmic cell constituents , immune
glohulins, thyroid and other organ specific antigen ^ ,
biological false positive reaction is scon in standard
tests for syphilis. The abundance and variety of
autoamibodies suggest a breakdown in tlie central
control of immunological homeostasis.
Copyrighted materia
174 * Textbook ot Microbiology
The fnsr immunological feature identified in
SLE was the LE ceil phenomenon described in
1948 . The LE cell is a neutrophil containing a
large* pale, homogeneous body ( LE body) almost
ELI mg the cytoplasm - [ III LE body is the
immunologically damaged nucleus of a leucocyte.
Sometimes, instead of being intracellular * the LE
body can be seen free, surrounded by a rosette of
neutrophils. The fact that LE cell formation in due
to an antibody ( LE factor) present in SLE can be
demonstrated by incubating normal blood with
serum from an SLE patient. The nuclei of some
leucuCytrs can be seen to swell and become pale
and spherical. Neutrophils can be observed to
surround these damaged cells , strip away the
cytoplasm and c; r:i;i:ll the free nucleus td form LE
cells- Giemsa stained smears of blood or bone
marrow can demonstrate LE cells, but its sensitivity
is so low that thin test has been replaced by other
antibody tests for diagnosis.
Immunofluorcscent tests for anT : nuclear
antibody ( ANA ) show up different patterns of
staining, such as homogeneous (diffuse ), peripheral
(outline ), speckled and nucleolar staining patterns.
ANA tests are sensitive but not specific for SLE,
as they may be positive in many other autoimmune
conditions, viral infections, chronic inflammatory
processes, as well as in persons using certain
medicines and in the aged .
Anti - DNA antibodies are tested by R1A or
ELISA, Three major types of these antibodies are
—
Seen - those reacting with single stranded fss ),
1
double stranded (ds) and both ss and ds DNA. Of
these, high titre anti -ds DNA antibody is relatively
specific for SLE. Another SLE specific anti I tody
is anu-sm antilhidy.
Klicuiiinioid nnbrills: This is a svmmerric
•at
polyarthritis with muscle wasting and subcutaneous
nodules , commonly associated with scrositis,
myocarditis, vasculitis and other disseminated
lesions, h i -s found more commonlv in women. The
synovial membranes of the affected mints are swollen
and edematous , wirh dense infiltration of
Lymphocytes and plasma cells. A striking feature is
the presence of a circulating autoantibody called
the ' Rheumatoid factor' ( RE) . This is usually a 19s
IgM , though IgG , and IgA RF have also been
demonstrated - RF acts as an antibody agtirst the
Fc fragment of immunoglobulins. They combine
usually with IgG though some types of RF are
directed towards other immunoglobulin classes, FIE
reacts wirh autologous* isologous or heterologous
immunoglobulins. RE is generally considered to
be an immunoglobulin behaving as anti body to
determinants present in the patient's own I
molecules, though some configure - i "Hal alteration
of IgG may be required before its reactivity with
RF becomes demonstrable.
RF is detected by agglutination tests using, as
antigens , particles coated with globulins. In the
Rose-Waaler test, the original technique for
detection of RE, sheep erythrocytes coated with a
subagglm inaiiiig dose of and erythrocyte antibody
( amboceptor ) are used M the antigen in an
agglut i par ion test, J n modifications of the test, latex
and bentonite are used as the carrier particles for
lgG. Antinuclear antibodies are frequently found
111 rheumatoid arthritis.
Polyarteritis nodata: T h i i s a nccrotising
angiitis involving medium sized arteries , ending
fatal tv due to coronary thrombosis , cerebral
hemorrhage or gastrointesrinal bleeding .
Polyarteritis is seen as a component of serum
sickness and other toxic complex diseases. Immure
complexes of hepatiris B virus antigen ( Hbs Ag ) in
affected tissues, including the kidneys, have beer
—
demonstrated i n 30 40 per cent of patients, 11i i JLLi> h
ii has been suggested that polyarteritis nodosa may
be an autoimmune disease, the autoantibodv
responsible has not been identified .
Sjogren syndrome: This is a triad of
conjunctivitis ncca, dryness of the mouth, with or
without salivary gland enlargement, and rheumatoid
arthritis. The syndrome may occur in association with
other collagen diseases. Ar:i nuclear antibodies and
rheumatoid (actor commonlv occur in sera,
M
LJ opyrighted materia
 < Autoimmunity
TRANSITORY AUTOIMMUNE PROCESSES
1 bese include Lund it lorni such as anemia ,
thrombocytopenia or nephritis that follow certain
infections or drug therapy. The infecting agent oi
drug induces antigenic alteration in some self-
antigens. Thc imimine response set up causes tissue
damage. The disease is transient and undergoes
spontaneous cure when the infection is controlled
or the drug withdrawn,
PATHOGENESIS OF AUTOIMMUNE DISEASE
Manv diseases arc considered to be of autoimmune
origin, based on their association with cellular or
humoral immune responses against self-antigens.
Autoanribodics are more easily detected than cellular
autosensitisation. However, the mere presence of
autoantibodtes during the course of a disease does
not prove rheir etiological role. Autoantibody
formation may be a result of tissue injury Lind the
antibody may help in promoting immune
elimination of the damaged cell Of tissue elements.
A typical example is lepromatous leprosy in which
175
large amount? of uutoanrihodics are regularly found ,
It has been said that but for the lepra bacillus,
lepmmatnus lepreuy may have been proposed as an
autoimmune disease .
The relative importance of humoral and cellular
immune processes in the etiology of autoimmune
diseases is not known. Antibodies may cause damage
by the cytolytic or cytotoxic ( type 2 ) and toxic
complex ( type T ) reactions. They are obviously
important in hcmocytoljtic autoimmune diseases.
Another mechanism of autoimmune tissue damage
is by sensitised T lymphocytes ( type 4 reaction ). It
is likely that humoral and cellular i m m u n e
responses may act synerglsticady in die production
1' some a u t o i m m u n e disease;. For example ,
experimental orchitis can he induced o n l y when
both types of immune responses are operative,
O ncc in i ri atedl, m ost autoimmu nc responses tend
to be self perpetuating . Their progress can be
arrested by immunosuppressive therapy, though the
degree of response to such therapy varies in different
diseases.

I u rt h er It e ad i i\ £
'

^Etakman>MMandand] Stewart
\ fcirI
. 2
^
1997 hanwnohgy- 8 cdfL Edinburgh: Churchill Livingstone
.
:
.
U Vetgani 1947 Basic nd Clinical Immunology. Edinburgh Churchill Ljfingjtooe.
Rcitt J et al. 1999. Immunology. 5 " edrc London: Mosby.

Copyrighted material
 Immunology of Transplantation and
S3 Malignancy
IMMUNOLOGY OF TRANSPLANTATION
When, AS a result of disease or injury, nn organ nr
tissue becomes irreparably damaged , or when an
OJgan is congcnital.1 v detective or ahnent ,
transplantation nr grafting becomes necessary tor
the restoration of function. The tissue or organ
transplanted is known as the transplant or graft.
The individual from whom the transplant is
obtained is known as the donor and the individual
to whom it is applied , the reopjertr.
Transplantation Is one of mankind s ancient
dreams-. L’ himcras, fanciful creatures composed cit
parts from different species, figure in the mythology
jmd pantheon of all ancient nations . Such
transplantations across the species barrier, however,
do not succeed. It was recognised very early that
transplants survive onlv when the tissue or organ
is taker front the recipient himself while grafts
from another individual of rhe same species or from
n different species would he rejected - TTie earliest
application of transplantation appears to have been
skin grafting for reconstruction of the severed nose,
using the patient's own skin - a technique
described in the SirshrufiF iiFm/irfa (circa 800 RC ) .
T1 lu reasons fur the rejection of exogenous grafts
were tor long suspected to he due to active immunity
hut it was onlv in the 1940s that the work of
m isograft. Grafts made between identical twins
Mcdawar and bis colleagues conclusively proved
its immunological basis.
Cr ASS I FIXATION OF TnANSPl AMTS
Transplants mav he classified in various wavs:
1. Hascd on the organ or tissue transplanted, they
arc classified as kidney, heart, skin transplant .
and so mi .
.
2 LSascd on the anatomical sice of origin of the
transplant and the site of its placement , grafts
are class] tied JS ‘orthoto pic and 'heteroiopic .
1 1
Orthntopic grafts are applied in anatomically
‘normal' sires, as in skin grafts . J letcrotopie grafts
are placed in anatomically ‘abnormal' sites, as
when thyroid tissue is transplanted in a
subcutaneous pocket.
3. Transplants may he of fresh tissues and organs
or of stored ones.
4. Transplants may he of living or dead materials.
Live grafts, such as kidney or heart, are expected
to survive and function physiologically in the
recipient and are called \dtaJ grafts'. Nonliving
transplants like bone or artery merely provide a
scaffolding on which new tissue is laid bv the
recipient. They arc called ‘static or ‘structural’
1
grafts.
5. Transplants may be classified based on the
genetic {and antigenic ) relationship between the ,
donor and the recipient ( Table 20, l ) An organ
r
or tissue taken from an individual and grafted
on himself is an autograft. A gruff taken from
an individual and placed on another individual
of the same generic constitution is called an
or between ngeneic memhers of highly inbred
^
strains of animals are examples of isografts.
Grafts between rwo genetically non identical
members of the same species are called allografts
(formerly called hoinografts). Grafts between
members of different species are called
xenografts ( turmerlv called heterografts).
Copyrighted material
 < -
l nmunology of Transplanlalion and Malignancy »
TEH : AI LIJCJIAFT REACTION
When a skin graft from an animal ( such as a rabbit )
is Applied on a genetically unrelated animal of the
same species , the graft appears to be accepted
Initially. The graft is vaseularised and seems
morphologically and functionally healthy during
the first two or three days. Huwever, by about the
fourth day, inflammation becomes evident and the
graft is invaded by lymphocytes aitd macrophages.
The blood vessel;; within the graft are occluded by
thrombi , the vascularity diminishes and the graft
undergoes ischemic necrosis . With extending
necrosis, the graft assumes a scab- Like appearance
and sloughs off by the tenth day. This sequence of
events resulting in the rejection of the allograft is
known as the first scf response {also known as the
1 addpn vr umODdisacfon.
"first set rejection or reaction’).
Ift in an animal which has rejected a graft by
the first set response* another graft from the same
donor is applied , it will be rejected in an accelerated
fashion . VuculiriiiDOd commences hut is soon
interrupted by the inflammatory response . Necrosis
sets in early and the graft sloughs off by the sixth
day. The accelerated allograft rejection is known as
the second seT response.
Mechanism of allograft rejection: The
immunological basis of graft rejection is evident
from the specificity of the second set response.
Accelerated rejection is seen only if the second graft
is from the same donor as the first. Application of
a skin graft from another donor will evoke only
the first set responsc.
Table 20.1 Terminology o gratis
Donor
Self
Different individual genetically identical
with recipient . Identical twin or member of
same inbred strain
Genetically unrelated member of same
species
Different species
177
An allograft will be accepted if the animal is
rendered immunulogically tolerant. It suitable living
CCLLH (such an splenic cells) from one pure line strain
of animal are injected into fetal or neonatal animals
of another inbred strain, the latter, when they grow
up, will accept grafts from the former animal. This
is due to the induction of specific immunological
tolerance against the donor tissues as a result of
contact with them during embryonic life . The
tolerance can be abolished by injecting lymphocytes
from a non tolerant syngeneic animal , or more
effectively, from a syngeneic animal sensitised
against the donor tissues by a prior
allotransplantation . This method of transferring
immunity by means of lymphoid ceils is known as
'
Transplantation immunity is predominantly cell
mediated. The first set response is, brought about
almost exclusively by T LYMPHOCYTEH . Humoral
antibodies are also produced during allograft
rejection . They can he detected by a variety of
methods including hemagglutination ,
lymphocytotoxicity , complement fixation and
immunofluorescence . Antibodies are formed more
rapidly and abundantly during a second set response
than during primary rejection . Antibodies are
believed to participate in the second set response
along with cell mediated immunity. When a graft
iv applied to an animal possessing the specific
antibodies in high titrts hyperacute rejection takes
3
place. The graft tern airs pale and is rejected within
hours without even an attempt ac vascukrisation ,
Name Synonym*
Autograft Autogenous or autogenic graft.
Isograft Isologous or syngeneic graft or
syrt graft
AHognfi Allogeneic graft. Formerly called
homograft
Xenograft Xenogeneic. Formerly called
heterograft
Copyrighted material
 173 * Te? j< ihao *
This is knwn as the ‘white grift response'. This
type of hyperacute rejection is sometimes seen in
human recipients of kidney transplants, who may
possess preexisting antibody as a resnJt of prior
transplantsiortr transfusion or pregnancy. The
glomeruli in such cases are choked by platelet and
leucocyte agglomerates . Donor specific blood
transfusions to recipients before kidney Transplants
have been found to favour graft survival. This may
he due to the enhancing effect of antibodies to
mismatched donor antigens , induced by the
transfusion.
Humoral antibodies may someumes act in
ppositK m to cell mediated immun i iy, by inhihit 11 Lg
graft rejection . This phenomenon , called
immunological enha /t cement was originally
described hy Kabss in tumour transplants. If the
recipient is pnetreated with one or more injections
of killed donor tissue and the transplant applied
subsequently, it survives much longer than in
control animals. The enhancing effect can be
passively transferred to normal animals by an
injection of serum from immunised animals ,
showing that the effect is due to humoral antibodies.
The antibodies may bring about the enhancing effect
in various ways . They may combine with the
antigens released from the graft so that they arc
unable to initiate an immune response {afferent
nth 11 L' ion ). The antibodies may combine with the
'
lymphoid cells of appropri . Lte specificity .md, by a
negative feedback influence, render them incapable
of responding to the antigens of the graft {central
inhibition ) . They may also cause 'efferent
inhibition * hy coating the surface of cells in the
graft so that sensitised lymphocytes are kept out of
contact with them.
-
Allograft immunity' 1 a generalised response
directed against all the antigens of the donor. A
-
-
ieciu i L:I L sen ^ itised by a ho L graft wi II reicct by the
second set response not only another skin graft but
also any other organ or [issue graft from the same
dim or.
of Microbiology *
I llSTi I C O M I ' VTIBII.I T Y A v j T I U E P J ^ i
Immune response against transplants depends on
the presence in the grafted tissue of antigens that
are absent in the recipient and hence recognised
as foreign . It follows, therefore , that if the
recipient possesses all LILC antigens present in the
graft , there will he no immune response, and
consequently no graft rejection, even when the
donor and recipient art nut syngencif . The first
generation { Fl ) hybrids between two inbred
strains possess antigens representative of both the
parent str .i i ns and will therefore accept grafts from
r iihtr of the parental strains. If the two parental
strains have genotypes AA and BB, respectively,
the Fl hybrid will be of genotype AB , It can
therefore accept tissues with genotvpc AA as well
as BB, as it possesses both alleles.Transplantation
in the reverse direct inn {from Fl to parent) will
not succeed as strain AA will react against antigen
B and strain BB against antigen A.
While transplants between members of a
JILLOILV inbred strain of animals are successful, an
,
except mr is seen when the donor is a male and
the recipient a female . Such grafts are rejected as
the grafted male tissue (XY ) will have antigens
determined by the Y chromosome which will be
absent in the female {XX) recipient. Grafts from
the female to the male will succeed - This
unilateral sac linked li i sti > i na > m patibil ity is known
as the EichwaldSilmseT effect
Antigens that participate in graft rejection are
called transplantation or h istocompatihiI i, ty
antigens. The blood group antigens are important
in transplantation. The term 'major
h ^TocoEnpatiliibcy system" refers to a system of
cell antigens that exert a decisive influence on
the fate of allografts. Major histocompatibility
systems have been identified in different species
,
~ El 2 in mice, AgB in rats, B in chickens, HI in
rabbits and DLA in dogs . The major
histocompatibility system in human beings is the
human leucocyte antigen { HLA ) system . A
Copyrighted materia
 i immunology of Transplaniaiion and Malignancy
description of the 11LA system is presented in
Chapter 15 .
FACTORS FAVOURING ALLOGRAFT
SURVIVAL
1- Next to AUO blood group compatibility;the most
importMt factor in allograft iLLrvival it- HLA
ciinipatihiiity This is Gated bv HLA typing and
tissue matching- III, A typing identifies the
ElLA antigens expressed on the surface of
leucocytes.
The standard typing method in the microcyte
toxidty test. Lymphocyte suspensions are added to
microwdls of tissue typing trays prtd.ispcnscd wirh
'
a panel of HLA typing sera, each containing
, L I loan til indies tO a specific HI , A antigen, and
incubated with complement. <..ZclU carrying antigens
corresponding to the J JLA antiserum arc killed by
complement mediated [nembrane damage. Tliese
can he detected by the addition dfcosin or trypan
hlue which stains only dead cells. The lymphocyte
is presumed to have HLA antigens corresponding
to the specificities of all the antisera that have
caused cell death , as indicated by the staining.
, ,
Antisera for HLA typing were original IT
obtained from nnultigravidae, placenta ] fluid and
from multiple blood Transfusion recipients, who
have antibodies against mismatched paternat nr
donor 11LA antigens . These arc now being replaced
by monoclonal antibodies .
More- discriminating molecular methods have
been developed for tissue typing. These include
restriction fragment length polymorphism (RFLP)
with southern blotting , and polymerase chain
reaction ( PCR ) amplification using sequence
specific primers.
OtlCe a Set of HLA compatible donors is
available (commonly; siblings of the patient ), the
best donors among them can be chosen by tissue
matching. This is done by the mixed lymphocyte
reaction or culture (MLR , MI .C ) . It depends or
the fact thatT lymphocytes in ai I ture, when exposed
to HLA incompatible antigens , will undergo blast
179
transformation, the intensity of the reaction being
a measure of the antigenic disparity between the
donor and recipient lymphocytes. The test, as
performed, is a one-way rest in which the donor
lymphocytes arc killed and only the recipient
lymphocytes ire permitted to be transformed in
response to the incompatible antigens on the donor
cells,
2 . As allograft rejection is an immunological
process, j /?imunosi! jppfcsL«jcinwL ]l inhibit ir . This
'
can he achieved in experimental animals by
neonatal thymectomy, chronic lymphatic
drainage or administration of ALS- proeedures
^
that will inhihit cell mediated immunity.
C Lin leal Transplantation employs a combi nation
of immunosuppressive drugs , including steroids,
anthiopiene and the fungal metabolite
cyclosporin A , which is currently the most
effective agent,
3, There appear to be certain privileged sites where
allografts arc permitted to survive, safe from
immunological attack . The fetus can be
considered an iotmitenne allograft as it contains
antigens which are foreign to tlie mother . The
reason why the fetus is exempt from rejection Is
not clear, though many explanations have been
offered. The placenta acts as an immunological
barrier by generating a hormone which is locally
immunosuppressive. Major histocompatibility
complex (MHC) antigens are present only in
low density on trophoblastic cells and the cell
membranes are relatively resistant to attack by
T or K cells. Antigen shedding by the fetus
blocks the aggressive T cells or antibodies by
an enhancement effect . An incomplete
mucopolysaccharide barrier rich in sialic acid
surrounds tire trophoblastic cells, protecting
them from cytotoxic lymphocytes , The high
concentration of alphafetoprotein in fetal blood
also may be a factor , as it has immunosuppressive
properties, which may protect the fetus against
itrnnurological dwmge from any TO sternal
leucocytes entering fetal circulation.
Copyrighted material
 ISO * Textbook of Microbiology
Anj site that is itn penetrable To
1
immunocompetent ceils (for examplef cartilage) is
an immunologically privLIegcd sire. Areas where a
lymphatic drainage system is absent such as brain,
hamster cheek pouch or ineffective such as testes
can accept allografts without rejection . Lack nt
vascularity at the site also prevents graft rejection.
This is the reason for the success of corneal
transplants .
-
GRAFT VERSI S - HOST REACTION
Gnift rejection in due to tln^ reaction of the host t[)
the grafted tissue (liost- versus-gnft response ). The
contrary situation, in which the graft mounts an
immune response against the antigens of the host,
is known as the graft-versus-hosi (GVH ) reaction.
The GVH reaction occurs when the following
conditions are present:
1. The grult contains immunocompetent T cells.
2- The recipient possesses, transplantation antigens
that arc absent iti the graft -
3. The recipient must not reject the graft.
Examples of situations leading to the GVH reaction
are:
a. Allograft in a recipient in whom Specific
immunological tolerance has been induced .
b, Adult lymphocytes injected into an
immunological tv deficient recipient . The
immunological deficiency may he due to
immaturity ( newborn ) or immunosuppression .
C - F, hybrid receiving a transplant from any one
parental strain.
The major clinical features of the GVH reaction
in animals arc retardation of growth , emaciation,
diarrhea, hepatosplenomcgaly, lymphoid atrophy
and anemia, terminating fatally. The syndrome has
been called runt disease.
IMMUNOLOGY OF MALIGNANCY
When a cell undergoes malignant transformation , it
acquires new surface antigens. It may al«i kwe some
normal antigens, This makes a tumour antigerucally
different from the normal tissues of the host. A tumour
can , therefore, he considered an allograft and be
expected to induce an immune response.
. CLINICAL EVIDENCE OF IMMUNE
RKSKONKE J N MALIGNANCY
Several clinical observations indicate the presence
of an immune response that prevents, arrests and
occasionally cures malignancies.
1 . Instances of spontaneous regression of established
tumours have been reported, especially with
neuroblastoma and malignant melanoma. On
the anjygy of the rede played by the immune
response in recovery from infections , it is
believed that recovery' from malignancy also may
represent an immune process,
2. Dramatic cures sometimes follow chemotherapy
of choriocarcinoma and Burkitt 's lymphoma.
Even a single dose of cytotoxic drug may, on
occasion, rcMih in n complete cure . Again, in
some types of rumours, such as hypernephroma
-
with pulmonary metastaae i, removal of the
primary tumour often leads to a regression of
the metastascs , These observations suggest that
once a large mass of tumour has been removed,
mopping up operations can be effected by the
immune process. The immune response appears
to be effective only when the tumour is below a
'critical mass'.
3. There is a higher prevalence of certain types of
Cancers observed unexpectedly al autopsy, than
,
rheir clinical incidence would suggest. This
indicates that the immune system is able to deal
with malignant cells as they arise and that only
some of them are able to overcome the defence
mechanisms and develop into clinical cancer,
4. Histological evidence of immune response
against malignancy is provided by the presence
of lymphocytes, plasma cells and macrophages
infiltrating tumours . The cellular response
resembles that seen in the allograft reaction.
Turnout? shewing such cellular infiltration have
a better prognosis than those that do not.
5. If the immune system plays a natural role in
Copyrighted material

* ImmurtO logy oF Transplaniaiion and Ma' ignancy
preventing tumour development , a high
incidence of malignancy should be expected in
immune deficiency states.This is indeed so. An
increased incidence of cancer, particularly
lymph ore titular malignancies, is found in
congenital immunodeficiency states, in AIDS
and in patients undergoing chronic
immunosuppressive therapy,
Tc MOUR ANTIGENS
Tumour antiijens arc antigens that are present in
malignant cells but absent in the corresponding
normal cells of the host.
Tumour specific antigens art present on the
membranes of malignant celts and induce an
immune response when the tumour is transplanted
in syngeneic animals. Such rumour specific antigens
which induce rejection of rumour transplants in
immunised hosts are termed tumour specific
transplantation antigens ( TSTA ) or tumour
associated transplantation antigens (TATA ).
In chemically induced tumours, the TSTA is
tumour specific. Different tumours possess different
TS FA, even though induced by the same
carcinogen. In contrast, the TSTA of virus induced
tumours is virus specific in thar all tumours
produced by one virus will possess the same antigen,
ever ifthe tumours occur in different animal strains
or species.
A second type of antigen is found in some
tumours . These are the fetal antigens which are
found :n embryonic and muiignant cells but not in
normal adult cells. The best known examples arc
alpha fetoprotein in hepatomas and the
carcinoembtyonic antigen found in colonic cancers.
Their m i l [ lies is represents a dedifferenti .LtJon of
malignant cells into more primitive forms.
The carcinoembryohK antigen is a glycoprotein
wh i ch can be detected i n the serum of many pat ic ots
With carcinoma of the Colon, particularly in the
presence of rnetastaSes. However, it also appears in
some other condiLions such as alcoholic cirrhosis,
and hcncc its diagnostic value i -. limited .
IP
181
AlphafetOpruteiu is an alphaglobulin srcietrd by
normal embryonic hepatocytes, Its serum level drops
sharply after birth and is hardly detectable in adults.
High levels arc present in hepatic carcinoma, in
which condition ir is of diagnostic value, Prostate-
specific anrigen (PSA ) has been used as a dignnstic
i ndicator for prostate cancer.
IuuUKfi RB&PONSB IN MALIGNANCY
Both humoral and cellular responses can be
demonstrated in malignancy. Anti-TSTA antibodies
can be demonstrated by indirect membrane
immunofluorescence. Delayed hypersensi r i v i fy to
turnout antigens can be detected by skin testing with
tumour cell extracts. Cell mediated immunity can P
be demonstrated by the stimulation of DNA
synthesis and lymphokiine production by the
patients leucocytes on exposure to the himour
antigens. The lymphocytes from the patients are
cytotoxic to the Cultured tumour cells.
Cell mediated immunity \$ believed to be the
mechanism of host defence against malignancy.The
humoral response may not be relevant, or miv even
be detrimental due to its facilitating tumour growth
by the process of enhancement.
IMMUNOLOGICAI SURVEILI ANCE
The concept of immunological surveillance had its
beginning in the observations of Ehrlich (1906) ,
It was revived by Lewis Thomas in the 195th and
developed by Burnet. It postulates that the primary
function of cell mediated immunity is to "seek and
destroyf malignant cells that arise by somatic
mutation. Such malignant mutations are believed
to occur frequently and would develop into tumours
but for the constant vigilance of the immune system.
Inefficiency of the surveillance mechanism, either
as a result of arcing or in congenital or acquired
immunodeficiencies^ leads to an increased incidence
of cancer.While this hypothesis is attract ive, it may
perhaps represent an ave r --- i.m p I i t’icaT it) n of a
complex ^ ituation -
If immunological surveillance iseffective, cancer
Copyrighted materia
 p
1E2 i lnxrbook O) M crobititofly
should not occur. The development of tumours
represents a lapse in surveillance. The mechanisms
of such lapses are not dear but several possibilities
have been suggested. Due to the very fast rate of
proliferation of malignant cells, thev may be able
to 'sneak through' before the development of an
effective immune response and once they reach a
Certain mass may be beyond the power o f
immunological attack . Circulating tumour andgens
may act as a 'smokescreen ', coaling the lymphoid
cells and preventing them from acting on the
tumour cells . The rumour antigens on malignant
cells may be inaccessible to sensitised cells, being
covered by some antigeidually neutral substance .
Humoral antibodies may / ause immunological
i melanoma has been reported to induce complete
1
enhancement, ‘Blocking activity has been
demonstrated m humoral factors. This may be due
to the circulating anLigen1 an Li body or antigen-
art ilwdy completes. Some rumours maybe of low
immunogeniclry or may form cytokines like
transforming growth factor (3 (TGF^ PI which
suppresses cell mediated ! mmunity.
lUMI NOTHERAPY O F CANCBA
Different approaches have been attempted in the
—
immunotherapy of -cancer passive, active and
adoptive immunotherapy, specific and nonspecific-
Passbe immunotherapy was the earliest method
of cancer immunotherapy. Antisera prepared by
i m mu n is ing an i mils wi th tumour b i opsy specimens
were used for the treatment of human cancer as
carlv as in 1#95. This method was found to be of
no use and therefore abandoned. A special type of
serotherapy has recently been found beneficial in
experimental tumours . Appropriate antisera that
possess 'deblocking activity in V itro have been found
1
IO Cause regress ion of tumours , apparently by
neutralising the circulating tumour antigens and
permitting the sensitised lymphocytes to act on
tumour cells . Monoclonal antibodies to tumour
antigens may play a mle 35 carriers in transporting
Cytotoxic or radioactive drugs specifically To the
tumour cells,
Specific acuvt immunotherapy by the in jeer ion
of tumour cell Vaccines' was tried carlv in last
a
century but was given up as unprofitable. The
method has been modi hied recently hy using purified
tumour cell membrane antigens and tumour cells
treated with neuraminidase to increase their
immunogenic potential.
Nonspec ific active immunotherapy employs
BCG and non 1 iv mg Cutyfitbactefium parvu m.
Mat he , the leading proponent of cancer
immunotherapy has reported very good results in
acute Irultem i a, fnliowi :ig combi ucd treatment with
BCG and allogeneic or autochthonous leukemia
blast cells . 1 ntralesional BCG in malignant
remission in a high percentage of pari curs [t has
,
also been used against mtradermaJ recurrence of
breast cancer following mastectomy.
Dimtrochlorobenxene has been tried in the
treatment of squamous and basal cell can-inomn of
the skin. Clucanh a pyran copolymer derived from
microorganisms , and levamisule , originally
Introduced as an anthelmintic, have been tried for
stimulating cell mediated immunity and
macrophage functions. Interferons have been
employed in the treatment of leukemias.
Specific adoptive immunotherapy has been
attempted with lymphocytes, transfer factor and
'immune RNA . The donors have been persons who
have been cured of their neoplasms or specifically
immunised against the patient 's tumour.
Lymphokine activated killer ( LAKJ cells obtained
bv treatment of the natural killer cells with
i nterkukin -2 have been found useful in the treatment
of certain malignandes1 such as renal carcinomas,
Immunotherapy is ineffective in the presence
of a large mass of tumour cells . Its role appears to
be more in getting rid of the residual malignant
cells after the gross tumour has been removed. The
best results in the treatment of cancer apparently
follow an integrated approach to therapy, employing
surgery, radiotherapy, chemotherapy and
immunotherapy.
opy righted material
 -
a liTBiiirirayrclatjy of Tf .inf.pi .inlfllion pnd Malignancy * 183

Further Reading
-
Barrett AJL 1987. G ^ft - wm - hns-T reaction a nrvkm J Royal Sot Med 80:368,
lilihfip JML 1987. THe molecular generic! of canter. SbiflC B JDfi.
^ ^-
Lachmann PJ ec aj. 1*393, CGLEWA} AjpCCtl of JjttrhirjWt y. 5* ecln Oxford; Blackwell .
Roitt 1 rt aJr 1998, / mmuncA y, 51* cdn. London; Mosby,
^
^
RtwnbCTE SA et d . 19#B. I^ fw tpfhMdn to immunotherapy of cancer. Anmlr Jnt Med lft8: 853.
Armrtrorig A, 200 L CelluLar immunotherapy for cancer. BMJt 323’12S9.

Copyrighted material
 Immunohematology

Blood has held a mysterious fascinat i on tor LLH from blood transfusion ? or mothers of infants with
the dawn of time. It was considered the essence of hemolytic disease. The main blood group system;
life and was believed to cure diverse diseases and with the dates of their discovery are shown below.
restore youth and vitality to the aged , Blood
transfusion has been attempted from very early antes ABO 1900 Duffy'
1950
bur such attempts were fruitless and often fraught MN 1926 Kidd 1951
with disastrous consequences. Blood transfusion P 1926 Dicco 1955
became sc ientitically feasible only after the discovery Rh 1940 Yt 1956
of blood groups by Landsteiner. Lutheran 1945 H 1962
In his original experiment, Landsteiner (1900) Lewis 1946 Do mbrock 1965
cross tested serum from himself and five of his Kell 1946 Colton 1967
colleagues against their red blood cells. Three
distinct patterns of agglutination were observed , Some antigens have been identified that occur
Cells which failed to agglutinate with any of the very rarely, being limited to certain individuals or
scrum sample? were des ignated group On while cells families.These have been termed 'private antigens .1

agglutinating in the two different patterns were

called groups A and B , respectively. The fourth
group AB was described later bv his pupils von
Dccastallo and Sturli (1902). In 1930, Landsteiner
was awarded the Xobel Prixe for his discovery of
human blood groups.
The ABO system is rhe most important of all
the blood group systems and its discovciy made
blood transfusion possible. No other blood group
antigens were discovered for rhe next 25 years. Using
rabbit antisera to different samples of human red
cetl*i Landste ii ier and Levine (1926) discovered the
MN and P antigens. Landsteiner and Wiener (1940)
rinsed rabbit and guinea pig antisera against Rhesus
monkey erythrocytes and tested them against human
red cells. This led ro the discovery of the "Rhesus
( Rh) factor^. Many more blood group antigens have
been identified subsequently, mostly hy studying
mtiboditS L I I patients who hid received multiple
ABO BLOOD GROUP SYSTEM
The ABO system contains four blood groups and
is determined by rhe presence or absence of TWO
distinct antigens, A and B , on the surface of
erythrocytes. Red cells of group A carry antigen A,
cells of group B antigen B and cells of group AB
have both A and B antigens, while group O cells
have neither A nor B antigen. The four group? arc
also distinguished hy the presence or absence of
two distinct isoantibodies in the serum.The serum
contains rhe isoantibodies specific for the antigen
that i s absent on the red cell The scrum of a group
A individual has anti - B antibody, group B has anu -
A and group O both anti-A and anti-B, while in
group AB both ami- A and am i-B are absent (Tabic
21.1 ).
Group A is subdivided into A1 and A 2-
Antiserum of group A agglutinates group Al cells

Copyrighted materia
 Immunohematology *
powerfully hut A2 cells only weakly. About StO per
rent of group A blood is Al and 20 per cent A2,
The subgroups of A antigen are represented ingroup
AR also. The recognition of group A subgroups
increases the number of ABO phenotypes from four
to si*; Al , A2 t B , A2B, A1B and O, Other A
suhgroups ( A3, A 4 , A5 ) have also been described
but they arc not clinically rdevant-
Blood group antigens are inherited according
to simple Mendelian laws . Their synthesis is
determined by allelomorphic genes A, B and O.
Genes A and B give rise to the corresponding
antigens, hut O is an JMORPH and dues nut produce
any antigen. The frequency of ABO distribution
differs in different peoples. Group O is the most
cummon group and AB the rarest. The ABO
distribution in Britain Is approximately 0-47 per
cent, A - 42 per cent , H - B per cent and AH - 3
per cent - In India, the distribution is approximately
- -
0 40 per cent, A 22 per cent, B - 33 per cent and
—
AR 5 per cent,
Anti-A and anti-B isoantibodies appear in the
serum of infants by about the age of six months
and persist thereafter . These are called ‘natural "
antibodies because they seem to arise under genetic
control without any apparent antigenic stimulation.
However, it is likely that they develop as a result of
unidentified environmental stimuli with the blood
group- like antigens present in bacteria or other
sources. Natural anti - A and anti - B antibodies are
IgAl saline agglutinating antibodies reacting
optimally between 4 'HT and 1.B °C but less active at
37 ' "C . Immune isoantibodics may develop following
A BO incompatible pregnancy or transfusion . More
Table 21.1 Dislribulion of ABO aniigens
Red cells
Group Afltjpen present Agglutinired by
serum of group
A A B, 0
B B A, 0
AB A arid Q A,B ,0
O None None
185
commonly they result from the injection of
substances containing blood group- like antigens,
such as horse serum or bacterial vaccin.es made
from media containing horse or hog extracts.
Immune isoantibodics are "albumin agglutinating'
IgG antibodies reacting optimally at 37 ‘t and
acting as hemolysins in the presence of complement.
They are clinically more important than natural
IgM antibodies and may cause more severe
transfusion reactions.
H uniigLti: Red cells of all ABO groups possess
a common antigen , the H antigen or H substance
which is a pieaireor for the formation of A and R
antigens, The amount of the 11 antigen is related
to the ABO group of the cell, group O cells having
the most and AR the least amount. Due to its
universal distribution , the H antigen is not
ordinarily important in grouping or blood
transfusion . Bhendc ct al {1952) from Bombay
reported the very rare instance in which A and B
antigens as well as the H antigens are absent from
red cells. This is known as "Bombay’ or OH blood.
Such individuals have anti- A, anti-B and anti - H
antibodies and their sen are i ncompatihle with all
red cells except of those with the same rare blood
group.
A, B and H antigens ate glycoproteins. They
are not confined to erythrocytes but can be detected
in almost all tissues and Quids of the body While
these antigens are always present in tissues, they
are found in secretions (saliva, gastric juice, sweat )
of only about 75 per cent of alt persons . Such
persons are called 'sccrctors' and those who lack
blood group antigens in secretions arc called
and antibodies In red cells and serum
Serum
Antibody pitsenr Agglutinates celt#
ofgroup
anti- B R , AB
anti- A A , AB
None None
Anti- A and anri-fl A , B, AJ3
Copyrighted materia
m * Textbook oJ Microbiology *

lnonsecretorsh , The secretion of ARH antigens in surface of erythrocytes:. Each agglutinogen is in turn
c&ncroUed by two allelic genes Se and ^c. Individuals .
m idc up of one nr morn antigens. Fisher, on the
who arc homozygous or heterozygous (or Sc arc other hand, postulated that Rh antigens are
secretory while rhose who are
iMQKCffetQCL
-
RC SC ;iie determined by three pairs. ol closely linked
allelomorphic genes , Cc , L> d and Ee . Every
A and El antigens are also found in certain individual possesses one member of each pair of
animals and pi Jilts. ITicy have Eieen extracted and ilieve j;eiiev derived from each parent. Each gene
purified commercially from the stomach of horses would be responsible for the production of a specific
and hogs. Blood group andbodio are al >o found in antigen* which could In: detected Eiy its Specific
some annuals. Substances spedfkiUy agglutinating antihody.
A or H antigens have lieen detected in HOfAt plants. The designations employed by the two systems
A potent anti A 1 agglutinin has been extracted tmm for the different Rh types arc as follows:
tWrvfnis bifltirux aru! ant ] Hfrom ( /fe.x eunj Kftus.
^
Blood group aggltitinins ol plant origin are known Fisher Wiener
Rh positive CDe Rhl
j -. icetins ' .
cDE Rh 2
cDe Rho
RH BLOOD GROUP SYSTEM CDE Rhz
Levine and Stetson { ] 939) demonstrated a new type Rh negative Cde rh/
of and body ni rhe serum of a WOtliatl who develo}>ed odE rh //
ede rh
severe re jet Lons- following trails! Uiion of her
CdE rhy
husband '1 ABO compatible bl 1 iod . Sh r had m-en tly
delivered a stillborn intacil with hemolytic disease. For routine purposes, the typing of pcfWOTS us
They suggested rh,u the woman may have been Rh positive or negative depends on the presence 01
sensitised by some antigen inherited by the fetus absence of antigen D ( Rim ) on red cells .and hence
from its father . [.arilsteiner aril Wiener ( 194!) )
identified iu the red cells ol the majority of persons
car lie accomplished hy testing with anti D {anti
RJL) serum. This is because D is the most powerful
-
tested, an arftigcn thatrac ted with rabbit antiserum RH antigen and acioun for die majority of
to Rhesus monkey erythrocytes. 'Ehis antigen was Rh incompatibility reactions. The distribution of
culled the ‘Rhesus' or Rh factor. The ‘new type o! 1
Rh positives difteni in different races. Among|jeople
antibody described by Levine and Stetson was of European descent , about 85 per Cent are Rh
identified as the anti-1th factor antibody. Wiener positive and 15 per cent negative. Among Indians*
and Rerers (1940) demonstrated anti-Kh antibody approximately 93 per cent arc Rh positive and 7
in some peisons who had received ABO compatible per cent negative.
transfusion. Levine and colleagues ( 1941) proved A variant of TJ is known as Du . fed cells of Du
that Rh sensitisation was the cause of hemolytic a
subtype react with some but not all tnti-D sera.
disease of the newborn. Though Du cells may nor be agglutinated by anti-
The Rh system is complex ami its study is 1 > scrj , they absorb the antibody on their surface.
complicated by the existence of two different The Du subtype can therefore be detected by
theories and nomenclatures for the genes und reacting reJ cell with anti - D Serum ami then doing
antigens . Wiener proposed that Rh antigens arc L direct Coombs test - For the purpose of blood

dete rm ined hy any one of several itllelk gene; which donation, Du cells are considered Rh positive. But
may appear at a single locus and govern the when it Du individual requires transfusion , it is
production of the appropriate agglutinogen on the advisable to use Rh negative blood hecause he is

Copyrighted materia
 * Immunobematolcgy
capable of being immunised by standard Rh positive
blond.
I’here arc antibodies in the
no natural unti ’ Rb
scrum. They arise only as a result of Rh
incompatible pregnancy or transfusion .
OTHER BLOOD GROUP SYSTEMS
The Lewis blood group system consists of two
antigens LeJ and Le\ It differs from other blood
group systems lit that the antigens ate preient
primarily in the plasma and saliva . Red cells acquire
the antigen by adsorbing them front plasma- The
Lewis phenotypes are doscly related ro the ABO
group and to the secretor status of an individual.
Narurally oreurring f ^wis antibodies arc frequently
found in the sera of persons lacking the
corresponding antigen.
In cbe MN system , using rabbit antisera, persons
were originally classified into three groups M, — cells may result. This is known as the 'dangerous
N and MN . An antigen, S , was later added to this
syitem. This system hte expanded to include at least
28 antigens.
Blood grump systems other than ABO and Rh
are of little clinical importance as they do not usually
cause transfusion reactions or kcmolvtic disease.
They have applications in genetics, anthropology,
tissue typing and torertsi-c medicine. As blood group
antigens are inherited from the parents, they are
often useful in settling oases of disputed paternity
MEDICAL APPLICATIONS OF BLOOD GROUPS
BLOOD TRANSFUSION
The existence of several different blood group
.mrigeni: makes it almost impossible to obtain
perfectly matched blood for transfusion. Bur in
routine transfusion practice, only the AEG and Rh
antigens are relevant . The other antigens ate H>0
weak to he of importance. Safety ill blood
transfusion requires that rbe following conditions
be satisfied LN choosing a donor
1. The recipient 's plasma should not contain any
anribody that will damage the dun.Or 's
erythrocytes.
187
2 . The donor plasma should not have any antibody
that will damage the recipient 's red cells .
!i. The donor tedcells should not have any antigen
that is lacking in the recipient . 11 the transfused
Cells posnevH a " foreign antigen', it will stimulate
an immune response in the recipient ,
ideally, the donor and recipient should belong
to the name ABO group. It used to be belt ] that O
group colls could be transfused to recipients of any
group as they possessed neither A nor li antigen.
Hence the O group wan- designated ;LH dlC 'universal
domr ' . The inti - A and snti - B antibodies in the
transfused O blood group do not ordinarily cause
any damage TO the ted cells of the A or U group
recipients because they will he rendered ineffective
bv dilution in the recipient ' plasma. But some O
*
group plasma may contain isoantibodics in high
titres ( 1 :200 or above) so that damage to recipient
O group'. Tlie ami- A antibody in the C group blood
is generally more potent than the anti - B antibody.
Hence the O blood group is more likely to cause
an adverse reaction when given to the A group
recipients than to those of tile .B group. While the
O group blood with low titre antibodies may he
transfused to a patient of any other group in dire
emergency, this practice should never be employed
as .a routine . Transfusion 0 I large quantities of the
O group blood to persons of any other group may
cause adverse reaction s-
Due ro the absence of isoatitibodies In plasma,
fhe AE group persons were designated 'universal
recipients' . A IS group donors may not always be
.
available -due to their rarity and ir may on occasion,
be necessary to use donurn of other groups. In Such
cases, group A blood is safer than group B , because
the anti- A antibody is usually mare potent than
the arti - B antibody.
Rh compatibility is important only when the
recipient is Rh negative. An Rh positive person
may safely receive cither Rh positive or negative
blood. But an Hh negative individual receiving Rh
jjosiTive blood may form antibodies against the Rh
Copyrighted material
IBS * TewrtJCIJk d Mir.ro biology
-
antigen. A subsequent tTunsfu .iun wilh Rh p< reitiv(
blood mav then cause an advene reaction . An
additional risk m women is Rh sensitisation le , LLLING
tci hemolytic disease of the newborn. Therefore it
is particularly Important that Rh negative women
who arc not past childbearing age receive only Rh
negative bloi >d .
Besides ABO grouping and Rh typing of the
donor and recipient, it k Lnvari.i.hiy necessary heftire
transfusion to perform a 'cross matching? to ensure
1 k . n the donor 's blood is compatible with the
recipient' blood. The routine procedure used in
most blood b?nk^ LS a rapid cross match by the tile
-
or slide method . Thi is done in two parts - the
major cross match where the dunor red cells are
tested against the recipient's scrum , and the minor
cross match where the recipient’s cells are rested
against the donor serum . One drop of a
suspension of donor red cells In saline is added to a
drop of the recipient's scrum on a porcelain tile or
a glass slide, mixed and observed for agglutination.
Though in most cases agglutination occurs cariy,
it mav4 sometimes he delayed 4
. 'lire result is to be
read , microscopically and under low power
micTuscope , after incubation in a moist chamber
—
for 10 15 nii notes at room temperature. In the
minor cross match , the same is repeated using
reel|'Lent cells and donor serum. Only the major
cross match is done ordinarilv, !
J
The saline slide test does not detect Rh and
o t h e r m i n o r incompatibilities . The most
discriminating method is the Coombs cross match
where washed donor cells and recipient sen mi are
incubatec in a water bath at 37 *C for two hours
and a direct Coombs test done. This detects all
incompatibilities, including incomplete annbotues.
Following an incompatible blood transfusion,
the red cells may undergo dumping and
intravascular hemolysin or they may be coated hy
antibodies, engulfed by phagocytes, removed from
circulation and subjected to cxtravascular lysis..
Incompatible transfusion may be accompanied by
symptoms Such as shLuring , tingling sensation ,
excruciating headache, constricting precordial
discomfort and severe lumbar pain . Hypotension,
cold clammy skin, cyanosis, feeble pulse and other
signs of collapse mav be seen. Jaundice, hematun.L,
oliguria and anuria may follow.
Some transfusion reactions may be due to
immunological processes other than blood group
incompatibility. Rigor, urticaria and other
manilestanons often occur due to the recipient bring
hypersensitive to some allcigcn present in the donor
blood. Serious reactions follow when hemolysed
or contaminated blood is transfused.
Whenever any react Lon OCCLITT, the transfusion
should be stopped immediately. The remainder of
the demur blood should be sent to the blood bank
for investigation .
The most common complications following
blood transfusion are of infectious origin .
Transfusion of blood contaminated by bacteria may
Lead to cndotnxiv shock or septicemia . Gross
contamination can be recognised in most cases by
inspection of the blood before transfusion* as
hemolysis is usually apparent. Such contamination
can be eliminated by proper Techniques of blood
collection .MLJ storage .
The most important infections transmitted at
present by blood transfusion are rhe HIV and
bepatiri '- viruses- Several cases of transfusion*
induced AIDS have occurred before HIV screening
of donors became mandatory. However, screening
may rot detect H ) V infected donors during the
window period when they are infectious. Hepatitis
B, C, D and possibly others can be transmitted by
transfusion. Screening for the hepatitis B surface
antigen can exclude most HBV carriers but the
available serological tests against other hepatitis
viruses are not quite satisfactory.
Despite diligent Screening, there exists a small
risk (about 1 in 300,000 ) risk of transfusion
associated HIV, HBV and HCV infections. The
vari . mt CJD fmon IS another risk in endemic arras
like the UK where it is mandatory to screen donors
fbr the prior and to remove leucocytes from blood
Copyrighted materi3
 * Immure hematology
before nan* fusion, as a precautionary measure.
Cytomegalovirus transmitted by transfusion may
cause in infectious mononucleosis-like svndromc.
-I
S>phili s may be transmitted by transfusion of fresh
blood from an infectious donor but not if the blood
tins been stored fot three days or more before
transfus ion, Malaria is another disease transm issiblc
by transfusion.
Whole blood transfusion is being replaced
increasingly by blood component therapy which
causes fewer com plications and results in more
npti m al uti I isa rion of hu man blood wh ich is a scarce
commodiry. For example, in anemia , packed red cell
transfusion is more beneficial than whole blood LLLi
it provides greater oxygen carrying capacity' with
less circulatoiy overload and minimal clecrrolytc
disturbance . Frozen red Cel is are available for
transfusion in patients with rare hi nod groups.
Similarly leucocyte and platelet concentrates are
available for specific needs. Plasma crynprecipi Cates
and Factor VII1 are routinely used in treating
hemophilia, Other coagulation factors are also
available for different coagulation disorders. Such
plasma products are manufactured from pooled
human blood and may transmit 11J V, hepatitis type
11 or C virus and other infectious agents unless great
care is taken in the selection of donors and in
manufacturing processes , An illustration of the
enormity of the risk was the tragedy in France in
the mid-1980s, when tainted blood caused HIV'
infection in over 4000 persons., of whom hundreds
died of AIDS in rhe next few years. Charges of
aHminai negligence were framed against the persons
considered responsible, From the Prime Minister
downwards. Though the Prime Minister was
acquitted, the Health Minister, the Head of the
National Blood Transfusion Service and many other
top officials wete cortvicted -
Almost ail adverse reactions of transfusion can
be eliminated by autologous blood transfusion
which Is rapidly becoming popular , Here, blood is
collected from rhe individual himself and stored
for use during elective surgery. Blood collection
m
may be started a month before the expected date of
transfusion. Autologous transfusion eliminates not
only infectious complications but also those due to
minor blood group incompatibilities and
hypersensitivity .
HEMOLYTIC DISEASE OP THE
NBVBOftN
When an Rh negative woman carries an Rh positive
fetus, she may be sensitized against the Rh antigen
by the passage of fetal red crib into the maternal
circulation . Minor transplacental leaks may occur
any lime during pregnancy but it is during delivery
that fetal cells enter the maternal circulation in Ljige
numbers . The mother is usually sensitized only at
the first delivery and, consequently, the first child
escapes damage ( except wllere the woman has been
sensitised already by prior Rh incompatible
transfusion ) . During a subsequent pregnancy. Rh
antibodies of the IgG class pass from the mother
to the fetus and damage its erythrocytes. This is
the pathogenesis of the hemolvtic disease of the
newborn , The cLin ical features may vary from a mere
accentuation of the physiological jaundice in the
newborn to erythroblastosis fetalis or intrauterine
death due ta hydrops fetalis.
Hemulvtic disease docs not affect all the
offspring of Rh incompatible marriages, Its
incidence is much less than the expected figures.
The following fucrors influence the incidence of
hemolytic disease due ro Rh incompatibility:
rmmunuln ical unreispomiveneu to the
^
Rh uiitigeiu Mot every Rh negative individual
forms Rh antibodies following antigenic
stimulation. Some Fail ro do so even after repeated
injection qf Rh positive cells. Thev are called
noniesponders / J lie reason for this immunological
unrcsponsivicnefts is not known.
1' etorunlcrnnl ABO incompntihility: Rh
Immunisation is more likely to result when the
mother and fetus possess the same ABO group.
When Rh and ABO incompatibility coexist, Rh
sensitisation in the mother is rare , In this situation
Copyrighted material
190 ^ Texlbook ol Microbiology *
the fetal cells entering the maternal circulation are
believed to be destroyed rapidly by the ARO
antibodies be tore thev can induce Rb antibodies
Number of pregnancies: The first child
usually escapes disease because sensitisation occurs
only during its delivery. The risk t o the icitaiU
increases with each successive pregnancy.
Zygosity t> f the father: All individual may-
be liomorygpus or heoeroiygoLis with respect
to [} antigen. When the tathcr in hurt ' > £Yg( Jus
all his children will be Rh positive . When he
is heterozygous , half his children will he Kh
positive
rbc fetus is Rh positive. In the case of hemolytic
disease oi the newborn, the maternal serum will
show Rh antibodies in the indirect Coombs test
and T1IL infantk red cells wQl give a positive m the
direct Conmhs test.
When hemolytic disease is diagnosed
antepartum , an intrauterine transfusion with Rh
negative blood may be indicated Red cells
introduced Into the fetal peritoneal cavity will find
their way into the circulation and will survive
normally. Rremature delivery followed by
transfusion may he necessary in some cases . When
(i baby i $ horn with hemolytic disease, exchange

transfusion with Rh negative ABO compatible

DETECTION OP Rh ANTIBODIES
Mod Rh antibodies are of the igt i class, and being
'incomplete antibodies’, they do not agglutinate Rh
positive cells in saline . A minority are complete
(saline agglunnating ) antibodies of the IgM class
These are not relevant in the pathogenesis of
hemolytic disease as they do not traverse the
placenta.
IgtJ anti - D antibodies miybe dcicered by the
following techni ucy: ( I ) using a colloid medium
^
such H 20 per cent bovine strum albumin ( 2 ) using
red cells treated with enzymes such as trypsin ,
pepsin , fidn or hromdin; and (3) by the indirect
Coombs test. The last is the moM sensitive method.
IDENTIFICATION OF Rh
INCOMPATIBILITY
Rh typing should :urm a part of routine antenati'll
cxaminiirinn . When rhe woman is Rh negative , and
her husband Rh positive, fecal complication's should
be expected . Women with Rh incompatible
pregnancies should be screened for Kh antibodies
-
hy the indirect Coombs test at 32 34 weeks of
pregnancy- and at monthly intervals thereafte r. The
appearance of Rh UOIIIMHIIC ^ during pregnancy or
their increase in titre, if they were present already,
would prove that the Ictus is Rh positive. If
amniocentesis is indicated demonstration of Rh
, A HO hemolytic disease is due to naturally occurring
antigen in the amniotic fluid would also prove that
blood is the treatment of choice.
PREVENTION OK Rh ISOIMMUNISATION
Remarkable success lias been achieved in the
. prevention of Rh isoi mmuimarion by the
administration ofanti- Rb lgG antibody at the time
when fhc antigenic stimulation is expected to take
place . The passively administered antibody may
prevent isoimmunisation by a negative feedback
mechanism or bv afferent inhibition . The
recommended practice is to inject 10D-300 Jig of
Hh immune BgG to (in Rh negative woman
immediately after delivery. To be effective, this
should be employed from the tint delivery onwards.
The Rh i m m u n e globulin for the purpose is
prepared from human volunteers.
AliO HEMOLYTIC DISEASE
Marernofctal ABO incompatibility is very common
and in a proportion of these, hemolytic disease
occurs in the newborn . In persons of blood group
A or Bj natural antibodies arc IgM in nature and
so do not cross the placenta to harm the fetus.
However, in persons of blood group O rhe .
isoantibodies are predominantly JgG in nature.
Hence ABO hemolyTic disease is seen largely in O
group mothers, bearing A or B group fetus. As
maternal isoanribodics, it may occur even in the

Copyrighted material
 * Imfnunoh&malolagy » 191

firstborn* without prior immunisation. AIJO
hemtdytic diseav? is much milder than Kh disease,
probably because erythrocytes of the newborn ham:
fewer A or ll antigenic sites; as tumpiLred to adult
erythrocytes. The direct Coomhs Test is therefore
often negative in this condition, while the indirec t
Coombs test ( neonatal serum with type specific
adult erythrocytes ) Ls more commonly positive.
Pettphnral blood smear characteristically shows
Spherocytosi H.
BLOOD GROUP AND DISIMSI S - -
It has been shown that some diseases may influence
blood group antigens. Blood group antigens have
been Imported CO become weal; in leukemia. The
reason for this is nor known- The acquisition of H
antigens by Group A perrons his been observed
following some infections. The antigen is believed
to oume hom the infecting microorganism.
Red cell suspensions contaminated with certain
bacteria, such as Pseudomonas aeruginosa,
become agglurinablc by .ill blood group sera and
even by normal human sera. This phenomenon,
IqlOWtl as the 77ionFsiejT— f ' renirirmchphcnamcium ,
is due to the unmasking of a hidden antigen normally
present on ill human erytbnocjtCS , This is called
the T antigen. Anti -T agglutinins art normally
present in human sera . Such panagglutiiLability cl
red cells has occasionally been observed in persons
suffering from systemic bacterial infections .
Several investigators have attempted to correlate
blood group and susceptibility to certain diseases.
It has Keen shnwn that duodena) ulcer is more
frequent in persons of blood group O than in others.
An association has also been established between
group A and cancer of the stomach.

Further Heading.
Andtrsun K< nj PM Nesi eds. 1994. SdatiiiC Bitit uflrajw/usunn MttUcirte - Philadelphia; S^undeft -
*
Sdtfaher OB el aL. 1996. [he rixL tvf tr iLifiixiinn- tc? iL»iiiLt ( cd IITIL infocticmi. Afeir Pnfff / MpJ!
"
. S 4c1Ht5.
^

Copyrighted material
 CM Staphylococcus
CM
Staphylococci arc Gram positive cocci that occur
in grape-like clusters. hl hey are ubiquitous and form
"
the mo*t common cause of localised suppurative
lesions in human beings. Their ability ro develop
resistance penicillin and other antibiotics
enhances their importance as a human pathogen,
especially in the hospital environment.
Staphylococci writ first observed in human
pyogenic lesions by von Recklinghausen in 1371.
Pasteur ( 1880) obtained liquid cultures of the cocci
from pus and produced abscesses by inoculating
them into rabbi ts. It was Sir Alexander Ogstun, a
.
Scottish surgeon who established conclusively the
causative role of the coccus in abscesses and other
suppurative lesions ( 1880 ). He also gave it the name
Staphylococcus ( Staphylc , in Greek , meaning
‘bunch of grapes'; kokkos, meaning a berry ) due to
the typical occurrence of the cocci in grape-like
dusters in pus and in cultures . Ogstort noticed [ hat
norvirulcnt staphylococci were also often present
on skin surfaces.
Most staphylococcal strains from pyogenic
lesions were found to produce golden yellow
colonies, anti the strains front normal skin , white
colonics on solid media . Rosenbach (1884) named
them Staph . aureus and Sraph . albus respectively.
Passet ( lkS5 ) described a third variety, Staph.
c/tFRH, producing lemon yellow colonics. However,
the association between virulence and pigment
production was not found to be constant.
Many other properties were proposed as
indicators of virulence. These included hemolysis,
gelatin liquefaction , Lipolytic activity' produedon of
urease, phosphatase and others, but none of these
was found reliable. The most constant association
was between virulence and production of the e nzvme
.
coagulase and to a lesser extent fermentation of
mannitol . Staphylococci were therefore classified
into two groups; Staph , aureus (formerly also called
Sraphpyogenes) containing scrams giving i positive
coagulate test , fermenting mannitol and usually
being pathogenic, and Staph - cpiiicrmkiis{formerly
also called Staph . dhirr) containing coagulase
negative , mannitol non fermenting and usually
nonputhogeuic strains. The genus Stapbpiocoecus
is now classified into 32 species and 15 subspecies
based on the chemical compoiition of their cell
wall components and other properties . Besides
Staph, jurrus, two cuagulaae negative species -
Staph epidermidis. Staph , baemohlraut and Stsph.
,
saprophyticus - can also cause human disease. Some
other coagulase negative species such as Staph ,
homink and SupA. capitus are part of the
LOrnoiensal flora of the human skin. Other species
arc parasitic on animals.
&TAPHVL.OCQCCUS AUREUS
Morphology: They are spherical cocci ,
approximately 1 Jim in diameter , arranged
characteristically in grape-like clusters ( Fig. 22.1).
Cluster formation is due to cell division occurring
in three planes, with daughter cells tending to
remain in close proximity. They may also be found
singly, in pains and in short chains of three or four
cells* especially when examined from liquid culture .
Long chains never occur. They are nonmotile and
noftfiporiilg. A few strains possess microscopically
visible capsules, particularly in young cultures .
Many apparently noncapsulated strains have small
amounts of capsular material on the surface. Thcv
Copyrighted material
 H StapMyloccccus
* 193

stain rcudlty with aniline \ and urc uniformly

Gram positive . Under the influence of penicillin
.
and certain chemicals they may change to L Forms,
c LITRLIR ;iI vhqtiictcffistic >ti They grow readily
i
^
)!ii nntltnarv media wlcliin a temperature range u-t

10-42 '’C, the optimum hclrg 37 UC and pH 7.4-

7.6 . They are aerobes and Facultative anaerobes.
On nutrient agar, aFlt'i incvblMD tor 24 hours ,
the colonies are large { 2- 4 mm diameter ), circular ,
cotnTV, smooth, shiny, opaque and easily

cmulsitiablc . Most strains produce golden yellow

pigment, though some may be white, orange or
yellow, The pigment does not diffuse into the
medium. Pigment production occurs optimally at
22 °C and only in acrohu- cultures , Pigment
production is enhanced when 1% glycerol
muTH laretatr or in ilk is l ncorpora ted i n the medium .

The pigment is believed to he a Lipoprotein allied

TO carotene.
On nutrient agar slope, [he confluent growth
presents a characteristic ' 0Ll- p.1 i.11t ' appearance. The
colonies on blond agar are similar to those on
nutrient agar. Most strains are hemolytic, especially
when incubated under 20-2 carbon dioxide ,
Hemoh'sis is marked on rabbit or sheep blood and
weak on horse blood agar.
They grow on MicConkev 's medium ,
producing smaller colonies that art pink due Co
1
lactose fermentation.
In liquid media , uniform turbidity is produced.
Several selective media have been devised for
isolating Staph. aureus from siiecbmens such as feces
containing other bacteria . rl he sc include media
containing 8-- 10 per cent Nad ( salt - milk agar, saEt
broth), lithium chloride and tellurite (Ludlam' s
Fig. 22.1 Staphylococcus in a smear of pus
ams but nut by other species. They are catalase
positive (unlike streptococci ) and usually hydrolyse
urea, reduce nirratrH tn nitrites, liquefy geLatin and
are MR and VP positive but indole negative. MOST
strains are lipolytic and when grown on media
containing egg yolk, produce a dense opacity.
Production of phosphatase can he demonstrated hr
culturing or nutrient agar containing
phenolphthatein diphosphate , VVhen such a culture
is exposed to ammonia vapour, colonies assume a
bright pink colour due ro the presence of free
pheiiolptirhalcin . This \ * a useful screening
procedure for differentinting Staph aureus from
,

medium), and pcblvrnyxin . For primary isolation,
sheep blood agar is recommends! Human blood
should not be used ns it may cntitam antibodies or
other inhibitors.
Biochemical react M i n s , They Ferment a
number of sugars, producing acid bur no gas. Sugar
ferine nratjon is > f no diagnostic value except for
[ clear hemolyris on blood agar; ( 4 ) produce a
mannitol , which is usually fermented bv Stitph.
Staph epidcrmkfa in mixed cultures, as the former
gives prompt phosphatase reaction , while the latter
is usually negative Or only weakly positive .
Staph- aureus strains usuallv exhibit the following
characteristics: (1) cnagulase positive; ( 3) greater
biochemical activity, ferment mannitc ; (3 ) produce
golden yellow pigment; ( ) liqucfr gelatin;
^
194 * Textbook ot Microbiology
( 6 ) produce p h o s p h a t a s e ; ( 7 ) i n a m e d i u m
_
containing potassium :i.]l LI r LI > , reduce tellurite to
form black colonics; and (8) produce thermostable
nucleases which can be dem < mstraied by tin: ability
of boiled cultures to degrade DNA in an agar
diffusion test.
Reautance Staphylococci are among the more
resistant of nnnsporing bacteri .L. Dried on threads,
they retain their x iability' for 3^6 months. They
^
have been isolated from dried pus after 2-3 months.
They may withstand 60 °C for 30 minutes. Their
r
II
thermal death point is 62 °C for 30 minutes. Some
staphylococci require 80 DC for one hour to be
killed . Heat resistant strains have the ability to
grow at a higher temperature, even aE 45 C . Most
strains grow in the presence of 10% NaCt and some
even in 15% NaCI , These features are of
significance in food preservation.
They resist 1% phenol for 15 minutes . Mercury
perchloride 1% solution kills them in 10 minutes.
Many aniline dyes are strongly bactericidal crystal
violet being lethal at a concentration of one in live
hundred thousand and brilliant green , one in ten
million.
Fatty acids inhihir the growth of staphylococci,
the highly unsaturated acids having a more
powerful action on cnigulasc positive than on
coagulasc negative strains. Staphylococci arc
uniformly resistant to lysoiyme but some
micrococci arc sensitive to it. Staphylococci are
—
gencrallv sensitive to Ivsostaphin q mixture of
enzymes produced by a particular strain of Staph ,
cpidcrnudss.
Staphylococci were uniformly sensitive to
penicillin originally though occasional strain - from'
the preantibiotic era have been found retrospectively
to he capable of producing penicillinase. Soon after
penicillin came to he used clinic ally, resistant strains
began ro emerge, first in hospitals and then in the
community at large.
ftmicillin resistance is of three types
1 . production of beta lactamase ( penicillinase )
wh i ch i nactivatcs peniciLlin by splitt i ng the beta
lactam ring. Staphylococci produce four types
of penicillinases, A to D. 1 Josp ital strains usually
form type A penicillinase. Penicillinase is an
inducible enzyme and its production is usually
controlled by plasmids which are transmitted
by transduction or conjugation . The same
plasmid may cany genes for resistance to a range
of other antibiotics and heavy metals.
2- Changes in bacterial surface receptors, reducing
binding of beta-lactam antibiotics to cells. This
change is normally chromosomal in nature and
is expressed more at 30 UC than at 37 ^ C . ThiS
resistance also extends to cover beta lactamase
resistant penicillins such as methicillin and
cloxacillins. Some of these strains may show
resistance to other antibiotics and heavy metals
B
also and cause outbreaks of hospital infection .
These strains have been called 'epidemic
methicillin resistant Staphylococcus atireu ot
EMRSA (as metiiii.ilLin is ail unstable drug,
^
cloxacillin is used for sensiri vity testing I it -4ead ).
3. Development of tolerance to penicillin, by which
the bacterium is only inhibited but not killed .
Staphylococci also exhibit plasmid-borne
resistance to erythromycins, tetracyclines.,
aminoglycosides and almost all clinically useful
antibiotics except vancomycin ,
lithogenicity and
Staphylococci produce two types of diseases
virulence:
infections and intoxications. 1 it the former the cocci
gain access to damaged skin, mucosal or tissue sites,
—
colonise by adhcruig to cells or extracellular matrix,
evade host defence mechanisms, multiply and cause
tissue dainage - 111 intoxi cations, thedisease i-s caused
by the bacterial toxins produced either in the
i nfectcd host or preformed in vitro, A number of
staphylococcal factors, both cell associated and
extracellular, have been identified, which may
influence virulence . However, apart from the
cmtnxins which cause specific ctinical syndromes,
no other factor has a decisive role in pathogenesis.
The virulence factors described include the
following:
Copyrighted materia
 * Staphylococcus *
CELL ASSOCIATED POLYMERS
1. The cell wal] polysaccharide peptidoglycan
confers rigidity and structure! integrity to the
bacterial cell. It Activates complement and
induces release of inflammatory cytokines,
2. Tcichoic acid , an antigenic component of the
cell wall facilitates adhesion of the cocci to the
host cell surface and protects them from
complement-mediated opsonisation .
1. Capsular polysaccharide surrounding the cell
wall inhibits opsonisation .
CEI L SURFACE PROTEINS
1, Protein A prese nt on most Sfapb. iiireus strains
has many bio Logical properties , including
che mo tactic , antiphagocytic and anti -
compkmentary effects. It also induces platelet
damage and hypersensitivity. Protein A binds
to the Fc terminal of IgG molecules (except
IgGJ ), leaving the Fab region free to combine
-
with its specific antigen . Protein A bearing
staphylococci coated with any IgG antiserum
will be agglutinated if mixed with its
corresponding antigen . This procedure known
as Cnagglu h nation has many up I i cations, as for
streptococcal grouping and gonococcal typmg -
Protciii A is a 11 cell mitogen. It has also been
used as a ligand for isolation of IgG.
2- Clumping factor, another surface protein is the
“ bound coagulase' which is responsible for the
‘slide onagulaae ' rest. When a saline suspension
of Staph , jureus is mixed on a slide with a drop
of human plasma the cocci arc clumped. The
slide coagulase test is routinely used for the
identification of Srajih . isolates ,
unreins
Capsulatcd strains may sometimes show a
negative test because the dumping factor may
be enveloped by the capsutar polysaccharide
EXTRACELLULAR ENZYMES
1 . Coagulase is an enzyme which hrings about
dotting of human or rabbi r plasma. It acts along
195
with a 'coagulase reacting factor' (CRF) present
in plasma , hinding to prothrombin and
converting fibrinogen to fibrin. Coagulase does
not clot plasma of guinea jugs and some other
species because they lack CRF- Cflldum or other
dotting factors are not required for coagulase
action. Eight types of coagulase have been
identified. Most human strains form coagulase
type A, Coagulase and clumping factor ( the so
called ' boundcoagulase') differ in many respects.
Coagulase is an enzyme secreted into rhe
medium. It requires rhe cooperation of CRF for
its action - Bound coagulase in a beat stable
constituent of the cel ] surface and its action is
independent of CRF. Only one type of clumping
factor has been identified . Staph a uncus strains
,
usually form both coagulase and clumping
factor. Coagulase test is the standard criterion
for the identification of Sfcfph , aureus isolates,
2. Lipases. Staphylococci produce n. number of
lipid hydrolases which help them in infecting
the skin and subcutaneous tissues ,
d, Hyaluromidase breaks down the connective
tissue. Staphylnkimse (fibrinolysm ), farcy acid
modifying enzymes and proteases help in
initiation and spread of infection.
4. Nuclease. A heat stable nuclease is a
.
characteristic feature of Staph aureus.
5. Protein receptors . Staphylococci possets
receptors lor many mammalian proteins such
as fibronectinj fibrinogen, IgG and Clip These
facilitate staphylococcal adhesion to host cell
and tissues.
TOXINS
Cytolytic toxins: Cytolytic toxins arc
membrane - active substances, consisting of four
hemolysins and a leucocidin.
Alpha hemolysin ( Alpha toxin, lysin ) is rhe
most important among them - It is a protein
inactivated at 70 DC, but rcactivarcd paradoxically
at 100 °C. This is because at 60-70 °C, the toxin
Copyrighted materia
19 ft * extdc c k
"
--
Combines with 2. heat labile inhi bitor wlnth is
denatured at 100 °C, leaving the taxm free.
Alpha tn^ in lyrics rabbit erythrocytes, bul is less
active against sheep and human red cells . It is also
leucocldal , cytotoxic , dcrmonccrot it (on
i ntradermal inoculation in fu I hbili , neurotOMC and
lethal (on intravenous inoculation in rabbits 1. Il is
'
toxic to macrophages, lysusomes, muscle tissues,
renal cortex and the circulatory system.
£ t 'fa hemahxin is a sphingomyelinase, hemolytic
for sheep cells, but not for human or rabbit cells . Lt
exhibits a ‘hot-cold phenomenon', the hemolysis
being initiated at 37 *C, but becoming evident only
after chilling .
Gamma hemoh^.' Ji L > composed of two separate
proteins, both of which arc necessary (br hemolytic
activity.
Delta hemolysin has a dctergent -likc effect on
cell membranes of erythrocytes , leucocytes ,
macrophages and platelets.
L^ucotrjd .- .T ( called the Panton-Valcnfine toxin
'
after its discoverer ) is also a two component toxin,
like the gamma lysin, hi' mg com poised of two
components ( S and P ) . Such bicomponent
membranc -acfive toxins as the staphylococcal
leucocidin and gamma lysin have been grouped as
svncfgnhymcnatmpk fnvins.
'
I ' nicmluxin: This toviu is responsible for rhe
mamlestac 1 ons of staphylococcal food poisoning
nausea , vomiting and diarrhea 2 - h hours after
consuming contaminated fond cunr . i i r. i ng
preformed toxi :i . The toxin is relatively heat stable,
resisting 100 for 10 to 40 minutes depending
on the concentration of the toxin and nature of the
medium. About two - tlv. rds of Staph. aureus strains,
-
growing in carbohydrate and protein food secrete
the Mo; in. Meat and fish or milk and milk products
cooked and left at room temperature after
concaminadon with staphylococci lor enough time
,
fbr the toiun to accumulate, arc the common items
responsible . The source of infection is usually a
food handler who is a earner. I’hc illness is usually
self limited , with recovery in a day or so .
or MicrotHO - cgy
Eight antigenic types of emerutoxin are currently
known, named A , R , C _ , D, E and H . They
.. . aTe
formed by io:\ igenic strai 1 is, M 1 igjy or in combinatiim .
The toxin is believed to set directly on the
autonomic nervous system to cause the illness ,
rather than on rhe gastrointestinal mucosa . The
toxin is antigenic and neutralised by the specific
antimxin. Type A Euxin is responsible for must cases.
Sensitive serological testa such as latex
agglutmai ion and ELISA are available for detection
of the toxin.
The tewm i ^ potent, mkrogram amounts being
capable of causing the illness. Some cases of post
antibiotic diarrhea are caused bv/ enterntoxin-
1
forming staphylococci . The toxin also exhibits
pyrogenic, mitogenic , hypotensive, thrombo-
cytopenic and cytotoxic effects .
Toxic shook syndrome toxin (TSST):
Toxic shock syndrome (TSS ) is a potentially fatal
multisystem disease presenting with fever ,
hypotcnki m, myalgia, vomiting , diarrhea, mucosal
hyperemia and an erythematous rash which
desquamates Subsequently, Hi is is associated with
infection of mucosal or sequestered sites by TSST-
produving Staphr aureus strains usually belonging
to bacteriophage group 1. TSST type -1 ( formerly
also known as enterotosin. type F or pyrogenic
exotoxin C) most often responsible , though
— enterotaxins B or C may also cause the syndrome.
TSS was first identified in 1978 in children and
adolescents, but became widely known only in 1980
following outbreaks in the USA in menstruating
women using highly absorbent vaginal tampons.
Thei r vag: nal swabs showed heavy growth of Staph ,
aureus, though blood cultures were mvariahly
negative. TSST- 1 antihndy is seen in convalescents
This is protective and absence of TSST- 1 antibody
is a factor in [ he pathogenesis of the condition .
-
Though tampon related TSS is now rare, the
syndrome occurs in other infections of rhe skin,
mucosa and other sites and also in some surgical
wounds.
Staphylococcal cntcrotoxins and TSST- 1 arc
opyrighted materia
 < S:aohpiQcoccLS-
whkh are potent actlvaton of T
IvmphocyteR. Being YfJ restricted T Cell mitogeny,
such mpcraiuigcns stimulate very large numbers
of T celts , without relation to their epitope
specificity. This loads to an excessive and
dvsrCgulated immune response with release of
cytokines interleukins 1, 2, tumour necrosis factor
and interferon gemma . This explains the
nuiltiiyittxn involvement and florid manifestations
in staphylococcal food poisoning andTSS.
Exfoliative ( epidermnlytia ) tn \ in: This
taxin, il» known as KT or "exfoliatin' is responsible
for the ' staphylocuCcal scalded skin syndrome '
,
(SSSSX exfoliative skin discuss in which the outer
layer of epidermis, gets separated from the underhing
tissues . The severe form of SSSS is known as
Ritter’;; disease m die newborn and toxic epidermal
necrolysis in older patients. Milder tin ms are
pemphigus neuiiuEoruni and bullous impetigo.
STAPHYLOCOCCAL DISEASES
Staphylococcal infections are among the most
common of bacterial infections and range from the
trivial to the fatal. Staphylococcal in Tedious are
character!Stic ally localised pyogenic lesions , in
control to the spreading nature of strep [ OCOCCal
infections. Common staphylococcal infections are
the following:
Skin arid soft ti % sutv: Folliculitis , furuncle
( boil ), abscess (particularly breast abscess), wound
infrtrion, carbti nclc, i mpetigo, paronychia, less < »ftoi
cellulitis.
Mtj euIo» keletal: Osteomyelitis , arthritis ,
*
burs ids, pyomyosltls.
kespiratonc Tonsillitis , pharyngitis, siniisiui,
oritis , bronchopneumonia, lung abscess, empyema,
rarely pneumonia.
Central nervous system: Abscess ,
meningitis, intracranial thrombophlehiris.
Kildm nsClllar: Bacteremia , septicemia, pyemia,
endocarditis
1 rinury: Staphylococci are unaiinmun in routine
urinary rract infections , though they do c? use
» 197
infection in association with local instrumentation ,
implintt or diabetes . Urinary isolates of
staphylococci are to be considered significant even
with low colony counts, as they may be related to
bacteremia.
bACTKR ]imiA (; i; Tt
StlphyloUCei mav be typed , bated OIL their
susceptibility tobacterinphages. An internationally
accepted set of phages is used for typing -
"
Staphylococcal phage taping is done by J PATTENL
method . "Hie strain to he typed is inoculated on a
[ dateof nutrient agar to form a lawn culture . After
drving, the phages arc applied mtr market! squares
in a fixed done ( routine test tLise ) . After overnight
incubation , the culture will be observed to be lysed
by some phages bur not by others. The phage type
of the strain is expressed by the designations of all
the phages that lyse it . Thus, if a strain is Ivscd
only by phages 52, 79 and 80, it is culled phage
type 52779 /SO. Phage typing is of great importance
in epidemiological studies of staphylococcal
infections .
international basic set of phases lor typing
Staph aureus of hyman origin
,
Group I 2% 52 . 52 A, 79, SO
Group 11 3iA. 3C. 55, 71
Group 111 6 , 42E, 47. S 3, S 4, 7 F . 77 , 63A ,
64, 65
Group 1V
Group V 94, 96
Not doomed Kl , 9.5
Not all cultures are typablc hv this procedure,
and the susceptibility patterns of circulating strains
vary in rime imd locality. Hert.Ce phages in tile
reference set require periodic revision .
Epidemiology: Staphylococci arc primary
parasites of human beings and animals, colonising
the skin , skin glands und mucous membranes . The
most common sources of infection are human
patients und carrier animals and inanimate objects
^
being less important . Patients with superficial
infections and respiratory infections disseminate
Copyrighted materia
19G * Taxlbook ol Microbiology
large numbers of staphyfocneri into the
environment - About 10-30 per cent of healthy
persons carry staphylococci. in rhe noie and about
10 per cent in the perineum and also on the hair.
Vagina] carriage is about 5-10 per cent, wfi i ch rises
greatly during menses, a factor relevant in the
pathogenesis of TS5 related to menstruation.
Staphylococcal carriage starts early in life,
colonisation of the umbilical stump being very
common in babies bom in hospitals. Some earners,
called ‘shudders', disseminate very large numbers
of cocci for prolonged periods. The cocci shed by
patients and earners contaminate fomites such as
handkerchiefs, bed linen and blankets and may
persist on them for day* or weeks. Staphylococci
may also come from infected domestic animals such
as cows.
Staphylococcal disease may follow endogenous
or exogenous infection - The modes oftrammissinn
may be by contact, direct or through fomites, by
dust or by airborne droplets.
Hospital infections by staphylococci deserve
spec id attention because of their frequency and
because they arc caused by strains resistant to
various antibiotics . Staphylococci are a common
cause of postoperative wound infection and other
hospital cross infections. Most of these are due to
certain strains of staphylococci that are present in
the hospital environment, the so-called ' hospital
1
strains . They belong to a limited number of ph gc
types and are commonly resistant to penicillin and ^
other antibiotics routinely used in hospitals. Some
of them, the ‘epidemic strains', cause epidemic? of
hospital cross infections. The first of these to be
recognised was phage type fHVSl , which accounted
for must of Staphylococcal infections io hospitals
throughout the world - They have since been
replaced by other strains of staphylococci and by
Gram negative bacilli.
Measures for the control of staphylococcal
infection in hospitals include:
1. isolation of patients with open staphylococcal
lesions:
2 detection of
, staphylococcal lesions among
surgeons, nurses and other hospital staff and
keeping them away from work till the lesions
are healed
^
3, strict aseptic techniques in theatres;
4, thcoldest, simplest and the most effective method
of checking hospital cross infection is hand
washing, which unfortunately is often
neglected.
If an outbreak of staphylococcal sepsis occurs,
a search tnay be made for carriers among the
ht i -spital stall.Those delected shou Id be Healed with
local applications of neomycin and chlorhetidine,
In some institutions in America, eradication of the
virulent resident strain has been attempted by the
deliberatedissemiuari on of a stra i n of low virulence.
The latter may oust the former by interference.
Antimicrobial prophylaxis by topical applications
of antiseptics such as hcaach [otophone has also
been found useful.
Laboratory diagnosis: The specimens to be
collected depend on the type of lesion (for example,
pus from suppurative lesions, sputum from
respiratory infections). In cases of food poisoning,
feces and rhe remains of suspected food should be
collected - For the detection o f carriers, the usual
specimen is the nasal swab. Swabs from the
perineum, pieces of hail and umbilical stump may
be necessary in Special situations.
Direct microscopy with Gram stained smears
i* useful in the case of pus, where cocci in cluster?
may be seen. This is of no value for specimens like
sputum where mixed bacterial flora axe normally
present.
Diagnosis may readily be made by culture. The
specimens are plated on blood agar. Staphylococcal
colonics appear after overnight incubation.
Specimens where staphylococci ore expected to be
scanty and outnumbered by other bacteria (for
example, swabs from carriers, feces in food pusoni ng
cases ) are inoculated on selective media like
LuJ I ani's or salt -milk agar or Robertson's cooked
meat medium containing 10 per cent sodium
Copyrighted material
 < Staphylococcus t 199

chloride, Smears aic LS-.IIIRITIED ( rum the cultures

and the eoagulase test done when staphylococci
arc isolated.
Coagulase test LH (lone by two methods ruhe —
and HJI LKC cnaguJuse Eesls . The tube oD gubsc tent
^
detects free cw ulut. About 0- 1 ml of a young
^
broth culture or agar culture luspension of the
itollle is Lidded Co abmiL ( 1.5 oil of human or rabbit
pkitria in a narrow test tube . FDTA , oxalate or
heparin may be used as the anti coagulant for
preparing the plasma. Citrate is not recommended
because it may be utilised by some continuum
bacteria , causing false positive RESULTH. Positive and
negative Controls are also set up. I ' he cubes are
—
meubated i n a wain bull at 37 "C for 3 6 hours. If
positive, the plasma clots and docs nor How when
the tube is tilted . Continued incubation is not
recommended as the clot may get lysed bv the
fibrinogen formed bv some strains.
The slide tu*r detecting bound coagulase is much
simpler and usually gives results parallel with the
rube test - When there is divergence, the tube test
will IK the deciding factor . For the slide test , the Fig. 22.2 Bacteriophage typing u1 staptiyIficdcCl
isolate is emulsified in a drop of saline on a slide.
After checking for absence of autoaggluunation, a Tre a i m e n t
drop of human or rabbit plants is added to the As drug resistance is so common among
emulsion and mixed. Prompt clumping of the cocci staphylococci, the appropriate antibiotic should be
indicatex a positive test . Positive and negative chosen bailed on antibiotic sensitivity tests . Benityl
controls also are set up . penicillin is the most effective antibiotic, if the
Antibiotic sensitivity tests should be performed strain in sensitive. Methicillin was the first
as 3. guide to treatment . This is important as compound developed to combat resistance due to
staphylococci readily develop resistance Co drugs. penicillinase [ beta lactamase ) production by
Bacteriophage typing may be done if the staphylococci. Due to the limitations in clinical use
information : detired I < IT epidemiologies] purposes. of tnethidllin, ckofacillins are used insteul against
Other typing methods include antibiogram pattern, penicillinase- producing strains . But methicillin
plasmid profile, DNA fmgerprinringh rlbotyping resistanf strains of Staph, auraa ( MRSA ) became;
and PCfbbwd analysis for genetic pleomoiphism. common, which were resistant not merely to
Serological tests may sometimes be of help in penicillin , but also to ill other beta lactam antibiotics
the diagnosis of hidden deep infections . and mint others besides . For life threatening
Antisraphylolyjsjn (antialphalysin ) ritrrss of more staphylococcal infections , vancomycin is the drug
than two units per ml, especially when the titre is of choice . Strain * rcs- i'- tant to vancomycin and
rising, may be of value in The diagnosis of deep tcicoplanin have appeared in hospitals where
seared infections such as bone abscesses, antibiotic UK is indiscriminate . For mild superficial

;r
200 i Tentbook of Microbiology

Lesions, systemic antibiotics itiay Cot be fiflSSSary,
Topics! applications of drugs not used systemic ally,
as bacitracin, chlotbaddiH or rnupirocir may he
suffioen[ .
Some strains show the phenomenon of drug
tolerance . These strains will be found to be
susceptible in the disc sensitivity test but their
minim urn bactericidal concentration will be very J
much higher than their minimum inhibitory
concentration . They are pot killed bv antibiotics in
but can cause disease when the host defences arc
breached. It is a common cause of stitch abscesses.
It ban a predilection for growth on implanted
fore ign bodies such as artificial heart valves, shunts,
intravascular catheters and prosthetic appliances,
leading to bacteremia . Hospital strains of Staph ,
cpidcftnkiis are usually multiple drug resistant . It
can cause cystitis. Endocarditis may be caused ,
particularly in drag addicts .
Staph bjprophyricus may be present on normal
,

the usual doses and persist, leading to failure in
eradicating the infection.
The treatment of carriers is bv local Application
of suitable antibiotics such as bacitracin and
antiseptics such as chlorhexidine. In resistant cases
posing major problems, rifampicin along with
another oral antibiotic may he effective in long term
suppression or elimination of die carrier state,
OTHER COAGULASE POSITIVE STAPHYLOCOCCI
Hesides S/apll tuiHJS, a few other StAphyluccmcal
species are coagulue positive , c . g . Staph ,
human skin and the periurethral area and r an cause
urinary tract infection, particularly in sexually active
young women. The infection is symptomatic and may
involve the upper urinary tract also. Men are
infected much ( css often, though it is sometimes wen
in older persons. The infecting strains arc usually
sensitive to most common antibiotics, except
nalidixic acid. Staph, saprophvticus is novobiocin
resistant.
Table 22.1 lists the features useful for
-
distinguishing the major Species of - taphylooucci .

intemahux anti Staph hyreus. These are animal

,
pffllfltcr ami do nut itilect humans.
COAGULASE NEGATIVE STAPHVL0C0CCI
Coagulsiyc negative staphylococci constitute a major
component of the norma ] flora of the human body.
Some species ofcoagulasc negative staphylococci
MICROCOCCI
These are Gram positive cocci which occur mostly
in pdn , tetrad ? nr irregular clusters- They arc
catalase and OJtidase positive. Tl ]eV are aerobic with
a Strictly respiratory metabolism . They air parasitic
on mammalian skin and arc ordinarily
nonpathogen ie. They resemble staphylococci but

Staph ,
-
can produce human infections Staph epidettnidu,
,

haemofyticus and Staph naprvphrticus.

,
Staph , epidermidis is invariably present on
normal human skin . It is nonpathogenic ordinarily
in stained smears the cells are generally larger and
more Gram variable than staphylococci . In cultures
they form smaller colonies. The common laboratory
test used to differentiate between micrococci and

Table 22.1 Fealures for distinguishing the major species ot staphylococci

ChmmrtGriMtic Staph aureus

, Staph * epidennidis Staph* saprophyticus
Coagulase 4 m
-
NovutriucLn sensitivity £ s R
Acid from manmti?] anaerobically + s
-
Phosphatase 4 4 =

Copyrighted materia
 i Staphylococcus 201

HiaphylchicKci is the Hugh and [.ciffinn's oxidation— oxidative and staphylococci show fermentative
fermentation test in which micrococci show patterns.

Pufthdr rtetidimijl,
Cmnlcjy KB and GC Arvher. 1997 . Stapfiybaocal human disease. New Yurt. Chiirchill -Lmn bbuiie.
^
Easnion . CSF and C Adlsuvi 1983. Staphyimwii tuu! Staphylococcal Infeaitm : London: Academic Press.
. .
Kk*uti WE jndlTi. RaniKrEnin 1994. Cliulcsi! japiditajitE erf' nsaiguJiutE Motive sc hylnvwn. Ota MknsAiaf Res - 7 t 117
,

^
Mtnrvdc P inrl J Kippk 1990- StAphylococad enteruCmins and their irhtivw. Science 24B : 705.

—
Rupp ME and GL Archer 1994. Coagdase nptm scaphvUxued, ,4m / Mtd 94 ; 313,
Sheaprtn JN. 19S4. STapJsyloociCLLiK iurum EIW persisieni pdugen, iVew jEsgfjF Med, 310, 136R,. 1437-

Copyrighted material
 CO Streptococcus
CM
Streptococci Gram positive cocci arranged in
ire
chains or pairs ( Fig. 23.1). They are part of the
normal flora ot humans and animals. Some of them
arc human pathogens , 'The most important of them
is ftnepmcuccuf pyogenes causing pyogenic
infections. with a characteristic Tendency m spread,
as opposed to staphylococcal lesions which are
typically localised. It is also responsible for the
nonsuppurative lesions, acute rheumatic lever and
glomerulonephritis which occur as sequelae to
infection.
Coed in chains were fust Seen In erysipelas and
wound infections by Billroth { 1874 ), who called
them streptococci ( strepfos , meaning twisted or
| %
/Kw
<
\ erythrocytes can he made out microscopically
- normal commensals in the throat, but may
Fig . ±3.1 Streptococci
Coiled ). OgStotL (1S81) isolated then ] from acute
abscesses, distinguished them from staphylococci
anti established their pathogenicity by animal
inoculation . Roscnhach (1SS4) isolated the cocci
from human sUp]Vurative les ]ons and gave them the
name StrcptoaH : rus pyogenes.
CLASSIFICATION
Several systems of classification have been employed
but in medical bacteriology the following method
is useful ( Fig. 23.2).
Streptococci are first divided into obligate
anaerobes and facultative anaerobes. The former
are designated pepiostreproeocci and are considered
in a later chapter . The aerobic and facultative
anaerobic streptococci arc classified nr the basis of
their hemolytic properties. Brown (1919 )
categorised them into three varieties based un tlieir
growth in 5% horse blood agar pour plate eolturcs.
Alpha (ft ) hemolytic streptococci produce a
greenish discolouration with partial hemolysin
around the colonies. The zone pf tysis is small (1
or 2 mm wide ) with indefinite margins , and unlysed
within tins zone. These are known as 'viridans
streptococci ' or .Streptococcus viridans (from
lviridisr meaning green ). The alpha streptococci arc
cause opportunist infections rarely. Pnemocoocus
fi fr. pneumoniae! is also an alpha hemolytic
'
streptococcus .
Bets ( [3 ) hemolytic streptococci produce a
sharply defined, dear , colourless zone of hemolysis,
2 ~ 4 mm wide , within which red cells are
loovricihted material
 « Sneplacoceus » 203

msiBooK w MiCHuBioLOov

SlrppNH. ih cL
' '

ft I ^LjUETLTIICn!;

f
Ach ihf -i aiM.I r-K.ullalivc
'
Obligate uia^ mK.\ 1

mmfcH Pcph KM re ptoc-rx:c i

Honulysih
I

—
11
j . AI phi
3. ( himmil
.
\ibt vintau freup.'} ( The cnDemonccyri grcHIp )
OiMififid unm fififctitt pliyiiB- Class-iFicd. into specie* by phy & iQ-
Inficnl And- tii'XtM:rnk:. al prvpcstic »- and Mncb lcil piuprrlics

2. Bela
cTlic hemolytic slrcpictLio i i
_
Smtajffcal GfCup tfKilk C Larhohvdraic aruigra
H r

3D LmeeficUi Gr&up* AABCDEFGHKL

MOTQUTUVI
Gmurp A - pWfi&Ul

Serological lypuig I M ft Mem }

'

-
Gnffuh types 41.2 , 3 etc , }

.
Fig. ZA Z Classification of streptococci
completely lysed.The term ' hemolytic streptococci '
strictly applies only to beta lytic strains . Most
pathogenic streptococci belong to this group .
Gammfl (?) or nonhemolytic streptococci
produce no change in the medium and so arc
sometimes referred to is ‘indifferent StTCptOCOCd'-
They include the fecal streptococci (en tenxacci,
$tr faecalis ) and related species, They are called
the 'enterococcus group'.
Hemolytic streptococci were classified by
Lancefield ( 1933 ) serologically into groups based
on the nature of a carbohydrate (C) antigen on the
cell wall. These arc known as Lance field groups,
twenty of which have been identified &o far and
named A~V ( without 1 and J ). The great majority
of hemolytic streptococci that produce human
infections belong to group A . Hemolytic
streptococci of group A are known as iir./yogeMS,
These may be further subdivided into types based
on the protein (M, T and R ) antigens present on
the cell surface (Griffith typi rig ). About eigh ty types
of Str. pyogenes hive been recognised so far {types
1, 2 n 3 and so cm).
Table 23.1 shows the medically important
streptococci and their characteristics.
STREPTOCOCCUS PYOGENES
Mi ir p l u p h \ : The individual cocci are spherical
or ova ] 0.5-1.0 pm in diameter Size variations
result from cultural conditions, lor example, when
grown anaerobically they are somewhat smaller.
They arc arranged in chains, the length of which

Copyrighted material
304 Textbook of Micmbiok y *
4
^
Table 23.1 Medically important streptococci and their characteristics

Species or LtJncvfk' ht Hemolysis Habitat iatxi rantry rests ffjmJftoif disease*

cofiimon mime Knmp m bumatt caused
Is

Str, pyogenes A beta Tlitoac, Bacitracin sensitive:; Upper respiratory tract

skin FYR test positive; infection!*; pyoderma
Rjbirie nor fermented rheumatic fever;
gJomerukmephrim
Sir. fMj[B(tfac R betai Fern ale CAMP test, hippurate Neonatal meningiiris,
genital hydrolysis wpcieemia
tract.,
rectum
Sk cqntiimilis C beta Throat Ribosc and trehalose Pharyngitis*
Fermentation endocarditis
Sir. anjinewus A, Q FP GY ku (alpha, Threat, Group A strains Pyogenic infections
untypablt jpimma ) colon, bacitracin resistant
female FYR negative; Minute
gedtal colony variant? of
1 r*ct other ghwps
Enterococcus $ p- i> Gamma. Colon Grcwnb in 6-?9ti NaCI; Urinary tract
{Str: fstctmlis and ( alpha, PYB poiinve infections,
other enterococci) beta ) endocarditis,
suppurative infections
tfofltnttHKOCCtJ i> gamma Colon No growth in 63% EodunMitii
Group D specie* NaCI
.
(Str bow )
*
Viridam Not typed alpha Mouth, Optocbin resistant , Endocarditis ( Str.
scicpiococci ( many (gamma ) colon, species classification jBJijpiii); dental caries
specie*) female on biochemical (Str; JHU&fls)
genital properties
tract

varies within wide limits and is influenced by the
nature of the culture medium , chains being longer
in liquid tlum in aoltd media . Chain formation is
due to the cocci dividing in fine plane only & nd the
daughter cells failing ro separate completely. 'ITierc
is often an appearance nt pairing within the chains.
Sigtiificince was once attached to rhe length of
the chains , and streptococci had been classified
accordingly { SET. inruns and brews) but this has no
relevance to virulence or other properties ^ III fact,
some nonpathngenic streptococci term the longest
chains, for example, bnr saLViriuj. ( Fig . 23.1 ).
Streptococci arc nonmotilc and nonspoting.
Some strains; of Sir. pyogenes and some group C
strains hitve capsules composed of hyaluronic acid,
while polysaccharide capsules are encountered in
members of group B arid D, These capsules are
best seen in very young cultures.
Cultural characteristic*; It is an aerobe and
a facultative anaerobe , growing best at a temperature
of 37 ( range TL-X1 ^C)- It is exacting in nutritive
requirements , growth occurring only in media
containing fermentable carbohydrates or enriched
w iiti blood or serum. On blood agar, after incubation

Copyrighted material
 * Streptococcus
for 24 hours , the colonics are small ( 0.5-1.0 mm )
circular scuu' ransparent, luw convex lines v. i 1 hi an
( protein and ( ipoteichoic .KHI , a middle layer of
area of dear hemolysis around them. Growth and
hemolysis are promoted by L 0 per cent CO?.
Virulent strains , on fresh isolation from lesions,
produce a ' matt ( finely granular ) colony, while
a^ irulcut Rtr.iins form
'
colon ies. Strainswith
well marked capsules produce 'mucoid' colon es,
corresponding in virulence to the matt type. Very
rarely * nonhemolytic group A streptococci are
encountered, which are typical of 5tr. pyrenes in
other respects.
Tn liquid media, such as glucose « r semm broth,
growth occurs as a granular turbidity with a
powdery deposit. [Vo pellicle is formed.
Bioohemioal reactions: Streptococci ferment
several sugars produt ing acid hut no gas.
Streptococci .Ire catalase negative. They are not
soluhle in 10 per cent bile, unlike pneumococci.
Hydrolysis of pyrmlidoryl naphthylamule ( PYR
test ) and failure to ferment ribose are useful in
differentiating Su; pyogenes from other streptococci.
Resistance: $fr- pyv encs is a delicate organism,
^
easily destroyed by heat ( 54 for 30 minutes). It
divs in a few davs .
Jr
in cultures- unless stored n a low
, r
, Several proton antigens have been identified in
temperature 4 C) , preferably in Robertson 's
( ?
cooked meat medium. It can, however , survive in
dust for several weeks, it protected from sunlight
It is rapidly inactivated by anfscptics. It is more
resistant to crystal violet than many bacteria,
including Staph , aureus. Crystal violet (1 mg/ L ),
nalidixic avid ( 15 mg/ L ) and colisrin sulphate (1C
mg/ T.) added tn blood agar provide a good selective
medium tor the ivolatii in of streptoeocs i . including
pnemnuLOccL. It ii susceptible to sulphonamides
and many antibiotics but unlike Staph , aureus dues
rot develop resistance TO < Irugs. Sens il ivi tv to bacitracin
is employed as a convenient method fbrdiffcrenti .Lting
Sir pyogenes from ocher hemolytic streptococci.
Antigenic structure: Fig. 23, 3 illustrates the
disposition of the various anti gens in 5tr. pyogenes.
The capsule when present inhibits phagocytosis
k is not antigenic in human beings.
t 205
The cell will is composed of an outer layer of
group spec irk carbohydrate and an inner layer of
pepddoglycan.
The pepridoglycan ( mLiCoprotein ) is responsible
for cell wall rigidity. It ban also some biological
properties such as pyrogenic and thrombolytic
activity.
Serological grouping of streptococci depends on
the C carbohydrate. Sir. pyugeres belongs to group
A. As this antigen is an iutqgTal part of the cell
will , it has to be extracted for grouping by a
precipitation test with group antisera - For the test ,
streptococii are grown in Todd-Hewiit broth and
extracted with hydrochloric acid ( Lancefield's acid
extraction method ) , or foritianiidc ( Fuller s
method ) or by an enTymc produced hv StnepfomyeeK
MISUS ( Maxted 's method ) or by autoclaving ( Rantx
and Randall 's method ). The extract and the specific
,
antisera are allowed to react in capillary tubes.
Precipitation occurs within five minutes at the
interface between the extract and the homologous
nnti -icrum. Grouping may also be done by agar gel
precipitation.
the outer part of the cell wall, Stn pyogenes can be
typed , based on the surface proteins M, T and R .
The M protein : s the most important of these- Tt
acts as a viralcncc factor by inhibiting phagocytosis.
It is an i i.L- eiiic . The antibody to M PROIEIIL promotes
phagocytosis of the coccus and is therefore
protective. Tire M protein is hear and acid stable
but susceptible to tryptjc digestion . Tt car he
extracted by the Lancefield acid extract!' in method
and typing is done with type specific sera. About
SO M protein types have been neoognised.
TheT prrnteir. is an fri id lahlie, ttyps i n n' -lstant
'
antigen present in many serotypes of Str. pyqgenes-
IE may be specific but many different M types
possess the same T antigen, lc is usually
demonstrated by the slide agglutinjtion test using
,
trypsin.- treated whole streptococci . Some types of
i’fr pvogenej ( types 2 * 3 , 28 and 48 ) and some
Copyrighted material
I JF W
206 4 .
Tesctbr Dk Dt Mu;robiology

strains ofgroups B, C and G contain a third antigen,
the R protein.TheT and R proteins have no relation
to virulence . A nontype-spetific protein, associated
with the M protein, has been identified. This is
known is M associated protein ( MAP).
I lair - - ike pili (fimbria ) project through the
'
capsule of group A streptococci. The pLLi consist
part ] / of M protein And are covered with
Lipoteictioic acid which is important in the
attachment of streptococci to epithelial cells.
Various structural components of $tr. pyogenes
exhibit antigenic cross reaction with different
tissues of the human body. Antigenic relations] lips
have been demonstrated between capsular
hyaluronic acid and Imm mi synovial fluid, cell will
protein and myocardium, group A carbohydrate and
cardiac valves, cytoplasmic membrane antigens and
vascular intima , and peptidoglycans and skin
antigens. It has bcc a postulated that these antigenic
cross reactions may account for some of the
manifestations of rheumatic fever and other
streptococcal diseases, the tissue damage being of
mi immunological nature
V O appears in sera following streptococcal infection .
J
. Esti matitm of this antibody (ASO titre) is a standard
* Following certain chemical treatments or bacterial
2m
2 p
:
2c
1 rest is now done by the serological method of latex
.
Fig . 33.3 Antigenic structure of S t f pyogenes
.
1 Hyaluronic Kid capsule. 2. Cell wall comprising
£ A . peptldoglytan , SB. group Speelfic carbohydrErte
and 2C . protein llpoteichcic acid fimbria 3 , Cyto-
.
:iasmic memtirane 4. Cytoplasm. 5 PHI cowed with
Ipotdcholc acid.
Toxins and other virulence factors; Srr .
pyogenes forms several exotoxin ? and enzymes
which Contribute to its virulence. Besides these,
the M protein also aoa as a virulence factor by
inhibiting phagocytosis. hfhe G polysaccharide has
been shown to have a toxic effect on connective
tissue in experimental animals.
Hemolysins Streptococci produce two
hemolysins, streptolysin "O' and 'S'. Streptolysin
O is so called because it is oxygen labile. It is
inactive in the oxidised form but may be
reactivated by treatment with mild reducing agents.
On blood agar, Streptolysin O activity is seen only
in pour plates and not in surface cultures. It may
be obtained in the active state hy growing
streptococci in broth containing reducing agents
such as sodium hydrosulphite . It is also heat Labile.
It appears to be important in contributing to
virulence. It is lethal on intravenous injection into
animals and has a specific cardiotoxic activity. It
has leucotoxic activity also. In in biological action ,
streptolysin O resembles the oxygen labile
hemolysins of CL perfringens, Gf. reran! and the
pneu mococcus.
Streptolysin O is antigenic and antistreptolysin
serological procedure for the retrospective diagnosis
of infection with Sir. pyogenes. The lytin is
inhibited by cholesterol but not by normal sera,
contamination, sera may develop inhibitory activity
due to some changes in the lipoproteins. Such sera
-2 arc unfit for the ASO test. Because of the
complexity of the hemolysis inhibition test, ASO
agglutination. An ASO time in excess of 200 units
is considered significant and suggests either recent
. or recurrent infeution with streptn-cocci -
Streptolysi n S and O are produced by groups A, C
and G also.
Streptolysin S is an oxygen stable hemolysin and
HO in responsible for the hemolysis seen around

Copyrighted materia
 i Slreplococcus *
streptococcal .colon ics on the nufin of blood agar
plates. Ic is called vtnptofywn S since it is soluble
in serum . It is a protein but la not antigenic .
Convalescent sera do not neutralise streptolysin S
activity. It is inhibited nonspccifically by scrum
Lipoproteins.
Pyrogenic exotoxin ( t> ytIm L nieh
Dick , tcnrlatinn.1 toxin ): This toxin was ^-
named 'erythrogeoie because its intradermil
1
injection into susceptible individuals produced an
erythematous reaction ( Dick test , 1924 ) . This test
was used to identify children susceptible to scarlet
fever, ,t type of acute pharyngitis with extensive
erythematous rash , caused by the Sir. pyvfienes
strains producing this toxin . Blanching of the rash
on lot a I injection of convalescent senim was used
as a diagnostic , test for scarlet fever ( Schultz
Charlton reaction, 191 H ). The Dick test and Schultz
Charlton reaction are now only of historical value
as scalier fever is no longer a common or serious
disease.
The primary effect of the toxin is induction of
fevtf and so it was ren amed Streptococcal pyrogen ic
exotoxin (SEE ). Three types of SPE have been
—
identified SPE ,ArR and C. Tvpes A and C are
coded for by bacteriophage genes , while type B
gene is chromosomal. SPE-s are ‘supefan tige ns’ ( like
staphylococcal entero toxins and TS-S toxin ), T cell
mitogens which induce massive release of
inflammatory cytokines closing fever, shock and
tissue damage.
Stfcptokinafle (fibrinotysin ) ; This roxin
prom ores the Lvsis of human fibrin dots bv activating
a plasma procursor (plasminogen ) . It is an antigenic
prorein and neutralising antibodies appear in
convalescent sera. Anti-Streptokinase antibody
provides retrospective evidence of streptococcal
infection . kihrinolysin appears to play a biological
role in streptococcal infections by breaking down
the fibrin harrier around the lesions and fkicilitaring
the spread of infection - Streptokinase is given
intravenously tor rhe treatment of carlv myocardial
infarction and other thrombocmboloic disorders .
207
Deoxyribonucleases { Streptodurnu &e .
DNAase ); These cause dcpolymerisation of
DNA . Pyogenic exudates contain large amounts of
DNA, derived from rhe nuclei of necrotic cells.
Strcptodornasc helps to liquefy rhe thick pus and
may be responsible fur rbe thin serous charterer ot
streptococcal exudates . This proper tv has heen
applied therapeutics LI v in Liquelying localised
collections of thick exudates* as in empyema. A
preparation containing streptokinase and
streprodornase is available for this purpose . Four
antigcnirallydistinct I JNAases, A , R, C and I ), have
been recognised , of which type B is the most
antigenic in human beings . Demonstration of anti -
DNAase R antibody is useful in the retrospective
diagnosis of $tr. pyogenes infection , particularly in
skin infections, where ASO fitres may he low.
Strcptodornasc Ft and 1 ) also possess tihonudeasc
activity.
Nicotinamide adenine tlinucleotidase
( NADaBet formerly drphosphopyridine
nudentidnse , UPNase ): This acta on the
coenzyene NAD and Lihe rates nicotinamide tmm
r
the molecule. It is antigenic and is specifically
neutralised by rbe aotibodv in convalescent sera.
The biological significance of NADasc is not
known * though it LH believed to be itUCOToxic.
Hynlumnidnn ^ This enzyme breaks down the
hyaluronic acid of the tissues . This might favour
the spread of inlection along the intercellular spaces.
Streptococci possess n hvalumnic acid capsule and
—
also elaborate a hyaluronidasc a seem ingly self -
destructive process. IT is, however* found that strains
( hut form hyalutoiiidwsv ill targe qoutitHt (M types
4 and 22 ) ire noncapsulated . The enzyme is
antigenic and specific antibodies appear in
convalescent sera.
Serum npmtiiy fucior: Some M types
pyogenes produce a lipoproteinase which produces ^
opacity when applied to aggr gel containing horse
or swine scrum. This is known as scrum opacity
factor ( SOI 1).
Many strains also produce proteinase ,
Copyrighted material
m -.
< TeKitwo of Microbiology *
phosphatase , amylase , N acetyl
esterases ,
glucusaminldiise , neuraminidase and other toxins
It is nett known whether, arid to what
OT CTlZVnncs -
extent, these contribute to pathogenesis.
tJ vrni U' % u : m
)
Stn pvogcncs produces pyogenic infect:OHS Vidl a
tendency to spread locally, along lymphatics and
through the bloodstream.
Respiratory infections! The primary site of
invasion of the human body by Stt pyogenes is the
throat . Sore throat Is the most common of
streptococcal diseases . Tt may be localised as
tonsillitis or may involve the pharynx more diffusely
( pharyngitis ). Virulent group A streptococci adhere
to the pharyngeal epithelium by means of
llpoteichoic acid covering the surface pili. The
glycoprotein fibroncctin on the epithelial cells
probably serves as the lipoteichoic acid ligand .
Tonsillitis is more common in older children and
adults than in younger cEiitdrcn , who commonly
develop diffuse pharyngin -i. Localisation is believed
to be favoured by hypersemi i i - ity due to pvior
contact.
From the throat , streptococci may Spread 10 the
surrounding tissues , leading to suppurative
complications, such as otitis media , mastoiditis,
quinsy, Ludwig's angina and suppurative adenitis
It may rarely lead ro meningitis, Streptococcal
pneumonia seldom follows thn > ;:. 1 infection but may
occur as a com plication of influenza nr other
respiratory viral diseases.
Skin and soft tissue infection ?: -S rr .
pyogenes causes a variety of S . J | - fniT.iti \ c infections
"
of the skin , including i nfecticm of wounds or bums,
with a predilection to produce lymphangitis and
cellulitis. Infection of minor abn- ioris may ar Limes
lead to fatal septicemia.
The TWO typical streptococcal infections of the
skin aic erysipelas and impetigo. The former is 1
diffuse infection Involving the superficial
lymphatics. The affected skin, which is red , swollen
and indurated , is sharply demarcated from the
surrounding healthy area. While erysipelas is rue
and seen only in older patilints , impetigo is found
mainly in young children . Impetigo is caused by
Srr. pyogenes belonging to a limited number of
serotypes , usually the higher numbered M types,
instead of the lower numbered M types which cause
throat infection*. Impetigo and streptococcal
infection of scabies lesions arc the main causes
leading to acute glomerulonephritis in children in
the tropics.
In pyoderma, antibody response to streptolysin
O 1-. nut iiiyb and ASO estimation does uol have
as much clinical significance as in pharyngeal
infect ions . Antibody to DNAase B and
hyaluronidase arc more useful in retrospective
diagnosis of pyoderma antccendcnt to acute
glomerulonephritis.
Streptococcal subcutaneous infections range
from cellulitic to necrotisitig fasciins. The latter
condition is more commonly caused by a mixed
aerobic and anaerobic bacterid i nice Lion but some
strains of Sfr. pyogenes ( more particularly M types
1 and 3 strains forming pyrogenic exotoxin A) may
alone be responsible. This is ordinarily a sporadic
condition and has been known since 1333 but small
outbreak?. in the UK and the USA have recently
caused much alarm because of their severity and
high fatality. These str.iinS have earned notoriety
under the namtllesh eating bacteria*. In such cases,
extensive necrosis of subcutaneous and muscular
tissues and adjacent fasci.i is associated with a severe
—
system I * illness a toxic shuck- like syndrome with
'
disseminated intravascular coagulation and multiple
system failure , Sir, pyogenes can be isolated from
the affected zite and rising : il res of antistreptolysin
and anti ‘DNAase B derronjurated - Though the
isolates are penicillin sensitive in vitro, treatment
with penicillin may not be effective , Vancomycin
n the drug of choice in life threatening infections.
Soft rissuc infections with %ome M types of Str
pyngrnGS ( 1 , 3, 12, 2;H! ) :nay sumeri rocs cause a toxic
shock syndrome resembling staphylococcal TSS,
Streptococcal TSS and necrotizing fasciitis occur
Copyrighted materia
 * SUepU) coccus
only in persons ntmirtlrtlune L :: Lht infecting M
types.
Genital infections: Both aerobic and
anaerobic Rtreptococn are norma] inhab:i ; ants of
the female genitalia. Str. pyngcnesvms an important
cause of puerperal Rep -. ii , with the infection usually
being exogenous, The emphatic demonstration by
Semmelweis in 1847 that hospital outbreaks of
puerperal fever could be prevented by the simple
measure of handwashing by those attending the
labour wards remains a landmark in clinical
microbiology. Puerperal fever is now much more
commonly due to endogenous infection with
anaerobic streptococci Streptococcal puerperal
sepsis used to take a tieaw tut! of lile before
antibiotic ? became available .
Other suppurative ! nfecth > n &: Str.
pyogenes may cause abscesses in internal organs
such as the brain, lungs, liver and kidneys, and also
septicemia and pyemia.
NonguppurfU i VC Cflinpliioatnjns; Sfr.
pyogenes infections lead to two important
—
nonsuppurative sequelae acute rheumatic fever and
acute glomerulonephritis. These complications
ensue J -3 week? after the acute Infection so that
the organism may not be detectable when sequelae
set in . They differ in their natural history in a
number of respects ('Table 23.2).
The pathogenesis of these complications is not
clearly understood. The C 5senti.il loio:i in rheumatic
fever is carditis , including connective tissue
degeneration of the heart valves and inflammatory
myocardial lesions characterised by Aschoff
nodules. Typically, rheumatic fever follows persi ' tent
or repeated streptococcal chroac infections with a
strong antibody response . The lesions are believed
to be the result of hypersensitivity to some
streptococcal component. It has also been suggested
that there may be an clement of autoimmunity
involved , and anrigcric cross* reach ' ms have been
demonstrated between streptococc i and heart
tissues. Lesions resembling rheumatic fever have
been produced experimentally In rabbits by repeated
209
i ofect ion with Str. pyogenes and in mice by in; action
of sonic tysates of the COCL i .
While rheumatic fever may follow Infection
with any serotvpe of Stt. pyogenes1 nephritis is
caused hy only a few 'ncphritogertic' types. In the
tropics, skin infections are perhaps more important
in this respect than throat infect ions. The nephritis
Is usually a self -limited episode that resolves
without any permanent damage. The pathogenesis
may be due to antigenic cross-reactions between
the glomerular membrane antigen and cell
membranes of ncphiitogcnic streptococci, or more
often it may be an immune complex disease. This
condition has been produced in monkeys and rabbits
by repeated infection with type \2 Str. pyogenes or
injection of bacteri .d products, and i:i mije with
soluble streptococcal products.
Epidemiology: The major source of Str
pyogenes is the human upper respiratory tract
throat, nasopharynx or nose - of patients and
-
earners . Carrier rates of up to 20 per cent have
been observed. Symptomless infection is common
and helps
community.
to maintain the organism in the
Transmission of infection is cither by
d i rect contact or through contaminated finger*, dust
or fbmites. In the tropics, streptococcal infection
of the skin is common and may be spread by
nonbitliig insects, particularly the eye gnat
Hippelares.
Streptococcal infections of the icsfur.Ltorv tract
arc mote frequent i:i children 5-8 yeai* «f age than
in children below two years or in adults. They arc
more common in winter in the temperate countries.
No seasonal distribution has been identified in the
tropics, Crowding is an important factor in the
tuns mission o I infection. Outbreaks ol infection
may occur in closed communities such a* boarding
schools or army camps.
Immunity is type specific and appears TO be
associated with antibody to the M protein .
Reinfections occur because of the multiplicity of
serotypes.
Laboratory diagnosis; In acute infections,
Copyrighted material
210 4 Textbook of Microbiology *
Table 212 Comparison of rheumatic fever and glomerulonephritis
Acute rheumatic fever
Site ofinicctjun Throat
Prior sensitisation
Serotype of
Str. pyogenes
Immune response
Essential
AMr
Marked
Complement level Unaffected
Generic sgsccpriluliry Present
Repeated aTtncks Common
Penicillin prophylaxis Essential
Course Progressive or static:
Prognosis Vari -abk
Htsbliihcd by CtJtUi'e1 while in the
iji ; i. jjj] irjsL > IS
nonsuppurative rnmplicatitinH., diagnosis is mainly
based on the demonstration of antibodies,,
Presumptive information maybe obtained by an
examination of f iram stained films from pus and
CIS F. The presence of 13 ram positive cocci i n chai ns
is indiraTivt of ItrepMcoCCal infect inn . However ,
smears are of mo value in infections of the throat or
etUTiliij, where Streptococci may lorm part of tire
^resident flora . are available commercially. The tests can be
For cultures* swabs should be collected under
vision from the affected site and either plated
immediate I v or sent to the laboratory in Pike's
medium ( blood agar containing 1 in 1,000,000
crystal violet arid 1 in 16,000 sodium aside). The
specimen IS plated on blood agar and humbated at
27 anaerobic ally or untier 5- L 0% CO , , as
hemolysin develops better umbr these conditions,
-Sheep blood agar is recommended b > r primary
isolation because it is inhibitory for Haemophilus
hsemoJy/ii -EJff, colonies of which may be confused
with those of hemolytic streptococci . I [cmolytic
streptococci are grouped by the LancefieJd
technique [’fie fluorescent antibody technique has
, i > NAasc B and iintihyaluronidase tests are very
been employed for the rapid identification of group
A streptococci A convcaienc method for the
* pyoderma, for winch ASO is of much less value.
identification of Str. pyogenes is baaed on M sorted 's
ohRcrvation th ,i t they are more sens i five to baci tiiici n
P
than other streptococci . A filter paper disc dipped
Acute iomenjJon ephri tie
^
Throat or skin
Not necessary
—
Pyodcroul types 4% S3 S5 59 61 B
and pharyngitis strains 1 and 12
Moderate
-
Lowered
Not known
Absent
Not indicated
Sponriiieouj resolution
Good
in a solution of bacitracin [1 unit/ ml ) is applied on
the surtuce of an inoculated blood agar . After
incubation , a wide zone of inhibition is seen with
Sir. pyogenes bur not with other streptococci.
Typing of Sir. pyogenes is requited only for
epidemiological purposes, if required, this may be
done by precipitation or aj Luti nation .
^
Rapid diagnostic teat kiln for [ he detection of
Streptococcal group A antigen from throat swabs
completed in 1 -4 hours and are nearly as specific
as. culture*, though less sensitive . 1
In rheumatic fever and glomerulonephritis, a
retrospective diagnosis of streptococcal infection
[ iiay be established by demonstrating hijfh levels
of antibody to strep EOLUCCAT toxins. The usual test
done is antistreptolysin (.1 titration. ASO titres
h ighc r thni n 200 arc in dicat i we of prior streptococcal
infection. High levels are usually found in acute
rheumatic fever but in glomerulonephritis, titles
arc often low, Anti deoxyribonuclease B ( anti-
i > NAase b) estimation is also commonly employed.
Titres higher than 200 are taken as significant . Anti-
Useful for the retrospective diagnosis of streptococcal
The streptozyme test , ,i passive slide
hemagglutination test using erythrocyte* sensitised
with a crude preparation of extracellular antigens
Copyrighted material
 < Straplacaccus *
of streptococci , is a convenient sensitive and specific
screening tst. Il becomes positive after nearly all
Iv yes of streptococcal infection!;, whether of the
^
throat or the skin .
Evmphylavi& The indication for prophylaxis in
streptococcal infection is only in the prevention of
rheumatic fever. This is achieved hy a long-term
administration of penicillin in children who have
developed early signs of rheumatic fever. This
prevents streptococcal reinfection and further
damage to the heart. Antibiotic prophylaxis is not
useful for glomerulonephritis as this complication
follows a single streptococci! infection , and
reinfections do not occur.
Treatment: All beta hemolytic group A
streptococci arc sensitive to penicillin G , and most
are sensitive to erythromycin . In patients allergic
to jnrnicillin, erythromycin nr cephalexin may he
used . Strains resistant to erythromycin hive brer
reported. Tetracylines and snlphonarirides are not
recommended . Antimicrobial drugs have no effect
on established glomerulonephritis and rheumatic
fever
OTHER HEMOLYTIC STREPTOCOCCI
Besides Str, pyogenes, streptococci belonging to
groups B , C, D, FH C and rarely H , K . O' and R
duty also t£ ii!ie human infections.
p
Data from Streptococcal Reference LdbontOiits
in India ( Lady Hardingc Medical College, New
l>lhi ; Christian Medical College, Vellore ) showed
that while approximutely 45 per cent of hcinnllvt i c
streptococcal isolates tested belong to group A , ID-
15 per cent belong to groups K and C each , about
25 per cent to group G and 5 per cent to group K
GROUP It
These are important pathogens of cattle , producing
bovine mastitis ( 5rr. itgaLietnie}. From the lyfeOs,
Group B streptococcus lias assumed great clinical
importance as rhe single most common cause of
neonatal meningitis in the West. Infection in the
newborn is classified is the early onset type
211
occurring vi thin a week of birth , and the late onset
type developing between the second and twelfth
weeks of life. The more common early unset type
presents as septicemia, meningitis or pneumonia ,
and is often fatal- Infection is acquired from the
maternal vagina during birth. In chc late onset type ,
infection is more often obtained from the
environment. Other Group B infections in neonates
include arthritis , osteomyelitis , conjunctivitis,
respiratory infections, peritonitis, omphalitis and
endocarditis. Group 1 ? streptococci may al -so cause
adult infections , including puerperal sepsis and
pneumonia.
Their ability to hydrolyse hippurate acts as a
presumptive identification method. They may be
identified by the GAMP reaction (Christie , Atkins
and Munch- PcLCTjonh which can be deulon &CfLted
as an accentuated / one of hemolysis when Str.
qgubctuc is inoculated perpendicular to a streak
of Staph. a uncus' grown on blood agar. Occasional
strains are bacitracin sensitive. Human pathogenic
Group R strains possess a polysaccharide capsule
which appears ro confer virulence. Nine capsular
semtypes have been identified, antibodies to which
confer type specific protection .
GROUP C
Streptococci of this group arc predominantly animal
pathogens and may be divided into ftiur species
biochemically. Group G rtnins isolated from
human sources usually belong to Strep, equisimiia
species . It can cause upper respiratory infections ,
,is well as deep infections such as endocarditis ,
osteomyelitis, brain abscess, pneumonia and
puerperal sepsis. Strain ^ arc often tolerant to
penicillin and serious infections may not respond
to penicillin treatment. The addition of gentamicin
is recommended in serious cases. It resembles Stz
pyogenes in fermenting trehalose but differs in
fermenting riboHe. It produce -? strcplulvsin OH
streptokinane ( antigcnicjilly distinct from that
produced by Sir. pyogenes ) and other entraceltular
substances. .Str ctjaiumilis is the source of
Copyrighted materia
212 H Ifevttucifc of Microbiology »

streptokinase used for thrombolytic therapy in
patients.
GROUP F
These grow poorly on blood agar unless incubated
under CO .,. They have been called the 'minute
K *
streptococci '. They are sometimes found in
suppurative lesions. One member of this group is
Sfrrpfoiucus MG which is an alpha lytic strain
isolated from cases of primary atypical pneumom .L.
Demonstration ofagglurinius to Streptococci MG
in sera of patients had been used as a diagnostic
blue milk. On MacConkey s medium they produce
tiny deep pink colonics, They arc relatively heat
resistant , surviving 60 C for .iO minutes .
Enterococci typically appear as pair? of oval cocci,
the cells in a pair arranged at an angle to each
other [Fig. 25.4). They are usually nonhemolytic,
though some strains may show alpha or beta
hemolysis .
The identification of enterococcus species is
made on biochemical grounds. E. Fsa^lii is the
enterococcus most often isolated from human
sources. E ikv. jj' s can be identified by its ability
'
j

test tor primary atypical pneumonia. to ferment mannitol, sucrose, sorbitol and aesculm ,
and to grow on tellurite blood agar producing black
GROUP G colonics.
These are commensals in the throats of human Enterococci are present in the intestine, genital
beings, monkeys nr dogs. 1 hey may occasiunally tract and saliva. They :uc frequently isolated from
cause tonsillidsn endocarditis and urinary Infections cases of urinary tract infection and wound infection.
in human brings. They may also cause endocardi us, infection of the
Groups H and K sometimes cause infective hi I iary tract, sept icen i ia, arid iiitraabdomiiial abscess
endocarditis. compEcating diverticulitis and peritonitis. Strains
Group O is isolated mainly from the healthy resistant to penicillin and other antibiotics occur
human throat and may cause acute tonsillitis and frequently, so it is essential to perform antibiotic
endocarditis. sensitivity for proper therapy.
Group R strains are natural pathogens of pigs. Nonenterococcal species of group D [ Str, bovk ,
They have beer reported from occasional cases of Str. njufnus) are generally susceptible to penicillin
meningitis, septicemia and respiratory infection in and are inhibited by 6.5 per cent sodium chloride
persons in contact with infected pigs or or bile . They may cause urinary infection or
contaminated meat. endocarditis rarely.

GltcHIF* D S ] hEPTOCnr : c :i THB VIRJOANS GROUP

These can be classified into two groups: {1) the Thu group, formerly called Streptococcus viflflta /lS,
enterococcus group (enterococci or fecal is a miscellany of streptococci normally resident in
streptococci ) , which have been reclassified as a the mouth and upper respiratory tract, and typically
separate genus called Enterococcus and containing producing greening (alpha lysis] on blood agm^
different species for example, E, facealis , E, iacciuin, hence the name viniiajM,
E. dunns; and ( 2 ) the nonenterocoocaf group, for Some of them may be notily tic.They cannot be
—
example, Str. horix, Str. eyujnus. categorized under the Lanccficld antigenic groups.
The enterococci possess several distinctive However, based on sugar fermentation , cell wall
features separating them from streptococci. These cumposi t ion and production of doctrans and Icvans,
include their ability tn grow in the presence of they have been das? itied into many species, for
40 per cent bile , 6 -5 per cent sodium chloride, at example, Str. mfos, Sfr. mutans, Str. safoanius, Str.
pH 9.fi, at 45 "C and in 0- 1 per «rt methylene

* 4.
J“
Ij opy righted materia
 i Sirepwcoccus e 2U

I * y* ?
fift #
v
\ i*
/

tv 4 f V #-.is- c v 4

^
"
OO r < . .±

& <
*J ?oI P / r
Er

Gram stem of a pus smear . Plate stows Gram Gonococci In urethral discharge. Gram stain.
positive violet coloured cocci in groups.
{ Staphylococci). in chains ( streptococci) and
pink rods (Gram negative bacilli}. Pus cells
show up pinfc slaincd.

ft /
% 4
© 8? *•$i i*l y«J V
.

_
rjt ,

4
* 6
%?
•
4
, JT e
*
49 /
% $> * ® v,» > i*
4 »
Q

<*
*
Acid fast slain ( Ziehl-heelsen stain) cl sputum.
The red rods are M (crfiereulosis.
^ «8»
V leprae; ZwW -Nwisen slain of section of lepra netful? showing
liprn cells and characteristic arrangeoienl of lepra bacilli.

Copyrighted material
214 * Texiaoo
*. o < Microbiology *

%
t*
vo . Vu ••
h

9 •P a '* f
"(FT
&

^
<

* *r Jf i
- *
*^ '
0
' • f
I• !* •
‘

%
e
A AOA D 7 *

«
Vtersjnia pe$ft& Sm f frflrti enlarged lymph glartd Kegrr bodies in dog' s brain in rabies [hippocampus
from a cast of plague- Leishman slain of dog). Alcoholic eosin and methylene blue stain.
CJwactarislic bipolar &;a-nmg,

|
r\;*
f T

Allantoic memb W showing Variola packs. Ai arttoic mcmornna showing Vaccinia pocks. Note
^
Clear cut and white irregularity in shape and sire

Copyrighted material
 ' Streptococcus * 215

responsible. Following tooth extraction or other

dental procedures, they cause transient bacteremia
and get implanted on damaged or prosthetic valves
or in a congenitally diseased heart, and grow to
form vegetations. Prophylactic antibiotic cover is
I advisable in such persons before tooth extraction
or similar procedures. While viridans streptococci
Lire generally penicillin sensitive, some ttriLius may

be resistant . It 15 therefore essential that in

endocarditis, the causative strain is isolated and its
an abiotic sensitivity determined so that appropriate
U
* antibiotics in adequate bactericidal concentration

*" u J*’
w* *
can be employed for treatment .
Sir. mijriEm- (so called because It assumes a
bacillary form in ,tcid environments! is important
in the causation of dental caries. It break.; down
1

dietary sucrose , producing acid and a rough

adhesive dextran. llic acid damages dentine and
"

Fig. £ 3.4 £ nl«r«occiu. Ova ! calls arangcc in pairs al
an angle, or in short chains .
They are ordinarily nonpathogenic bur can on
occasion cause disease. In persons with preexisting
cardiac lesions , they may cause bacterial
endocarditis., Srr. sanguis being most often
the dextrans bind together food debris , epithelial
cells, EiiLicus and bacteria to torm dental plaqt s,
^
which lead to caries, Experimental caries in
monkeys has been prevented by a £ fr. /nutans
vaccine, but its extenfion to human use is fraught
with problems.

Further Eteuding
Brighton D et iL 1591, A scheme for identification ofviridaru streptococci , / Afn/ A-frcrutuoJ 35 ;367
L

BISRW AL - 1951. Gftttip A ntpptrNewe ) infections Hid Jcvtc Hwninnk; fever. Afcw t'-ngj Med 325;7fS3
* r

Bisno AL and DL Stevens 1996. Streptococcal infections of slcin and soft tissues. Ne\vEnglJ Med 334:240.
( .
Chaelet [ J and H Liratn 1936 . Suspiocrtecal puerperal atpsis ud JIIKICIILL InfecdtHi: A Instc rinL perspective. .-
L JJ ;

Microbiol Rev 2 1315.

Mocllering RC . 1592. Emergence: of Enterococcus as a significant pathogen. Ciui Infer Dis 14: 1173.

Copyrighted material
 Pneumococcus
{ Dipiococcus pneumoniae: Sir:. pneumoniae)
P n e ULIHKTUL CLLSP ii Gram positive lanceolate
' pneumococci
dipiococcus , formerly classified as Dipiococcus
pucn mom iac . has been re classified as Sir.
pitcumonrae because of its genetic relatcddlcss to
streptococcus. Pneumococcus differs from other
Streptococci chiefly in its morphology, bile
solubility, optochiu sensitivity and possession of a
specific polysaccharide capsule. Pneumococci are
normal inhabitants of the human upper respiratory
tract . Iliey are the single most prevalent bacterial
agent in pneumonia and in otitis media in children.
They can also cause sinusitis, bronchitis, infections
bacrercmw , meningitis and other infections,
Pneumococci were first noticed in ] SSl by
Pasteur and Sternberg independently. They
produced a fatal septicemia In rabbits by inoculating
1 uLui .ni saliva and isolated pneumococci from the
blood ot the animaU, Hut the relationship l’etwccn
'
r
« preparations or may be stained directly by special
H. .JVW.*
f.G
*. V
J
\
<*% >n,
J
., i
#?
-
i L.'
*0 ' .«
Fig. 24.1 Pneumococci in pus
and pneumonia was established only
liter by Fraenkcl and Wcichsclbauin independently
in 1886.
Murpholo jt Pneumococci arc typically small
^
(1 fim ), stighdy elongated cocci, with one end broad
or rounded and rbe other pointed, presenting ,i flame
shaped or lanceolate appearance . They occur in
pairs ( diplococcr ) , with the broad ends in
apportion , the long axis of the coccus parallel to
the line joining the two cocci in a pair. They are
capsulated, the capsule enclosing each pair. The
capsules are best seen in material taken directly from
exudates and may be lost on repeated cultivation.
In culture , the typical morphology may not he
apparent and the cocci are more rounded, tending
to occur in short chains. They are nonmotiic and
nunsporlng.
They uric readily stained with aniline dyes and
arc Gram positive . The capsule may be
demonstrated as a clear halo in Indian ink
techniques.
' Cultural charactsmticsc Pneumococci have
complex growth requirements and grow onty in
- enriched media. They arc aerobes and facultative
anaerobes, the optimum temperature being 37 “ C
( range 25-42 °G ) and pi 1 7.3 ( range b. 5-8.3) .
—
Growth is improved by 5 10% CO .
On bhjod agar, after incubation ^ for 18 hours,
the colonies are small (0. -1 mm), dome shaped
^
and glistening, with an area of green discoloration
( alpha hemolysis ) around them , resembling
colonies of Stt, vitidsns. On further incubation the
colonics become flat with raised edges and central
Copyrighted material
 Pneumococcus
umbonation, so that concentric rings arc seen on
the surface when viewed from above (draughtsman
or curom coin appearance) . Some strains that
develop abundant capsular material ( types 3 and 7 )
form large mucoid colonics .
Under anaerobic conditions, colonies on blood
agar arc surrounded by a zone of beta hemolysin
due to oxygen labile hemolysin O. In liquid media
such as glucose broth, growth occurs a* uniform
turbitliry. The cocci readily undergo autolysis in
cultures duo to the activity of intracellular enzymes.
Autolysis is enhanced by bile salts, sodium Jauryl
sulphate and other surface active agents. Heat killed
Cultures do OUT undergo autolyris.
Biuehemi[;ul reactions: Pneumococci ferment
several sugary forming acid only. Fomentation is
tested in Hiss's scrum water or serum agar slopes .
Fermentation of 7 imitin by pneumococci is a useful
test for differentiating them from t&eptococd as
ihfi latter do not ferment ir.
Pneumococci arc bile soluble. Jl a few drops of
10% sodium dcoxydiolate solution are added TO
1 ml uf an overnight broth culture, the culture dears
due It ) the lysis of the oOcci . Alternatively, if a Iwpful
of 10% deoxychoEatc solution is placed on a
pneumococcus colony on blirod agar the colony
lyses within a few minutes. Bile solubility is a
constant property of pneumococci and hence Is of
diagnoseic importance . The bile solubility test is
based H > H the presence in the pneumococci of an
autolyric amidase that cleaves the bond between
alanine and mimmUC acid in the pepfidoglycan . The
am id Jsc is activated by surface active agents such
as bjfe or bile salts, resulting in lysis of ihe
organisms, The test should be carried out at neutral
pH using dcoxycholatc and live young cells in saline
suspension.
Pneumococci arc catalase and oxidase negative.
Resistance: Pneumococci are delicate organisms
and are readily destroyed by heat ( thermal death
point 5 2 eC b( 15 minutes ) and antiseptics. In
cultures, they die on prolonged incubation, perhaps
due t« an acaimulation of (mk peroxides , Strains
217
e« f
Fig . 2 M Pneumococci . Indian ink preparation to
show capsules .
.
may be maint uiied on semisolid blood agar or by
lyophili &ation .
They are sensitive to most antibiotics, beta
lactams being the drugs of choice. Almost ; LIL strains
were sensitive to (1.05 mg penicillin till 1967, when
resistant strains began to appear . The resistance may
be Intermediate ( MIL’ 1 pg) or high ( 2 |ig or more )
and due to mutation or gene transfer.The mode of
resistance is not production often lactamase, but
alteration in the penicillin binding proteins on the
bacterial surface. Such strains are also resistant To
multiple drugs . A drug ter- i slant Strep , pneumoniae
( DRSP) strain originating in Spain has. spread to
mo( t parts of the world posing problems in
Or ament .
The sensitivity of pneumOCocd to optuchin
(ethyl hydrocuprein) 1 /500 ,000 is useful in
differentiating them from streptococci . When a disc
impregnated with optocbm is applied on a plate of
blood ugar inoculated with pneumococci, , L wide
Mine of inhibition appear on incubation .
Antigeniu properties; I he most important
antigen of the pneumococcus is rhe type specific
capsular polysaccharide. As this polysaccharide
diffuses into the culture medium or infective
exudates and tissues, it LS also called the 'specific
Soluble Huhht .Lnce ( SSS), tincumococti arc
1
Copyrighted material
21 B
classified into types haned on the antigenic nature
of the capsular polysaccharide. Pneumococci
isolated from Lobar pneumonia were originally
classified info three types* I , II and Til , and a
heterogeneous group JV , Members of group ] V
were Later classified into types , and now more than
% different serotypes are recognised, named 1, 2 ,
and so on.
Typing may be carried out by (1) agglutination
of [ he cocci with the type specific antiserum ;
( 2) prod pitation of the S5S with the specific Rcnim;
or (3 ) by the capsule swelling reaction described
hy Neufrld (1902). In the capsule swelling or
Lquellung" reaction ( qucllung = swelling ) , a
suspension of pneumococci is mixed on a slide with
a drop nf the ty'yio specific anti -serum and a Icapful
of methylene blue solution . Ln the presence of the
homologous antiserum , the Capsule becomes
apparently swollen , sharply delineated and tefVactile.
The quellung test can be done directly with sputum
from acute pneumonia cases. It used to be a routine
bedside procedure in the past when the specific
antiserum was used tor the treatment ofpncumonia.
The antigenic icy of the capsular polysaccharide
varies in different species. Ic is antigenic in human
beings and rabbits. But in mice, large doses
( 500 fig ) induce no immunological response
(immunological paralysis), while small doses
(0.5 |uLg) are antigenic.
Pneumococci contain other antigens also
v* f. i
t
«
4
-
Fig . 24.3 Draughtsman appearance of pneumococcus colonies. Left: View Irom above to show concentric
rings . Right : side view In cross section .
* Taxlbook of Microbiology *
a nudeoprotein deep inside the cell and a somatic
‘C’ carbohydrate antigen, both of which are species
specific.
An abnormal protein ( beta globulin] that
precipitates with the somatic ' C antigen of
pneumococci , appears in the acute phase sera of
cases of pneumonia but disappears during
convalescence , li also occurs in some other
pathological conditions. This is known as the 'C-
rcactive protein' (CRP ). Its apparent antibody-like
relation to the f antigen of pneumococcus is only
fortuitous. It Is not an antibody produced as a result
of pneumococcal infection. It is an 'acute phase 1
substance, produced in hepatocytes. Its production
is stimulated by bacterial infections, inflammation,
malignancies and tissue destruction. Tt din appear ; 1
when the inflammatory reactions subside. CRP is
used as an index of response to treatment in
rheumatic fever and certain other conditions. CRP
testing, by passive Agglutination using Latex particles
coated with anri- CRF antibody is a routine
diagnostic procedure.
\ iiH cititwr: On repeated subculture, pneumococci
--
undergo i smooth to rough (S-R) variation . In the
R form , the colonies are rough and the cocci are
noncapsulated, autoagglutinible and a virulent. R
forms atix v spontaneous mutants and outgrow
ibe parental 5 forms in artificial culture; in tissues,
such R mutants are eliminated by phagocytosis.
— Rough pneumococci derived from cap&ukted
Copyrighted materia
 4 Pneumococcus
cells of one serotype may be made to produce
capsule -5 of the same or different serotypes, on
treatment with DNA from the respective serotypes
of pneumococci . Th i s transforms!ion, which may
be demonstrated in vivo or in vitro, was discovered
by Griffith (1928) and is of consnlcrable historical
interest as the first demonstration of generic
exchange of information in bacteria.
Tooting and other virulence factors:
PneiiintMiDCL-: produce ait oxygen labile hemolysin
and a Icucocidin bait these arc weak and make no
contribution to virulence . The virulence of
pneumococci depends on irs capsule and the
production of a toxin called pncumolysin . The
capsular polysaccharide, because of its acidic and
hydrophilic proper ics, protects the cocci from
phagocytosis. Capsulated pneumococci are not
phagocytosed efficiendy in fluid media or exudates.
1 :iey are however , Susceptible to 'surface
phagocytosis', hung engulfed against a firm surface,
such as fibrin clot or epithelium .
The enhanced VLTulencc of type 2 pneumococcus
is due to the abundance of its capsular maferial -
Noncapsuiatcd strains are av:.rulcnt. The antibody
to che capsular polysaccharide affords protection
against infection.
Pncumolysin, a membrane damaging toxin
produced by pneumococci has cytotoxic and
complement ac - ivaiing properties and so may be a
virulence factor. It is immunogenic. Pncumolysin
negative mutants show reduced virulence - o
'
experimental animals. Pneumococcal autolysins, by
releasing bacterid components in LNFECTED tissues
'
- .
mav , LL : M contribute to virulence
jf
’
] atInigenici ty; Expei i mentally, fatal i nfeci i on
can be produced in mice or rabbits by intraperitoneal
inoculation of pneumococci - Death occur? ut 1-1
days, and pneumococci can be demon - trated in laigic
numbers in the peritoneal exudate and heart blood.
Pneumococci colonise the human nasopharynx
and may cause Infection of the middle ear, paranasal
sinuses and respiratory tract by direct spread .
Infection of the meninges can also occur, by
f 219
contiguity or through blood . Pneumococcal
bacteremia may also lead to distant infections as in
the heart, peritoneum or joints. Infection is
commonly endogenous, but exogenous injection may
also occur, especially wirh highly virulent strains.
The commonest pneumococcal infections are
oriris media and sinusitis. Prior respiratory infection
or allergy causing congestion and blockage
predispose to these conditions. Serotypes 6, 14, J 9p
and 1. ll ire eornTn -mi. lv/ encountered in these
'
i
conditions, :.n the West.
Pneumococci are one -of the most common
bacteria causing pneumonia, both lobar and
broncbopncumnniiL. They also cause acute
tracheobronchitis and empyema.
Aspiration of nasopharyngeal secretions
containing pneumococci into the lower respiratory
tract is a common event and may occur even in
sleep. Normal mucosal defence mechanisms such
as entrapment, expulsion and the cough reflex, aided
by the ciliary escalator effect prevent establishment
nf infection. When the normal defences are
compromised by viral infection, anesthesia, chilling
or other factors, pneumococci nultiply, penetrate
the bronchi .il mucosa and spread through the lung
along peribronchial tissues and lymphatics.
Bacteremia is common during the early stage of
lobar pneumonia. Toxemia is due to the diffusion
of the capsular polysaccharide into the btaod and
tissues. The fall of temperature by crisis and relief
of symptoms coincide with the neutralisation of
the SSS by anti capsular antibodies.
In adults, types 1-B are responsible for about
15 per cent of cases -of pneumococcal pneumonia
and for more than 50 per cent of all fatalities due
tD pneumococcal bacteremia . In children, tvpes b,
14, 19 and 23 are frequent causes.
Bronchopneumonia is almost always a secondary'
infection. Tins may be caused by any serotype of
pneumococcus . The damage to the respiratory
epithelium and excessive bronchial secretions
caused by the primary infection Facilitate the
invasion of pneumococci along the bronchial tree .
Copyrighted materia
220 + Texibgak jiH.
BioncKopneurfwnii is frequently a terminal cwnt
in aged ami debilitated patients.
MiiL'innoLocu are- cuautiuiilyuudued with the
acute exacerbation? in. chronic bronchitis. The
copious respiratory KcietkHU in chronic bronchitis
aid pneumococcal invasion. Another bacterium
cnmmonlv associated with this condition is
Haemophilus influenzae.
Meningitis in the most serious ot pneumococcal
infections . ] r is usually secondary to other
pneumococcal infections such as pneumonia . otitis
media, sinusitis or LOI ij Linctmtis but in a pn>portion
of ca?es , other foci of infection may not be
demonstrable . Pneumococcal meningitis occurs ir
11 ages. Untmltd case;; sue almost invariably fetal .
*Ever with antibiotic therapy, the case fatality nttt
is about 2 ? per cent .
Pneumococci may also produce suppurative
1 > ions in other parts ot the body — empvema ,
pericarditis , cniris media, sinusitis, conjunctivitis
suppurative arthritis and peritonitis , usually as
complications of pneumonia ,
ftpidtimology; Natural infection with
pneumococci has been reported in some species of
animals such as guinea pigs but they have little
relation to Homan disease . The source ot human
infection is the respiratory tract of carriers and less
often, of patients . PnturnOcocci occurin the throat
of approximately half die population sampled at
any one time , They are transmitted from one to
.mother bv fingers or hi inhalation of contaminated
droplets or droplet nuclei . Dissemination is
facilitated bv crowding.
Infeciion usually leads only T O pharyngeal
carriage . Disease results only when the host
resistance is lowered by contributory factors such
as respiratory viral injections , pulmonary
congestion, stress, malnutrition , immunodeficiency
or alcoholism- Splenectomy and sickle cell disease
arc important predisposing conditions.
Pneumococcal serotypes vary greatly in
virulence. I hfi case totality rates it pneumonia
( mav be ubtained from blood culture in glucose
Microbiology
*
may vary According to The virulence of the infecting
scrorypc. Type 3 is the most virulent,
Lobar pneumonia is usually a sporadic disease
but c ] >i Jcinics i nav occur anKmg dosed communit ies
a ? in army camp? - The incidence of
bronchopneumonia increases when an epidemic of
influenza or ocher viral infection of the respiratory
tract occurs. Cases are more common in winter and
affect the two extreme age groups more often.
1. Llhi >mttirjy di -. if{ iUiMi !lt The clinical diagnosis
yt pneumonia is easy but as the disease may be
caused by several different microorganisms ,
etiological diagnosis should be marie hy laboratory
tests- d 'h is is of great importance in treatment.
In the acute phase of Lobar pneumonia, the
rusty sputum contains pneumococci in large
numbers, with hardly any other kind of bacterium.
E’hev mav hr demonstrated bv Oram stain. In the
preanti biotic eta, direct sc retyping ol pneumococci
in wet films of sputum by the qucllimg icsr was a
routine IKMJHILIC Test because success of treatment
depended on administering the specific antiserum .
In later stages of the disease , pneumococci arc less
abundant.
The sputum , after homogenisation if necessary,
is iiurculated on blood agar plates and incubated at
37 C under 5-lti per cent CO ,. Growth occurs
"
after overnight tncLihatiu-n. Where sputum is not
available, as in infants, scrum- coated laryngeal swab *
may be used for culture. Isolation from respiratory
secretions is facilitated by using blood agar
containing gentamicin 5 pg/ml.
l- rom specimens where pneumococci are
expected to be scanty illation may be obtained by
intraperitoneal inoculation in mice, even if cultures
me licgaliie . lnuculated mice die in 1-3 day ?, and
pneumococci may be demonstrated in the peritoneal
exudate and heart blood . The test may be negative
wirh occasional strains that nr avirulcnt tor mice
( type 14 strains).
In the acute stage ot pneumonia, the organism
L t opy righted materia
 * Pneumococcus * 221

TfrUl* 24.1 DifrEren|ip|ign bglwegn $ fr. prteymOrtte

* ami Sitf- vMfliML
Sit. pneumonia* Sir. viridwns
Morphology Capsulited, LftcptilatG NoACipHilaKd, oval
diplococcj or round cells in chaiins
Quetfung rcs- t Positive N egativc
Colonics litidally dome- shaped ., Dome - shaped
Later,'draughtsman colonics
Growth in liquid media Uniform turbidity Granular turbidity,
powdery deposit
Bile solubility Invariably positive Invariably negative
lmiliin fermentation Positive Negative
Optnchin sensitivity Positive Negative
Intrapcnioneal inoculation Fatal infection Nonpathogenic
in mice

hroth . [foliation of pneumococci from hi nod polysaccharide . I he existence of some 90 serotypes

indicates bad prognosis.
In acute otitis media pneumococci may be
demonstrated in the fluid aspirated from the middle
car
In case of meningitis presumptive diagnosis
may be made fnirn Grant Stained films of CSF. Cram
positive diplococi.i can be seen both inside the
polymorphs and extricettulirlv. Diagnosis is
confirmed by culture. In cases which are negative
by may be possible to establish the
vulture , it
diagnosis by demonstrating the SSS in C 5 F by
precipitation with antisera.
Capsular }Hilysaccharidc can be demonstrated
in the blood, urine and cerebfHpinll fluid by
voLinterimmunoelevtrCiphcii¥sis. Antibodies can be
demonstrated by agglutitiition, pnecip itaCun, mouse
protection tests and bactericidal tests with whole
blood. Indirect hemagglutination * indirect FA test
and radioimmunoassay have been employed .
Prophylaxis immunity is type specific and
associated w i t h antibody to the capsular
makes a complete polyvalent vaccine impracticable,
A polyvalent polysaccharide vaccine representing
the capsular antigens if 23 most prevalent serotypes
has been stated to give 80-90 per cent protection,
lr is nut meant fur general uw, but only in persons
at enhanced risk of pneumococcal infection such
as those will ] absent Or dvafunvrioo.il spleen, sickle
cell disease, coeliac disease, chronic renal * lung,
heart and liver diseases , diabetes mdfitvi and
immunodeficiencies including HIV infection. It in
not recommended in children under two years of
age and those w i t h Ivmphoreticular malignancies
and immunusuppressive therapy..
, Tre&lrnentThe antibiotic of choice is parenteral
penicillin in serious cases ,md uncatyafia in milder
ones, provided the infecting strain is penicillin sensitivc-
Many penicillin resistant strains are also resistant to
other antibiotics like erythromycin and tetracycline,
A third generation cephalosporin is indicated in such
.
csss Vancomycin is to be reserved for life threaten!
iUnenes with hisrhlv resistant strains. ^
•

Furtlur Reading
American Academy oFPlcdiatria 1997 . Therapy for children «nrh iimrivc pneumococcal infection , fttfatrin W:289.
jvnbs MR and PC Applebaum 199S. Antibiotic resistant pneumococci , Rev Mft / Mjarbiof 6 i 77-
Turima >»rn El Ct *1 , IW 5- n <>cnoLa] iriicL tmiL Ativ ting! J McJ .3.12:12 B0.
'
Ilf|
) nfij[

EVpVS MR- 2001 The

. renaissance of c - reactive piotein. BMJ. 322 : J .
Quit PC ei al . 19BI . Symposium on die Pneumococcus , Ref Jnlict Pi* 3:183*

Copyrighted material
 in Neisseria
CM

The genus Neisseria consist Gram negative

aerobic nonsporuhtmg , nonmutile, oxidase positive
cocci typically arranged in pairs (dipiococd ) .
Besides the two important pathogeny JV.
.
*n , *
*
menj ng'rriJFj; and JV. go /iorr/iaeae , the genus
'
«
rt
.
P
contains muny other species such as JV kiCTAjn rci |

‘JO* / .,:
I
that occur as ccunmensab m the mouth or the upper
respiratory tract .

NEISSERIA MENINGITIDIS
( Meningococcus ; Diplocdccus intraCtUaftris
meningitidis ) Meningococci^ first de -scnbfd
and isolated in 19S7 by Weichselbaum from the
-•*» .*s .a
, .2
‘

* ® l»
spinal fluid of a patient. f *
N . meningitidis causes meningococcal
meningitis [ formerly also known an cerebrospiral >
fever) which may occur sporadically, as localised
outbreaks or as epidemics, and also septicemia. Fig. £ 5.1 Merlin gecuceUs In Hold- Inset
Mnrphol y; Meningococci are Gram negative
^
oval or spherical cocci 0.6-08 pm in size ,
- enlarged view to show flat
cocci .
adfi sidn of the

typically arranged in pairs, with rhe adjacent sides

Battened (Fig. 25.1), The long axis of the coccus is
at right angles to -a line joining the two cocci in
a pair Considerah1e variations occur in size,
shape and staining properties, especially In older
cultures, due Co autulysis. In Smears from legions,
substances in bine media mrhrr rl i b ( n ... II - -,
additional nutritional needs.
-
They are strict aerobes, no growtl occurring
anaerobically. The Optimum temjlerarun for growth
is 35-36 '’C, No growth takes plai- e below 30 *C.
Optimum pH is 7.4^7.6. riTiwth is facilitated by
1

the cocci arc more regular and generally
intracellular TheJT? arc non motile . Most fresh
isolates are capsulated .
t inltm- ul charLicteristics: Meningococci
have exactinggrowth requirements and do not grow
on ordinary media. Growth occurs on media
- r
5-10 per cent CO, and high humidity.
On solid media , after int ibatfon for 24 hours ,
rbe colonies are small (about 1 HUM in diameter )
translucent , round , convex, I - M H - I grey, with
smooth glistening surface and . li entire edges.
The colonies are typically lertTlCi l . r in shape ,

buryrous Ln consistency and . L il -. ernulsifiablt .

enriched with hJood , serum or asdtie fluid , which
promote growth by neutralising certain inhibiting Weak hemolysis occurs on HICHKI agar. Smooth ami

OfJVt rh
"
d material
 * Neisseria
rough types of colonies are found. Growth is pour
m 1 i.c ]vimedia, producing a granular tutbidi tv with
little or no surface growth.
By .LU;HT , chocolate agar and Muelle H niton
^
starch casein hydrolysate agar ire the media
cii mm -. m I v used for culturing men ingncocci .
-
Modified Thayer Martin { with vancomycin ,
nilislir and nystatin ) i > a useful selective medium .
Eliocheitiivd rCACtions: They arc catalase and
oxidase positive. The prompt oxidase reaction helps
the identification of ncisseria. ( both meningococcus
and gonococcus) in mixed cultures. When freshly
prepared 1% solution of oxidise reagent ( tetrameihyl
pataphenylene diamine hydrochloride) is poured
on the cuicure media, the neisseria colonies cum
deep purple. Subcultures should be made
immeilLately, as the organism dies on prolonged
exposure to the reagent. The test may also be
performed by rubbing a little of the growth with a
loop on a strip of filter paper moistened with the
oxidase reagent ( Kovach method ). A deep purple
colour appears immediately.
Indole and hydrogen sulphide are not produced
and nil rates axe not reduced - Glucose and maltose
are utilised, but not sucrose or lactose, producing
acid but no gas (gonococci acidify' glucose but not
maltose ). Arid formal ion by neisseriae is weak,
being oxidative and therefore best tested on peptone
scrum agar slopes containing the sugar and
indicator.
Am iconic properi ica and DIBBM float ion:
Meningococci are capsulated , unlike other
ni ' isseriae . Rased on their capsular polysaccaride
antigens, meningococci are classified into at least
13 scrogroups, of which Groups A, B and C are
the most important. Group A is usually associated
with epidemics and Group L mostly with Idealised
outbreaks, while Group R causes both epidemics
and outbreaks. Groups 29-E, W-135 and \ also
frequently cause meningitis. Any serugroup may
colonise the nasopharynx, but these six groups
account for rhe large majority of meningitis,
Serngmups are further classified into serotypes and
223
siihtypeS based on outer membrane proteins and
poLynaccharides.
Resistance: Memngococci arc very delicate
organisms , being highly susceptible to heat ,
-
dessication, alterations in pi I and to di aufectants.
-
They were uniformly sen .:rive to penicillin and
other antibiotics, but resistant strains have emerged
and become common m many arexs-
Pathogenicity: Cerebrospinal meningitis and
nieningpcoccai septicemia are the two mu in types
of meningococcal disease . Meningococci are strict
human parasites inhabiting the nasopharynx .
Infection is usually asymptomatic. In some, local
inflammation ensues, with rhinitis and pharyngitis.
Dissemination occurs only in a small proportion .
The manner in which the cncci Spread from
the nasopharynx tn the meninges may he directly
along the perineural sheath of the olfactory nerve,
through the cribriform plate to the subarachnoid
space, or more probably, through the bloodstream.
In certain cases the -. itc of entry of the
meningococcus may be the conjunctiva. Cases of
meningococcal purulent conjunctivitis occur. On
reaching the centra! nervous system, a suppurative
lesion of the meninges is set up, involving the surface
of the spinal cord as well as the base and cortex of
the brain. The cocci are invarmhly found m the
spinal fluid, both free and within the leucocytes .
Case ferity is variable but in untreated cases may
be as high as SO per cent. Survivors may have
sequelae such as blindness and deafness. Some cases
develop chronic or recurrent meningitis
Mcningocooicinia, presents -as acute fdver wiith
chills, malaise and prostration. Topically a petechial
=
rash occurs early i i the disease- Meningococci may
be isolated from the petechial lesions. Metastatic
-
involvement of the pun ears, eyes, lungs and
,
adrenals may occur. About 10 per cent develop
pneumonia.
A few develop fulminant meningococccmia
(formerly called Waterhouse- Friderichsen
syndrome ) which is an overwhelming and usually
fatal condition , characterised by shock, disseminated
Copyrighted materia
224 * Tflsitbook of Microbiology *
intravascular coagulation. and multisystem failure.
Rarely chronic meningococcemia may be seen.
Meningococcal disease is favoured by deficiency'
of the terminal complement components (C5 C9).
The pathogenic agent in meningococcal disease
- large numbers in the spinal Auid and , in the early
appears to be the endotoxin [ LPS) released by
-
autoJy js. The vascular endothelium is particularly
-
sensilive to the endotoxin. All itt.ii' it mLl .imm iUirv
.
cascade systems as well is cytokines and nitric oxide
are triggered and uprcgulated. In fulminant cases
adrenal hemorrhage and profound shock are
present . divided into three portions . One portion is
Natural infection is limited to human beings,
[ ntraspinal inoculation of Large numbers of cocci
may produce a picture of meningitis in monkeys-
Irtraperitoncal inoculation of the cocci suspended
in hog gastric mucin brings about a fatal Infection
in mice.
Epidemiology: The human nasopharynx is the
1
-
only n crvi u r of the mci i i ngococcus, Asympn ? mat ic
nasopharyngeal carriers rarely contract the illness
hut serve to infect their contacts. Transmission is
essentially hy airborne droplets or less often by
fomhes. During intcrepidcmk periods, the carrier
-
race is about 5 10 per cent . An increase in carrier
rate heralds the nr Ret of an epidemic . During
epidemics the carrier races in closed cummumiiL' E
-
may go up co VO per cent, Meniiigi.ii is common
m children between 2 months and 5 years of age.
Epidemics usually occur in semidosed communities
living in crowded conditions, as in jails and ships
formerly, and in army camps in recent times.
Prevalance of mei:ingiii > is highest in the
' meningitis betr of Africa' stretching from Ethiopia
to Senegal. Frequent epidemics have occurred here .
One of the largest was in lV9ft , when 150,000 cases
and 15,000 deaths were reported.
Laboratory (JUL IKKUS: The primary agents
^
causing purulent haeierial meningitis are
meningococci , pneumococci and Haejnopfi .' fus
influenzae type h. Other important causative agents
air group B streptococci, staphylococci, Escherichia
coir and Listeria monocyfqgenes-
It is necessary to establish the specific etiology in
purulent meningitis for proper treatment . In
meningococcal meningitis, the cocci are present in
stage, in the blood as well. Demonstration of
meningococci in the nasopharynx helps in the
detection of carriers,
1. £ jtnnii| ] fii >fi of £ .’.S7 The fluid will be under
j
pressure and turbid, with a large number of pus
cells . For bacteriological examination, if a
sufficient | L uarttity is available , the CSF is
centrifuged and Cram stained smears are
prepared from the deposit. Meningococci will
he seer mainly inside polymorphs but often
cxtraccllulariy also. This presumptive diagnosis
is sufficient to start antibiotic treatment. The
supernatent will contain meningococcalamigen,
which may be demonstrated by latex
agglutination or counter Immunoelectrophoresis
using meningococcal antisera. Similar tests are
also available for pneumococcus, H . influenzae
type b and Group B streptococcus antigens.
Antigen detection is particularly useful in
parII. illy treated patients in whom smear and
culture tests may be negative . The second
portion of the CSF is inoculated on blood
agar or chocolate agar plateR and incubated at
35-3fi T under 5-10%. CO{. Colonics appear
—
after IS 24 hours and may be identified by
morphology and biochemical reactions. It is
i mportant to remember that morphologically
.
similar organismR such as iV, flavesccns JV, fljrva
and Acinetobtcter may also cause purulent
meningitis occasionally. The isolated
meningococcus may he grouped , if rcqiiired* by
agglutination with the appropriate sera . The
third portion of CSF is incubated overnight,
either as it in or after adding an equal volume of
glucose broth and then subcuitured on
chocolate agar . This method I N .LV sometimes
succeed where direct plating fails.
2 . Blood culture: In menulgocuCccniia and in early
Copyrighted material
 * Natasaria
cases of meningitisP blood -culture is often
positive Cultures should be incubated For 4^7
..
days , with daily subcultures. Meningococcal
. with chemoprophylaxis .
antigens can be found in che blood in active
disease. .
3 . .Vasap/isiyngBjil swuh: This is- useful for the
detection of carriers. Sampling should be done
without contamination with saliva. The swab
should be held in a suitable transport medium
( for example, Stuarts ) till it is plated .
4 . Petechia/ lesions: Meningococci may
.
sometimes be demonstrated in petechiaJ lesions
by microscopy and culture .
5. /lijtppvy;At autopsy, specimens may be collected
from the meninges , lateral ventricles or the
surface of the brain and spinal cord for smear
and culture ,. Meningococci may die il specimens
are not collected within twelve hours of the
death of the patient.
6. fietrapectTH: evidence: Retrospective evidence
of meningococcal infection may he obtained by
detection of antibodies.
7. Mo/ecdir diagnosis: Group specific diagnosis
ol infection Can be made bv detection of
meningococcal DNA m|uence in CSb or blood
by PCR amplifications.
Treatment: Prompt treatment IH essential To
ensure recovery without sequelae . Sulphonamides,
once the mainstair, are not used now due to
widespread resistance . Intravenous penkillin G LR
the trcfltmcn t of choi cc. Chlora mphen i oo! is equally
effective . One of the liter cephalosporins
(ceftriaxone , ceftazidime ) may he used for the
iniriation of treatment before rhe eriology of
meningitis is known.
After the initial course of treatment, cradicarivc
therapy is to be given with rtfampicin or
ciprofloxacin to free [he nuophuyni of the cocci
and prevent carrier state .
Prophylaxis: Sulphonamides are not effective
-
due to pyjitUHe. Pfcnicillin is unihk to eradicate
the carrier state. Rifampicin or cLprotloxacin LS
recommended for chemoprophylaxis. As attack rates
225
arc very high in the household or close contacts of
meningococcal patients, they should be provided
Monovalent and polyvalent vaccincs arc mMk
containing the capsular polysaccharides of groups
A , L\ W-135 and V . The vaccines induce good
immunity after a single dose in older children and
adults hut arc of lirtLe value in children below
3 years. The immunity is group specific . There is
no Group B vaccine available ar present.
NEISSERIA GORORRHOEAE (GONOCOCCUS )
JV . gonorrhocie causes the venereal disease
gonorrhea. The gonococcus was first described in
gonorrheal pus by Ncisscr in 1879 . Hu mm in 1885
cultured the cocCUs and proved its pathogenicity
lw inoculating human niluiuetrs . Gonococci
resemble meningococci very closely in manv
properties.
Morphology : In smears from. 1 be urethral
disduuge in acute gonorrhea , the organism appears
as a diploeoccus with the adjacent sides concave ,
being typically kidney -shaped . It is found
predominantly within the polymorphs, some cells
containing as many as a hundred cocci .
Gonococci possess pill on their surface. Pili
facilitate adhesion of the cocci to mucosal surfaces
and promote virulence hv inhibiting phagocytosis.
Piliated gonococci agglutinate human red blood
cells hut not red. cdk from other mammals . The
hcmagglurination is nor inhibited by mannose ,
( iulturai characteristics: Gonococci arc
more difficult to grow than meningococci . They
are aerobic but may grow anaerobically also .
Growth occurs h-esi at pH 7 . 2 -7 . fi anil at ; L
temperature of 35-36 “ C. Jt is essential to provide
5- 10% GOj. They grow well on chocolate agar
and Mueller-Hintcni agar . A popular selective
-
medium is the Thayer Martin medium ( containing
vancomycin , colistln and nystatin) which inhibits
most contain in ants including nunpithogen i c
neisseria .
Colonies are small , round. Translucent , convex
Copyrighted materia
22b 4 Textbook of Microbioiagy
or slightly umbonate, with finely granular surface
and lobate margins. They are soft and easily
cmul ' i liable.
Four types of colonies have been recognised -
Tl to 7 4.Types 1 and 2 form small brown colonies.
The cotip .LTL " jiiIi .L1 CL ] , autoaggluLinsble and virulent.
Types 3 and 4 form larger, granular, nonp igrocnted
colonies. T3 and T 4 cocci are nonpiliatcd, Form
smooth suspens i i ms and are avi rulcnt. Fresh i ^ oktes
from acute cases of gonorrhea generally form Tl
and T 2 cnlnni.es. On senaf subculture, they change
to T3 or T 4 colonial morphology. Tl and T2 types
.
arc also known as P" and P" respectively, while
1 > and 14 are known as P . "
E i cwhemi L'al react inns- Gonococci resemhle
meningococci except in the effect on maltose-
Gonococci acidify only glucose and not maltose .
Antigenic properties Gonococci are
antigemc& lly heterogeneousc 1 hey arc capable of
changing their surface structures in vitro, 7*hcy
probably do so in vivo as well, to avoid host defence.
The surface structures include the following;
Pifj which are hairlike structures several
micrometres long, act as virulence factors by
promoting attachment to host cells and inhibiting
phagocytosis, Pili are composed of repeating peptide
subunit ?. ( pilins) consisting uf conserved (uonstanl !
and variable regions. Fill undergo antigenic and
phase variation.
The trilaminar outer membrane of gonococci
contains many different proteins. Protein I is the
major constituent ind shows antigenic diversity,
which helps in typing gonococcal strains. Protein I
of a '- ingle strain is antij emcally constant, though
^
It shows considerable heterogeneiry between
different strains, There we two variants of protei n L,
called IA and IB, Any one strain carries only either
LA or IB but not both. Using monoclonal antibodies
to protein 1 epitopes, gonococci can be classified
Litfo several setovars, Al to 24 and Bl to 32 .
Proteins I and III act as ligands attaching the
coccus to the host cells. They also form a
tratsmembrane channels (porms ) which play a role
in the exchange of molecules across the outer
membrane.
Protein II is related to the opacity of the
gonococcal colonies and so is called the 'opacity
associated' (OPA ) outer membrane protein. Strains
with the OPA protein form opaque colonies and
those hi . king it transparent colonies. A strain may
express 0 to 3 serological varieties of the OPA
prorein at a rime- OPA may be responsible for
attachment to the host cells and also for the
clumping af cocci seen in urethral exudate smears.
I’ hc outer membrane also contains
lipopolysaccha ride (endotoxin ) which may be
responsible for the toxicity in gonococcal infections.
Many other proteins of poorly defined roles in
pathogeniciry have been described. Both gonococci
and meningococci elaborate IgAl protease that
splits and inactivates IgA.
RuistAAne The gonococcus is a very delicate
organism , readiily killed by heat , drying and
antiseptics. Tt is a strict parasite and dies in 1-2
hours in exudates outside the body.
—
In cultures, the coccus dies in 3 4 days but survives
in slant cultures at 3? °C if kept under sterile paraffin
oil. Cultures may be stored for years if frozen
quickly and left at -70 °C.
lJHthojJi;n , , - iiy ? Gonorrhea is a venereal disease
which has been known from ancient times. The
name gonorrhea ( mcaniTig> flow of seed ) was first
employed by Galen in 130 AD.
The disease is acquired by sexual contact . The
first step in infection is adhesion of gonococci to
the urethra or other mucosal surfaces. Fill are
involved iti this adhesion. Adhesion is rapid and
firm so that micturition after exposure offers no
protection against infection. The cocci penetrate
through the intercellular spaces and reach the
subej4cbeiial connect i ve ti HSLLC by the th Lid day after
infection, The incubation period is 2-8 days. In
men , the disease starts as an acute urethritis with a
mucopurulent discharge containing gonococci in
large numbers . The infection extends along the
urethra to the prostate , seminal vesicles and
Copyrighted material
I JF W
 * NetSBflria *
epididymis. Chronic urethritis may lead DOSTRICTURE
forma r ion . The infect - on may spread to the
periurethral tissues, C. IIKIIIC uhseenses and multiple
dischargm sinuses ('wacercan perineum").
^ , the initial infection involves the
In women
urethra and cervix uteri , "i"hc vaginal mucosa is not
usually affected In adults because the straiificd
squamous epithelium is resistant to infection by the
cocci and also because at the acid pH of vaginal
secretions. { Vulvovaginitis occurs in prepubertal
girls ). The infection may extend to Bartholin's
glands , endometrium and fallopian tubes . Pelvic
inflammatory disease and salpingitis mav Lead to
sterility. Rarely, peri tor it is may develop with
perihepatic inflammation ( Pitz — Ifugh -Curtis
syndrome ) , Clinical disease Is as a rule less severe
in women, many of wliom may cany gonococci in
the cervix without any symptoms. Asymptomatic
carriage of gonococci is rare in men,
Proedv. - occurs in both sexes. It may develop
by direct contiguous spread in women but in men
i s usually the result of anal sex . Conjunctivitis may
occur, usually due Co aufr ijnocuf ation by the pat i ent 's
fingers. Blood invasion may occur from the pri maty
site of info. don and mav lead to metastatic lc-- Ii > n^
, adults . The reasons for the Increase in gonococcal
such u arthritis , ulcerative endocarditis and verv
rarely meningitis. Occasional cases of pyemia have
been reported.
A nonvencreal infection is gonococcal
ophihilmoa in che newbornr which results. from
direct infection during passage through the birth
canal. Gonococcal bacteremia leads to skin lesions,
especially hemorrhagic papules and pustules on the
hands, forearm , feet and legs * and to tenosynovitis
and suppurative arthritis, usually of the knees, ankles
and wrists.
Gonococci contain several plasmids . Nincty-m
per cent of the strains haw a small cryptic plasmid
of unknown function . Two other transmissible
plasmids contain genes that code for beta lactamase
which causes resistance to pcnicillin -
: Epidemiology: Gonorrhea is an exclusively
human disease , there being flu natural irtiectiort in
; In chronic infections, there may not be any
227
animals . Experimental disease may be produced in
chimpanzees by urethral inoculation . A lethal
infection can he produced in mice by intracerebral
inoculation.
The only source of infection is a human carrier
J
or less often a patient. The existence of asymptomatic
carriage n women makes them a reservoir serving
to perpetuate infection among their male contacts.
The made ni infects in Is almost exdu:- ivclv venereal.
"
if
Fomices do not play any significant role, as the rood
die rapidly outside the human body. The only
nonvenereal infection is ophthalmia neonatorum.
Ort.ee very arniman , thin has been controlled by
the practice of instilling 1 % silver nitrate solution
into the eyes of all newborn babies (Grade's method ).
When sulphonamides, and later penicillin, were
found very effective for the treatment of gonorrhea,
it was hoped that the disease could be eradicated.
However, after a temporary decline, its incidence
has been rising steeply. In 1970, the global incidence
of new cases was estimated at 16 million , making
h one of the most common infective diseases. In
same areas, gonorrhea has reached epidemic
proportions , especially in adolescents and young
infection are largely social and cultural. In the
19B0s, with the AIDS scare, there was a noticeable
decline in the incidence of gonorrhea, but this has
not been kept up , A higher incidence of gonorrhea
has been observed in persons belonging to blood
group B. The bans for tins not known.
Laboratory diagnoita In the acute stage ,
diagnosis can he established readily bill chronic
cases sometimes present great difficulties , Ill acute
gonorrhea the urethral discharge contains
gonococci in large numbers. The meatus is cleaned
with & gauze soaked in saline and a sample of the
discharge collected with a platinum Loop for culture ,
or directly on slide for smears . In women, besides
the urethral discharge, cervical swabs should also
be collected - This should be done carefully, using a
speculum. High vaginal swabs arc not satisfactory
Copyrighted material
22%
urethral discharge. The 'morning drop' of secretion
may be examined or some exudate may be obtained
after prastatic massage . It may also be possible tit
demonstrate gonococci in the centrifuged deposits
of urine in cases where no urethral discharge is
available.
The demonstration of intracellular Gram
negative diplococci Ln stained smears provides a
presumptive evidence of gonorrhea in men. It has
to be emphasised that diagnosis of gonnfthea by
smear examination is unreliable in women as some
of the normal genital flora have an essentially
similar morphology. The use of fluorescent
antibody techniques for the identification of
gonococci in smears has increased the sensitivity
and specificity of diagnosis by microscopy.
For culture, specimens should he immolated on
prewaimcd plates, immediately on collection. If this
is out possible, specimens should be collected with
charcoal impregnated swabs and sent to the
laboratory in Smart's transport medium . In acute
gonorrhea , cultures can be obtained readily on
chocolate agar or Mueller-i bn ton agar incubated
k
1i .
oe .tr
I
"5?
4
-
.8
u
ITJTI LJ
1
4 : tfV
_ From I 97fi , gonococci producing p- lactamase
IT'
TI
yiLr
Fig . 25.2 Gonococci In urethrai pm Inset: enlarged
view lo shew kidney Shaped cells with adjacent
surfaces concave .
^ TemtiOO* 01 Microbiology
at .35-36 *
c under 5-10% CO .,. In chronic cases,
where mixed infection is usual and in the
e&rmin &tioi] of lesions such as proctitis, however,
it is better to use a selective medium such as the
Thayer^ Martln medium . The growth is identified
hv morphology and biochemical reactions.
It may not be possible to obtain gonococci in
culture from some chronic cases or from patients
with m cl astatic lesions such as arthritis. Serological
tests may be of Value in Such instances. The
complement fixation test has been used with varying
degrees of success. IT becomes positive only some
weeks after the infection is established and may
remain positive fur mouths or years after the disease
has been cured . The test may also be positive
following meningococcal infections. It is necessary
to use a polyvalent antigen because of the antigenic
heterogeneity of the gonococcal strains.The test is
not suitable for routine use. Many oilier serological
tests have been attempted, including precipitation ,
passive agglutination, immunofluorescence* and
radioimmunoassay using whole-cell lysate, pllus
protein and lipopolysaccbaride antigens. However,
no serological test has been found useful for routine
diagnostic purposes .
I refitment In 1935 , when sulphonamides were
introduced for treating gonorrhea , all strains were
sensitive to the drug but resistance developed
-.*£> j.O** *
1
rapidly Again, when penicillin was introduced, all
strains were highly sensitive ( \1IC 0.005 unit/ ml ) .
From 1957, strains with decreased susceptibility
( MIC higher than 0,1 uniit/ml ) became common .
i As patients infected with such strains did not
m
F"
respond to the usual doses of penicillin , vciy large
.
doses of penicillin, 2.4-4 .fi million units were used .
-. yCr -
r
( penicillinase ) have appeared, rendering penicillin
- treatment ineffective. These penicillinase- producing
IV, onrrrrllOCJe ( PPNG } have spread widely.
^Tlic Centers for Disease Control anil Prevention ,
. USA in 1993 recommended the following schedule
for uncomplicated gonoirhca: Ceftriaxone 125 mg
single INI Jose or Ciprofloxacin .500 mg (or
Copyrighted materia
 * Neisseria 229

Ofloxacin 400 nig ) single oral dose , plus
Doxycyoline 100 mg twice daily for 7 days or
Erythromycin 1 g single oral dose . The regimen is
costly hut wmrltH very well against gonococci and
due to gonuCOccal infection, the coed persisting us
L-forms and hence undetectable by routine tests.
The majority of such cases are , however , the result
of infection s ofdivcrsccriology. Thc most important
'

the frequently coexisting chlamydial infection.
Prophylaxis: Control of gonorrhea consist* of
early detection of cases , contact tracing , health
education and other general measures , As even
clinical disease does not confer any immunity,
vaccination has no place in prophylaxis ,
NONGONOCOCCAL {NONSPECIFIC} URETHRITIS
Along with an increase in the incidence of
gonorrhea, there has also been an increase, in recent
years, of eases of chronic urethritis where gonococci
cannot be demonstrated . This has been called
lurlgOtluCoccd Or notlgpcdfr: urethritis. In some
of these , urethritis forms part of a syndrome
consisting of conjunctivitis and arthritis in addition
( Reiter's syndrome ). Some of these cases may be
Table 25.1 Differential characteristics of Neisseriae
of these arc CAfam. Cndumitii , UrcapIaMma
^
uia ticum and iWm /a ma bonuais. J lerpesvinis
^^
and cytomegalovirus may also account for sonic
cases . Urethritis may also be caused by other
bacteria ( for example , Gardncrcllt nginilis ,
Ac- jnerohuL- rer IwofTi , Ac. cilccActdcitsX fungi
( Candida albicans ), pmtotna (Trichomonas
vaginalis ), or even by mechanical OJ chemical
irritation . As etiological diagnosis is soldum
achieved , the management of this syndrome is
difficult ,
COMMENSAL NEISSEHIAE
Several species of Ncisscriac inhabit the normal
respiratory tract . The characteristic features of yjroc
of the common specie: arc bated in Tabic 25, 1 -

SpCciiGM Colonies Growth FrrrnrntMtinn Serologicaii

On At GJuocve Mubvre SutTOtt c/ jsji /knfiofT
nutrient 22"C
IgSLT

N. mtnigindis Ryu lid, MTicxalh , A A Thirteen

shiny, ettamy antigenic
Consistency groups
N, gononhocMC Same as above, A Antigenically
bui smaller and heterogenous
_
JY flarescens
more opalescent
Resemble + + Antigenically
meningococcus but distinct
pigmented yellow homogeneous
gfrtUp
.
jV sicca Small , dry, opaque, if + A A A Aumaggjucmahlc
wrinkled * brittle
j\r. Autoa
/aaJjs Variable, smooth
^^dnabk
cafjur # +
and xriRtluant or
itraifTftiiftsJ
t’ adherent and
opaque, not easily
emultifiibLe

Copyrighted materia
230 * Textbook of Murobiology >

Their pathogenic significance is uncertain though colonisarion by N- Jacranncs in young children may
'

some of them ( for example * N , fi& ve&cen& y N .
citarrhzli * ) have been reported occasionally as
having caused meningitis.
.V . iaetsmjL’ jf frequently isolated from the
nasopharynx is closely related to meningococci ,
though it is virtually aviinilcnt . It dirTers from
pathogenic nei &aeriar in being positive in the
ONPG test tor beta galactosiiiase. Nasopharyngeal
be responsible for the presence in them of antibodies
protective against mcniitgococcal iitfecdon.
jVeiiieru catarrhaUy is now classified as
Maraxclli { BranhumcHa } catarrhxtis . It i * an
opportunistic pathogen capable of causing laryngitis *
bronchopncumoria, men i rgitis, n, nus i i is and middle
ear disease .

Further iteailjnj
Britigan BE and PF Spading 1965. Gonococcal infection . New Engl J Med . 1 . ( <? ! . '

Cartwright fc {H3 ) 1995. Menifigecoccil Disease Chichester: john Wiley.

Mootc SA et at 19S9. EVrfpeL ilvci im [ Kithiigm: . Ncissetin -spp. C/ip MNTIIIIKJJ Ke ^ 2 (5uppl - t5) .
h

Rcscntstcm NC et il. 2001 . Mci in tjcwca] diseases , .’Vew tngiJ Med , 334: 1376
^
Tunlcel AR and MW Schrld 1995 . Acute bacterial meningitis. fjnerf , 346: 1675.

LJ opyrighted materia
 Corynebacterium
Coryneb nutria j-rt- Gram pusitive h ornnidd fast,
iqonmptilc rods with irregularly Plained. segments,
and somerimes granules. They frequently show dub
Heaped swellings and he nee the name
coiyncbacteria ( from cvryne , meaning club) lire
, bacterial cell - Stained with LoefflptV methylene
most important member of the genus is
C- diphtheriae, the causative agent of diphtheria.
Diphtheria has been known from ancient times.
Aretacus, the Cappadocian, in the second century,
described the Egyptian or Syriac ulcer, which most
medical historians agree can he identified as
diphtheria. The disease was first recognised as a
clinical entity by Rretormcau who called it
' diphtherirc . The name is derived from the tough,
' They 3 re usually seen in pairs, palisades (resembling
leathery pseudo-membrane farmed in the disease
(drphrhe/os, meaning leather ) . The diphtheria
bacillus was first observed and described by Klebs
( 1 RS3) but was fust cultivated by Lirefflef (1884) .
Jt Is hence known as the Klehs-1 .oeffler bacillus
or KLB , Loeffler studied the effect of the bacillus
in experimental animals and concluded that the
disease was due to some diffusible product of the
bacillus . Roux and Yersiu (1863) discovered the
diphtheria exoEmin and established its pathogenic
effect. The antitoxin was described by vOrt Behring
(1090) .
CORY NEB ACTERIUM DIPHTHERIAE
Morphology: The diphtheria bacillus is a slender
rod with a tendency to dubbing at one or both
ends, measuring approximately 3-6 * 0.6-0.5
Jim. The bacilli arc pleomorphic . They are
nonsporing, noncapsulated and nemmotik - Cells
often show septa, and branching is infrequently
observed. They are Gram positive but tend to be
decolorised easily. Granules composed ut
polymctajihnsphare air seen in the cells. They air
more strongly Gram positive than [he rest of the
blue, the granules take up a bluish purple colour
and hence they are called me [ achromatic granules.
They are also called vohjrijior Babes Einst granules.
They 3 re often situated nr the poles of the bacilli
and are called polar bodies. Special stains, such as
Albert’s, Ncisscr’s and Ponder ’s have been devised
for demonstrating the granules clearly. The bacilli
ate arranged in a characteristic fashion in smears .
stakes of a fence) or small groups, the bacilli being
at various angles to each other , resembling the
letters V or L. This has been called the Chinese
Jctfcr or cu/ieiform arrangement. This is due to the
incomplete separation of the daughter cells after
binary fission (Fig. 26- 1) .
Cultural characteristics: Growth is scanty'
on ordinary media . Enrichment with blood, serum
or egg is necessary for good growth. The optimum
temperature for growth is 37 DC (range 15-40 °C)
and optimum pi I 7.2 - It is an aerobe and a
facultative anaerobe.
The usual media employed for cultivation of
the diphtheria bacillus arc Loeffler 's strum slope
and tellurite blood agar . Diphtheria hacilli grow
on Loefflefs serum slope very rapidly and colonies
can be seen in 6- 8 hours, long before other bacteria
grow. Colonies art flt firet small, circular white
opaque discs but enlarge on continued incubation
and may acquire a distinct yellow tint. Several
Copyrighted material
232 4 Textbook
/A mu
= f -
\ fc
\ *s c
\ <£
\ o~
/ A- ! V
»\ ^ l
!s
^
>
i .
& at different times. There is evidence to show that
\ u- , / «/;
* mitis in populations naturally immune or srtifiddly
f
%
Fig . £ 6,1 Lett : normal lorms ol C.d rftefie
Rig hi : Involution Forma ^
modifications of tellurite blood agar hive been
milledh such as McLeod’s ind Hoyle’s media
Tellurite (0,04%) inhibits the growth of most other
bacteria, acting as a selective agent. Diphtheria
luLlJi reduce tellurite to metallic tellurium, which
i > incorporated ITI rhe ^ninnies giving them a grey
.
or black colour The growth of diphtheria bacilli
may he delayed on the tellurite medium and colonies
may take two days to appear. Based on colonial
morphology On the tellurite medium and Other
properties, McLeod classified diphtheria bacilli into
-
three types gracjs, infemredfus and nuris, The
'
names were originally proposed to relate to the
clinical severity of the disease produced by the three
types - gravis , causing the most serious , and mills
the mildest variety, with intermedins being
responsible for disease of intermediate severity.
However, this association is not constant . The
necessity for typing an isolate in the laboratory' has
been superseded by the need to know whether the
strain is toxigenic or not . Certain biological
characteristics of these individual types have some
value.
or Microbiology *
The gravis and intermedius types; are associated
with high case fatality rates, while mitis infections
are less lethal . Paralytic complications arc most
common in gravis , hemorrhagic complications in
gravis and intermedius, and obstructive lesions in
the air passage in mitis Infections, 111 general, mids
is the predominant strain in endemic areas, while
gravis and intermedius tend to be epidemic. The
mitis type is better able than the more virulent types
in establish a commensal relationship with the
host Wide variations have been noted in the
frequency of the different types in different places
the gravis and , ro a lesser extent, the intermedius
strains ate able to spread more readily than the
immunised. Tabic 26.1 lists the characteristics of
.
the three types
Diphtheria bacilli ferment with rhe production
of acid, (but no jps) glucose, galactose, maltose
and dextrin (but not lactose, mannitol or sucrose).
. Some strains of virulent diphtheria bacilli have been
found to ferment sucrose.It is necessary to employ
Hiss's serum water for resting sugar fermentation,
FVoteolytic activity is absent. They do not hydrolyse
urea or tbnn phosphatase.
TOXIN
Virulent strains nf'
dLphrheria bacilli produce a veiy
powerful exotoxin, The pathogenic effects of the
.
bacillus arc due to the toxin Almost all strains of
gravis and intennedi.ua ( about 95-99 per cent} are
toxigenic, while only about 9D-f!S per cent of the
mitis strains arc SO. The proportions vary with the
.
origin of the cultures tested Strains of all three
types arc invariably virulent when isolated from
acute cases, A virulent strains arc common among
convalescents, contacts and carriers, particularly in
those with extrafaucial infection. There is
considerable variation in the amount of toxin
produced by the different strains, some strains
.
producing it abundantly and others only poorly But
the toxin produced by all the strains of diphtheria
Copyrighted material

Table 26.1 Type
Grtvis
Morphology Usually short rods, with
uniform scaining, few or no
granules. Some degree of
pleomorphism., wirh iireguladv
barred, snow-shoe and (ear -
drop forms
Colon v on tellurite In hours, colony is 1 2 mm
blood Hgur in size , with greyish black
, 1mm in size* misty. Does
centre* paler, semitranslucent
periphery and commencing
crcnation of edge- Tn 2-3 days*
-
3 S mm in si- sw, fUr colony
with raised dark ten ( re and
crcnared edge wirh radial
-
srriarion ‘daisy head colony
Consistence of
V
Like cold margarine * bri ( tEe,
1
colonies moves AS ii whole on the plate,
not easily picked out or
emultifiable
Hemolysis Variable
Growth in broth Surface pellicle, granular
.
deposit Little or no turbidity
Glycogen and stanch Positive
fcrmenraiion
bacilli is qualitatively similar. The sfrdin almost
universally used for toy in production is rhe ' Mark
Williams 3' straiji , which has been variously
JcM- rilKul as a initis- ( Tiipltry and. WiluKi ]
and ail
irfermeditLs strain Cmickshank .
( )
The diphtheria toxin is ; L protein and has lieen
crys- tahayecl. It has a molecular weight of about
62 n 000, It is ratnemcly potent aTid the lethal dose
For A 250 g guinea pt is 0.0001 mg. It consists of
^
wo fragments, A and B, of MW 24 n (XX> and 3? ,000,
respectively. Both fragments are necessary for the
roxie effect. When released bv rfie bacterium, the
Jr
4 Corvi ' Efbadtmum 233
differentiation of diphtheria bacilli
furenudfur M/rif
Long barred forms writh Ijnng, curved ,
dubbed CM kb; poor pleomorphic rods wirh
granulation, very prominent granules
pleomorphic
* IK hour colony small* Size triable* shiny
black. In 2-3 days,
not enlarge in 48 hours, colonies become flat *
dull granular centre with with a central
smoother, more elevation poached egg
glistening periphery and
a lighter ring near The
colony
1
-
edge "frogs egg" colony
„ Intermediate between Soft, buttery, easily
gravis and mirif HuibafiaUe
Nonhemolvtic
t
Usually hemolytic
Turbidity In 24 hours* Diffuse TUffcidicy with
clearing in 49 hours* soft pellicle later
with line granular
sediment
Negative Negative
toxin is inactivt because the active Hite on fragment
A is masked. Activation is probably accomplished
by proteases present in the culture medium and
infected tissues. All the enzymatic activity of the
tone in is present in. fragment A. Fragment B is
responsible for binding the toxin to the cells. The
antibody to fragment B is protective by preventing
the binding of the toxin to the cells. The toxin LH
labile. Prolonged storage, incubation at 37 C for C’
—
4-/> wceksp treatment with 0.7 0.4 per cent formalin
or acid pH converts it to toxoid. Toxoid is toxin
r that has lost Its toxicity but not its antigenicity, It is
Copyrighted materia
234 * Textbook al Microbiology
capable ot inductiij< antitoxin and reactiii
"
specifically with ir . ^ specificity and other characters.
The traigenicity of the diphtheria bacillus
depends on the presence in it of corynephages
which act as the genetic determinant
controlling tOJfin production. NontOXigeinic strain!;
may he rendered toxigenic by infecting rhe[]i with
beta phage or some other toxlargcr phage. This is
known as lysngcnii- or phage COnWfSj'oo. The
-
toxigcnk icy re mains only ns long ns the bacillus is
Ivsogentt. When the bacillus is cured of its phage,
as by growing it in the presence nf antiphage serum,
it loses the toxigenic capacity.
Toxin production h also influenced by the
concentration nfiron in the medium. The optimum
level ol iron for toxin production is 0.1 nig per
litre, while a concentration of 0.5 nig per litre
inhibits the formation of toxin . The diphtheria
toxin act* by inhibiting protein synthesis .
Specifically, fragment A inhibits polypeptide chain'
elongation in the presence of nicotinamide adenine
dinucleotide bv inactivating the elongation factor ,
-
EF 2 . It has a special affinity' for certain tissues
such as the myocardium, adrenals and nerve
cmlings -
Re*istance: Cultures may remain viable fer two
or metre weeks it 2S-3Q "C . It is readily destroyed
by heat in 10 minutes at SK :> C and in a minute at
r
100 ^C . It is more resistant tn the action of light,
desiccation and freezing than most nonsporing
bacilli . Ir has been cultured from dried bits of
pseudomembrane after 14 weeks. It remains fully
virulent in blankets and floor dust for five weeks . Ir
is easily destroyed by antiseptics. Li is susceptible
tn penicillin, erythromycin and broad spectrum
antihintics.
Antigenic structure: Diphtheria bacilli arc
anrigeideally heterogeneous . By agglutination ,
gravis strains have been classified into 13 types ,
intermedins into 4 types and mitis into 40 types .
Gravis strains of types I and 111 have been reported
to be common in Great Britain , type II worldwide ,
type IV mainly in Egypt and tape V in the USA .
*
No connection has hecn established between type
Bacteriophage typing : About 15
bacteriophage types have been described. Type 1
and III strains arc mitis, IV and VJ intermedins,
VII uvirulent gravis and the remainder virulent
gravis, The phage l pefi are apparently stable. A
^
svstem of bacteriocm ( diphthericin} typing has
also been described. Other methods of typing
include bacterial polypeptide analysis, DNA
restriction patterns and hybridisicion with DNA
probes.
Pathogenicity: The incubation period in
diphtheria is commonly 3-4 days hut may on
,
occasion he as shore as one day. In carriers , the
incubation period may be very prolonged. The site
ol infection mav he ( 1) faucial; ( 2 ) laryngeal;
( 2 ) nasal; {4 otitic; ( 5 ) conjunctival; 16) geriHxH
)
vulval, vaginal or prcpucial ; and { 7 ) cutaneous,
which is usually a secondary infect ion on pre-
existing skin lesions . Sometimes diphtheritic
whitlow or ulcer mav occur. Cutaneous infections
/
ate commonly caused by nontoxigenic strains of
diphtheria bacilli .
Faucial diphtheria is the commonest type and
mav vary from mild catarrhal inflammation to
verv widespread involvement . According to the
clinical severity, diphtheria may be classified as:
1. Malignant nr hypertox ic i n which there is severe
toxemia with marked adenitis ( buUneck). Death
i « due to circulatory failure. There is high
incidence of paralytic sequelae in those who
recover .
2. Septic, which leads to ulceration , cellulitis and
evert gangrene around the pseudomembrane;
and
3. Hemorrhagic , which is characterised by
bleeding from [ 3 ie edge of the membrane,
cpistaxis, conjunctival hemorrhage, purpura and
generalised bleeding tendency.
The common complications are:
I. Asphyxia due to mechanical obstruction of the
respiratory passage by the pseudomembrane for
Copyrighted material
 i CoryMtiLrlLiltdi
which an emergency tracheostomy may become
necessary.
jr
2, Acute circulatory failure , which may he
peripheral or cardiac,
3, Pbstdiph thei me paralysis, wl ic h tyf > ic ally occurs
m the third or fourth week of the disease ;
palatine and ciliary but not pupillary paralysis
i -; characterise < , and spontaneous recovery' is the
rule.
4, Septic, such as pneumonia and of . n -j mcdin -
Kelapse may occur in about 1% of cases.
Diphtheria is a toxemia. The bacilli remain
contined to the site of entry', where they multiply
and form the to \ in , Thc to \ i:i causes local ncctor: c
changes and the resulting fibrinous exudate ,
-
together with the di integraring epithelial cells,
leucocytes, CTythrocytCS and bacteria, constirutc the
pseudomembrane , wh . ’ h is characteristic of
'
i different ated from some commensal coryncbactcria
diphtheritic infection. The mechanical
com pile it ions of diphtheria are due to the
membrane, wh: k the system ic effects arc due to
the toxin.
Nonloxigenic str.iins of diphtheria hacilii may
cause infection even in immunised individuals, as
immunirv. with the toxoid does not confer
Jf
antibacterial immunity, Such infection is mild
J
though pseudomembrane formation may sometimes
occur ,
Diphtheria does not occur naturally in animals
but infection can be produced experimentally ,
Susceptibility varies in different species .
Subcutaneous inoculation of a guinea pig with a
culture of virulent diphtheria bacillus will cause
—
death in 1 4 days . At autopsy, the following features
can be observed:
1. gelatinous , hemorrhagl -, edema and, often,
necrosis at the site of inoculation;
2 . swollen and congested draining lymph nudes;
3. peritoneal exudate which may be clear, cloudy
or bloodstained;
.4 congested abdominal viscera;
5- enlarged hemorrhagic adrenals, which is the
pathognomonic feature;
235
6- clear, cloudy or bloodstained pleural exudate;
and
7. soTnetlines, pericardial effusion.
I nbrotatory diagnosis Laboratory
confirmation of diphtheria is necessary for the
initiation of control measures and for
epidemiological purposes but not for the treatment
of individual cases- Specific treatment should be
instituted unrnftintrefy on suspicion of diphtheria
without waiting for laboratory tests. Any delay may
be fatal.
-
Laboratory diagnosis con i '- ts o!isolation of the
diphtheria ba . itlu -- and demonstration of its toxicity.
One or two swabs from the lesions are collected
under vision, using a tongue depressor. Diphtheria
bacilli may not always he demonstrable in smears
from the lesion, nor car thev be confidcntlv
normally found in the rhroac . Hence smear
examination alone is not sufficient for diagnosing
diphtheria but is important in identifying Vincent’*
anyma. for this, a Gram or Ln- hmurt. stmned smear
is examined for Vinocnl s spinochetes and fusiform
bacilli. Toxigenic diphtheria bacilli may be identified
in smears by immunofluorescence. For culture, tlxe
swabs are inoculated uiiLoeffler 's serum slope, tellurite
blood agar and a plate of ordinary blood agar, the last
for differentiating streptococcal or staphylococcal
pharyngitis, which may simulate diphtheria . If the
swab cannot be i r.oculated promptly i t should kept
moistened with sterile serum so that the bacilli remain
viable. The scrum slope may show growth ML 4-B
hours hut if negative, will hive to be incubated for 24
hours. Smears stained w: rh methylene blue or one of
the special stains ( Ncisscr or Albert stain ) will show
the bacilli with metachromadc granules and typical
arrangemeiit.TeUuriiie plates will have to be incubated
as growth may sometimes be delayed. The tellurite
-
for a; least two days bd ore heii ig con i .lien d nc p'l ive,
^
medium is particularly imjvirtant in the isolation of
diphtheria bacilli from convalescents, contacts and
earn era as in these cajscs they may he outnumbered
bv. other bacteria,
-' n
f
opy righted material
KW.1
256 i Textbook. ot Microti ology
, »

Vlt! l I KMCR TESTti
Any isolate of the diphtheria bacillus should he
betted fur virulence or ttuigenicity for the
bacteriological diagnosis ro be complete, Virulence
testing may be bv in vivo or in Vitro methods, the
former hv. the subcutaneous or i ntradermsl test and
X
dish while the medium is still fluid. If hnree
scrum is not available, sheep or rabbit scrum
may he used . When the agar has set, the surface
is dried and narrow Streaks 0-f the Strains are
made at right angles to the filter paper strip. A
positive and negative control should be put up .

the litter' by [ he precipitation

culture tesr.
test or the [issue The plate is uicubaled at
—
fur 24 4 fi hours.
Toxins produced by the bacterial growth will
diffuse in the agar and where it meets the
In vivo tests antitoxin at optimum concentration will produce
/. Suhctilfttieous rt '.vj; The growth from in a line of precipitation ( Fig. 26- 2 )- The presence
-
overnight c u l t u r e n n 14 o c f c r ' -slope is
emulsified in 2 -4 ml broth and 0- R mE of the
of such arrowhead lines of precipitates indicates
that the strain is toxigenic. No precipitate will
emulsion injected subcutaneously into two fonil in the case ot uoTtlmHgtmic strains. This
test is very convenient and economical hut some
guinea pigsh one of which has been protected
with 500 units of the diphtheria antitoxin brands of peptone and some samples of scrum
13-24 hours previously. If the strain it virulent, do nor give saiisfacroty results,
the unprotected immal will die within four days , 2. Tissue c u f f i s i c The Euxigenicity nf
showing the autopsy appearance described d i p h t h e r i a bacilli can be demonstrated by
earlier . The method is not usually employed as incorporating the strains in the agar Overlay of
if is wasteful of animals. ctdl c u l t u r e monolayers. The toxin produced
Isitr: iL' ufiuictnss trWi' The broil] emulsion of diffuses into the cells below and kills them.
[ he culture is inoculated intracutaneously into Kpidemiolugyj Diphtheria was formerly an
[ wo guinea pigs (or rabbits ) so [ hat each receives
O
0.1 ml in two different sites. One animal acts as
the control and should receive 500 units oi
antitoxin the previous day, "iTic other is given
50 units of antitoxin intraperiioneally four hours
atter the skin test, in order tu prevent death .
Toxigcniciry is Indicated by i n f l a m m a t o r y
reaction at the site of injection , progressing to
necrosis in 4fi-72 hours in the test animal and
no change in the control animal , An advantage
in the intraeutaneous test is that the animals do
not die, As TTia ny as ten ^ trai n s can he tester! at a
time on a rahhir .

In vitro test
i . Kick !y t;vf pn iphtifum teat: A rectangular
^ FI -9 - 2E .S Elgk £ test. A - Fitter papsr
strip of filter paper impregnated with diphtheria impregnated
antitoxin (1000 -units ml ) is placed on [lie surface wliti A. ftlphihtfiw intitorin, Toxigenic sirs in ,
of a
^
normal horse serum agar in a petfi C. Nonto* igenic strain . 0 . T strain shoeing
lemlgonletly .
 Coryn-^u a clorlum
Important pediatric di-tase all over the world but
followi ug the development ofeffect i ve prophylat tics
and mass Immunisation , the disease has been
virtually eradicated from most advanced countries.
In those dcwlo]'ing countries where childhood
Limnui i isation programmes have been i nplemented
[
effectivdy, diphtheria has become rare but in others
it continues to be a serious problem. The prolonged
and extensive epidemic of diphtheria in parts of
the erstwhi ’e Soviet Union in the 1990s , involving
several thousands, with a mortality of up to 20 per
Lent is
a warning of what can befall countries that
neglect : mmunisation and let living conditions
deter iorutc-
endemic area -s, it is mamlv a disease of
] r.
childhood. It is rare in the first year litv due to
the passive immunity' obtained from the mother,
reaches a peak between two and five years, falls
slowly between five and 10 years, and rapidly
between 10 and 15 years with only verv low
incidence afterwards because of active irnmunitv V
acquired by repeated subclinical infections.
Asymptomatic carriers who outnumber eases by
a hundredfold or more in endemic areas arc the
most important source ofinfection. In the temperate
regions, carriage is mainly in the nose and throat.
Nasal carrier harbour the bacilli for longer pcri-iids
than pharyngeal earners. In the tropics, diphtheria
bac i Hi infecr the -- kin more often titan tlie respiratory
tract - Toxigenic diphtheria bacilli may persist m
the skin for over three .vcarcr Cutaneous infections
may stimulate natural immunity ro diphtheria but
may also lead ro fatn iaJ diphtheria in non immune
contacre, Fomnet do not seem to play an important
role though in special situations toys and pencils
mav act .LS vehicles MI infection.
if
In nature, diphtheria iiR virtually confined to
human beings., though cows- may on occasion be
found to have diphtheritic infection of the udder.
The infection in such casec Is invariably transmitted
jf
bv the milker. The infection may be spread through
tlitr milk uf iiifcited cows.
237
PROPH’i L wis
Diphtheria can he controlled by immunisation .
Three methods of immunisation are avi . liable ; , 1
active, passive and combined. Of these, only active
immunisation can provide herd immunity and lead
to cradic;i tion of the disease. Pass: vc and combi ned
immunisation can only provide emergency
protection to siw;ep1 ihUL i 11Llii. ILIUS!- ex loosed to risk.
'
The objective of : mmunisation is to increase
protective levels of antitoxin in circulation. Early
in the development of diphtheria prophylactics,
when immunising agents were scarce and not free
from rL -ilc , it was customary to test for susceptibility
before active irnmumsut unx was given [’he ,
susceptibility test used was the Schick test
Titroduced in 1913, The Schick test is no longer in
use, (Older editions of this textbook may be
consulted for details of Schick test, if required).
The availability' of safe and effective toxoid
preparations has made susceptibility tests
unnecessary. If, for any spe . i .il reason , the
circulating antitoxin level is to be assayed , it can
now be done by serological tests such as passive
hemagglutination or by neutral Nation in cell
culture. Antitoxin level of 0- 01 unit or more per ml
of blood is considered as index of immunity.
Active immunisation: Diphtheria
immunisation in children was initiated in 1913 by
von Behring using a toxin- antitoxin mixture
containing only enough antitoxin ro leave the toxin
just underneuiralised , Tlii- was a hazardous
preparation because the final toxicity of such
mixtures was determined not merely by the relative
proportions of toxin and antitoxin but also by other
variables, including the manner i:i which the toxin
and antitoxin were mi ^eth If to a iven amount of
^
antitoxin, the equivalent amount of toxin was added
ail at once, the mixture remained nontoxic. If instead,
the same amount of toxin was added in two or more
instalments .u intervals o f l S minutes or more, the
resultant mixture was toxic . This paradoxical
occurrence was known a* the Dam^z phenomenon
>pyrighted material
H dr w
 * Texmoo* ol Microbiology >
and was due to rbc ability.' Lit'thc toxins and antitoxins
to combine in vatying proportions. When the toxin
v/ , L >. IDELJ in instalments, the toxin added first
js
combined with imnrethan i equivalent of Antitoxin ,
laving insufficient antitoxin behind to neutralise
[ he toxin added HuhvequeiLily.
Several other preparations were introduced for
active immunisation (for details, the third edition
of this textbook may be consulted ) . Only rvvu
preparations are in Line nmv , Fonnol toxoid ( also,
known as fluid toxoid ) and adsorbed toxoid. For moi
toxoid is prepared by inctibating the train with
formalin at pH 7.4 7.6 for three to four weeks at
37 'C until the product is devoid of toxicity while
retaining immunogcnicity- Adsorbed toxoid is
purified toxoid adsorbed onto insoluble aluminium
—
compounds usually aluminium phosphate , lens
often the hydroxide. Adsorbed toxoid is much more
immunogenic than the fluid toxoid . Due to anxiety
bout ID enhanced risk of provocative poliomyelitis,
it was replaced by fluid toxoid tar
, SLITTIC time .
However, as the risk is considered small and as the
immunogenicity of fluid toxoid waa unacceptably
low, adsorlsed toxoid is now used almost univenally
us the- preferred agent [ t is advisable to give
, standardisation or measurement should be with
adsorbed toxoid by intramuscular injections as
subcutaneous injection may be painful.
Diphtheria toxoid is usually given in children
as a trivalent preparation containing tetanus traoid
and pertussis vaccine also, as the DTP, DPI’ or
triple vaccine . A quadruple vaccine containing in
addition the inactivated poliovaccinc is also
available , For young children , diphtheria toxoid
given in a dose id 10-25 Lf units is recommended.
Smaller doses ( 1-2 I . F units ) are used for older
children and adults to minimise adverse reactions.
Jo toxoid preparations the lower dost of toxoid is
indicated In the small lelter 'd ' and the full dose by
'
capital LD \ For example, the tetanus diphtheria
vaccine for adults containing low dose diphtheria
tOXuid is referred to as 'Td ".
The schedule of primary immunisation of infants
and children consists of three doses at DPT giver
at intervals nf at least tour weeks, and preferable
six weeks or mom , followed by a fourth dose about
a year afterwards. A further booster dose is given
at school entrv.
Paative tmm uni station: This is an
emergency measure to be employed when
suHccptibles are exposed to infection, as when a case
o ( diphtheria Is admitted to general pediatric wards.
It consists of the subcutaneous administrarinn of
500-lQQti units of antitoxin (imtidiphtheriric serum ,
ADS), As this is a horse scrum, precaution against
hypersensitivity should be observed.
< lonihincil immunisation: This consists ot
administration of the firsr dose of adsorbed toxoid
on one arm , while ADS is given nn the other arm ,
to be continued by the full course of active
immuiLLSatioo. Ideally, all Cases that receive ADS
.
piophyl Ktically should receive combined
immunisation.
STAMIUKDI ^ ATIO ^ OF
A N T I T< I \
-
I NS
TOXINS A\M
1 lit: toxin content of culture filtrates varies
consul lira Id v from batch to batch. As such their
reference to rheir biological activity. Ehrlich
'
defined the minimum lethal dose ( MId ): of the
diphtheria toxin as the least amount of the toxin
required to kill a guinea pig weighing 250 g within
% hours alter subcutaneous inoculation. One unit
of antitoxin was defined as the smallest amount of
Antitoxin required loneutraltee 100 MLD of toxin.
Keeping a labile substance like the toxin an the
standard led to inaccuracies. Toxin undergoes
spontaneous denafuration into toxoid which wilJ
combine equally well with the antitoxin. Thus, any
sample of toxin will contain a variable quantity ot
toxoid which will vitiate standardisation of
antitoxin . The antitoxin, on the other hand, is
permanently stable in the freeze - dried state .
Therefore, the antitoxin has hccn adopted as the
reference preparation , EhrlichV original antitrain
is accepted ns the international standard. One
Copyrighted materia
 i Corynebacie'ium *
antitoxin unit {AU ) is defined as rhe amount of
antitoxin that has the time mill combiningopacity
far taxi n and toxoid together as one unit ofEhrEch's
original aniitcmn .
Since toxin always contains some toxoid, two
other units for measurement of toxin have been
introduced, the LO and L + doses. The LQ (Limes
nul ) dose ul the diphtheria toxin is the largest
amount of toxin that, when mixed with one unit of
antitoxin and injected nbcutuieously into a 250 g
guinea pig, will on the average cause no observable
reaction. As 'no reaction is nor a definite end point,
in actual practice, the end point is taker as mini mum
local edema.The L* (Limes tod ) dose oi diphtheria
toxin is the smallest amount of toxin rhat when
mixed with one unit of antitoxin and injected
subcutaneously into a 250 g guinea pig will on the
average kill the animal within 96 hours. If toxin
combines with antitoxin in constant proportions , it
tvould be expected that the ditference between the
I A d wc and the I $ d* we would be ex|ual to 1 MI ,l >,
"
Blit when the estimations arc actually made, it is
found to wary from 10-100 MLD or more. This
discrepancy is due to the presence in toxin
preparations of varying amounts of toxoid and to
rhe ability of the toxin and antitoxin to combine in
J
varying proportions. This is known as the Ehrlich
phenomenon.
The use of death as an end point for the titration
of toxin is wasteful of animals. Romer intn hduced a
method of titration employing the erythematous
swelling produced by the intiaJenmal injection of
toxin , and its neutralisation by antitoxin. The
minimum reacting dote ( MRDJ is the leutuioint
of toxin that when iniectrd intradermallv in a guinea
, to nitrite . It produces two types of toxins, one
pig, causes an erythematous flush 5 mm in diameter
visible alter 36 hours. 'Hie Lr dose is the smallest
amount of toxin which, after mixing with 1 unit of
antitoxin , will produce & minimal skin reaction when
injected irUrndcrmally into J guinea pig.
Ramon introduced a test tube method for
titrating toxin and antitoxin based on flocculation.
The flocculating or Lfunit of diphtheria toxin is
239
the amount of toxin which flocculates most rapidly
with one unit of antitoxin. The Lf unit has several
advantages. It is inexpensive and rapid and docs
nut need animals, Ic is also the only method available
for the titration of toxoids . The amount oi toxoid
in prophylactics is expressed in Lf units. Many
other in vitro teats have been developed for antigen
assay. These include cell culture neutralisation tens
using rabbit Iddncy cells, passive hemagglutination
test with roxi n coated ta nned sheep RHC , and HI A -
Treatment: Specific treatment of diphtheria
consis.rs of antitoxic and antibiotic therapy. Antitoxin
should be given immediately when a case is
suspected as diphtheria , as the fatality rate increases
with delay in starting antitoxic treatment. The
dosage recommended is 20,000 to 1 ,00,000 units
for ferrous cases , half tile dose being given
intravenously. Antitoxin treatment is generally not
indicated in cutaneous diphtheria as the causative
strains are usually nontoxigenic.
.
C diphtheria? ft sensitive to penicillin and cun
be cleared from the throat within a few days by
penicillin treatment. Diphtheria patients are given
a course of penicillin though it only supplements
and docs not replace antitoxin therapy,
Eiythramyrin ft more active than penicillin in rhe
treatment of carriers-
OTHER PAmOGEWC COflYNE BACTERIA
C. / //. CEJtA.XS
This bacillus related to C. dlphr/tcnac can cause
dLphtheriii -like lesions . It resembles the gravis type
of the diphtheria bacillus but it liquefies gelatin ,
fcrroenis trehalose slowly and does not reduce nitrate
prohahly identical with the diphtheria toxin and
the other resembling the t o x i n o f
C. pscvdatubawlaiiz It is pathogenic to guinea
pigs , the lesions produced resembling those caused
by C. diphthen«, It lias been found to cause
in lection in cows, ami human infectw may be
transmitted through cow 's milk. It is sensitive To
.
erythromycin , Diphtheri i anritoxin is protective .
Copyrighted material
240 * Textbook of Microbiology *

IT lias been suggested that C’. ukem /ii may be
considered i subgroup of diphtheria hacilU rather
than a separate species,
j4ioiiafiiictBHiiii ( formerly Corynebmcteriun}}
hairmoh-tiLunr ea.n cause phuivngitiB and skin ulcers.
C jakdinn can cause cuta neous and blood i nfeetin ins
m immunocompromised hosts. IT is usually
multi resistant, nespnncling only tci vancomycin.
Cofynebacterlj of veterinary importance are rhe
Preisa Notard Lae ill LIS ( C. juL' utforubervLjJosjfi ),
which causes pseudettuberculosis in sheep and
suppurative Lymphadenitis in horses, C. renafo
causing cystitis and pyelonephritis in cattle and
C, equf , isolated from pneumonia in foals.
Com NEB At :TERIA CAL SING
SI r t:i t E H ; ] A I . SKIN INFECTIONS
Erythra Rina , a Localised infection of the stratum
corneum usually affecting the axilla and groin, is
caused by C nuiuiUHnnum , This is a lipophilic
coryncbactenum and can be grown readily in media
containing 20 per cent fetal calf scrum .
C. renujs has been associated with trichomycosis
'
axillaris , characterised by the formation of
pigmented nodules around uxitlaiy and pubic hair
shafts.
DtPHTHBROiD3
CaryDcbactcria resembling C- diphtheria occur a*
nnmiiil commensals in the throat, skin, conjunctiva
and other areas.These may sometimes be mistaken
for diphtheria bacilli and arc called diphtheroids.
In general, diphtheroids stain more uniformly than
diphtheria bacilli, possess few or no mccachromatio
granules and tend E u b e arranged i n parallel rthn
( palisades) rather than cuneiform pattern. However,
some diphtheroids may be indistinguishable from
diphtheria bacilli microscopically. Differentiation
is by biochemical reactions and more reliably by
virulence tests, The common diphtheroids are
C. paeudodiphthcritiL-um ( C. hairnannri) found in
the din ' ll and C. xenonJ.t found in the conjunctival
sac. The former is unease positive and does not
forment glucose , while the latter is unease negative
and foments glucose . Both are pjTar.inamltl3. Re
positive, unlike diphtheria bacilli .
OTHER CORYNEFORM UACTEHIA
Besides genus Ctujwbflcflenuin, a number of Other
genera of coryuefujuj bacteria have been defined .
Among them , the genus FroprumfMetierftJjn is of
medical interest as three species, T! aeries. E
jnauJonm and F wvixfam, are constantly present
on human skin. Thev are anaerobic and .icrotoJeraiit,
growing well in lipid containing media. P. £ rnes is
often isolated from acne lesions bur its pathogenic
role LR uncertain.
CrtrrrttbtfCfttrrum parvum which in frequently
used as an immunomodulator is a mixture of
Hropiombacterium species.

Further It ending
.
Coyle MB and BA Lipjlcy 1990 Coryneform bacteria in mfretious disease. Clin Mkmhinf iiev 3:227 .
. 9%L R (SLiTi;Knce of diphtheria in the fnDlerSHtelUnm lancet JJ 7:t 99 l
Hardy E K I i v i at| .
. .
Hotier W 1991, Cutaneous diphtheria SntJ Ctrmjml 30: fiJ 5
. .
Zainiri 1 PWN Cprynebacteri urn , 1 n Collet , JG et u ( eda ), JVactrcaJ Medical Alitmlaoingy 1^" edn. Edinburgh: Churchill -
LivifttfHfK ,

]) opyrighted material
 1^ Bacillus
CM
Sporogeuous , rod-shaped bacteria are classified
into two genera , the ucruble Bacilli and the
anaerobic Civslriiliz. The genus Bscillus consists
of aerobic hseiili forming hear resistant spores. They
are Gram positive but rend to be decolourised easily
so ns to appear G mm variable, or even frankly Gram
negative . They are generally motile with
pcritrichout flagella , the anthrax bacillus being :a
notable exception , Members of this group exhibit
great diversity in their properties. The genus
includes psychroph ilic , mcsophilic and
thermophilic species , the maximum temperatures
for vegetative growth ranging from about 25 G to
above 75 aC and the minimum from about 5 “ C to
45 C . Tlirir salt tdlrrute varies from less than
"
2% to 25% NiCl .
Their spores are ubiquitous, being found ir ] soil,
dust, water and air and constitute the commonest
contaminants in bacteriological culture media.
BptUftit a:J fhr.ii j.^ , the causative agent of anthrax,
'
is the major pathogenic species. H . txmis can cause
tundhnrrLC gastroenteritis. Some species may lie
responsible for opportunistic infections ,
BACILLUS ANTHHAClS
Considerable historical interest Is attached to the
anthrax hacillus. It was the first pathogenic
bacterium to be observed under the microscope
( Fbllcnder , 18J9), the first communicable disease
shown to be transmitted by inoculation of infected
blood ( Havainc , IS5U ) . the first bacillus to be
isolated in pure culture and shown to possess spores
( Krvh, lE 7f >} and the First bacterium used for the
preparation of an attenuated viccine ( Pasteur, 1881).
Anthrax was for long feared as a potential tool
in biological warfare. This fear became an actual
fact in 2001, when anthrax in the form of weapons
grade spores, having enhanced dispensability and
virulence was seni hv mail to various destinations
P
in the USA, causing disease and death in many
persons.
Morphology: The anthrax bacillus is one of the
largest of pathogenic bacteria, measuring 3-10 flm
* 1-1 - b |jm . In tissues, it is found singly, in pairs or
in short chains, the entire chain being surrounded
by a capsule. The capsule is polypeptide in nature,
being composed of a polymer ofd (-) glutamic acid.
Capsules are not formed under ordinary conditions
of culture but only if the media contain added
-
bicarbonate or are incubated under 10 25% CO,.
If grown in media containing serum , athumin ,
charcoal or starch , capsule formation may occur in
the absence of CO , .
In cultures, the bacilli are arranged end to cud
in long chai ns . The c nds of the bacil it arc truncated
or often concave and somewhat swollen So that a
that n of bacilli presents a 'bamboo stick’ appearance ,
bporcs arc formed in culture or in the soil but never
in the animal body during life. Sporulation occurs
under unfavourable conditions for growth and is
encouraged by distilled water, 2% NaCl or growth
in nxalatcd agar. Oxygen is required for speculation,
but not for germination. Spoliation is inhibited
by calcium chloride . Spores are central or
Kulrfcrnliual, elliptical or oval in ib.ipc, .irtd are of
the -same width a? the bacillary hotly so that they
do not cause bulging of the vegetative cell ( Fig.
27.1k
Copyrighted material
 242 * Textbook or Mtatobioko y
^
while a virulent or attenuated strmir form smooth
colon ies . OTI gelatin st:th I run a characteristic
a ‘
inverted fir tree' appearance seen * Willi slow
liquefaction commencing from ' top ( Fig , 27.3),
On blood agar , the colonies n nonhemolytic,
rhough occasional strains jimduc < a narrow zone
of hemolysis. In broth growth as floocular
ilrpoiiit , with little or no- rbiditl
When U- anthneis is grown n the surface of a
i solid medium containing "
-
5 ) units of
'

perLLcillJ ci/ ml , in 3 b hours the ce i become large*

sph etical , a ud occur i I . I : "11 11 Hurtace of the
F

agar , resembling a string of pe iris. This 'string of

.
Fig 27.1 AL.i.hidi Ddtnl - pearls reaction differentiate^ .
1
B. xnthtau*
'

from B . ccrcus and other ucrobic spore bearers.

Thc anthrax bacillil# it Grain positive and
Another USefultCstto different!, L between tlie two
'

nnriaciti fast , The spores J : > nut stain by ordinary it the susceptibility of if . '
is gamma phage .
methods bur can be stained differentially by special
A selective medium ( PLET medi J l . consisting
techniques. When stained with Sudan black B, fat of polymyxin, lysozyme , lent diamine tetrn
globules may he made out within rhe bacilli . When
acetic acid ( EDTA ) and rl acetate added to
blood films containing anthrax bacilli are stained
heart irthtsion agar, h&s been to isolate iJ .
with polychrome methylene blue for a tew seconds
and examined under the microscope, an amorphous
aathneis from mixtures "tii .i ng other spore -
beating bacilli .
purplish material is noticed around tbc bacilli. This
represents the capsular material and is characteristic
of the anthrax bacillus. This is called the
M’ Fndycans reaction and is employed for the
presumptive diagnosis of anthrax in animals.
"

Hie anthrax bacillus is nonmotile, unlihe most

oilier members of this genus ,

i . . , L L L . - . : : charm. ttnsties: It is an aerobe ,

ami a facultative anaerobe * with a temperature range

for growth of 12-45 °C (optimum 35 ~ 37 rjC). The
optimum temperature for sponilation is 25-30 aC,
Good growth occurs on ordinary media.
On agar plates, irregularly round colonics arc
formed * 2-3 mm in diameter* raised * dull , opaque *
greyish white, with a frosted glass appearance .
Under the low power microscope, the edge oi the
colony is composed of long, interlacing chains of
bacilli , resembling locks of matted hair. This is
called the ' Medusa head appearance' ( Fig. 27.2 ) . Fig . 57.3: Medii &a ntaa
Virulent capsulated strains form rough cultures * bed Hi

my righted mafern
 * Bacillus * 243
_
- i ' h e 11 < ] IKIL: -. I i r i - isulion *u Glucose , maltose , L:UI
sucrose arc fermented producing acid but no gas .
Nileaiis art reduced to nitrites. Catalase is formed.
-
R t K i s l art c L : The v e ge rat E V e b a c L l 5 i ii re nor
particularly resistant and are destroyed at ftO QC LCI
30 minutes. In the carcasses of animals which have
died of anthrax, the hacilfli remain viable in the
bone marrow for a week and in the skirt for two
weeks . Normal hem fetation of smears may not kill
the bacilli in blood films. The spores arc highly
resistant to physical and chemical agents. They
have been isolated from narurally infected soil after
as. long as 60 years . They resist dry heat at 140 *C
for 1-3 hours and hoiling for 10 minutes . I’hey
survive in 5% phenol for weeks . HgC in a 1/
1000 solution may tail to kill anthrax spores in less
than 70 hours . Four per cent potassium
^
permanganate kills them in 15 minutes. Destruction
of the spores in animal products imported into
nonendemic countries is achieved by 'duckerinjr ' in
which formaldehyde is used as 2<Hi solution at .
Fig 27.3 Anthrax badbus In gelatin stab cullute
30-40 QC for 20 minutes for disinfection of wool showing- Inverted fir tree appearance
and as 0.25 per cent at 60 gC for six hours for lesion , heart blood and spleen ( more than 10* bacilli /
animal hair and bristles. The anthrax bacillus is ml). The bacilli are &een confined to the interior
susceptible to tulphotumulcs, penicillin , of the capillaries, where their numbers may be so
erythromycin , streptomycin , tetracycline and great as to obstruct the flow of blood .
chloramphenicol. Occasional strains res is ran r ro
penicillin have been met with.
Two virulence factors have been identified
the capwtfarpQjypeptkk and the infhnur roxiji, each
—
Pathogenkdty: In nature, anthrax is primarily ot which is encoded by a separate plasmid.
,

a disease of cattle and sheep, and less often of horse ?
and swine hut experimentally most animals are
susceptible to a greater or lesser degree. Rabbits,
guinea pigs and mice are susceptible. Infection can
be produced with difficulty in birds. Frogs are
resistant, while toads are very susceptible ,
Following the subcutaneous inoculation of a
culture into n guinea pig, the animal dies in 24^72
hours, showing a local , gelatinous, hemorrhagic
edenu at the site of inoculation , extensive
subcutaneous congestion and characteristically, an
enlarged , dark red, friable spleen. The hlood is dark
red and coagulates less firmly than normally. The
bacilli are found in large numbers in the local
The capsular polypeptide aids virulence by
inhibiting phagocytosis. Loss of the plasmid ( pxflJ )
which contrail Capsule production leads to lots of
virulence . This; is how rhe live attenuated anthrax
spore vaccine ( btenie strain ) was obtained.
Anthrax toxin was identified bv the finding that
the injection of sterile plasma of guinea pigs dvmg
of anthrax into healthy guinea pigs killed them and
rhit deith could be prevented by immune serum.
The toxin is a complex of three fractions: tlie edema
factor (OFor Factor I), the protective antigen factor
( PA or Factor 11) and the lethal factor ( LF nr Factor
IJI ), llicy are not toxic individually but rhe whole
complex produces local edema and generalised
344 ^ Textbook ol
shock. The three factors have been characterised
and cloned- PA IS the (Vaclicm which hinds to the
receptors on the target cell surface, snd in turn
provides attachment sites for OF or LF, fa * ilitating
their entry ii Lto the cell. Antibody to PA is proCrc1i vc
because it blocks the first step in toxin activity*
namely* its binding to target cells. Ob is an udenvl
cyclase which is activated only invide the target
cells, leading to intracellular accumulation of cyclic
AMP. This Is believed to be responsible for the
edema and other biological effects of the toxin.
Entry of LF into the target cell causes cell death
bur the mechanism of action is not known. Loss of
the plasmid (p>dl 1) wb i. l L encoder the anthrax tun in
renders the strain avimlcnr. This is believed to have
been the basis for the original anthrax vaccine
developed by Pasteur. The aviruleni Sterne vaccine
Strain is devoid of the plasmid coding for the
capsular polysaccharide .
ANTHRAX
Anthrax is a 70onnsis. Animals arc infected by r
ingestion of the spores present in the soil, Direct
spread front animal to animal Is rare. 'Hie Mi - case
is generally a fatal septicemia but may sometimes
be localised * resembling the cutaneous disease in
human beings. Infected animals shed in the
discharges from the mouth, none and rectum, large
numbers of bacilli , which sporulstc in soil and
remain as the source of infection.
Human anthrax is contracted from animals
*
directly or indirectly The disease may be ( 1)
cutaneous * ( 2 ) pulmonary* or ( 31 intestinal, all types
leading co fatal septicemia or meningitis.
Cutaneous anthrax follows entry of the
infection through the skin- The lace , neck, hands*
arms and back arc the usual sites . The lesion starts
as a papule 1- 3 days after infection and becomes
vesicular, containing fluid which may he dear nr
bloodstained . The whole area is congested and
edematous and several satellite leniom filled with
*
serum or yellow fluid are arranged round a central
necrotic lesion which is covered by a blaek eschar.
MisrutaWOfiv
.
(The name anthrax , whi h means coal , comes from
the black colour of the escliar.) The lesion is called
a maJjjjrjinntpusfir/e . The LII. sease used to be common
in dock workers carrying loads of hides and skins
on their bare backs and hence was known as the
hidepnfrers diseasc . Cutaneous anthrax generally
resolves spontaneously but 10- 20 per cent of
*
untreated parents may develop fatal septicemia Or
meningitis .
Pulmonary anthrax is called the >J miters
disease because it used to be common in workers
in wool factories, due to inhalation of dust fiom
infected wool . This is a hemorrhagic pneumonia
with a high fatahiy rate. Hemorrhagic meningitis
may occur as a complicarinn .
Intestinal anthrax is rare and occurs mainly in
primitive communities who eat the carcasses of
animals dying of anthrax. A violent enteritis with
bloody diarrhea occurs, with high case fatality.
Hum ait anthrax mav , be industrial or non
§r
-
industrial (agr ' cultural}. The former is found in
workers in industries such as meat packing or wool
factories . Mon - industrial anthrax is often an
occupational disease in those who associate
frequently with animals , such as veterinarians *
butchers and ferment. Tl may also he found in the
general population . Cutaneous anthrax used to be
caused by shaving brushes made with animal hair.
Stomoxys cidcittans and other biting insects may
occasionally transmit infection mechanically
Anthrax IS enzootic in India , the number of
ar. imals infected running into tens of thousand
*
annually. The disease is rare in some countries such
.
as Britain * where infection is imported through
contaminated hides, hone meal fertiliser and other
animal products- The extent of anthrax in human
beings is nor dear but about 20,000 to 100 *000
cases are believed to occur annually throughout the
world* mostly in rural areas- Large epidemics of
anthrax were reported from Russia and Zimbabwe
during 197S-80. An epimotic of anthrax in sheep
hufi been active near the Andhra-Tamil Nadu
border, causing many cutaneous and
Cropyrighted materia
 i Bacillus
iitoifi oeficephaliLC human infection with high
[
^
mortality rate. There have heen nuthieab in ^
Karnataka and Wear Bengal- Anthrax inferrinn in
human being* provides permanent immunity and
second attack* are extremely rare.
Laboratory diiignosiu Anthrax may be
diagnosed by micruscopy, izulfUft, animal
inoculation and serological demonstration of the
anthrax antigen in infected tissues . Acute and
convalescent phase sera should be obtained, since
and bodies Co the orji .iimm Can be demon-stmted hi
L'LI diffusion complement fixati > jnh antigen coated
* of factory hygiene and proper sterilisation of animal
tainted red Cell agglutination and ELISA
techniques . The type of test to be employed depends
on the nature of the material available .
When an animal i -; suspected to have died of
anthrax, autopsy is not permissible, as the spilt blood
« ilJ lead Co contamination of the soil . An ear may
be cut oft' from the carcass and sent to the laboratory.
Alternatively , swabs soaked in blood or several
blood smears mav be senr. The demonstration of
Grim positive bacilli with the morphology ot
anthrax bacilli drill a. positive M'FadyeHrts RUttion
will enahle the presumptive diagnosis to be made.
ImmuiiiotluoreKcenr microscopy can confirm the
identification. I solation of the hacillus is easy if gross
contamination has not ouutied. The anthrax
bacillus cun often be isolated from contaminated
tissues by applying them over the sh avert skin of a
guinea pig. It is able to penetrate through minute
abrasions and produce fatal infection , If the sample
received is putrid so chat viable bacilli ate unlikely,
diagnosis may be established by AscoLiV
thermoprccipitin test by demonstration of the
anthrax antigen in tissue extracts.
After the biotermrism experience in the USA
in 2001, the CDC (Centers for Disease Control )
have prepared guidelines for identification of anthrax
bacillus.. Any large Gram positive bacillus ivith the
-
general morphology and cultural features of
anthrax; nomnoiile, nonhemolytic on blood agar
and catalase positive can be given a presumptive
report of anthrax. For initial confirmation., lysis by
245
ganimaphage and direct fluorescent antibody tesr
ID FA ) tor capsule specific staining and. for
polysaccharide cell wall antigen are sufficient.
For a further confirm ationT PCR tor anthrax
bacillus specific chromosomal marker? can he done.
For epidemiological studies and strain
characterisation, MLVA { multiple locus variable
number tandem repeat analysis) or AFLP ( amplified
fragment length pohmorphis- m ) can be used,
Frophyhxk Prevention of human anthrax is
'
mainly hy general methods such as impruverrieriC
products like bides and wool. Carcasses of animals
suspected to have died of anthrax are buried deep
in quicklime or ere mated to prevent soil
contamination ,
Prevention of anthrax in animal* is aided bv d
ULtivc nnmuni ^.iLion . The original P. i ^teurV anthrax
vaccine Is nf great historical importance. It was
Pasteurs cocKvimnn demonstration of the protective
^
effect of his anthrax vaccine in A public experiment
ul Pouillv - lr - Foft in lSSl that marked the
Jr
beginning ol scientific icriniunu prophylaxis-.
Past mr 's vaccine was tbe anthrax bacillus attenuated
—
by growth at 42 43 nC-
As the spore is the common infective form in
nature, vaccines eonsis-tmg of spores of attenuated
-
strains were developed The Sterne vaccine
contained spores of a noncapsulated avimlmf
mutant strain . The Mazucchi vaccine contained
spores of stable attenuated Carbazoo strain an 2%
-
saponin The spore vaccines have been used
extensively in animals with good results. They give
protection for a year following a single injection.
They are not considered safe for human use, though
they have been used for human immunisation in
-
Russia Alum precipitated toxoid prepared from the
protective antigen has. been shown to be a safe and
effective vqccin-c for human use. It has been used in
persons occupationally exposed to anthrax infection .
Three doses intramuscularly. at intervals of six
weeks between first and second, and six months
between second and third doses induce good
Copyrighted materia
246 * Tpjfibook of Microbiology *

Immunity, which ' an he reinforced if nrcessarv with

'
annual booster injections -
Treatment Antibiotic therapy is eFfe -ciive HI
human cases but rarely succeeds in animals as
therapy Is not started sufficiently cally, Antibiotics
have no effect on the toxin once it is formed
fancillm and streptomycin urc no longer used for
treatment . They H ,tve been replaced by djoJtytlcluie
und dprcifhixiLm, winch are effeciixe in prophylaxis
and treatment.
ANTHGLACOIU BACILLI
Many memhers of the genus bacillus. Other than
the anthrax h.ic i LLLIH have occasionally caused huniirt
infections. Of them, the most important is B. cercus
which from 1970 has been recognised as a frequent
cause of foodburne gastroenTeriris. It has also been
associated with septicemia, meningitis, endocaidj tis,
pneumonia, wound infections and other suppurative
lesions, particularly as an opportunist pathogen . B.
subtilis, if . lichcniformis and a few other species
have also been occasionally isolated from such
lesions. These and a large number and variety of
nonpathogemc aerobic S p o r e bearing bacilli
appearing as common contaminants in cultures and
Table 27,1 Dillerenllatlng features belw«n anthrax and anthracoid bacilli
haiing a general resemblance to anthrax bacilli have
been collectively called p&cudomiEhfiix or art rftracojiJ
baeiJii . Table 27.1 lists the main differentiating
feature's between them.
,
BACILLUS CEHEU 5
IS , rencu? has become important as a cause of food
poisoning . It is widely distributed in nature and
may be readily isolated from soil , vegetables and a
wide variety of foods including milk , cereals , spices,
meat and poultry. B. cenetis is generally motile but
non mo rile strains may occur . It resembles B.
anthiacj's except that it is not capsuiated and not
susceptible to gamma phage and docs not react with
anthrax fluorescent antibody conjugate . Animal
pathogenicity test also diffexendates between the
two .
B. cereus produces
'
two patternsof foodboriie
disc ase . O ne is associated with a wide range of foods
including cooked m e a t and vegetables . It is
characterised bv diarrhea and abdominal pain,
H-lfi hours after ingestion of contaminated foods.
Vomiting is ram . B. osreus is not found in targe
numbers in fcca! specimens from these patients.
The scCOnd type associated almost exclusively

Anthrax bacilli Atithttcoid bacilli

1, Nonmotik Generally motile
2, Capsukted No- neapsutared!
' ,
S Ciw in lopR chains Grow in short chains
4. Medusa head colony Not present
5. No growth. Ln peniriJJini agar Grow usually
(10 units/ ml )
6 . Hemolysis absent or weak Usually well marked
7. Inverted fir tret growth and Rapid liquefaction
slow geLatin liqucfacrion
8. No turbidity in bsroth Turbidity usual
9. Salscln fermentation negative Usually positive
10 . No growth at 45 *C Grows usually
] ] , CmwTh tnblhncd by chlonil hydrate Nor inhibited
12 . Susceptible to gumma phage Not susceptible
11 , Pathogenic to Laboratory animals Not pathogenic

rv
U opyrighted materia
 Bacillus 247
*
with ihe consumption of cooked rice * usually fried POCUffnubtCT in ligated rabbit ileal loopN resembling
rice from Chinese rest u unites The illness is
r the hear labile enterotoxin of jEscfiencftia coli
characterised by acute nausea and vomiting 1-5 Strains causing the emetic type of disease produce
hours after the meal. Diarrhea is not common. a toxin which causes vomiting when fed to Rhesus
B. emeus is present in luge numbers in the cooked monkeys , resembling staphylococcal enterotoxiti.
rice and fecal samples from these patients. Both The emetic toxin was produced only when B. Cereux
types of illness are mild and self limited, requiring was grown in rice but not in other media. Two
no specific treatment . mechanisms of action have been described for the
It has been shown that the two types of disease enterotoxin of if , cereuf, one involving stimulation
are caused! by strains of J5 cereus belonging to of CAMP system and the ocher independent of it.
—
r

different serotypes. The dkldMll disease is mostly A special mannitol-egg yolk phenol red “ -
caused by serotypes 2* 6, 8 9P 10 or 127 while the
B polymyxin agar ( MYPA ) medium is useful in
,

rice associated emetic illness is caused by serotypes isolating B. ccirus from feces and other sources,. B ,
1* 3 or 5, Isolates from the diarrheal type of disease etfeu& producer leeiihiitAfit Mid lernients glucose

produce an enterotoxin which causes fluid but not mannitol.

1' iirther R e a d i n g
Ak&aray N ec al _ 1990 Cueaneous anthrax. Trop Gtqgnph Med 42: 163
. .

Rrachman PS. 19S0. lnfadpliiHi anthrax. Ann New Yotk Acid Sd 353:83.
Dixon TC rt al. 1999. Andirax. New Eng J Med 341:815
Indira Devi K.. 2001, Anthrax ], Acad Clm Microb 3:55.
LaJicha MK and A Kumar 1996, Anthrax in South India. Lmcci 348:553.
LaforccFM. 1994. Anthrax. Clin /nTcctDrj I 9A 0O9.
MaseLson, M et ad. 1994. The Sverdlovsk anthrax outbreeJk of 1979. Science 266:1202 .
Mock M and A Foret 2001 Anthrax. Ann Rev Microtoof 55:647..

opy righted material

 CO Clostridium
CM
The gcntis Clostridium consists of Gram positive,
anaerobic , spore tunning bacilli . The spores art-
wiiier than the bacillary bodies, nving the bacillus
^
a swollen appearance , resembling a spindle. Hence
.
tbe name Clostridium ( AJcJ-sTer meaning a spindle ) .
The genus contains bacteria tWpwiliblc tor three
major diseases of huimr beings - gas gangrene,
food poisoning and tetanus. Some ot the pathogens,
for example, Cf . perfh ngrnnLand Q teram are found
'
normally in human and animal intestines, Mam
species arc pathogenic hot most are saprophytes
found in .soil , warcr and decomposing plant and
animal matter. Intestinal flfirtridia rapidly invade
the blood and tissues of the host after death anil
initiate decomposition of die cadaver. Some (for
example Cl acefrib( JtybYum ) are lit industrial
importance, used for rhe production of chemicals
such as acetone and butanol.
Clostridia are highly pleomorphic. Thev are rod
shaped, usually 3-K|lm it 0.4-1.2 pm in siie. Long
filaments and involution forms are common. Spore
formation occurs with varying frequency in different
sjutcies . Some [ 07. iporogej'ie.d sporulite readily
while others ( 07. ywrfrjngejrs ] do SLJ inconstandv,
Spondadon takes place in the animal hody also .
The shape and porition of spores vary in different
species and these arc of use in rhe identification
and class i heat uni of clostridia , Spares may be:
1 . Central o r equatorial , giving the bun II Us a
spindle shape ( Cl. bifcnnctiniu),
2. Subrermimi, the bacillus appearing club sh aped
[Cl perfj-JJTj eJi.s ) .
^
3. Oval am! rerminah resembling a tenni* ran ker
[Cl ferriurn ).
4. Spherical and Terminal , giving a drumstick
appearance ( 07. renm).
Closrridja aie motile with peritrichate flagella,
with few exceptions such a*. Cl perfringens and
Q rtianr type VI which are iwnmotilccThe motility'
is slow and has- been described as ’stately ' , 0-7,
pertrfn erih and 07. butyricum art capsulated, while
^
others arc not .
Clostridia are easily stained . They arc Gram
positive hut in older cultures , cells are often Gram
variable , m even frankly Cram negative.
Clostridia are anaerobic. The sensitivity to
oxygen varies in different species. Some ( for
example, Cl jrovrt) are exacting anaerobes and die
on exposure rn oxygen , while no me others (for
example, Cl. Jbs'fofyrjL’ unT) are aerotoferant and may
even grow aerobic ally. More important than the
absence of oxygen is the provision of a sufficiendy
low redox potential ( Eh ) in the medium , ITiis car
be achieved by adding reducing substances such as
unsaturatvd fatty acids, ascorbic acid, glutathione,
cysteine , t hi ogive cdhe acid , alkaline glucose,
sulphites or metallic irqn . A small concentration
ufCO , appears to enhance growth. The optimum
temperature for pathogenic Clostridia is 37 ' C .
Some saprophytic clostridia are thermophilic and
others psyrhmphylic. The optimum pH is 7-7.4.
Growth is relatively slow on solid media .
Colonial characteristics arc variable. Some species
are hemolytic or blood agar. A very useful medium
15 Robertson 's cooked meat broth . It contains
msaturated fatty acids which take up oxygen , the
reaction being catalysed by hemrir in the meat ,
and also sulphydril compounds which bring about
Copyrighted material
 * Clostridium
a reduced OR potential. Clostridia grow in the
medium, rendering the broth turbid. Mott HJKM:LL"S
produce gas . Slccharalvtic species turn the meat
pink. Proteolytic species turn the meat black and
produce tnul and pervasive odours. In lilmLLS milk
medium, the production of acid, clot and gas can
be detected .
1 lie vegetative veil;; of dnRtridiu dn not diffitr
from no ns poring bacilli i.n their resistance to
physical and chemical agents . The spores exhibit a
pronounced but variable resistance to Iteai, drying
and disinfectants. Spores of CL hotulinum survive
boiling alter 3-4 hours and even at 105 C are not
killed completely in less than 100 minutes. Sports
ot most strains of Q. pejfjfiyjejif art; destroyed by
boiling for less than five minutes but spores of some
Type A strains that cause food poisoning survive
fur several hours CL fetanj spnrcs persist for vtars
, and organs ofa cadaver. This is the most important
in dried earth . Spores of some strains of Cl. tetarn
-
resist boiling fur 15 90 minutes, though in must
cases , they arc destroyed within five minutes. All
species are killed by autoclaving HT 1 'C within
20 minutes. Spaces arc particularly resistant to
phenolic disinfectants Formaldehyde is not verv
active and spores may sometimes survive immersion
in 2% solution lor upfo five days. Halogens are
effective and 1% aqueous iodine solution kills spores
within three hours. Clutaraldc hyde { 2% at pH 7.5-
8.5 ) is very effective in killing spores. In general,
dns ' ridia IfC susceptible tu metronidazole ,
penicillin, cephalosporins and chloramphenicol ;
lets sO to tetracycline , and resistant ro
*
aminoglycosides and quinoloncs.
Clostridia can produce disease onlv when the
conditions are appropriate . Their invasive powers
are limited . Pathogenic elostridin form powerful
ototoxms. CL bomirnttn is virtual! v nuniuvasive and
nonmfectiu-us. Botulism is due to the ingestion of
preformed toxin in food. Cl. ftefuirhu little tsvaffime
property and is confined to the primary site of
lodgement. Tetanus results tram the .LCILOTI of the
potent exotoxin it produces. The gas gangrene
Clostridia , besides being toxigenic, are also invasive
249
and can spread along tissues and even cause
septicemia.
Many methods have been adopted for the
classification of clostridia. These include
morphological features such ; LS the shape and
position id spores and biochemical fortunes such
'
as saccharolytic and proteolytic capacities (Table
28- 1 ) . Clostridia of mcdicaJ importance may also
he considered under the diseases they produce (see
classification below}.
CLOSTRIDIUM PERFRINGENS
( CL wdchii, Bacillus acrogcncs apni/tiuf,
B. phlegmonic emphysematwee)
The bacillus wiu originally cultivated by AchaJme
(1891 ) hut was first described In detail by Welch
and Muttall ( 1 f$ 92 ), who isolated it frnin the blood
ot the dostridia Causing gas gangrene . It also
produces food poisoning and necrotic enteritis in
human bei ngs an d many rerious discares i n jni mal s.
CL pcrUingcns is a normal inhabitant of the
large intestines of human beings and animals h is ,
found in the Iteccs and it contaminates the skin of
the iHrrinetim. buttocks and thighs. 'tTe spores are
commonly found in sod , dust and air ,
Morphology: It is a plump , fJrant positive
bacillus with straight, parallel sides and rounded
or truncated ends , about 4- ft pm « 1 tim . usually
occurring singly or in cttiaius or smull bundles- . It Is
pleomorphic, and filamentous and involution forms
arc common - It is capsulatcd and non motile- Spores
arc central or subicrminal but arc rarely seen in
artificial culture nr in material from pathological
IcsiiOlU, and their absence is tine of the chanacteristic
.
morphologkal features of Cl pcrfmtgnns,
Cultural characteristics: It is an anaerobe
but can also grow under mkrttttbpihUit conditions.
Oxygen is not actively feme to the bacillus and
cultures do not die on exposure ro air, as happens
with some fastidious anaerobes. It grows over a
-
pH range of 5.5 8.0 and temperature range of
20*0-50 :: C . Though usually grown at 37 C , a "
Copyrighted material
2 SQ i Tuxtbook of Microbiology

Temperature of 45 °C is optimal many strains.

tor
The generation time at this temperature maybe as
short iis ten minutes. This property can he utilised
for obtaining pure cultures oi CL perfringens.
Robertson 's cooked meat broth inoculated with , production of acid and gas. It is indole negative,
rrutturei of Q pertuns ena and other bacteria and
^
incubated at 45 °C for 4-6 hours serves ns nn
enrichment. Subcultures from this cinro blood
agar plates yield pure or predominant growth of
CL pcrfringcns .
Good growth occurs in Roller [son’s cooked meat
medium - The meat is turned pink hut is rot
digested . The culture hat in acid reaction and i
sour odour.
In litmus milk, fermentation of Lactose leads to
formation of acid, which is indicated by the change
in the colour of litmus from hlue to red.. The acid
coagulates the casein ( acid clot ) and the clotted
milk is disrupted due to the vigorous gas
production . The paraffin plug is pushed up and
shreds of cloE ate seen Slicking to [ he side?; of [ he
tube, 'this is known as ’stormy fermentation ' ,
After overnight iiLLLihation on rabbit , sheep or
human blood 3gUT, colonics of most strains show a
"target hemolysis’, resulting from a narrow zone of
complete hemolysis due to theta toxin and J much
wider zone of incomplete hemolysis due to the alpha
Table 33.1 A morphological and biochemical classification ot Clostridia
toxin . This double zone pattern
of hemolysis may
fade on longer incubation.
Biochemical re action stChjcow, matt-ose,
lactose and sucrose arc tormented with the
MR positive and VP negative. H ,S in formed
abundantly Most strains reduce nitrates.
Resistance: Spores arc usually destroyed
within five minutes by boiling but those of the
'
htod poisoning' strains ofTypc A and certain Type
C strains resist boiling for l -!i hours . Autoclaving
of 121 "C for IS minutes is lethal. Spores are
resistant to the antiseptics and din in feet ants in
common use.
Glassification: CL pertringena strains are
classified into five types , A to Eh based oil the tmdris
they produce . Though the bacillus produces a large
number ot toxins, typing depends on the four ' major
toxins' - Typing is done by neutralisation tests with
specific antitoxins by intracutaneous injections in
guinea pigs or intravenous injection in mice.
Toxins; CL perfringens is one of the most prolific
ot toxin - producing bacteria, forming at least 12
distinct toxins, besides many other cnzvmcs and
biologically active soluble substances. The four
’
major TOXINJ , alpha, beta , epsilon and iota , arc
predominantly responsible for pathogenicity. The

Bath proteolytic and faccAanfrtrc

Position Proteolytic Sicchiroiytic Slightly prtneolyTie Stcchtl&lybc Neither
of spores predominating predominating but Mit but nor proteolytic nor

—Central or
—
ti&
CL jineiifatis Cl pcffnntgen?
—
mcchimlyrit : proteolytic

a fiffw
stcehmrofytic

tubfcrminaJ CL hotulinum CL Kp&CCMl

ABE CL chauvoei
CL histoiyticum CL JTOVVJ CL bomlifiujn
CL sordeih CM
CL sporogcnc.3
Oval and CL difficile CL tertium O.CwWsutJm
terminal
Spherical Cl . Xrfarrr CL Tetunomorphum
and CL sphenoid?*
terminal

Copyrighted materia

Clostridia « human palhogens
A . The gti gangrene group:
] . Established.
pathogens
2. Los pathogenic
3 . Doubtful pathogens
B . Tetanus :
* CloslritJium * 251
yolk. The opalescence in the egg yolk media may
he produced by other lecithinase forming bacteria
also (O. miiji, Cl hiJernierifans, some vibrios, some
CLpctfhngGM aerobic spore bearers.] . In the case of these bacteria ,
Cl aep6cum the react inn is not neutralised by the CL pcrfruigens
Cl novyi
antitoxin, except with O hr fermentsn $ which
Cl histolyricim}
CL talk* produces a serologically related ld. iTliinasc .
Cl bilesmentans The alpha toxin is hemolytic for the red cells
Cl sporogznes of most species, except horse and goat, due Dc^ its
Cl rctimi action on the phospholipids on the erythrocyte

C . Food poisoning :
,
membranes . The Ij'v.ls is of the hot-cold variety,
being best seen after incubation at 37 °C followed
1. Gastroenteritis Cl pertringens
( Type A )
by chilling at 4 JC , It is relatively hear stable and is
2 . Necrotkkig enteriti* CS. pettiringmi only partially inactivated by boiling for five minutes.
( Type C ) Beta (]3), epsilon (s) and iota (l ) toxins have
3 - BmEut CL tt 'rwJjVjMirr lethal and necrotising properties. Gamma ( y) and
D* Acute colicifi a diffkfc eta ( n ) toxins have minor lethal actions. The delta
ifi ) toxin has a lethal effect and is hemolytic for
iL ]: HL>. (a ) toxin i$ produced by all types of Ci.
perftrn rnf and most abundantly hy Type A stm i ns.
-
the red cells o-f even toed ungulates (sheep, goats,
pigs, cattle ) . The theta ( GJ H>xm is an oxygen labile
^
Thb is the most important toxin biologi«JIy and hemolysin anligenically related to streptolysin O-
is responsible for the profound toxemia of gas lr ic aliio lethal and a general cytolytic toxin . The
gangrene. It is lethal, dermonecniLi ^. and hemolytic kappa ftc ) toxin is a collagcnasc. 'the lambda ( X)
I: is a phospholipidase [lecithinase C) which, in toxin is a proteinase and gelatinasc. The mu ( ji )
the presence of Ca ” and Mg" ions, splits lecithin toxin is a hyaluronidane and the mi ( v ) toxin a

into phu & phoryl choline and d lycende. This
reaction is seen as an opalescence^in serum or egg
yolk media and is specifically neutralised by the
antitoxin When Cl. perfringens is grown on a
deoxytn lM? nudcasc .
.
Besides ibe toxins, Ci perttingeia also produces
other soluble substances, some of which possess
enzymatic properties. TTtese include the enzymes

medium containing 6% agar, 5% Filde -. pepuc
digest of sheep blood and 20% human semen , with
the antitoxin spread on one half of the plate, colonies
on the other half without the antitoxin will he
surrounded by a zuilc of opacity, There will be no
opacity around the colonies on the half of the plate
with the antitoidnh due to the specific neutralisation
of the alpha toxin. Thiv specific lecirhinaae effect,
known as the Nagler reaction , is a useful test for
the rapid detection ui Cl. pcrfnngens m clinical
specimens ( Fig. 28 - 1 ) . The incorporation of
neomycin sulphate in the medium makes it more
selective, inhibiting coliforms and aerobic spore
bearers. Human serum may be replaced by 5% egg
which destroy the blood group substance, A and
H, a neuraminidase which destroys myxovirus
receptors on red blood cells, a substance which
renders red blood cells panagglutinable by exposing
TheirT am igena, a hemagglutinin active against the
red blood cells of human beings and most animals,
a Hbrir.olvsin , , i hemolysin distinct from alpha ,
theta and delta toy i ns, histari ine, a "bureti ng factor*
which has a specific action on muscle tissue and
may be responsible for the characteristic muscle
lesions in gas gangrene and a circulating facror'
which can cause an increase m the adrenaline
sensitivity of the capillary bed and also inhibit
phagocytosis.

Copyrighted material
252 « Ti&xibook of Microbiology *
J \\T H O C R N r C I T Y
CL perfrifigcftS produces the following human
infections:
Gns gangrene CL perfringem Type A LS the
predominant agent causing: gas gangrene- It may
occur as the sole etiological agent but is more
commonly seen in association with other clostridia
as well as nondostridial anaerobes and even aerobes.
All clostridial wound infections do not result in
gas gangrene. More commonly, they lead only to
wound contamination , or anaerobic cellulitis, It Is
only when muscle tissues arc invaded that gas
gangrene (anaerobic ntyosicis) results.
I' OuJ poisortiftg: Some strains of Type A
(food poisoning strains) can produce ftiod poisoning.
They are characterised by the marked heat
resistance of their spores and the feeble production Ffcfl, ES-1 Nngter reaclion. Cl- perfringens colonies on
of alpha and theta toxins. They have been shown ttie right lull ol Ihe plate are surrounded fay haloes,
to produce U beat labile enteroroxin which, lib; wWle colonies on the left half ( containing srtltoeram
the enterotoMins of V choierae and enterotoxigenic to alpha to * in ) have no haloes around them,
£ . coli , leads to fluid accumulation in the rabbit Gangrenous appendicitis: Cl . perfringena
ileal Loop. Type A (and occasionally Type D ) strains have been
Food poisoning by CL peffringtns is usually isolated from gangrenous appendicitis. The
caused bv a cold or warmed up meat dish. When demonstration of antitoxin in these patients and die
contaminated meat i* cooked , the spores in rhe beneficial effects of the administration oFaniitoxin aLso
interior may survive. During storage or warming suggest the etiological role of tbe bacillus in this
they germinate and multiply in the anacrohic condition. It has been proposed that die toxemia and
environment in rhe coobd meat . Large numbers shock in some cases of intestinal obstruction and
of Clostridia may thus be consumed * which may peritonitis may bedue to the toxins of CL pertriiigEns.
pass unharmed by the gasinc acid due to the high Ntrerolising enteritis: This is a severe and
protein in the meal and reach the intestines where often fatal enteritis known by different names in
they produce the encerotoxin. After an incubation different countries: Germany ( Darmbrand) , New
period of ft-24 hours, abdominal pain , diarrhea and Guinea ( pigbel ) . East Africa, Thailand and Nepal.

—
vomiting set in - The illness in self-limited and
recovery occurs in 24 48 hours. Diagnosis is made
by isolating heat resistant CL perfringens Type A
from the feces and food . As this maybe present ut
normal intestines, isolation from feces, except in
large numbers is not meaningful. Isolation from
food has to be attempted by direct plating on
selective media , as the bacillus is present in food
mainly as the vegetative cells.
It is caused by CL perfringens type C Strains with
heat resistant spores which germinate in the
intestine producing beta toxin causing mucosal
necrosis. The evocative name 'pigbel’ is New Guinea
pidgin for abdominal pain and diarrhea following
unaccustomed feasting on pig meat along with
trypsin iithibitors like sweet potatoes . Immunisation
with type C toxoid has been shown to protect
against this condition.

Copyrighted material
 Table 261 : Toxins produced by Cl . parfringens. types

Major toxins Minor toxins

Typc Pathogenicity

S
a l T T\ K
* M V

-
A Ca g-infcficne: WALW infecrions, Mpritemia. + ++
* - - - -
4

B
Food poisoning

Lamb dysentery
4 ++

+ ++
-
+++ * 4
4
*
-
4
~

- 44
- 444 444
++

4+
Closndjm
P

C Enteritis Ln animals
Enteritis necroticans in. human beings
+ 4+
44 +-
44
4+
* -
=
-
4#
=

-
*4
-
# 44 4

-
-
-
-
- + 44

D Enterotoxemia of sheep +4+ - 4 +4 - - 44 44 44

E Doubtful pathogen of sheep and cattle + #"

* — +++ — >

— — ++ ++ ++

Copyrighted
materil
bJ
a
254 i Taxlbook of M c'obioogy *
Biliary tmcsl infection: Cf p&rningens
beer reported to produce two ore but serious
i nfections ( "t the lull , iry tfact - 1CUK trilptjwemanm >
s hokCYHNtis SILLI pOStcholcCVSteCtOlTiy SCptLCCmiU.
'
Bndi enous gns gangrene of tntra -
^
uhJmiiinal origin: Gas gangrene of the
abdominal wall has been imported as an infrequent
complication oi abdominal surgem The infection
is endogenous, the organism being derived from
the gut and contaminating the abdominal wall
during surgery. Gas gangrene of the thigh as a result
of infection tracking from the abdomen 11 LIS also
been reported.
Brain abscess and meningitis: Brain
abscess and meningitis doe to Cl pcriiingcns have
been reported very rare I v.
Panophthalmitis: Panophthalmitis DUL to Cl.
perfringen* has occasionally followed penetrating
- widely distributed in soil. It is u strict anaerobe,
eye injuries.
Thoracic infections: Clostridial infection of
the chest cavity may follow penetrating wounds of
the thorax . This, is more often seen in batrlc
casualties than m driliui situations .
Urogenital infections : Infection of the
urinary tract may occasionally follow surgical
procedure such as nephrectomy. Clostridial
infection tit tlie uterus in a serious and not infrequent
condition , commonly associated with septic
abortion . Septicemia i * common in this condition.
CLOSTRIDIUM SEPTICJM
This bacillus was first described hv Pasteur and
. J
Joubert {1887) and called Vifcritwi septique.. It is a
—
pleomorphic bacillus ibout 3 8 jim * 0.6 jim in
fiLM, iuntiin oval, central or snbtermirLal spores- It
^
is motile by peritrichate flagella. Growth occurs
ainacrohicallv on ordinary media. The colonics are
M r
irregular and transparent initi.dlv. turning opaque
on continued incubation . Hemolysis occurs on
horse blood agar. Growth is promoted bv glucose .
It is saccharolytic and produces abundant gas.
Six groups have been recognised, based on
somatic and flagellar antigens. CJ . rcptioUB
produces at least four distinct toxins and a
fibrinolysin. The alpha toxin is hemolytic ,
dermonecrotic and lethal , the beta toxin is a
leucotoxic deoxyribonuclease, rhe gamma roxin a
hvaluroniduse and the delta toxin an oxygen labile
hemolysin .
J
Cl icpOcum is found in the soil or in animal
in res tines. It is associated wirh gas gangrene in
human beings , usually with other clostridia . It also
dU 9« " braxy ' in sheep and 'maJignanT edema' in
cattle and sheep .
CLOSTRIDIUM NOVYI
( CJ, oedemartensj
This is i large, stout, pleomorphic, Gram positive
bacillus with large , oval, tubiermmal spores. It is
readily inaL- rtvattd by exposure of cultures to air.
Four types ( A to D) are recognised, based oo the
production of toxins . Onh type A is of medical
importance , as it causes gas gangrene. Gas gangrene
caused by CJ. novyi is characterised by high
mortality and large amounts of edema fluid with
little or no observable gas in infected tissue. Other
type* produce veterinary disease, There was a lethal
outbreak of Cl. HOVIT type A inleCtion in heroin
addict* in Britain in 2000-
CLOSTRIDIUM HI3T0 LYTIC UM
This is an actively proteolytic clostridlum, forming
oval , subterminal , bulging spores . This is
acroroletant and some growth may occur even in
aerobic cultures . It forms at: least five distinct toxins.
It is infrequently associated with gas gangrene in
human*.
GAS GANGRENE
Oakley ( 1 S4) defined gas gangrene as a rapidly
^
spreading, edematous myonecrosis* occurring
characteristically in association with severe wound*
of extensive muscle masse * that have beer
contaminated with pathogenic clostridla ,
particularly with Cl /vr/rmgrns. '[Tie disease has
Copyrighted material
 * Cl D51n di u m
been referred to in the past as 'malignant edema'
Other descriptive terms that have heen used are
.macmbit fdostriLli.il) myositis’ and 'clostridial
'
myonecrosis' .
Gas gangrene is ctiaracreriaticaUy a disease of war,
in which extensive wounds with heaw .s
contamination
re very common. In civilian life, the disease generally
follows road accidents or other types of injury
involving crushing of large muscle misses. Rarely, it
may even follow ^irgical operations.
The bacteriology of gas gangrene is varuid-
R a rely is this due to infection with a single
Clostridium. Generally several species of clostridia
are found in association with anaerobic streptococci
and facultative anaerobes Fnich an i . . atli , proteus
and staphylococci. Among the pathogenic clostridia,
£7- yjerfh jigiens LR the mnsr frequently encountered
'
(approximately GQ per cent), and Cl. no and Ci .
scpficum being the next common ( 20-40 per cent),
and CL histoh^L-um less often . Other clostridia
usually found are Cl . sporogenos, CL hllax ,
.
CI. bifetmen rails Cl. aotdelh ; , CL Jtnjtivtidumaod
.
Q terr.' Lrm, These may not be pathogen1. ’ by
themselves . The relative incidence of the different
species varies m dliferent Series of cases and may
he a reflect : an of the distribution of the species in
different Sails.
Cfesp idia usually enter the wound .LIUIIL; with
*
implanted foreign panicle such a soil (particularly
*
manured or culnvated soil), road dust , bus of ,
clothing or shrapnel. Clostridia may also be present
on the normal ski:i, cspec i ally on the pc i i -icum and
thighs. Infection may at times he endogenous. Gas
gangrene may occasionally follow clean surgical
procedures (especially amputations for vascular
disease) and even inject inns (especially adrenaline j ,
The mere presence of clostridia in wounds does
not constitute gas gangrene. MacEcnnan has
distinguished three types of anaerobic wound
infections:
Simple wound contamination with no int .i >:on
of the underlying tissue, resulii ig in Hide more than
'
some delay in wound hcaliug-
255
. Anaerobic cellulhh in which clostridia invade
the fascial planes, with minimal toxin production
and no invasion of muscle rissues . The disease is
gradual in onset and may vary from a limired Lgas
abscess’ to the extensive involvement of a limb.
Anaerobic myositis or gas gangrene, which is
the mint serious, associated with clostridial invasion
of healthv muscle tissues and abundant formation
jf
of ex-ntoxi ns. Gas gangrene results only if the
conditions favourable for clusterhaJ multiplication
exist in the wound. The most important of these is
low oxygen tens! i to.'Hiis is achicvcLl ideally in battle
wounds in winch there are : mplanted bullets or
shell fragments, along with bits of clothing and
soil particles. The ionised calcium salts and silicic
acid in the soil cause necrosis . Crushing tissue or
tearing of the arteries produces anoiLp. of the
muscle. Extravasation of blood increases the
pressure on the capillaries, reducing the blood
supply s Iill further.The Eh and pH of tile damaged
tissues fall, and these changes along with the
chemical changes that occur within the damaged
and anoxic muscles, mcluHmg breakdown of
carbohydrates and liberation of aminoacids from
proteins, provide an ideal pabulum for rhe
.
proliferation of anaerobes. Ex IT uvasated hem iglobirt
and myohe mnglohin are reduced and cease to act
as oxygen carriers. As a result, aerobic oxidiLTinn is
halted and anaerobic reduction of pyruvate to lactate
leads to a further fell in Eh .
The clostridia multiply and elaborate toxins
- .
wfe.Lih cau *e further ti ^-uc damage The lecithinascs
damage cell membranes and increase capillary
permeability, leading to extravasation and increased
tension in the affected muscles, causing further
anoxic damage. The hemolytic anemia and
hemDglohmun.L seen m CI. perinngvns infections
are due to the lysi of erythrocytes by the alpha
*
to in. The collagenases destroy collagen barriers
*
in the tissues and hyaluronidases break down the
intercellular substances furthering invasive spread
by the clostridia. The abundant produedon of gas
reduces the blood supply still further by pressure
Copyrighted material
256 4 TgKfbggk g1
eBern, extending the irci ofanw damage. It thuf
^
becomes possible for the infection to spread from
the original site, making the lesion a progressive
one .
The incubation period may be as short as seven
hours or as long as six weeks after wounding , the
average being 1 (H-S hours with Cl , perfringtris+
2- 3 days with Cl scpticum and 5-6 days with
Cl , novyi infection The disease develops with
increasing pain , tenderness and edema of the
affected part along with systemic signs of toxemia.
There is a thin watery discharge from the wound ,
which later becomes profuse and serosanguinous.
Accumulation of gas makes the tissues crepitant .
In untreated cases, the disease process extends
rapidly and inexorably. Profound toxemia and
prostration develop and death occurs due to
circulatory failure .
r
Laboratory diagnosis: The diagnonis of gas
gangrene must be made primarily on clinical
grounds, and the function of the laboratoiy is only
to provide confirmation of the clinical diagnose as
well as identification and enumeration of the
infecting organisms. Bacteriological examination
also helps to differentiate gas gangrene from
anaerobic streptococcal myositis, which may be
indistinguishable from it clinically in the early
stages. In the latter, Gram stained films show large
numbers of streptococci and pus ceils , but noc
butiHi , contrasting with the scanty pus cells and
diverse bacterial flora seen in films from gas
gangrene.
The specimens to be collected arc: ( 1} Alms
from the muscles at the edge of the affected area,
from the tissue in the necrotic area and from
the exudate in the deeper parts of the wound;
( 2) exudates from the parts where infection appears
to be most active and from the depths of the wound,
to be collected with a capillary pipene or a swab;
and ( 3) necrotic tissue and muscle fragments.
Gram stained films give presumptive
information about the species of dostridia present
and their relative numbers. The presence of large
Migrgfciiolggy
numbers of regularly shaped Gram positive bacilli
without spores is strongly suggestive of Cf .
perfriugtns infection. "Citron bodies" and boat or
leaf shaped pleomorphic bacilli with irregular
Staining suggest Cl- sepbeum. Luge bacilli with
oval , subterminal spores indicate Cl novyi. Slender
hadlii with round, terminal spores maybe Cl tetani
or Cl tetanQmaiphunt .
Aerobic and anaerobic cultures are made on
fresh and heated blood agar, preferably on 5-6 per
cem agar to prevent swarming , A plate of serum
or egg yolk agar , with Cl periiingens antitoxin
spread on one half is used for the 'Nagler reaction'.
Four tubes of Robertson's cooked meat broth are
inoculated and heated it 100 ’C for 5, 1C, 15 and
20 minutes , incubated and subculturcd on blood
agar plates after 24-48 hours, to differentiate the
organisms wi th heat resistant spores. Blood cultures
are often positive, especially in Cl pcriUngcni and
CL sfpacwn infections. However Cl . perfringent
bacteremia may occur without gas gangrene. The
isolates are identified baaed on their morphological ,
cultural biochemical and toxigenic characters.
Prophylaxis find therapy: Surgery is the
most important prophylactic and therapeutic
measure in gas gangrene . All damaged tissues
should be removed promptly and the wounds
irrigated to remove blood dots, necronc tissue and
foreign materials. In established gas gangrene,
uncompromising excision of all affected parts may
be life- saving . Where facilities exist , hyperbaric
oxygen may be beneficial in treatment.
Antibiotics are effective in prophylaxis, in
combination with surgical methods. The drug of
choice is metronidazole given IV before surgery
and repeated S hourly for 24 hours. As mixed
aerobic and anaerobic infections arc usual , a more
broadspectrum antibiotic prophylaxis, such as a
combination of metronidazole , gentamicin and
amoxycillin is advisable .
Passive immunisation with 'anti -gas gangrene
serum' ( equine polyvalent antitoxin in a dose of
10,000 IU Cl- per&ingcns, 10,0001U Cl norland
GODvrianift - - nateria l
ru
 < C iQstridiuFri
5 ,0tK) IU Cl . tepdcum antitoxin given 1M or in
j The optimum temperature is 37 and pH l .A . It
emergencies W ) used to be the common practice
EH prophylaxis. However , in view of its uncertain
efficacy and availability, it.H use has become rare .
CLOSTRIDIUM TEtANl
Cl . tetani is the causative organism of tetarms.
Tetanus has been known from verv early limes ,
* m
having hecn described by Hippocrates and Aretaeus -
Carle and Rattone { 1094) transmitted the disease
to rahbits . Nicolaier ( 19 )14 ) , studying the
experimental disease , suggested that the
manifestations of tetanus were due to a strycbninc -
likc poison produced by rhe bacillus multiplying
loyally. Rosenhaeb ( 1 Fifth ) demonstrated a slender
bacillus with round terminal spores in a case of
tetanus. The final proof of the etiological role of
the bacillus in tetanus was furnished by KitasatO
( 1889 ) who isolated it in pure culture and
reproduced the disease in animals by inoculation
of pure cultures.
C/. tctxni is widely distributed in soil and in the
intestines ofhuman beings and animals . Iris ubiquitous
and lias ijeen tmuTied from a wide variety of oilier
smimrs , including street and ha Kpitill dust , onttoti WKKII ,
piaster of Paris, bandages, caCgut , talc, wall plaster
and clothing . It may occur as an apparendy harmless
contaminant in wounds.
Morphology: It is a Gram positive , slender
bacillus, about 4- B * Q.5 Jim though there may
be considerable Variation in length. Lt has a straight
axis, parallel sides and roundedends. It occurs singly
and occasionally in chains. The spores arc spherical ,
[ terminal And bulging , giving ( h
* bacillus the
characteristic dmnstick appearance ( Fig. 28 - 2 ).
‘ '
'Hie morphology of the spore depends on its stage
of development and rhe young spore may be oval
rather than spherical it is noncapsulated and motile
,
by peritrichate flagella. Young cultures are strongly
Gram positive but older cells show variable staining
and may even be Gram negative .
Cultural chflracterittjci: It is an obligatory
anaerobe that grow* only in the absence of oxygen.
E 257
grows on ordinary media . Growth is improved by
blood and serum but rot by glucose. Surface colonies
are difficult to obtain as the growth lias a marked
tendency to swarm over the surface of the agar,
especially if the medium is moist . An extremely
fine, translucent film or growth is produced [ hit is
ytactically invisible , except at the delicately
filamentous advancing edge . This property enables
the separation of C7. fef*ni from mixed cultures . If
the water of condensation at the bottom of a slope
of nutrient agar is inoculated with the mixed cell
culture, after incubation anaerobically far 24 hours ,
subcultures from the top of the tube will yield A
pure growth of the tetanus bacillus (Fi1dg*h
technique ).
In deep agar shake cultures, the colonics arc
spherical fluffy balls , 1 -3 min in diameter, made
up of filamcnts with a rad i at arrangement . I n gelari n
stab cultures a fir mce type of growth occurs, with
KIQW liquefaction.
It grows wrl ] in Robertsons cooked meat broth ,
with turbidity and some gas formation . The meat
is riot digested but is turned black on prolonged
iocubatiorn -
V
V| s *
P
\ .
I i
J
fl 9
X 1 , \ \S
Ww
;
A \\
t /
Fig . £fi . 2 Ci. IL";* CHTJL some with spares and some without
253 4 T xtbM- k fif Mitrfi&nilagy
^
On blood agar, a hcmoly^ ]' is produced , which
Liter develops into 3 hemolysis, due to the
production of hemolysin ( tctanolysin ) .
Bine hemic a I reactions: Cl- reran; has feeble
proteolytic but no siccharolyriL property; It docs
not attack ary sugar It forms indole It is MR and
VP negative . H3S is not formed. Nitrates arc not
reduced. Gelatin liquefaction occurs very slowly, A
greenish fluorescence Is produced on media
containing neutral red ( as on MacConkflys
medium }.
Resistance: The resistance of tetanus spores to
ke.it appears lo 1 ^ subject ti ? strain differences. Most
are killed by boiling for 10-15 minutes but some
resist bo i I ing for upto three hours. When destmet ion
of spores is to be ensured, autoclaving at 121 °C
for 30 minutes is recommended. On the other hand,
when heat is applied in order to free cultures of CL
refan i from minsporirg contaminants, it is important
not to exceed @ 0 "C for 10 minutes, as even this
nil Id treatment can cause considerable destruction.
Spores are able to survive in soil for years, and are
resistant ro most antiseptics.They are nor destroyed
by 5% phenol or 0.1% mercuric chloiuie solution
i n two weeks or more lodi ne ! 1% aqueous solution )
, 130 nanograms. There Ls considerable variation in
and hydrogen peroxide (ID volumes) kill the spores
within a few hours.
(Classification: Ten serological types have been
recognised based on agglutination ( types 1 to X).
Type VI contains nonfligelkted strains. All other
types posses* type specific flagellar antigens. All
the types produce the same to;vin , which is
neutralised by antitoxin. produced against any one
type.
—
"logins
: Cl tefsni produces at least two distinct
tQxins a hemolysin ( tctxnotrw'.t ) and a powerful
neurotoxin ( feranojpas /njn ) . The two are
antigenically and pharmacologically distinct and
their production is mutually independent. A third
toxin , a nonspasmogenic , peripherally active
neurotoxin , has been identified. It is not known
whether this plays any role in the pathogenesis of
tetanus.
Tecanolysin is a heat Labile, oxygen labile
hemolysin, anti gen ically related to the oxygen labile
hemolysins produced by Cl. pttiinng&is, Cl. jrayyi
and Str. pyogens. It is nor relevant in the
pathogenesis of tetanus.
Tetanospasmin is the toxin responsible for
tetanus, [T is oxygen stable but relatively heat labile,
being inactivated at 65 °C in five minutes. It is
plasmiill coded. It gets toxoided spontaneously or
in the presence of low concentrations of
formaldehyde. It is a good ant igen and is specifically
neutralised by the antitoxin . The toxin has been
crystallised. It is a simple protein composed of a
single polypeptide chain . On release from the
bacillus, it in autolysed to form a hetcrodimer
consisting of a heavy chain (93 ,000 MW ) and a
light chain (52*000 MW ) joined by a disulphide
bond . Tetanus and botulinum toxins resemble each
other in their i l i u m -! IILLLLI sequences.
The purified toxin is active in extremely small
amounts and has an MLD lor mice of about 50-75
“ lfla mg- The amount of twin produced depends
on the strain of bacillus and the type of culture
medium used. Its MLD for human beings is about
the susceptibility of different species of animals to
the toxin.The horse is the most susceptible. Guinea
pigs, mice, goats and rabbits arc susceptible in that
descending order, birds and reptiles ate highly
resistant. Frogs, which are normally Insusceptible,
may he rendered susceptible by elevating their body
temperature.
lithogenicity: Cl . reran; has little invasive
power Wished spores injected into experimental
animals do not germinate and arc destroyed by
phagocytes. Germination and toxin production
occur only if favourable conditions exist, such as
reduced O-R potent id, devitalised tissues* foreign
bodies or concurrent infection. The toxin produced
locally is absorbed by the motor nerve endings and
transported ro the central nervous system
i ntraxonally.The toxin is specifically and avidlyfixed
by gangliosides of the grey matter of the nervous
Copyrighted materia
 * ClDSU dium
tissue. [ ctanospasmiit resembles strychnine in its
effects. The tetanus to* in specifically blocks synaptic
inhibition in the spinal cord , presumably at
inhibitory terminals that use glytn ^e and GABA ; LK
neuroTiansnl inters. The toxin acts piesynaptially,
unlike strychnine which acts postsynapri .- ally. The
abolition nf spinal inhibition causes uncontrolled
spread of impulses initiated anywhere in the central
nervous system . Tins results in muscle rigidity and
spasms due to the simultaneous contraction of
-
agon i -its and antagonists , i n the at ence of reciprocal
inhibition. The convulsion pattern is determined
by the most powerful muscles at a given point, and
i:i most animah i -. characterised by tonic extension
of the body and of all limbs.
The toxicity of tetanospasmin is influenced by
the route by which it is administered . Given orally,
it is destroyed by the digestive enzymes and is
without effect . Subcutaneous, intramuscular and
intravenous injections are equally effective.
Intrancural injections are more lethal and injections
direedy into the central nervous system very much
more so. The route of administration also modifies
the clinical picture . Experimental tetanus may
accordingly, be of the Hlocal ', 'ascending' or
descending' variety. These differences arc related
to the manner in which the toxin reaches and is
disseminated in the central nervous system . . onset ).
When the toxin is inoculated intramuscularly
in one of the hlndlimhs, tonic spasms of the muscles
of the inoculated limb appear first, This is known
as local tetanus and is due to LIII- toxin acting on
-
the segment of the phial cord containing the motor
neurons of the nerve supplyi ng the Lnocu I . Lted area.
Subsequent spread of the toxin up the spinal cord
causes 'ascending tetanus'. The apposite hindlimb,
rmnk and fore!imbs are involved in an ascending
fashion . If the toxin is injected intravenously,
spasticity develops first in the muscles of the head
and neck and then spreads downwards [descending
tetanus ). This type resembles the naturally occurring
tetanus in human beings.
r 2Sy
TETANUS
Tetanus is characterised by tonic muscular spasms,
usually commencing at the site of infection and in
all but the mildest cases becoming generalised,
invoking the whole of the somatic muscular system.
.Most frrquendy, the disease follows injury, which
may even be too trivial to be noticed. Puncture
wounds are particularly vulnerable as they favour
the growth of the anaerobic bacillus. Rarely it may
follow surgical operations, usually due to lapses in
atepsis. Sometimes the di -^eise may be due to local
suppum it m, such as Ot it is med ] a [otogenic tetanus).
Tetanus i * an important complication of septic
abortion. It may be caused by unhygienic practices,
such as application of cowdung on the umbilical
stump or rituals Such as earboring or circumcision.
Tetanus may also be caused by unsterile injections.
Tire i ncubation period is variable, from two days
-
to several weeks, but is oommotily 6 12 days. This
is influenced by several factors, such as the site and
nature of the wound, the dose and toxigenic ity of
the contaminating organism and the immune status
of the patient . The incubation period i ; of
prognostic significance, the prognosis being grave
-
-
when it is short . Ol - imilar significance is the
interval between the first symptom of the disease,
usually trismus, and the onset of spasms ( period of
Tetanus was a serious disease with a high rate
of mortali 1 y, 80-90 per cent, before spec die
treatment became available . Even with proper
treatment the case fatalitv rate varies from 1S 50 -
per cent. Tetanus neonatorum and uterine tetanus
have very high fatality rates (70^100 per cent ), while
otogenic tetanus is much less serious.
Tetanus is more common in the developing
countries, where the climate is warm , and in rural
areas where the soil is fertile and highly cultivated,
where human and animal populations are
substantial and live in close association and where
unhygienic practices are common and medical
facilities poor- In mrul India, tetanus was a common
opy righted material
260 v Taxlbook ol Microbiology
cause of death , particularly in the newborn , But
immunisation of infants and expectant mothers hits
reduced the incidence tr> 5 large L^ vtcnt .
Laboratory diagnosis: The diagnosis of
tetanus should always be made on cluneal grounds.
Laboratory tears only hel[i in confirmation. Not
inficquciitfy ir may not IK possible to establish a
laboratory diagnosis at alf
Laboratory Lliagirosis may he made by
demonstration of Q, refartr by micrwcopy, culturv
or by animal inocnlation. Microscopy is unreliable
and the demonstration oi the typical drumstick '
'
bacilli in wounds in itself is not diagnostic of
tetanus, The bacilli may be presem in some wounds
without leranui developing, i t may not also be
possible to distinguish by microscDpv between Cl.
tcttinr and morphologically si mi I nr bacilli such as
Cl. aanosnotphum and LV. ohouicb Diagnosis
^ .
by culture is more dependable. Isolation i $ more
likeEv from excised hits ot tissue from the neurotic
r
depths of wounds than from wound swabs, The
material is inoculated on one half of a blood agar
.
plate. Cl tetwiproduces a swarming growth which
may be detected on the opposite half of the plate
after 1 -2 days incuh- utioo anaerobically . Tlie
material is also inoculated into three rubes or
et>okrd meat broth, one of which is heated to
SO cC tor lo minutes, the second tor ri miitutts,
and the thud left unhaUetL The purpose of heating
tor different periods is to kill vegetative bacteria,
while leaving undamaged tetanus sjwires, which vary
widely in heat resistance . The cooked meat tubes
urt iucubaled at 37 "C and SubcullUted On one half
ot blood agar plates daily for Uplo four Javs. Cl.
reran / may be isolated in pure culture by
subculturing from the swarming edge of the
growth. The incorporation of polymyxin li , to
which clostridiu are resistant, makes the medium
more selective.
For iide ntification and Iwatiptmirity testing , blood
'
agar plates ( with 4 agar to inhibit swarming),
^
having tetanus antitoxin (1500 units per ml ) spread
over one half of the plate are used. The Cl . tetani
strai ns are s tab- inoculated on each half of the plate,
which is then incubated anaerobically for two days.
Toxigcuic Cl. reraru strains show hemolysis around
tbe colonies, only OIL the half without the antitoxin,
l .vsih is inhibited by the antitoxin on the other half.
This* may help in identification of the culture as
Cl. tetani but is unreliable as a test of toxigenic in '
since it indicates the production only of tetaiudysin
and not necessarily of teranospastuin, which is the
pathogeniv toxin .
Toxigenkiiy is best rested in animals. A two to
four -day-old cooked men culture (0- 2 ml ) is
-
inoculated into rhe root of the rail of a mouse. A
second mouse that has received the tetanus
antitoxin ( 1000 units ) an hour earlier serves a> the
control. Symptoms develop in the test animal in
12-2-1 hours , tiegi ruling with stiffness in the tail
Rigidity proceeds to the leg on the inoculated side ,
the oppos i re- leg, tr u ok a nd forelimbs, in that order .
The animal dies within TWO days r
Inlt may be killed
r
earlier as the appearance of ascending rctanus is
diagnostic.
Prophylaxis: Tetanus is a preventable disease ,
As the spores are ubiquirons , wound contamination
is unavoidable. The disease is due To the action of
rhe toxin . Therefore the obvious and most
dependable method of prevention is to build up
antitoxic immunity bv active immunisation by
routine Immunisation of children and booster doses
where appropriate.
The nature of prophylaxis depends Largely on
rbe type of the wound and the immune status
of the patient . Tlie available methods of
prophylaxis axe (1) surgical attention; ( 2 ) antibiotics;
—
and (3) immunisation passive, active or combined.
burgical prophylaxis aims at removal of foreign
bodies , necrotic tissue and blood dots, to prevent
an anaerobic environment favourable for the
tetanus bacillus. The extent of surgical treatment
may vary from simple cleansing to radical excision,
depending on the type of wound.
Antibiotic prophylaxis aims at destroying or
inbihiting tetanus bacilli and pyogenic bacteria in
Copyrighted material
 4 Clostridium »
wcmnds so thiLt the production of toxin is prevented.
In experimentally infected animals tetanus can be
prevented by antibiotic:*- when adminis- Eered four
hours after infection but not alter eight hours . This
, Bovine and ovine ATS were introduced to
emphasises the need for prompt administration ot
antibiotics. Long- acting penicillin injection ib the
drug of choke. An alternative: is ciythrumydri 500
mg b.dL for five dap, Antibiotics are to be starred
before wound toilet* Baoifucin or neomycin may
be applied locally also. Antibiotics have no action
on the toxin . Hence , antibiotic prophylaxis does
,
not replace immunisation but serves as 3 useful
adjunct.
Passive immunisation is by injection of tetanus
antitoxin, Anti tec anus serum ( ATS ) Irom
hyperimmune horses was the preparation originally"
used . The dose employed was 1500 1LJ given
subcutaneously or inTramtiscularly in nonimmune
persons MKWI after receiving any" tetanus prgne injury
ATS was useful not only in reducing the incidence
of tetanus but also in prolonging the incubation
period and reducing the mortality when it did not
prevent the disease. However, equine ATS carried
, means whereby tetanus following unnoticed injuries
rwo disadvantages implicit in the use of any
heterologous: serum "immune elimination and 1
-
hypervciuithity. The half life of ATS in human
beings is normally about seven days bur in persons
who have had prior injections of horse serum , it is
eliminated much more quickly by combination with
pre -existing antibodies. Prior sensitisation also leads
to hypersensitivity reactions which may range from
mild local reactions To serum sickness , and even
fata] anaphylaxis. It is* therefore, obligatory that a
rest for hyperaemitiviiy should invariably be made
before administration of ATS The intraderma] tc^t
, third dose 6 months later [or as per
for hypcrsensirivicyp which m in common use -, has
been reported to be unreliable, A "trial dost given
1
subcutaneously would be a belter index of
hypersensitivity A dose of 0.5 ml of ATS is given
subcutaneously and the patient observed for at least
half an hour for general reactions . As even this
dose may precipitate anaphylaxis in some cases., a
Anion loaded with adrenaline ( 1 /1000 ) must be
261
kept ready. In persons- with i history ot any allergy,
the trial dose should be 0.05 ml of a 1/ 10 dilution
of ATS.
overcome reactions to horse serum but these in turn
nan also produce hypersensitivity.. Passive immunity
without risk of hypersensitivity can be obtained by
rhe use of human antitetanus: immunoglobulin
(TIG). This is effective in smaller doses (210 units)
and. has a longer half- life (3“ 5 weeks ). As TIG in
prepared by immunisation of human volunteers, its
n.vai I ability" is limited.
FHHVC immunisation is an emergency procedure
to be used only once. The former practice of persons
receiving ATS every time thev arc wounded was
not only useless and wasteful hut alsro positively
dangerous . It is better to eliminate the use of ATS
altogether, tetanus being controlled by active
immunisation * with human ITG being moved for
emergency use In the nonimmune.
Active immunisation is not onlv the most IF
effective nuthod of' prophylaxis bur also rhe only
can be prevented . Th : $ is achieved by spaced
injtetioivs of fanned hm>idh which is available cither
as ' plain tONo d ' , nr adsorbed nr aluminium
'
hydroxide or phosphate . 1 In adsorbed 10X 0 id is a
better antigen . The tetanus toxoid is j- iven either
alone or along wirh the diphtheria tiwcoid and the
pertussis vaccine as the ‘triple vaccine', in which
pertussis vaccine acre as art adjuvant also. A course
uf immunisation consists of three doses of tetanus
tfJXQi . 1 given intramuscularly, with an interval of
4-6 weeks between the first two injections and the
recommendations of the National ITOTTH] nisarion
Programme ). A full course of immunisation confers
: nainu - 11 ry for a pc i i.od of at least ten yean, A "booster
dose * of toKoi d i R recommended after ten years. ATS
nr TIG Rhould not be given to an immuni -ed
individual. Instead, a booster dose of toxoid i - given
if wounding occurs three years or more after the
full course cif immumsation. Too frequent injection
opy righted material
8 dr w
v>% * Textbook o! Microbiology *

of toxoid shoti [d he avoided as hypersensitivity
rcucticmii ]ruy occur occasionally.
An illustration ol' the efficacy of active
immunisation that in World War LI , onlv 12 eases
of rotaries occurred in 2 , 7J? 4 , K 1 hospital
admissions for wounds or injuries , among the
^ Tncatmen t cons isrs of ensuring quiet, control Ling
American soldiers who had been previously
immunised .
Combined immunisation consists of
administering to a rnnimmure person exposed 10
the risk of tetanus TIG injection at one site, along
with the first dose of toxoid at the contralateral
site , followed Hy the second and third. JTNKCIH of
toxoid ac monthly intervals. It is important to use
adsorbed toxoid as the immune response to plain
toxoid maybe inhibited by TIG . Ideally, combined
immunisation should be employed whenever
passive immunisation is called for.
Table 2? , 3 shows the recommended integrated
prophylaxis of tetanus following injuty.
Trealiment: Tetanus patients should he treated
in hospitals, preferably in special units . The reason
for isolating them is to protect them from noise
and light which may provoke rhe convulsions.
However , because the patients are isolated, there is
a common impression that they arc highly
infectious. This is not true. TelirtUs- patienis ate
hardly ever infectious , and person to person
transmission does not occur at all.
spasms, maintaining airway by tracheostomy with
intermittent positive pre -Saure respiration and
attention to feeding . Human TIG 10,000 LU
suitably diluted may be given by slow IV infusion,
tollowed , if needed, by another 5,000 IU later. Ever
though TIG may not neutralise the toxin already
bound to the nervous tissue, it can inactivate the
unbound toxin and any further toxin that may he
produced. Antibacterial therapy with penicillin or
metronidazole should he started at once and
ootitinued for a week or more. ATS used to he given
intravenously in massive doses as part of the
treatment . Several controlled trials have been
undertaken to assess the value of antitoxin and the
Optimum dose. The results indicate that antitoxin
is of value in treatment but that 10 ,000 1U
intravenously gives as good replies as much higher
dciSCE.

Table 20-3 Tetanus prophylaxis In (he wounded

Nature 0/ wound" forsnune statu* of fho person
Immune Partially immune Nonimmune
Clean (wound toilet performed within six hours ) Toxoid * 1 * Toxftid * 1 Toxoid * 3-
Contaminated {mil or other foreign or
Mere tic material present ) Toxoid * l * Toxoid x 1 Toxoid x J
TIG TIC
antibiotic* antibiotics
Infected Toxoid K 1* Toxoid * I Toxoid * 3
Antibiotic? TIG TIG
antibiotics antibiotics

Mote : Immune - Patient has had a full course of three injections of toxoid.
Fortuity immune
- - Patient has had iwy injections of toxoid.
.
NorLi. n : rmint: Patient bjs had on** cr m: irtjKtidit of fcHtiwd , (if irtirtULnisarion status is rutlotown.
TIC - Tpianui Immune Globulin .
* The toxoid needs to be pwen only if three years or mure have elapsed after active immunisation or

the last booster injection .

Copyrighted materia

- Clostridium *
Patients recovering from tetanus should receive
u fiill course of active immunisation , as an attack of
the disease docs not confer immunity. Second
attacks of tetanus have been recorded.
CLOSTRIDIUM BOTULINUM
Cl. fjoniffnuru causes botulism, a paralytic disease
usually presenting as a form of food poisoning
The name botulism is derived from LH a usage"
( hofu/ us, Latin for sausage ) formerly associated with
this type of food poisoning. C7. botufoiuin was first
isolated by van Ermertgem ( 1896 ) from a piece of
ham that caused an outbreak of botulism . The
bacillus LS a widely distributed saprophyte, occurring
.
in vitgin sojln vegetables, hay silage, animal manure
and sea mud.
Morphology : It is a Gr ^ m positive bacillus
about 3 p x 1 Jim , noncapsulated , motile by
.
pcritrkhiBe flagelL producing subierminal,, oval,
bulging spores .
Cultural characteristic!: It is a strict
anaerobe. Optimum temperature is 35 but some
strains may grow even at 1-5 °C . Good growth
occurs on ordinary media . Surface colonics arc large,
irregular, semitransparent, with fimbriate border.
Biochemical rcactionsvaiyin different types, Spores
are produced consistently when grown in alkaline
glucose gelatin media at 20-25 ^ C. They art not
usually produced at higher tcmper^ turcs-
Resistance: Spores are heat and radiation
resistant, surviving several hours at 100 C and for
upto 10 minutes at 120 ^ C. Spores of nonproieolvtic
types of B , E and F are much less resistant to heat .
.
Classification: Eight types of Cl botulinum
.
have been identified (Types A* 13, Cl C2 , D, E, E,
G ) based on the immunological difference in the
toxins produced by [Item. The toxins produced by
the different types arc identical in their
pharmacological activity but arc neutralised only
by the homologous antiserum. An exception is C 2
toxin , which shown cntcmtoxLC activity, while all
the others are neurotoxins .
Toxin: C7, botulinum produces a powerful exotoxin
I
263
that is responsible for its pathogenicity. The toxin
differs from other exotoxins in that it is not
released during the life of [ lie organism . It is
produced mtTacellnlarlv and appear; in the medium
only on the death and autolysis of the cell . It is
believed to be synthesised initially as a nontoxic
protoxin or progenitor toxin. Trypsin and other
. proteolytic enzymes activate progenitor toxin to
active Toxin .
The toxin has been isolated as a pure crystalline
protein which is probably the most toxic substance
known lr has a MW 70.000 and a lethal dose for
,
mice of 0,000,000,033 mg. The lethal dose for
human beings is probably 1-2 |ig. It is a neurotoxin
and acts slowly, taking several hours bo kill.
—
The toxin is relatively stable, being inactivated
only after 30 40 minutes at SO and 10 minutes
at 100 ° G. Food suspected to be contaminated with
botulinum toxin can be rendered completely safe
bv pressure cooking nr boiling for 20 minutes- It
resists digestion and is absorbed through the small
intestines in active form. It acts bv blocking the
production or release of acetylcholine at the synapses
and neuromuscular junctions . The onset is marked
by diplopia, dvspllagia and dysarthria due to cranial
nerve involvement - A symmetric descending
paralysis is the characteristic pattern, ending in
death by respiratory paiuhnis.
A small quantity of Cl- botulinum type A toxin
injected into a muscle selectively weakens it by
blocking the release of acetylcholine at the
neuromuscular junction. Muscles so injected atrophy
—
hut recover in 2 4 months as new terminal axon
sprouts form and restore transmission .
Intramuscular injection of the toxin, first used to
treat strabismus , is now recognised as a safe and
effective symptomatic therapy for many
neuromuscular diseases.
The botulinum toxin can he toixoided . It is
specifically neutralised by its antitoxin and is a good
antigen . The- toxins produced by the different types
a
of botulinum appear to be identical , except for
immunological differences . Toxin production
Copyrighted material
 264
1
appears to be dcremaned by the presence of
bacteriophages, at least ir types C arid D.
Pathogenicity: O. ht Ir :JJ' IV ? -: JM is noninvasive and
virtually nonrtfectinus, Its pathogenicity is -due to
the acrion of its toxin , the manifestations of which
arc collectively called botulism , Botulism is of three
-
types foodborne botulism, wound botulism and
infant botulism.
FfMxJhttrnc botulism in due t[ j tht
indention ' if
preformed toxin The types ot the bacillus and the
. svndrome
nature of rbe food responsible var\ in different
regions. Human disease is usually caused by type*
A, B, E and very rarely F. Types C and D arc usually
associated with outbreaks in eatde and wild fowl .
Type C has been assoc iated with sudden death in a
—
few patients. The source of botulism is usually
preserved lotaJ meat and meat products in Europe,
canned vegetables in America and fish in ^ apan.
Type !; is iHsociatcd with fish and other seafoods-
.
Proteolylic varieties of Cl bondmuzn can digest
food, which then appears spoiled . The cans are
often inflated and show bubbles on opening.
Nonprorcolytic varieties leave food unehanged.
-
Symptoms begin usually 12 36 hours after
ingestion of food. Vomiring, thirst , consr - pahon,
ocular paresi >, difficulty in swallowing, speaking
anti brtathing constitute the common fcatuneFC Coma
or delirium may supervene . Death is due to
respiratory f nlure and incurs 1-2 days after onset.
Case fatality varies from 25— 70 per cent.
Wound bofu /rsm is a very rare condition
resulting from wound infection with C7. fwtuirmtm.
[ ; : xicl i & produced at the site of infection and is
absorbed. The symptoms are those of foodbome
botulism except tor the gaNtnunteNtiiLxl components
which are absent. Type A has been responsible for
most of the cases studied .
-
Infant botulism was recogm ed as a clinical
entity in 1976. This is a toxico-infection. C’J.
bomlirtutn spores arc ingested in food , get
established in the gut and there produce the to \ i.n .
Cases occur :n infants helow six months. Older
children and adults are not susceptible . The
Of WiCfObiQ
^y *
manifestations are constipar .on, poor feeding,
Icthaigy, weakness, pooled oral secretions, weak or
altered cry, fioppiness and loss of head control.
is not generally demonstrable in blood .
-
Pati i;nt& excrete t n and spores i n th i : r feces. Toxin
Management consists of supportive care and asnstml
feedingi - Am itoxiita and antibiotics ins not inch cared.
Degrees of severity vary horn very mi lid illness to
fatal disease. Some cases u1 sudden infant death
.
1
have been found to be due to infant
botulism. Honey has been incriminated as a likely
food item through which the hacillus enters the
Laboratory diagnosis: Diagnosis may be
ennfirmed by demonstration of the bacillus or the
toxin In food or feces. Gram positive spuring bacilli
may be demonstrable in smears made from the (bod .
Cf tafvhnuFTT mav be isolated from the food or
the patients feces. The food is macerared in sterile
.
sisJir&e, and the fi.lrrite inoculated info mice or guinea
pigs inCraperLtoneally. Control animals protected bv
polyvalent mtiioxiti remain healthy. Typing it done
by passive protection with type- specific antitoxin .
The toxin may occasionally be demonstrable in the
patients blood, or in the liver postmortem.
A retrospective diagnosis may be made hy
detection ofnttkoDon in the patients scrum but it
may not be seen in. all cases.
Control ? As most cases of botulism follow
consumption of inadequately canned or preserved
tood , control can be achieved by proper canning
and preservation. When an outbreak occurs, a
prophylactic dose of antitoxin should be given
Lntnmutmkffy to all who consumed the article of
food.
Active immunisation has been shown to be
effective . It immunisation is needed, as in laboratory
workers exposed to the risk, two injections of
aluminium sulphate adsorbed toxoid may be givers
at an interval of ten weeks, followed by a booster a
year later Antitoxin may be cried for treatment.
Polyvalcnt antiserum to types A, B and E may be
administered as soon as a clinical diagnosis is madcr
L> opyrighted materia
 * Clostridium * 265

Supportive therapy with rn^irtTETianOt Cit ropilflti<Wl
is of equal or greater importance-
CLOSTfllDlUM DIFFICILE AND ANTIBIOTIC
ASSOCIATED COLITIS
CL ( Hfficik wus first isolated in ly.i.S frym the fcL es'
of newborn infant*- . It was HO named because of the
unusual difficulty in isolating it. It is a long, skndcr
C rrajn positive h .uillus with a pronounced tendency
to lose its Gram reaction . Spores are large , oval
and terminal. It is mm hemolytic, sticcbarolytic and
weakly proteolytic , It was not considered
antibiotic therapy. Many antibiotics have been
incriminated including ampicillin, tetracycline and
chloramphenicol but linaunYviii ml cfodamyrin arc
puiticuliiily prone to cause pseiLtlnmerribFjnous colitis.
It has now been shown that antibiotic associated
colitis is due to the active mu triplication of Q
dtfBa'fe and lies production of an cnteioroxin as well
as a cytotoxm . Diagnosis can be made by
demonstrating the toxin in the feces of patients by
itscharacteristic effect on Hcp-2 and human diploid
cell cultures nit by ELISA . The toxin is specifically
neutralised bv the C7, sordelii antitoxin. CL difficile
X

pathogenic till iy ?7 when it was found ro he
responsible tor antibiotic nissociaccd colitis.
Acute colitis, with ur without me mb t .me
formation , is an important complication of oral
can aJso be prown Jrom the teces o-f patients.
CL difficile strains are usually sncsista.nl to most
antibiotics Metronidazole is the dru ot choice .
*

Vancomycin and bacitracin arc also useful. ^

Further Keiidiin
Bartlett JG . 1994. Clostridium difficile . Clin Infect Dis, 18;S265 ..
^
Brazier JS. 1995. Laboratory diagnosis of CL difficile associated disease . Rev Med AHcrohial fr:236.
L uerdcn BI and BS DraBar {ede ) - 199 L Amkansbes jji Airman disease. London; Arnold
^ .

Hathava CL. 1995 Botulism . Cun Topics Mkrohiol Immunol , 195:55 .

»
'

Sakurai J» 1995. Toxins of Cl, PcriringjeRs* Rev Med Microtia\L 6:175

. »

Sanford JP. 1995 . Tetanus - forgotten, bur not pone . Sew Entf J Med , 332:812,

Copyrighted material
 o> Nonsporing Anaerobes
CM

Anaerobic bacteria have been known since the T . Cocci

original observation of Pasteur that bacteria which A. Gram positive
produce butyric acid, his Vihriiin bulrriyut:, were a. Fepiostrtptococcus
rendered nonmotilc on exposure to air (1B63), b. I ptococcus
Though many anaerobic bacteria may be ^
B. Gram negative
pathogenk fur human beings, [ hey are generally Veillonelli
neglected in diagnostic laboratories, This, neglect IL Bacilli
is not because they .
arc uncommon Indeed , they 1. Endospore forming
outnumber aerobic bacteria in many habitats , Clostridia
including most sites of the human or animal 2. Nonsporing
hndy. Even in such seemingly aerobic situations A. Gram p^ tsitivc
as the month and the skin , anaerobic bacteria ate a. Eubaeterium
ten to thirty times more frequent than aerobes. In b. Propiorubacterium
the human intestines, they outnumber aerobic c. Lactnharillus
bacteria a thousandfold . ' [ he numbers of anaerobes d. Mohiluncus
'

-
present have been estimated to be 10* llGVml in
the small intestine. ItfVm! in saliva and 10 IL /g in
e. Bifidobacterium
f Aetirtomyeeii
the colon - B. Gram negative
Afuerobk bacteria differ widely in the degree a. Haeteroida:
of anaerobiosis required for their growth, borne b. Prevotcllla
species fail to grow if the atmosphere contains as c . Pbrphyromonas
little as 0.03 per cent oxygen, while at the other d. Fusobacterium
extreme , some arc aerotolerant and may grow e. Lcptotrichia
sparsely on [ Ire surface of aerobic places . III . Spirochetes
Consequently the techniques employed for the a - Treponema
propagation and study of anaerobes vary in b. Borrelia
complexity. Besides the medically important species listed
Eatly methods of classification used such above, there ate several anaerobes that occur in soil
unstable criteria as cell and colony morphology, and water and which may be of industrial and
biochemical reactions and antibiotic sensitivity agricultural importance (for example ,
patterns. Current (dassifLcatitm is based on DNA methamobacterLa , butyrivibrios).
base composition and analysis pf the fatty arid end
products of metabolism . Medically important ANAEROBIC COCCI
anaerobes may be broadly classified as fotlowsr ANAEROBIC cocci represent a heterogeneous

Copyrighted material
 s Nonsporipg Anaerobes
collection of cocci whose classification and
no menu l a Lure have undergone several
modifications. J hev can he divided into Gram
' '
P
posir :.vc and Gram negative groups.
Anacrnbic Gram positive cocci had been
classified into the genera Pcptostrcptococcus and
JVptococcus ori inally, based on morphology
^
chain- forming and paired cocci placed in the
former and duster-forming cocci in the latter .
However, DNA base ratio studies have kd to most
of the species formerly considered as peprococci
being reclassified as peptostreptococci . They are
cocci of small size ( 0.2-2.5 pm ). Many of them
are aejotoLerant and grow well under HWfc CO . in
an aerobic atmosphere.
Thev are normal inhabitants of the vagina,
intestines and mouth . They may cause several
clinical infections such as puerperal sepsis and other
genital infections , wound infections, gangrenous
appendicii ]^ , urinary tract IIIFECLI : ms, osteomyelitis
and abscesses in the bra m, lungs and other internal
organs. They arc often seen in large numbers in
pus from suppurative lesions, so a Gram stained
smear may be helpful in diagnosis. Infections are
usually mixed, the cocci being present along « uh
clostridia or anaerobic Gram negative bacilli
ftptoirreprococcus anaerub/ ejs is most often
responsible for puerperal sepsk and JV-
for abscesses . i\t . asacchajolvticus. Fst. tetrad) us
and Put- prevoti arc some other species- commonly
present in clinical specimens .
Vcilloncllae are Gram negative cocci of varying
s i res occurri ng as d i ploeocci , short chains Or groups.
They arc normal inhabitants of the mouth, intestinal
and geuiTil tracts. Veillunella psrvuii has been
reported from clinical specieftens hut LK pathogenic
role is uncertain .
All anaerobic cocei are generally sensitive to
penicillin, chloramphenicol and metronidazole, and
resistant to streptomycin .md gentamicin.
ANABRQEIC GUAM POSITIVE BACILLI
This group contains many genera , of which the
2fi 7
medically relevant are £’ u bacterium ,
Propion j'bacferju m „ Luctobacillvs , Mnkiluntus and
Bifitlubacteirum. Other genera m thi -s group ,
Acn nornvcei
'
tr - and Arachnid, are dealt with
J
, elsewhere .
Members of the genus Eubaclrnum are SI nClly
anaerobic and grow very slowly. They are members
of the normal mouth and intestinal flora . Some
species ( £. bnachy, E, rim , Jam, E. nods turn ) are
commonly seen in periudontiris. E. kntuni Is
commonly isolated From notional clinical spc< imens-
AupiAnikrrMuai is constantly present on the
skin. E acncs is a common contaminant in blood
and CSF cultures.
Lartobarjlius is present in the mouth , intestines
and , typically, in the adult vagina ( Dodcrlcirfs
bacilli ). It is generally nonpathogemC, though l..
catcnaforwc has been associated with
bronchopulmonary infections .
iJjfldobacreriu /ti is a pleomorphic rod that
shows true and false branch i ng. It is presem i n large
numbers in the intestines and in the mouth.
Mobiluncus species ate motile , curved,
anaerobic bacilli that may appear as Gram variahle
.
rods, M. muiieris and M curtisii have been isolated
, from the vagina in bacterial vaginosis, along with
GardncrelJa vtginulis. .Bactcri .i ] vaginosis is a
polymicrobial infection characterised by a thin
malodorous vaginal discharge. Its ' rotten fish ' smell
is accentuated by mining :i with a drop of KOH
solution. The vaginal pH LS leas than 4.5 . Clue cells
(cpitlvcli .L1 cells with surface covered by adherent
bacilli ) are seen in stained or unstained films.
AN AEROBIC GRAM NEGATIVE
BACILLI
Medically important anaerobic Gram neganve
bacilli belong ro the family ihrdtnokfecieae and are
classified into the genera iiacrernideff ,
Fusobacterium and Lepfomdria,
-
ti ;Titc njuit\< are the most comment anaerobes
isolated from clinical specimens. They are
nonsporing, non motile, strict anaerobes, 11sually very
opy righted material
m i Textbook of Microbiology *
|IIL" < ' n '
- i
.
: K , appearing iiH \ 111 i rods, I r iik Ling
forms or ooceobaeilii, seen F- iogly, in pair * or in
short chains - They grow well or media such as
brain heart infusion agar in an anaerobic atmosphere
containing 1094 CO , . They possess capsular
polysaccharides which appear to He virulence
factors, and antibodies to them can he detected in
patients . Thev are normal inhabitants of the
intestinal, respiratory and female genial tracts-
Bactcroides species have been classified based
on their u ace ha roly tic effects. Asiceharoly nc
pigmented specicH have been separated IS the genus
lsii|
_
ph>rnomcinasn containing Pgingivalis reRp u > il le
for periodontal disease , ft cndodontulis causing
denta ] root canal infections and other species.
Moderately Hacchaioiytic speties inliibiTed by 20%
bile are placed in the genus Prcvotella. containing
F. mehninogetiica, F buccafis, F. dentieok and
Others. The genus TtatrernideR proper now includes
the important species ft- foigifts, and others Ruch
as fl , vutfiaitit , B . chs ra si on j( and B.
theta / otaom.vron.
ft . fragi / iR is the most frequent of the
nonsporing anaerobes isolated from clinical
specimens. It is often recovered from blood,
pleural and peritoneal fluids, CSF, brain abscesses,
wounds and urogen ::al infections - P
mtkimiotfemci is easy to recognise because of
the black or brown colour of the colonies.. The
colour is not due to the melanin pigment as was
once thought but to a hem - it derivative, It has been
- _
i IJL .H > . I from various infections luludimt lung or
( ,
-
liver abscess, mastoiditis , intestinal le mns and
lesions of the mouth and gums. Cultures of ft
jne/ jmrto enK'a and even dressings from wounds
^
infected with the bacillus give a characteristic red
fluorescence when exposed to ultraviolet light.
The genus FusofwcfejTum contains fong, thin
or spindle shaped bacilli with pointed ends . E
nurfeatum is a normal inhabitant of the mouth and
is found in oral infection and pleuropulmonary
sepsis . F, rtcei'ophtJfMim produces a wide range of
exotoAins and has been responsible lot liver abscess
and other abdominal infectious in animals and less
often in humans .
The genus Leplulr ichia. Contains the SLftgle
species, E. fujccairs which was formerly known as
Vincent 's fusiform bacillus or Fusobacferforn
.HLRIFTI n i .' L . FI L' V are long, straight or slightly curved
1
nods, often with pointed ends. They are part of the
normal oral flora and arc seen in acure necroticng
le ^ iMftR in the mouth.
-
A common condi t ion i Vincent 's angi:ia, wli i ch
may resemble diphtheria , with the inflamed
pharyngeal mucosa showing a greyish membrane
which peeb easily. Stained aments show large
fusildrm and spiral bacilli .
ANAEROBIC INFECTIONS
There has been a reawakening of interest m
anaerobic infections during recent years . This is
due to the availability of improved and simplified
techniques for the isolation and identification of
anaerobes.
Anaerobe infections are usually endogenous and
are caused by tissue invasion by bacteria normally
resident Oft the respect ive body surfaces. Anaerobic
bacteria arc normally present on the skin, mouth,
nasopharynx and upper respiratory tract, intestines
and vagina {Table 29.1) . Anaerobic infections
generally follow some precipitating factor such as
trauma , tissue necrosis , impaired circulation ,
hematoma formation or the presence of foreign
bodies . Diabetes, malnutrition, malignancy or
prolonged treatment w i l h aminoglycoside
antibiotics may act as predisposing factors .
Anaerobic infections are typically polymicrobial,
mote than one anaerobe being responsible besides
aerobic bacteria. While the infection is usually
localised, general ms- semi nation may occur bv
bacrerem ::i . Tabic 29.2 lists the common sines and
type of anaerobe infections and the bacteria
responsible.
Iherc are some clinical features which suggest
the presence of anaerobie infection. Pus produced
by anaerobes is characteristically putrid, with a
Copyrighted materia
 a Non &poting Anaerobes
Table 29,1 N&nnal anaerobic Hare ri the human body
Anaerofcf
CIosmdiujYi
Siin —
JVfou th njtsophMiynx
Actinomyces
Bifidobacterium
lJropionibacterium
ff&yvnoidcs trwg&i
R inchninagenrcu
FuBob&c Ecrin m
Gram positive cocci . -+
Gram negative cocci
Spirochetes
pcriltivc, nauseating odour. However, there roav
he exceptions; infections solely due ro B tragi!is
may he free of this imelL Pronounced cellulitis is a
common feature of anaerobic wound infections;.
Toxemia and fever are not marked.
Lrtbornlory diagnosis: As anaerobes form
pan of the normal flora of the skin atid mucous
surfaces, their isolation from specimens has lii be
interpreted cautiously. The mere presence of an
anaerobe does not prove 1 causal role - Specimens
should be collected in such a manner as to avoid
resident flora . For example , the sputum is
unsatisfactory' for culture from a suspected case of
lung abscess; only material collected by aspiration
would be acceptable.
As some anaerobes die on exposure to oxygen,
earn should be exercised to minimise contact with
air during collection , transport ami handling of
specimens. A satisfactory method of collection is
ro aspirate the specimen into an airtight syringe,
plunge the needle into a sterile robber cork to seal
it and send ir immediately to the laboratory. E\is
and other fluids may be collected in small bottles
with airt ight caps and transported qu ickly, cn suri ng
that the specimens fill the bottles completely, bwabs
are generally unsatisfactory hut where they arc to
^ 269
Intestine ViginM
+4
* >
4 +4 +
44
4 4 4 +4
4 4 4
1 4 4- +4
+4 4 -
4 4
4
be used , they should be sent in Stuart’s transport
medium -
ln the laboratory, exposure should be Limited
to the minimum. Examination oh a Cram stained
smear is useful. Pus in anaerobic infection usually
shows a large variety of different organisms and
numerous pus cells . Rarely, as in brain abscess, H
single type of organism alone may he seen .
Examination of the specimen under ultraviolet
light may show the bright red fluorescence of
"
R mehninngcniai. ( las liquid chromatography of
the specimen rriav vie Id presumptive information
on the types of anaerobes present .
Several special media have been described for
anaerobes but for routine diagnostic work, freEblv
prepared blood agar with neomycin , yeast extract,
heroin and vitamin K is adequate . Plates arc
incubated at n 7 ' CT in an anaerobic jar , with 10%
CO ,. 'Ehc Caspak system provides a convenient
method nf routine anaerobic cultures . Plates are
examined after 24 or hours . Some anaerobes, inch
:LS fusobucteria , require longer periods of incubation.
Raralld aerobic cultures should alwavTi be set up. This
is ncccsaxy as a control for the growth on anaerobic
plates and also because in most anaerobic infections
aerobic bacteria are also invoked.
^opy righted material
270 * Textbook of Microbiology *
Table 29.2 Common anaerobic infections and Ihe bacteria responsible

Site siiidf type of infection flstterjj commonly rcsponeible

Centra} nttrvom system
Brain abscest B. fmgiiis; f
BPFOJtrcptocoCCm
Earp fKVdi riiroafc
Chronic sinusitis, otitis media ,
mutoiditia, orbital cellulitis Fusobacteria ( aerobes frequently responsible}
AJoufft and / aw
Ulcerative gingivitis ( Vincent a ) spirochetes
Fusobacteriap
Dentil abscess, cellulitis; Mouth anaerobes,
Abscess and sinus of jaw Actinomyces, other mouth anaerobes.
Respiratory:
Aspiration pneumonia, Lung abscess, Fusobacteria, /? mdanine nic*,
bronchiectasis , empyema ^
anaerobic cocci; B. frMgilis rarely
Abdominal:
SubphrcniCj hepatic abscess '

appendicitis, peritonitis;
ischiorectal abscess; , JJ. fragiUs
wound infection after
cnlorecraJ surgery
Femaie genitalia:
Wound! infection Mowing
genital surgery;
Puerperal sepsis; P mehminogemca ,
tubo-ovarian abscess, anaerobic cocci; K fragitis
Bartholin's abscess,
septic abortion Genital aruuernbes and Cl perfringens
Sirin and raft timm
Infected sebacious cyst. Anaerobic cocci
Brcast abscess, axillary abscess Anaerobic cocci; jRme /aninogertj-ca ( Staph , sureu#
' '

commonest cause}
Cdlulitkj. diabetic ulcer , gangrene B. rragjJjs and others.
'

Further Rending
Duerden DL and BS Dnumr ( eds ), 1991. Ajisfcirohttr in Hiimitn Disease- London: Arnold.
,

Finenld M . 1995.. Overview of clinically important anaerobes. Clin Jm/ect Dis. 30: S2QS,
KiiMf DL. 1993. InJfeetLoisa due in mixed anawebie organisms. In Hamsons Principles of Internal Modidnc^ 14 edn. New
H

York MdCnw Hill,

Copyrighted material
 8 Enterobacteriaceae I: Conforms - Proteus
The predominant aerobic bacterial flora of tht jjr.ee
'
-
111 r L i mes of human be i ngs .11 nl animals is composed
uf nonspuring, noma .- bl fast , (. iram nej lLve bacilli
rhry exhibit general morphalugi cal and ^ ,
biochemical similarities and me grouped together
in the large and complex family Enrero'iMCfcrjJce*?.
Members of this family may or may nor be
capsulared and are morile by peritrichale flagella *
nr ate non motile - They arc acmh is - and facultatively
anaerobic, grow readily in ordinary' media, ferment
glucose producing avid and gas or acid only, reduce
nitrates to nitrites and form catalase but not oxidase.
Within the family * they exhibit very wide
biochemical and antigenic heterogeneity. T ltougb
the family i ^ s ibdivided into groups or tribes, genera,
subgenera , species and types* many strains are met
with that possess every conceivable combination
of characters and do not fall into any such arbitrary
taxonomic category . The frequency of genetic
mechanisms fluch as conjugation and transduction
in these bacteria contributes to their infinite variety .
Classification of these bacteria into well demarcated
compartments, though necessary for their systematic
study would therefore be artificial . The
*
-
classification of Entcmhacti riaceae has hecn
controversial and there hive been successive changes
in their grouping and nomendaturc.
The oldest method was to class ' ty these bacteria
into three groups based on their action on lactose.
T. Lactose fermenters (for example
Kschcrichia,
Klebsiella )
II . Late Lactose fermenters (for example
Shigella sonnei
Paracolons )
‘ 1
III . NortlactoSe fermenters (fur example
Salmonella ,
Shigella )
This method of classification was derived from
the use of lactose in MACCDHKEY's medium the ,
most popular medium for the isolation uf fecal
bacilli . Though taxononucally unacceptable, this
scheme had practical value in diagnostic
bacteriology. The majority of the commensal
intestinal bacilli arc lactose fermenting ( LF). As
the most common member of this group Is the colon
bacillus* or Escherichia coh. all lactose fermerumg,
enteric bacilli were called coliform bacilli . The
major intestinal pathogens, Salmonella and Shu^rl/a
are nonlactose fermenters ( NLF), and hence readily
detectahlr by the colourless colonies they form on
MacConkey's medium. There remained a small
group which showed delayed fermentation of
I .iceo^' and with the creep t ion o t Shigella sonnei,
they were all commcnsLta. This heterogenous group
of late lactose fermenters was called panaco/on
bacilli.
Classification based on a single property, such
as lactose fermentation ., is contrary to modern
taxunoimdal concepts. The current practice is to
group together bacteria that possess a number of
common morphological and biochemical
properties, and similar DNA base compositions,
While the three widely used systems for the
classification of Entewhacteriaceac ( Bergtys
manual* Kauffmann, Edwards-Ewing) have certain
differences, the general approach is the same. The
family is fust classified into its major subdivision -
Copyrighted materia
272 i TgxIbOOk 01 Mizrahitjlogy *

group or
-
h trill* cnn iisth gf One or MOT*
tribe, Rjs
genera and each genus one or more suhgenen and
ftQgUfnnrV, F.. hcrmznii and F.. whOUtthkh have
been isolated infrequently from clinical specimens ,

species.. The species arc classified into types

biotypes, serOtvpeS, bacteriophage types, colicin
— if . Martae found in rhe gut of cockroaches is
biochemically different in being Judole and beta -
typei, galattosidasc negative. It has not beer isolated from
clinical specimens..
EHTEfiOBACTEfllACEAE Unlike other cnli forms, E . coii is a parasite living
Tribe h Eschi- richiae only in the human Of animal intestine . Voided in
Genus 1 . Escherichia feces, it remains viable in the environment only for
2 , Edwardsiclla some days. Detection of El coii in drinking water,
j . Citrobiicter therefore, is taken as evidence of recent pollution
Salmonella
4. with human or animal frees.
5. SEugelk Morphology: E. coii is a Gram negative,
Tribe 11: Klcbsitlleac vtraightj rod measuring 1“ 3 * 0.4-0.7 pm arranged
Genus 1. Klebsiella singly or in pan* h is motile by periuichate flagL-lhi ,
,

2 . Enterubacter though some strains may he nonmotilr. Capsules

1 Hafhia
Serratia
4,
Tribe HI - Pfocecac
Genus ] . Proteus
2. Horganclla
d . Frovidencia
Tribe IV: Erwuiieae
Genus 1 . Envinta
Tlie genus Vfff.sjnJi, including [ he pUgUc
bacillus , has been placed in rite family
EnrrrabflCivrjatiCac but because of the special
importance of plague, the major disease caused by
yerslniue md ire lick of sinukrily to enteric disease ,
ir is dealt with separately.
ESCHERICHIA CQLI
This genus is named after Escherich who was the
first to describe tbr colon bacillus under rhe name
Bacterium coii commune (!Sft 5 ). based on minor
differences ][] biochemical characteristics, colon
bacilli were described under various names but ir
view of the nr. J rabilitv of the biiKhcm ical pnoperries
in this group, they have all been inducted in one
species Enllrridiia coii which is further
subdivided into biotypts and serotypes. A few other
species have been described in the mentis bur they
are of little medical importance. These include E.
nod fimbriae ar* found in smne strains - Spores arc
nor formed.
Cull oral characteristics: h is an aerobe and
L facultative anaerobe . The teiii|ierature range is
1CM0 aC {optimum 37 aC), Good growth occurs
on Ordinary media . Colonics are large, thick , greyish
white , rtMsi. smooth oriaquc or pinially translucen [
discs. This description applies to the smooth ( S )
torm seen on tresh isolation , which is easily
cmulsitiable in saline. I lie rough { RJ forma give
rise to colonies with , m irregular dull surface and
sre often autoagglutiaible in saline . The S R
variation occurs .is a result of repeated subcultures
-
and is associated with the loss of surface antigens
and usually of virulence. Manv pathogenic isolates
have polysaccharide capsules. Some strains may
occur in the him cold' form.
Many strains, especially those isolated fro in
pathologic conditions, are hemolytic on blood ag-an
On MacConkejfs medium, colonies art bright pink
due to laetoM fermentation . Growth is largely
inhibited cm selective media such as DC A, or SS
igar used for rhe isolation of salmonellie and
shigellae . In broth , growth occurs as general
turbidity and n heavy deposit, which disperses
completely on shaking.
Biochemical reactions: Glucose , lactose .

Copyrighted material
 4 Erigrobacienacoae lr
nnir I , maltose and many other sugars arc
fermented with the production M acid jnd gSt$,
Typical strains do not ferment sucrose. The trior
biochemical tests widely employed in rite
classification of enterobacteria are tile indole ,
methyl red ( MR ) t Voges-Froskauer ( VP ) and
citrate utilisation test's, generally referred to hy the
.
mnemonic ' IMViC , E coli ! H indole and MR
positive, and VP and citrate negative (IMViC + 4
). Gelatin is not liquified * H ,S is not formed,
unci is not split and growth does not occur in KCN
medium.
Antigenic stcwCturtj berotyping of E coli is . Fimbriae also promote virulence. 'Tire common
based on three antigens - the somatic antigen O,
the capsular antigen K and the flagellar antigen H .
So tar some 170 tvpes ofO antigens, IDO K antigens
acid 75 11 antigens have been recognised . The
antigenic pattern oi a strain is recorded as the
number of the particular antigen it carries., as for
example 0111 : K5S: H2-
Tlic K antigen is the acidic polysaccharide
antigen located in tile ‘envelope’ or ToicTCHiapsute.
( K for Kup^ cl, German for capsule ). It endows the
O antigen and renders the strain insgglunnable by
the O antiserum . It may also Contribute to virulence
by inhibiting phagocytosis . Formerly K antigens
were subdivided into three kinds - the tlienookIdle
L antigens, the thermostable A antigens ami the K
antigens found on enteropithogenic strains
associated with infantile diarrhea. Later it was
shown that the B antigen was not a separate entity'.
K intigens are therefore currently classitied into
two groups , 1 and 1!, generally corresponding to
the former A and L antigens.
Several different serotypes ot E. ctili are found
in the normal intestine Most of them do not have
K antigens . The normal colon strains belong to the
'early ' O groups (1, 2 , 3 , 4 etc . ) , while the
entcropathogenic strains belong to the ’ later ' O
groups ( 26, 55, 86, 111 , etc}.
Virukrtue factors: Two types of virulence
antigens .ind toxins.
—
factors have beer recognised in E- coli surface
Colilorma - Proteus 273
The somatic lipopolysiicchiridc surface Q
antigen, besides exerting crtdiUtode activity, also
protects the bacillus from phagocytosis and the
bactericidal effects of complement The envelope
or K antigens also afford protection against
phagocytosis and antibacterial factors in normal
serum, though it is not effective in the presence id
antibody to Q or R antigen . Most strains iff E. coii
responsible for neomtil meningitis and septicemia
carry the Kl envelope antigen which is a virulence
factor resembling the group B antigen ot
meningococci.
fimbriae seen in most enterobacteria , which arc
chtomosnmally determined , present in large
numbers and causing mannose sensitive
hemugglulination, in probably not relevant in
pathogenesis. A different kind of fimbriae which
are plasmid coded, found only in send! number -'
and mediating mannose resistant hemagglutinins
have been shown to act as virulence factors. Some
oi them may no? occur as morphologically separate
structures but only as surface antigens - tor example,
K 33 and K 99 antigens in strains causing diarrhea
in animals, or the Eoloniaxiion Fattur Aotjjgem
.
( CFA ) in enterotoxigenic E coli causing human
diarrhea. Fimbriae arc also nfimporrarice in urinary
tract infection , as for example the P fimbria which
binds specifically ro the F blood group substance
or human erythrocytes and uiocpitheliid cells.
Er coli produce two kinds of ercotoxina -
Aeznofyiov and enteranudns. Hemolysins do not
appear to be relevant in pathogenesis though they
arc produced more commonly by virulent strains
tli .LI 1 by svirulentnnidt Fflterotauuiih are inifjortanl
in the pathogenesis of diarrhea. I'hrce distinct types
of E. coli cntcrotoMins have been identified - lie ^ f
labile toxin ( LT ) , lie at stable toxin ( ST) and
verotox. in ( VT ) also kn0wn an Shiga-like toxin
( SLT).
.
Thc E coii cnterotoKin LT was discovered in
1956 by De and Colleagues in isolates from adult
di .L’ ihea ciL'- cii In Calcutta, bv the rabbit ileal Loop
Copyrighted material
 274 < T& x.tCiook of Mcrobicnogy

Table 30 1 Enterabacteriacefie ; Important di & tsng rushing leaturEs ol ihe dillerenl genera

I i 1
'
a
| 1a I|
*
I s I
G
45
LU * w I l £
— —
Motility 4 4 —
»
4 4 4 4 4 4 + 4
Gas from glucose- + * 4 + 4 + + d d + +
Acid from lactose 4 4 — “ 4 4
Add from sucrose d d 4 4 d ^ d
Growth in KCN - + d - + 4 + 4 4 4 4
Indole + 4 d - d - d + 4

MR + 4 -
1 f f “ 4 + +
VP + 4 +
Citrate
H2S
Urease
Phenylalanine
+
+
-
1
4 -
— 4
+
4
4

d
4 4 d.
+
4
d

+
d

d
deaminase ( PPA )
Arginine
- — — - “ 4 + 4

dehydro! ase d d + - d .

Lvsine
Jr

decarboxylase
Ornithine
+ + — + - d d 4 4 - - -
decarboxylase d + d t d - 4 4 4 d + -
(d
-. pewits
different in different specie* nr strain )- )
Jinporunr exceptions:
lS cyphi do< ¥ not produce g» from sugm,
’Sh . sonncL ferments Eactose and sucrose lat-c

method which they lud earlier used for identifying leading to increased outflow of water and
the cholera enremtnxin ( CD , vtr injection of E.
% electrolytes into the gut lumen, with consequent
entf culture filtrates into dosed ligated loops of rabbit diarrhea. Though the mechanism of action of LT
ileum induced outpouring of fluid and ballooning and CT is the same „ she Latter is about a hundred
of the ]oops, E, toll IT rrsemMc & the chotera tostin times more potent than the former. LT is a powerful
, .

in its structure , antigenie propetficn and mode of antigen and can therefore,, be detected by a number
,

action. It is A complex of polypeptide subunits - of serological as well as biological tests (Table

each unit of the toxin consisting of one subunit A
( A for active) and five subunit * R ( B far hindipg ) .
Tile toxin binds ro the Gnil gangliosido receptor
on intestinal epithelial cells by means of subunit B,
following which rhe subunit A is activated to yield
. two fragments - A1 and A2 , l hc Al fragment
" '
activates adenyl cyclase In tltc emerocyte 10 form
cyclic adenosine 5 ' monophosphate ( cAMP ),
30.2 ) ..
The heat stable toxins of E- «di (ST), first
identified in 1970, are low molecular weight
polypeptides which are poorly antigenic. Two types
of ST are known, STA (or ST 1) and STfi (or ST
II ). STA acts by activation of cyclic guanosine
monophosphate (cGMPJ in the intestine. 1c acts
very rapidly and induces fluid accumulation in the

Copyrighted material
 i Entprabactermceao I : Cdlprurms
intestines of infant mice within four hours of
intragastric administration . This mLinl mouse test
LR the standard method fur demonstration of ST , .
It also induces fluid accumulation in the intestinal
loops of neonatal hut rot weaned piglets. ST , is
methanol soluble . ST,, causes fluid accumulation
in young y inlets ( uptn nine weeks) but not in infant
mice . The mode of action of ST„ is not known but
: it is not through cAMP or cGMP. It is not
methanol soluble. SI genes are earned on plasct i ids
which may also cany other genes, such as for IT
and drug res stance . However, ST . and ST & genes
are not seen to be carried on the same plasmid.
E , colt Vc rocyiotoxin or Veroroxln ( VT ) was
so named because H was first detected (1977) by Us
cytuUixit: effect on Veto cells , a cell line derived
from African green monkey kidney cells. It is also
known as Shiga-like toxin ( $ LT ) because ir is
similar to the Shigella dt-senreriae type 1 toxin in
its physical, antigenic and biological properties .
Besides cytotoxicity in Vero or HeLa cells , VT also
shows cnterotmricitv in rabbit ileal loopc-- and mouse
paralytic-lethality as does the Shiga toxin . VT is
also composed of A and B subunits. VT genes
appear to be phage encoded . An antigenically
different VT, called VT . has been identified, which
is not neutralised bv the Shiga antitoxin, unlike
VTr
CLINICAI INFECTIONS
Four main types of clinical syndromes are caused
by E. coll : 1) urinary tract infection , 2 ) di.irthcA,
3) pyogenic infections, and 4) -septicemia.
I I r t n n r y tr -act infection :. E . troti and o t h e r
eol [forms account for the large majority of naturally
acquired urinary tract infections. Those acquired
in the hospital, following iiiRmnnentarion , are more
often caused bv other bacteria such as Pseudomonas
.a
asid. PfuTcui.
The E. coh\ $emtypet commonly reipooiible For
urinary tract infections lire chose normally found in
BJ ‘l1
the feces, O groups I , 2, 4, G , 7 h etc. Only one
scrofvpe is generally isolated from infected urme at
- ProlfluS t 275
a time* though recurrences may be due to different
serotypes.
Infection may be precipitated by urinary
obstruction due to prostatic enlargement , calculi
or pregnancy. About 5-7 per cent of pregnant
women have been reported to haw urinary infection
without any symptoms. Such .!> vmpttur.' .rt re
bdcftmiru undetected and untreated may lead to
symptomatic infection later in pregnancy,
pyelonephritis and hypertension in the pregnant
woman, as well as to prematurity and perinatal
death of the fetus.
Whik infections of the lower urinary tract seem
to be 'ascending Infection caused by fecal conforms,
pyelonephritis is probably due CO hematogenous
infection . Strains canning K antigens are more
-
commonly rcspon ible for pyelonephritis, while
most isolates from cystitis lack K. anrji cns ,
^
Bacteriological diagnosis of urinary tracr
infection has undergone a marked change following
the development by Kass of the concept of
'significant bacteriuri . L . Normal urine is sterile but
1
during voiding may become contaminated with
genital commensals . In order to avoid such
con rami nation , urine used to be collected by
catheterisation for culture. Any bacterial growth
from cathetcri ^cd urine was considered to denote
infection . Even under ideal Conditions ,
catheterisation leads to urinary infection in at least
two percent , and when pTecaulumK are inadequate ,
the risk is much higher. Hence catheterisaLion is
no longer considered justifiable for diagnostic
purposes. Instead, clean-voided midstream samples
H >f]r 11
' .sre employed I > r eulI :irL . S . H 11 -.- ic .
L' ' '
sbould be collected carefully to reduce
contamination to the minimum. In men, lr is
sufficient if midstream urine is collected after the
prepuce i :i retracted and the glans pcnb cleaned
with wet cotton . Tn women, anogenital toilet is more
Important and should consist of careful cleaning
with soap and water. Noniiriiant antiseptics such
as chid r ho ! dine have been recommended for vulval
cleaning. Uni re should be passed keeping the labia
Copyrighted material
276 * Textbook of Microbiology

Table M.2 Methods lor detection ol ETEC entertfwlns

A& SRjr LT ST
In rests
Ligated ttbbiT ileal h>op
Read at ft hours # +
Read it IS hours 4

Infant rahbir howcl 4 4-

Infant mouse intragasihc {4 hours}
Adult rabbit skin (vascular permeability factor)
-
*
4"
-
In vitro tests
Tissue culture mm
Rounding of Vi mouu adrenal celk -
EkfUHtum of Chinese haunittr grvwj ( CHO ) cells + =
Serological rests
' ELISA f 1 ( ST- mSA
wnh mono-
T
colonial
antibody
Passive agglutination tests., passive immune -
hemolysis, precipitin ( Eikens) test *
Genetic trsts #

ON A probes 4

- .^
s- L p ^i r L
^L^ cJ bv tm
Tbc fiisl pnrtiijn of urine that
rK.
flushes out commensal bacteria from the anterior
urethra is discarded. The next portion of the urine
( midstream sample;J i -s Collected directly in CO a.
Sterile wide mouthed container and transported to
the laboratory without delay. Urine is a good
medium for the growth of coliforms and other
urinary pathogens, and hence delay in processing
will vitiate the results of quantitative Culture. If
delay of morn than 1-2 hours is unavoidable , the
specimen should be refrigeruted -
In quantitative cultures , midstream unite
samples w i U give a biphaslc diatribu Lit m of colon i L :H ,
most specimens containing either less than 10,000
or more than 100,000 bacteria per ml. Kass and
other investigators have established that in the
presence of active infection in the urinary tract the
urine will contain 104,004 bacteria or more per
ml. This level is, therefore, considered to represent
significant bacterinria. Counts of 10,004 bacteria
or Cess per ml are due to contamination dunmg
voiding and are of no significance. Counts between
the two levels are infrequent when the sample is
collected properly and processed promptly. Such
results should he considered equivocal and the
culture repeated. Needless to say, interpretation of
b’acteriuria, should always he with reference to the
condition of the patient. In patients on antibacterial
or diuretic drugs and with some bacteria like Staph ,
aureus, even low counts may be significant -
For quantitative culture, serial ten fold dilutions
of urine are tested hv the pour plate or surface
culture methods. This, however, is too complicated
for routine diagnostic work, for which
semiquandradve techniques are more convenient.
The most widely used techn ique employs a standard
loop which transfers a fixed, small volume of urine.
One loopful of urine is placed on a non inhibitory
medium (blood agar] and another loopful on an
indicator medium ( MacConkey ) . The former
medium gives a quantitative measurement of
bacteriitria* while the lattet enables a presumptive

~
y\
Lj opyrighted materia
 • Efiierobactenaceae I : CoiiEorms '
diagnosis o- f the bacterium . The isolates ire
identified hy their properties.
Bacteriological investigation of urinary tract
infection is not complete without an antibiotic
sensitivity test of the isolate . E. coii and other
common urinary pathogens develop drug resistance
so frequently that no antihactcm therapy can be
instituted meaningfully without testing individual
strains. Resistance is often to multiple drugs and is
of the transferable variety. Antibiotic sensitivity tests
may be done directly using the uri :ie samples as
inocula and the results confirmed by repeating the
test with individual isolates.
Because urinary Luc L injection i £ £Uth a common
problem and bacteriological facilities are not always
availableh several screening techniques hive been
introduced for the presumptive diagnosis of
significant bacterium . These include the following:
1 ) Gricss nitrite test - based on the absence of nitrite
in normal urine . The presence of nitrite, detectable
by a simple test , indicates the presence of mtiate -
reducing bacteria in urine, 2) catalase test — the
presence of catalase as evidenced by frothing on
addition of hydrogen peroxide indicatesbaclcriuria,
though a positive result is obtained also in
hematuria, 3) tri jihcnyltctraioliijni chloride { ETC )
test - bised on the production of a pink - red
precipitate in the reagent caused by the respiratory
activity of growing batteria + 4) microscopic
demonstration of bacteria in Gram stained films of
urine, 5 ) glucose tesr paper - based on the utilisation
of the minute amounts of glucose present L:I normal
urine, by bacteria causing the infection, and 6) dip
slide culture methods - ig*r coated slides are
immersed in urine or even exposed to the stream of
urine during voiding, incubated and the growth
estimated by colony counting or by colour change
of indicators. None of the screening methods is as
sensitive or reliable as a culture .
The antibody coated bacteria test has been
employed for the localisation of the sice af urinary
infection . This is based on the assumption that
bacteria coated with specific antibod ies are present
Protejs 277
in the urine only when the kidneys are infected
and not when the infection i * confined to the
bladder. Antibody coated bacteria are detected by
immunofluorescence lining fluorescent tagged
antihuman globulin or by staphylococcal
coagglutmlTiim .
Uiorrbeat Right from 1885 when Escherich
fust isolated the bacillus from the feces of infants
with enteririis, E . coii had been suspected to be a
causative agent of diarrhea. However, as there was
no way then of differentiatingdiirrheagenic E . coii
strains from the welter of commensal strains
Invariably present in normal feces, it remained
unconfirmed till serotyping schemes for E . coii
were developed. It was only in 1945 that Bray
established the etiological role of a specific type of
E.culi subsequently recognised as type Cl 11) during
^
a hospital outbreak of childhood diarrhea in
London. Soon many other emeropathogemc
serotypes of E , coii came to be recognised as
responsible for infantile diarrhea. Subsequently,
other varieties of E . coii diarrhea came to be
identified in children as well as in adults. At least
five different types of Jiarriieagemc E . coli are now
recognised - entcropathogcnic, enterotoxigenic,
entero invasive , entero - hemorrhag ic
(Shigatovigeuic or Verotoxigenic ) and entero-
aggregarive E , coti-
Entcropathogen ic Ei . cnli ( EPEGfc These
have been associated m & i nly wn h di ,irrhea m i nfants
and children usually occurring as institutional
outbreaks but they can also cause sporadic diarrhea
in children and less often iii adults , EPEC diarrhea
was common worldwide from the late 1940* to the
1960a . Afterwards, it has become less common,
EPEC were identified by serotyping, initially
by their O and B antigens (for example , 026:B6,
055:B5 t 0I 11:B4 and so on ). After the existence of
the B antigens became suspect , only Q typing i =
practised.
The diagnosis of EPEC diarrhea is relalively
easy during outbreaks but very difficult in sporadic
cases. Fresh diarrheal feces is plated on blood agar
Copyrighted material
278 i Tejcibook
qnd MacC-grikey media. After ( mrnight incubation,
K oIi colonics arc emulsified in saline on a slide
and tested for agglutination hy polyvalent and
monovalent EPEC O antisera . At least ten colonics
per plate should be tested as many serotypes are
present in a single culture. If isolated colonies are
negative,, the confluent growth is emulsified and
tested. During outbreaks, if the causative serotype
is- known , cultures need be tested only with the
j ,
particular antiserum. EPEC antisera are now
difficult to obtain and so specific diagnosis is
available only in few laboratories, When the
outbreak is caused by a strain with some readily
,
a1
demonstrable feature such as ' ANUT to ferment
sorbitol, rapid identification is possible by using
appropriate culture media.
The pathogenesis of EPEC di .urhca is not fully
understood. EPEC do not ordinarily produce
enterotoxins, nor arc they invasive. In infantile
enterids, the bacilli are seen ro be adherenr ro the
mucosa of the upper small intestine , intimately
attached to cup-like projections (' pedestals 1 ot the
enterocyte membrane, causing disruption of the
brush border microvilli . The name enfcncwdfwrenf
E. coli has been proposed for these strains, which
can be identified by their adhesion ro HEp-2 cells.
Enterotoxigenic H . cnli ( ETBC ):
Diarrhea caused bv £TEC is endemic in the
•li
developing countries in the tropics, among all age
groups in the local papulation Its seventy vanes
, and given names such as
from mild watery diarrhea to fatal disease
indistinguishable from cholera . Persons from
developed countries visiting endemic areas often
-
suffer from ETEC diarrhea a condition known
as "traveller’s diarrhea’. ETEC d:.irrhea came into
prominence from the late l%Os.
Though plasmids with enteroloxir: genes may
, entrrainvasiir
he present in any strain of E. coli , in practice only
a small number of serotypes become
enterotoxigenic (for example, 06, OS , 015, 025, 027,
0167). Toxin production alone mav not lead to
illness.The strains should first be able to adhere to
intestinal mucosa. This adhesion in mediated by
of Microbio- logy »
ticiihn.il ur culonisaliun factor aTiligertii, of which a
number have been identified (CFA I, II , ITI ,[V ).
Diagnosis of El EC diarrhea depends on the
demonstration of entcrotctvin in E . coli isolates by
anv of the methods listed in Table 30.2 . A strain of
jf
ETEC may produce either IT or ST or boThB
Defection of LT is easy as many in vitro methods
arc available, such as tissue culture tests ( rounding
of Yj mouse adrenal cells and elongation of CHO
cells due to intracellular increase ol cAMF
concentration ), and serological tests, ( ELISA ,
passive agglutination and Immunofysin tests ). In
vivo tests such as rabbit loop or intradermal tests
may be used when in vitro tests are not available.
The deceedon of ST is more difficult. Tine infant
mouse test is still widely employed - The poor
antigenicity of ST has prevented the development
ol serological cescs ,, chough ST ELISA using
mnnndnnal antibody has been introduced. Genetic
probes art available for detection of ST and LT in
.
Ecoli culture, or directly i.n feces food or water.
Enterdiivitivt E. coli ( ElEG): These
resemble shigcllac in many respects . Many of these
strains are nonmotile, do not ferment lactose or
fiernienr 3 r late with acid, but without producing
any gas and do not form lysine decarboxylase. Many
of these show O antigen cross reaction with
shigellae. These ‘atypical’ E . coli strains had earlier
-
been grouped under the 'Alkalescens Dispar Group'
a /Jca /escens '
( resembling Sh. fJaneri except in fermenting
dulcitol and forming alkali in litmus milk} and Sh
dispar' (late lactose fermenter Like Sh, sonme i but
indole positive ). Besides these 'atypical strains’ many-
-
typical E. LCI1: trains can also cause clinical illness
resembling shigellosis These have been termed
.
E.coli because they have the capacity
to invade interstitial epithelial cells in vivo and
penetrate HeLa cells in tissue culture. EIEC strains
LI ^ULI 11 v I hduiig Lo serogroups 02 Et , LC. 0112 ac, Q 124,
.
0136, 0143 0114, 0152, 0154 .
Clinically Li EC infection resembles shi ellos::- ,
ranging from mild diarrhea to frank dysentery, and
^
Copyrighted materi3
 * EntufobactGiiJceae l : ColifutmB - Proteus
OCClUtt in children as well M adults . Foi laboratory
(li nosis of FI EC, the Screny lest used to be
^
employed ( that is , instillation of a suspension of
freshly isolated EIEC or shigella into the eyes of
guinea pigs leads TO mucopurulent conjunctivitis
and severe keratitis). Mice may be used instead of
guinea pigs-. Cell penetration of Hel .a or HEP-2
cells in culture is a more humane diagnostic test.
Tliin ahijitv to penetrate- cells in- determined hv a
large plasmid , detection of which can also he a
diagnostic tent. The plasmid to ties for outer
membrane antigens called the 'virulence marker
antigens’ { VMA ) which can be delected bv the
EUSA ( VMA ELISA ) tear
Flnl crnlicmc )Trhii (ji [: Ft .vtjli ( EHEC )
S bigamy!genic K , coli ( STEC ) dr
Verotoxigcnic K . coli ( VTBC ): E. coJi
strains producing vcrocvloroxm ( VT) or Sliiga - like
toxin (SIT ) can give rise Co diarrheal disease ranging
in severity from mild diarrhea to fatal hemorrhagic
colitis and hemorrhagic uremic syndrome ( HUS)
particularly in young children and the elderly. The
primary target for VT appears to be the vascular
endothelial cells.This may explain rhe pathogenesis
of HUS, in which a characterisuc renal lesion is
capillary microangiopatbv. VTEC also produces
diarrhea in cattle and pigs. The typical EHEC is
serotype Ol 57: H 7 hut a few others such as 02 b:
? countries. Most of the are 0- untypahte, but many
Hi also belong to this category.
The disease may occur sporadic ally or as
outbreaks of food poisonong . The source of
infection is contamination by human or animal
feces, directly or indirectly. Changing life stv1es and
citing habits, with growing popularity of fust thud*
have led to a remarkable increase in EHEC food
poisomng. Jn the USA, about 20,000 cases of 0:157
food poisoning occur every year, many with
hcinorrliagic complications . A Large outbreak of
food poisoning caused by E. troli 0 :1 57 „ with QVW
10,000 cases occurred in japan in mostly
affecting school children . Investigation of this
epidemic revealed the source of infection to he salad
vegetables such as radish and alfalfa sprouts, in
» 279
which the bacteria were found beneath the skin
and in the deeper tissues . Washing would not
remove the bacteria from such vegetables and
cooking alone may ensure safety Thin finding
extends rhe scope of EHEC food poisoning to
vegetarians also,
Laboratory diagnosis of VTEC diarrhea can he
made by demonstration of rhe havilEi nr VT in frees
diltCtly or in culture. As VTEC forms only a
minority of focal conforms in infected cases, testing
individual colonies may not be successful. The
sensitivity can be considerably inert- used by using
DNA probes tor VT1 and VT . genes. VT can be
detected by its cytotoxic effects on Venn or HeLa
cells. Demonstration of VT neutralising antibodies
in convalescent sera may help in retrospective
diagnosis.
Most VTEC strains belong to the serotype
0I57:H7 which does not lemient sorbitol , unlike
the majority ot E. CQI L SO the use of sorbitol
'
MieConkey ntedium helps in screening for 0:157
VTEC.
Em ernuggregutix e E. mill ( EAEC ):
These strain? MC SO named because they appear
aggregated in a Stacked brick’ formation on Hcp-
2 cells or glass . They have been associated with
persistent diarrhea* especially in developing
arc H-tvpable. Tn animal experiments they cause
shortening of villi , he morrhagic necrosis and mild
edema with mononuclear infiltration of the
gubimicHL They form a low molecular weight heat
stable enterotoxin called EASTl
( rrtcrciajrjTFTcjriifjvc bvni- *tnbie enttr taxin - l )
"
^
l*yo enic infections:E. coir form the most
^
common cause of intra -abdominal infections, such
as peritonitis and abscesses resulting from Spillage
ot bowel contents . They also cause pyogenic
infections in the perianal area. They are an
important cause of neonatal meningitis.
SepticeTiiia: Blood Stream invasion by £ coli r
mav lead to fatal conditions like septic shock and
'systemic inflammatory response syndrome’ (.SlRJj),
Copyrighted material
280 * Textbook
AR R. coll commonly show multiple drug
resistance, antibiotic sensitivity testing of strains is
important in Treatment.
EDWARDSIELLA
The genus contain! the species £dwareJsi>Ua
fands which is a nnnc .LpHulatcd, motile bacillus with
weak fermentative powers , The name tarda refers
to its lardy or weak fermentation of sugars. Of the
sugars commonly used , only glucose and maltose
are fermented. It forms irdnle and HjS, utilises
citrate and decarbaxylates lysine and ornithine.
.
E tarda is a normal intestinal inhabitant of
snakes and other cold blooded animals. It has been
cultured from normal tfld diarrheiiic human teccs.
Its pathogenic role is uncertain but it has been
isolated from wounds, urine, blood, and from CSF
in cases of fatal meningitis.
CITRQBACTEfl
These arc motile bacilli which utilise citrate, grow
in KCN medium , produce H3S and ferment lactose
late or not at all. Three species are recognised, Citiv,
frrundii which gives typical reactions and Citru,
ioseri ( formerly Cirro. diverses ) and Cifro.
amaJonaticirs which do not form H S- Cltrp.
^
freundii strains were formerly classified as the
-
'Ballerup Bethesda group . They exhibit extensive
’
antigenic sharing with satmoncllac- This may cause
confusion in the diagnostic laboratory Some strains
(for exmiple, the Bhainagar strain ) have a Vi
Table 30.3 Differentiation oi Klebsiella species
It, pneumoniae
Gas from glucose
Acad from lactose
4
+
- d
MR
VP
—
+
Citrate 4-
Urease
Malonate
+
4
Lysine 4 d
gi Microbiology *
antigen serologically identical the antigen nt S.
Co
jyphi and 5* paratyphi C- These may he used for
the estimation of VI antibodies or for raising Vi
antisera.
Cirrobactcr is a normal intestinal inhabitant.
Many strains formerly called paracolons belong ro
this group . It has been isolated from a few eases of
enteric fever hut its etiological role is not established.
It may cause infections of the urinary tract, gall
bladder, mitldle ear and meninges.
KLEBSIELLA
The genus Klebsiella consists of nonmotile ,
capsulatcd rods that grow well on ordinary media
forming large , dome shaped, mucoid colonies of
varying degrees of stickiness.They are short, plump,
straight rods, about 1-2 K 0.5-0.8 mm in size. The
capsule is often prominent and can be made out
even in dram stained smears as haloes around the
,
bacilli. KlrhsieUae are widely distributed in nature,
occurring both as commensals in the intestines and
as saprophytes in soil and water. Their classification
has undergone various modifications- They have
been classified i n t o three species based on
biochemical reactions and into over 80 serotypes
based on the capsular ( K) antigens (Table 30.3).
KLEBSIELLA PNEUMONIAE
( Friedlanders bacUhrs , Bacillus mucufus otpsuiatus)
This bacillus was first isolated by Fried Under
K, ozaeoae K. rhifJos cie.rD.m JI tis
'
d
——
+
— ——
d
d
— +
—
—
Copyrighted materia
 * Fnterptjgdefiacaae l : Conforms Proteus » 283

( 1 B93) from fatal nf pneumonia. It fermems

casts
sugars glucose, lactose, CUOVR, mannitol ) with
{ hy the presence df a plasmid .
the production of acid and abundant gas. I [ is mdolc
and MR negative and VP and titrate positive {IM
Vic ) . Biochemically variant strains arc
* *
common . Ir forms mease, Strains, formerlylabelled
as oonmotile Aembactcr aerogenes [ K . aerogenes ) y
are row considered to he K . pneumoniae subspecies
acrogcncs, It is the second most populous member
of the aerobic bacterial flora of the human intestinc-
lt has become a very important cause of
nosocomial infections, even replacing E . ctili in
some centres. J r causes pneumonia, on nary infection,
other pyogenic infections , septicemia and rarely
diarrhea -
Kicbait’ lfa pneumonia is a serious disease with
high case fatality, It occurs in middle aged or older
E. coii. The production of this toxin is determined
Diagnosis is made by culturing appropriate
specimens and identifying the isolate by
biochemical reactions, Antibiotic sensitivity should
invariably be done , Many strains cany plasmids
determining multiple drug resistance.
K . vzacifzc is a bacillus associated with ozena,
a disease characterised by foul smelling nasal
discharge. Identification LS difficult due 10 wide
variations in the biochemical reactions of individual
strains, ft. ozaenae belongs to capsular types 3-6.
K . rhinosclemmatis causes rhinoseleroma , a
chronie granulomatous hypertrophy of the nose
prevalent in southeastern Europe, India and Central
America. The bacilli are seen imracellularly in
lesions It can he identi tied by biochemical reactions
,

persons who have medical problems such as and belongs to capsular type 3.
aJcoholism , chronic bronchopulmonary disease or The species A'. o Tnca may he rarely isolated
diabetes meliitus. The disease is characterised by from clinical specimens. ^
massive mucoid inflammatory exudate of lobar or
m

lobular distribution, involving one or more lobes ENTEROBACTCR

of the lung. Necrosis and abscess formation arc
more frequent than in pneumococcal pneumonia ,
Serotypes 1, 2 and 3 are usually responsible for
pneumonia. Positive blood cultures can he obtained
m about 25 per cent of the cases.
K , pireumoiwue is a frequent cause of urinary
infection. As most strains are resistant to antibiotics,
treatment poses serioUG problems, ll also causes
pyogenk infections such as abscesses, meningitis
and septicemia.
Some strains of A. pneumoniae isolated from
cases of diarrhea have been shown to produce an
cntcrotrain very similar to the heat stable toxin of
jr
Formerly known as Aerobaeter these ax motile,
capsulatcd , lactose fermenting bacilli which are
indole and MR negative and VP and citrate positivc.
TWt> clinically relevant species are E . clovac and
F . zemgenes (Table 30-4).
They are normally found in feces, sewage , soil
and water and rarely in urine , pus and other
pathological materials, They may be responsible
for hospital infections.
HAFNIA
This is a motile, nonlactose - fermenting bacillus
which is indole and MR negative and VT and citrate

fault 30.4 Differentialion between E , cloacae and E. aerogenes

E. cloacae E . acrogcncs
Gas from gjyceml - +
Aesculin hydrolysis - +
] .yimc decarboxylase
Arginine dihydiulaK
-
+
i

Copyrighted materia
282 * Tgxlboofc 01 Micmtuoiogy *
positive , Biochemical rca *cti < > n ^ arc evident best
at 22 LC ; n 37 C they may be negative or irregular.
Only one genus is recognised , tl , aim lr is
found in human and animal feces, sewage, soil and
water .
SERHAT1A
This ditfcis from Hatnia in farming a pint, red or
magenta , nondifllfoiblc pigment called prodigies in
which is formed optimally at room temperature.
Only one specks is of medical importance - 5,
/nareescej] .? ( £# iic > //trs prodigiosus' ) , Jr is
l
pleomorphic, with minute coccobicillaiy and
normal haciliury forms-. It i ^ a saprophyte found in
water , soil and find . Ir may grow in sputum utter
collection and may suggest hemoptysis because of
the pigment formed Cpseuduhetnupivsis ' ).
Nosocomial infections due to S, rthucescttis are
heing reported with increasing frequency, The
bacillus lias been associated with meningitis *
endocarditis, septicemia , peritonitis , respiratory
.
infection and many oilier conditions Multiple drug
resistance is common in hospital strains.
TfilBE PROTEEAE
( Fnrteua bacilli )
Proteus bacilli constituting the tribe Pmtrexe are
lactose nonfermenters and so do not strictly belong
n> the group of 'co.li form bacitli , However, they
1
are also normal intestinal commensals and
Opportunistic pathogens like coliforms, and so are
included in this chapter. The name ' Proteus refers
Table 30.5 Biochemical features of Proteus bacilli
Tetr Pr Pr.
tttirnbilit Igtiia
v morgtnii
Urease 4 4
Ornithine 4 -
decukiyliie
Indole - +
PermencaEi ^ n. of adi*iiiTi>] - -
Fermeniarion of frehaloi* 4 t
ro their pleomorphism, after the Greek god EYoteui
who could assume any shape.
'
The tribe Profcrac is classified into three genera
- Pro re us, Moig-AncUa and Provkfcnria . Most of
them except for cume Pruvindencia strain --... produce
a powerful urease which rapidly hydrolyses UTea
fo ammonia and carbon dioxide. A characteristic
feature which distinguishes Pro ferae from Other
1
enterobacteria is the presence, in all members of
the tribe, <> t the nuitnc phenyl alanine deaminase
which converts phenyl danine to phenyl pyruvic
acid (PPA reaction ). All of them with few
exceptions show the following features:
Gram negative , noncapsulated, pleomorphic,
motile rods,
* resistant to KCNt
* degrade tyrosine;
* tail to acidify lactose, dulcitol or malonate;
' do not form arginine Or lvsirte Jec.Lrhox vlasc or
beta galactosidasc;
MR positive, VP negative .
*
The itiaior dlfFer-Cii Mating features of medicallv
important species of pnoteus hacilli arc shown in
Tabic 30.5 .
Proteus bacilli possess somatic O and flagellar
II antigens, which arc of considerable historical
interest . Weil and Felix { 1916} studying Proteus
bacilli observed that flagellated strains growing on
agar formed a thin surface film resembling the mist
produced by breathing on glass and named this
variety the JJauchr form (from ffaurfr, meaning
4
film of breath ) . Nonflagellaled Variants grew as
M o f g. Prov, Prov. Prov,
ih-ulifitiens stumrtii rcttgcn
* 4
+ - - -
# 4 4-
- + ± ±
t 4 -
Copyrighted material
 ^ EnlerobacifiriaCEfEie I : Colilorms-
isolated colonies without rhe vurface t’llTTi and were
called 'Ohne 1
Hauch ( meaning without Film of
breath ). These samts came ro he abbreviated as
rhe H and 0 forms . Subsequently, the 13 and O
were extended to refer to the flagellar and somatic
-
antigens of Otllct bacilli AS well. I In tHW ate also
OKed to designate the type of hacteriid agglutinat ion ,
the 11 type refer ring to the loose, fluffy masses
formed when flagellated cells are agglutinated, and
the 0 type ro the fine granular appear* ncc of
somatic agglutination.
Weil and Felix also observed that certain
non motile strains of Pt. vulgarcalled rhe lX
-trains ' , wtre agglutinated by sera from typhus fever
parienCS- This heternphilic agglutination due to the
sharing of a carbohydrate hipten by certain strains
of Proteus and iiJcettsLae forms the basis of the
Wei I -Felix reaction for the diagnosis of some
TickertsiaJ infections . Three nonmotile Proteus
strains 0X 2 , OXIV anti OXK are used in the
agglutination tost .
Proteus baLilli are widely distributed in nature
as saprophyte*, being found in dccompotmg animal
matter, in sewage, in manured soil and in human
and animal feces. They are frequently present on
the moist areas of the skin . They are opportunistic
pathogens, commonly responsible lor urinary and
Septic infections , often nO£tic0fnul
The genus ADMIX contains rwo species of
medical importance - Pr. mirabilix , which is an
important urinary and nosocomial pathogen , and
Pr. vulgaris which is found much less commonly
in human infections. Pr. mfrabjJrs ! H indole negative
and Pr. vulgnrrt. nidufo positive.
Cultures of proteus bacilli have a characteristic
putrefactive HMJOUT described as 'fishy ' Of 'senutuT
Pr. mirtbiln aod / V. vujgarirswarm on solid culture
media . Discrete colonics are seen in young cultures
but thereafter actively motile cells spread on tlie
surface of die plate in successive waves to font ] a
thin filmy layer in concentric circles. Swarming
growth is a problem in the laboratory when mixed
growth is obtained in which proteus bacilli are
- Froteun r 283
.
present with other bacteria Several methods have
been used Co inhibit swarming - increased ( b (X» )
concentration of agar, incorporation of chloral
hydrate (1:500), sodium aside ( 1:500) , alcohol (5’
6%i), sulphonamidc, surface active agents or boric
acid (1 : 1000 ). Swarming does nnt occur Qn
MacConkey 's medium, On which smooth colourless
colonies are formed .
The genus Mdipidla has only one species,
. .
M. tnoiganii (formerly Pr morgnnii) It docs not
Swarm in culture. Tt is oommordv found in human
and animal feces and causes urinary infection sr
infrequently. Nosocomial wound infections also
occur .
The genus Providencia ( formerly known as
Aofetrs inconstant} con tains three species seen in
clinical infeiTioiu. PPyv akalifacieos is some times
seen in normal human feces but far more frequently
in rHirrhftdl stools though its etiological role is
uncertain. Prov. sEtWtji is a common cause c4 mitUUV
infection and if infection in burn. Prov. rettgerii is
i
part of the normal fecal flora of reptiles and
amphibians, and sometimes causes nosocomial
infection of the urinary tract, wounds, bums and
blood,
Proteus bacilli arc resistant to many of rhe
common antibiotics. An exception is Pr. mimhilis
which is sensitive to anupLdilijrt Lind cephalosporins.
]
Provident ia are the most resistant, particularly Am
stiwrtii which is also resistant to disinfectants such
as ehlorhexkline, Cctrimide, hcnyMlkonium chlun.de
and heavy metal compounds such as silver
sulphonarnide., making it a major pathogen in bum ®
units. Jt is sensitive to phenol and gluiaraldehyde-
Amikacin and dprofioxodn arc generally effective
in tmtnwnt
ERWINIA
These ire anaerogenic bacilli forming a yellowish
pigment, usually found in soil and causing plant
infections E , hcrhk' O'b has occasionally been
*
isolated from respiratory and urinary infections in
predisposed or hospitalised patients.
Copyrighted material
284 4 Tftxtboofc of Microbiology

Further U . Gliding
BlIMlllJf 1990. The pathogenicity ofenteropathogcnic E#cAmcAia coL ] ModMiaobioL 47:383.
Barnwell JG . 19901. PadiiDphy5®log> of Larrhed. disorders . Her Jn/iet DJS. l 2 S.3fl.
^
'
s “

Cooke EM. 1985. EsrAfriciji coli, an overview. J Hjg (Cmb) 95: 523 .
Ewing WH , l 9B6, Bdmtxb and &mng $ Modfiotioa o f E r-r e; 4^ sdn New York Bbener,
.

Feng R 1995 t ou/i tempt 0157 Nov* vehk

, : 1 k o
'| IsletiML EfflflpJij Imfeef OES. 1:3.
Fitzpatrick ML 1999. Haemolyiu; uremic iyiviroitit ami E. ctxfa 0157. BAfj. 310 L684.
_ _ .
Gnthiim JC and A Galhw?iy. 2LXjL Laihutf aiuEY JiagtHutia oi iiriniLry tract LnieecLdcL J Clin i ' i fh 54:911 .
Grinnkn WR 1990. Bacteremia due t« ftthefkhi* n*K. -ftfv /nJecr Di*. 12:OOS
,

GIOR Hj . 1991. The puhogeneti* ef Eicheridtii coir diarrhea- RewMed MksebkL 237 .
Kawr* P and JB K-aper . 1998. Diarfhjjgenic E .ccli. Qnn Microbial Reis 1 L742.

uo
y py righted materi 3
 CO Emerobacteriaceae II: Shigella
DyHiteryis ;L -li nicaJ condition of multiple etiology,
L months- fhey remain viable in moist environments
characterised by the frequent passage of blood
stained, mucopurulent stools. The two common
types of dysentery arc bacillary and amebic. The
causative agents of hiL iHary dysentcTy belong to
'
the genus Shigella* so named after Shiga, who in
1896 isolated the first member of rhivs genus from
epidemic dysentery in Japan. Some other bacilli,
such as entcro invasive E- cofi .Vibrio
parthtcmotytiiis and CafnpyJobacrer, can also cause
’
the clinical picture of dysenterv.
Morphology : ShigeElac are short, Crain
-
negative rods, about 0, 5 pm * 1 3 pm in size.They
are nonmofile, nnnsporing and noncapsulated.
Fimbriae may be present .
(lulturAl characteristics: They are aerobes
and facultative anaerobes, wh a growth tempe -
rature range of 10- 40 °C and optima of 37 <S. and
r
pH 7.4 . They grow on ordinary media bur less
readilv than other enterobacteria. After overnight
incubation, colonies are small , ahout 2 mm in
diameter, circular, convex, smooth and translucent .
Occasionally on primary' isolation and I reqoent Iv
in subcultures, a proportion of the colonies may be
of the rough type. Colonies on MacConltcv agar
arc colourless due to the absence of Lactose
ferment attorn An exception is Sh, sonner which
ferments lactose late and forms pale pink colonies.
Deoxycholate citrate agar ( DCA) i $ a useful
selective medium. Growth is inhibited on Wilson
and blairs hismuth sulphite medium.
iCcBtNtnnee Shigelkc arc not specially resistant.
They arc killed it 56 in one hour and by 1%
phenol in 30 minutes. ! n ice they last tor 1-6
'
.
for days,but die rapidly on drying In feces they die
within a few hours due to the acidity produced by
.
the growth of coliforms Sh, senna is in general
more resistant than other shigella species .
Hioohemical relictions: Shigellas are MR
positive and reduce nitrates to m trites. They cannot
utilise citrate as the sole source of carbon, do not
form I IS and are inhibited by KCN Catalase is
produced except by Sis . dysmtoile type 1 . Glucose
,
is fermented with the production of acid, without
gas, except for the Newcastle and Manchester
biotypes of Sh. flexnen type 6, and some strains of
Sh . bovJii tvpes 13 and 14, which form gas.
Fermentation of mannitol is of importance in
classification and shigellae have traditionally been
divided into mannitol fermenting And
nonfermenring species . .Sh, flexneri , Sh , bovdii and
.
Sh Monnei ferment mannitol, while Sh , djventciiat
does not . Exceptions are not infrequent. Lactate
and sucrose are not fermented, except hy LSJJ. smaa
which ferments them late . Adonitol, inositol and
salicin are not leriUentod-
Antigenic structure: Shigcllae possess ore
or more Lmaior ' antigens and a large number of
‘ .
minor" somatic O antigens Some strains possess
K antigens. These arc not relevant in typing but
may sometimes interfere with agglutination by O
Antisera . Fimbria! antigens arc also present. In
general, the antigenic structure of shigellae is
simple, compared to the complex structure of
salmoncllae.There 15 considerable antigenic sharing
between some members of rhe genus as well as
between shigellae and £. coii. Common fimbria!
Copyrighted material
2&> * Textbook off Microbiology
an ripens may also occur, particularly in Sh. ScxnCii-
li is , therefore, important that: the identification
of shlgcllae should he made by a combination ot
antigenic and biochemical properties and nor by
slide agglurbiiiNDn alone.
CLASSIFICATION
Shigcllac arc classified into lour species or
subgroups based on a combination of biochemical
and serological characteristic? Serotypes are
r named after Flexner, who described the first of the
distinguished within the species , -Sir . aonnri is
serologically homogeneous and is danificd by
Cclicin typing.
Sh . dyscnterinc ( subgroup M i This species
of mannitol nontermenting bacilli consists ot ten
serotypes . Type 1 is the bacillus originally
described by Shiga ( ifi . sfrfgar}, Jr is indole negative
and is the only member ot the family that is always
catalase negative .. (Si. tehmitzi and Sh. so/incr arc
invariably catalase positive , while among other
shigella species , some strains may be catalase
negative, )
Sh . Jyrejifrriiir type 1 forms a toxin (Shiga
toxin ), the earliest example of an exo toxin produced
by a Gram negative bacillus. Three types of toxic
activity have been demonstrated in shigella culm re
filtrates : ( 1 ) neurotoxicity, demonstrable by
paralysis and death on injection into mice or
rabbi ra. JI Tintigh known as ‘henrotovi n', the primary
site of its action appears to be not the nervous
tissue hut the blood vessels, iniainlv of the central
nervous system, with the neurological effects being
ton » ndar v ; (2 ) eilTcruUlxicity, with induction < 11 tluid
accumulation in ligated rabbit ileal loop . Two
new shigella enterotoxins have hern identified ,
designated as Sh- ET- 1 and 2, the former
confuted to Sh . flexneri 2a and the latter more
widespread; and ( J ) cytotoxicity, causing cytopathic
changes in cultured Vero cells . This appears to be
the same as Verotoxin 1 ( or Shiga -likc toxin )
produced bv certain strains of E cali ( VTEC) . The
toxi n consists of binding ( fl ) and acti ve { A subun i is.
' mild diarrhea. 1 lowever, Sh . sojinei infecrion persists
Subunit A is divided into two fragments Al and
t
A2 . Fragment Al appears to inactivate host ceil tiO
b ribosome , interfering with protein synthesis .
-
Sh thsevjHTFje type 2 (5b, schmils. i) forms
indole and ferments sorbitol and rhamnnsc .
Serotypes 3-7 were described by Large and Sachs
in India and hence used to be known as the l .aige-
Sachs group . Three fiirther serotypes have been
dusi- ribud making a total of ten.
Sh . Ilexntri ( subgroup H ) i This gioup is
mannitol fermenting shigellae from Philippines
( 19 D0 ). This group is hiocheinically heterogeneous
find antigen ic ally the most complex among
shigcllac. Based on type specific and group specific
antigens , they have been classified into six serotypes
( 1 -6 ) and several subtypes ( la; lb; 2a , 2b; 3a , 3b.
3c, 4a , 4bn 5 a , 5b). In addition , two antigenic
"
variants' culled X and Y are recognised , which lack
the type specific antigens. Serotype t> is always indole
negative and occurs in three biotypes, some ot which
form gas from sugars.
Sh . Itovdii ( subgroup Cfc 'Hiis group consists
of dvsenterv bacilli that resemble Sh . tlexneri
biochemically but not antigenieally. Tlie group in
named after Boyd , who first described these strains
from India ( 1931 ) , Fifteen serotypes have been
identified . X!J . boydii are isolated least frequently
from cases of bucillarv dysentery.
Sh . sonnei ( subgroup Dfc litis bacillus , first
described by Sonne ( 1915 ) in Denmark, ferments
lactose and sucrose late . It is indole negative. It is
antigcnically homogeneous bait may occur in two
forms - phase J and phase II - the latter forming
colonies that are larger, flatter and more irregular
On subculture , phase I produces both types of
colonies but phase II is considered to be a Joss
variation. Organisms in phase 11 maybe isolated
from patient^ bait are more common in convalescents
and carriers.
Sh . Mtnnci causes the mildest form of bacillary
dysentery, In many cases the disease may only be a
'
as the most common shigellosis in the advanced
Copyrighted materia
 * Emerobactefiac*ae
eoimtries. For epidemiologici! purposes, Sh.. sonjwj
has been classified into many colicin types,
P \ THOGBPJIC 1 T"i
fihigcllac cause bacillary dysentery. Infection occurs
by ingestion. The minimum infective dose is ] < iwL
OS few its Li I 100 Willi being Capable of iiii dating
die disease, probably lwvjusr they survive gastric
acidity better than -other enterohxLttria. Their
pathogenic mechanisms resemble those nt
. .
enters invasive E coli The bacilli infect the
epithelial cells of the villi in the krge intestine and
multiply inside rhenn , spreading laterally to involve
adjacent cells and penetrating into the lamina
propria. Inflammatory reaction develops with
capillary thrombosis, leading to necrosis of patches
-
of epithelium, which slough olff, letting behind
IrartHyersc Hiipcrticial ulcers. Bacteremia nuty occur
in severe infections , particularly in malnourished
children and in AIDS .
1 hough Sh . dyscntetiie type 1 forma an
e \Hiti*xin it appears to be much less impirtartl in
7 disease may vary from acute fulminating dysentery
pathogenesis [ ban the ability of the bacillus to
penetrate and multiply in colonic mucosa ,
Nontoxigcnic mutants can still cause dysentery but
rot noninvasive ones . The invasive property' of the
bacillus can be demonstrated by its ability to
penetrate cultured I IcLa or Hcp-2 cells or by the
Table 31.1 Diflinguishing features ol Shigella
Subgroup A B
Species Sh_ dywentcrixe
Mann Lhi ]
Lactose
Sucrose
——
Dulcitol
1 rvdole
=
d
Ornithine decarboxylase
Serotypes lfi
A - Acid
-
d Variable
II . Shigella » 207
Congo red binding rest. Invasive property is related
to the presence in the bacillus of large plasmids
( M.W. 140 « 10") coding for the outer membrane
protein responsible for cell penetration , These
proteins are called 'virulence marker antigens'
( VMA ) . Detection of VMA by ELISA serves as a
virulence test for Shigellaef as for ctuerouwosive
fcall .
Baci 11 ;in (JysciUUJ h as A sh ort i m Lthat N m period
" '
'
( 1-7 days, usually 48 hours ) . 'Die onset and clinical
course arc variable and are largely determined by
the virulence of the infecting strain . The main
clinical features are frequent passage of loose, scanty
fcccs containing blood ind mucus, along with
abdominal cramps and tenesmus . Fever and
vomiting may be [ ^ resent . Complications are most
otten j;een in infection with Sh* dweiilerixt: ti u; I
and include unbinds, toxic neuritis, conjunctivitis, ^
parotitis and , in children, intussusception .
Hemolytic uremic syndrome may occur as a
complication in severe cases. The severity of the
to mild diarrhea. AH the term bacillary dysentery
refers only to the more severe cases, the term
'shigellosis has been employed to include the
7
whoit spectrum of disease caused by sbigellae.
\ [ uni an beings arc the only natural hosts for
shigellie . Captive monkeys have been found
species
c D
Sh >
ffcxncri Sh. baydn Sh. sonnei
——
A
——
A A
A Late
A Late
—A
d
d
*
-
- -
6 + variants 15 Only one
Copyrighted material
m <
Table 31.2 Blotypes cl Sh. Itexneri Type 6
Biotypt Frmacii fa troii of
Ghicote Mi nnitoi
RU>yd RK
A A
Manchester
AG AG
Newcastle A or AG
A = Avici
-
AG Acid and GM
infected but such infections may h.ivc hecn of
hudlixi origin , Experimentally, dysentery cm be
produard only ]]i monkeys. Human volmweer studies
huve clarified the spectrum of shigellosis . Of H group
of volunteers ingestin 10 , 000 Sh . tlexneri 2a
^
bacilli , a quarter remained ibymptqattttic, another
quarter had transient fever a day or two Later, a
quarter developed fever with watery diarrhea, while
tuiJv OrtC quarter developed topical dvscntcrv.
Evpldemiulngy : Epidemics of bacillary
dysenrery have always accompanied wars and ufreu
influenced [ fieii outcome. In several campaigns,
more men have died of dysentery than were killed
LO bardc. A recent instance was the major epidemic
affecting many thousands , with high case fatal itv,
which occurred during the Rwandan civil war Jit
1994, Epidemics in civilian communities are
associated with poverty and Each of sanitation
The only source of infection are human bongs
- cases , ( if less ^ftctl carriers . Chronic carriage is
rare , the bacilli disappearing from feces within a
few weeks* except in some malnourished vhidren
or AIDS patient*. ishigcllac exhibit a high rate of
secondary household transmission . The modes of
transmission may he as fellows : (1) direct, ill rough
-
contaminated fingers : hand -to mouth' infection;
( 2) through fomibes such as door handles , water
taps, lavatory seats; (3) through water; ( 4 ) through
contaminated fiw > d or drink. Skigelhisw , cspcciallv
Sh. sonnei infection , may occur as food poisoning:
and ( 5) through flies which may transmit the
infection as mechanical vectors ; (6) Shigellosis may
occur in young mate homosexuals ns part of the
gay bowd syndrome.
TextbwK of Microbiology
Shigellosis is worldwide in distribution hut
.
epidenuolpgicaUy there arc a lumber nfdifferences
herwren the nature and extent of the infection in
the affluent and in rhe poor countries . Where
environmental sanitation is good , as in Britain,
shigellosis is mainly seen in young children and in
special situation* like mental hospitals (asylum
dysentery ). Sh. SOJinej' it the predominant infecting
agent. In the USA , Sh. sonnei is the main type in
the north , while Sh. Hanoi is more common in
the south . In countries where environmental
mutation is poor, endemic shigellosis is found in
all age groups and is caused by all species. In India,
Sh - tfexnerr has been the predominant species,
having formed .50-B5 percent of isolates in different
series. Sh. dysenteriae ( 8-25 per cent ) and Sh.
sonnet ( 2 ~ 24
per cent ) are the next common specicS-
Sh . toidii (0-8 per cent ) has been isolated least
frequently.
I he picture baa changed in recent years. After
a long period of quiescence, Sh . dysenticrj je type 1
'
suddenly appeared in an extensive and virulent
epidemic form in Central America in 1968, In 1973,
;L similar outbreak started in Bangladesh and later
in Sri Lanlca. Several localised outbreaks were
observed In India from 1974, followed by extensive
epidemics in various Stiles from the early 19ft0?.
I he epidemic strains showed plasmidbornc
multiple drug resistance -
Laboratory diagnosis: Diagnosis is made by
isolating the bacillus from feces. Fresh feces should
be inoculated without delay or transported in a
sulmhle medium such as Sachs' buffered glycerol
-
saline, pi i 1.0 7 . A. Highly alkaline transport media
used for vibrios are inhibitory for shigcllac . Rectal
swabs are not satisfactory.
For Inoculation i r i s best to use mucus flakes if
they (ire present in the sample. MacConkcy and
DC A plates are inoculated. A f t e r overnight
,
incubation at 37 "C, the plates ate inspected for
nonlactose fermenting colonies, which are tested
for motility and b i o c h e m i c a l reactions, Anv
nonmotile bacillus that is urettSC, citrate, H S and
^
Copyrighted material
 4 Eriterabacianacgae ( I: Shig&lla h 289

KCN negative should be curt her i nvest i gated by widely prevalent in shigellae . Indiscriminate
biochemical tests (Table 31.1). Identification is antibiotic treatment will only worsen the problem
confirmed by slide agglutinsbon with polyvalent
. of drag resistance in intestinal bacteria. Antibiotics
and monovalent sera - Demonstm tion of antibodies should therefore be 1 i mited to severe or toy i q gases,
in sera is not usefiil.
, and in the very young, debilitated and the aged.
Trcntmcntz Uncomplicated shigellosis self is a The choice of antibiotic should be based on the
limited condition in which the patient usually sensitivity of the prcvailitig stTain. Many strs i ns are
recovers spontaneously in a few days. However, in still susceptible to nalidixic acid or norfloxacin and
acute cases , particularly in infants and young other fluoroquinoloncs-
children, dehydration has to be corrected promptly. Conlrol; Control consists essentially in improving
Oral rehydration is adequate in most cases. persona] and environmental sanitation. Antibiotics
Routine antibacterial treatment is nor indicated have no place in prophylaxis. No effective vaccine
in dysentery. Multiple drug resistance plasmids axe is available .

further HIM ding

HaleTH . 1999. Bacillary dysentery. In Topley ind Wikon'i Microbiology indMicrobiMilnfccrions . edn . vol . 3. London:
Arnold. ^
-
kru i 11 GT. 199$. Shigellosis. In Harrison's Primdpin oflazcrntl MrcticJu. VoJ.l . 14 edn., New York: Midlriw Hitt .
'

LCWIF MJ - 1997 - Sh igella - In Greenwood D {cd }, JWedica/ AfifiubioJcKy, 15* edn , Edinburgh: Church ill - L -'iugrtDfK -

opy righted material

 CM Enterobacteriaceae III: Salmonella
CO
The b cmis Sitrn&neilx couii -sts ot bacilli that
^
parasitise the iurevrincs nf a large numhcr ol-
ve rtrbratc species and inlcct hum in beings, leading
to enteric fever , gastroenteritis, septicemia with or
without focal sjppLiriiiiiii, and the carrier state .
The most important member ot the genus is
S .-iirrutneHa lyphi , the can native agent ot typhoid
Fever. The typhoid bacillus was first observed by
Ebertb ( IfitfO) in the mesenteric nudes and spleen
oF fatal cases of typhoid fever and was Isolated by
tJafFky ( 1884 ) . It came to be known as the Ebcrth-
tiaffky bacillus or Ehcrthelh lyphi. Salmon and
Smith ( 1K 85 ) described a bacillus which was
believed to cause hog cholera ( mistakenly, as It is a
-
virus di.Hcahc ) . nu: bac illus, later called Y choScrac -
"
HUH, was the first of a series of si mi Ur organ isms
to be isolated from animals and human beings -
-
the genus S^isimut Ha. It was CubHetjUently realised
that the typhoid bacillus also belonged tu this
group, in spite of minor biochemical diFferences ,
and it was: redesignated S . typhi, the genus
Eberthclla having been abolished .
Salmonellae currently comprise above 20 UQ
h c r o t v p e s or Species , :LII uf them potentially
pathogenic, hor practicaJ purposes, they may be
divided into two groups : (1 ) The enteric fever
group, consisting of the typhoid and paratyphoid
bacilli that are exclusively or primarily human
parasites and f 2) the food poisoning group, which
^
are essentially animal parasites but which can also
-
infect human being :, producing patrocntcritis,
septicemia or localised infection!.
Morplmli kgy:5almoncliae ait Cram negative rods,
about 1-3 pm * 0-5 pm in size . They arc motile
with perilrichate flagella , except lor one type,
.
,Y giiltin iriim - pultorum , which is always
non motile. Non motile mutants of other types mav
sometimes be found . They do not Form capsules or
spores but may possess fimbriae.
Cultural characteristics: Salmonellae are
ae n > bi £ and fitultf tively arise n itaic, growing ie adi ly
on simple media over a range of pH 6-S and
—
temperature 15 41 ' C {optimum 37 ' C ) .
C olonies are large, 2-3 mm in dianteter, circulai,
low convex and smooth. Thev are more translucent
I
tfian coliForm colonics . On MicConlcey and
deoxycholate citrate media , colonies are colourless
due to the .absence of lactose Fermentatiou. On
Wilson and Blair bismuth sulphite medium , jet
hl:iL- k cnIonics with a metallic sheen arc formed
.
due t < > production of 11 S. S paratyphi A and other
^
species that do not form H .S produce green
colonies.
Selenite 1' and tetrathionute broth .ire commonly
employed as enrichment media .
EiioobcTmenl rtnOtiMIfc Salmons llav ferment
glucose, mannitol and maltose, forming acid and
gas . An important exception is S. typhi which is
anacrqgenk. Lactose, sucrose and salicin are nor
fermented hidok is not produced They art MR
,
positive, VP negative and citrate positive. Y lyphi
and a lew other sal monel lac do not grow in
Simmons' citrate medium as they need tryptophan
as the growth factor. Urea is riot hydrolysed. H S
is produced , except bv Y paratyphi A, S. cholene
stris and some other stjjecics.
^-
The enteric fever group may he separated
biochemically ('fable 32.1 ) ,
Copyrighted material
 Entenobacieriaceae III : Salmonella t 291

Rcsiyfcmce: The bacilli are killed at 5 ? ‘C in The O antigen is unaffected by boiling, alcohol
one hour or at 60 " U in 1 ? minutes, Boiling fir or weak acids . When mixed with antbcru , O
chlorination of water and pvteurisation o f milk antigen suspensions Form compact, chalky, granular
destroy die bacilli . In polluted water and soil, LILCV clumps . O agghitination takes place more slowly
survive for weeks anti in ire tor months. Cultures and at a higher temperature optimum (St >— 5.S ^C)
may he viable for years if pCCKnul from fining. than H agglutination ( H 7 *C)- The O antigen is
They sue killed within five minutes, by mercuric less immunogenic than the IT antigen and the litre
chloride ( 1 : 500) or 5% phenol. uf the O antibody induced after infection or
Antigenia structure : Salmoncllnc possess the immunisation is generally lower than that of the
following antigens based on hi eh they arc classified ft antibodyr
- .
and identified: (1) flagellar antigen 11, (2) somatic The O antigen is not a single factor but a mosaic
antigen O, and (1) a surface antigen Vi, found in of two or more antigenic factoid. Sulmonelllae aie
some species Several strains carry fimbriae . classified into a number of groups based on the
Fimbrial antigens are nut important in identification 1 n e sei ice of duncteristic O ai i rigens on the banerial
but may cause confusion due tu their nonspecific surface.
nature and widespread sharing among \ i urUigcTi: Many strains of $. typfii tail ro
enterobacteria. agglutinate with the O anti serum when freshly
LI Ulltj £ L:li: This antigen present on the flagella isolated. This is due to the presence of a. surface
is a heat labile protein. Ir is destroyed by boiling <ir poly saccharide antijtcn enveloping the O antigen .
by treatment with alcohol but not by formaldehyde . Felix and Pitt, who first described this antigen ,
When mixed with antisera , H suspensions believed that it was related to virulence anti gave it
agglutinate rapidly, producing large , loose , fluffs the name ' Vi antigen'. It is analogous to the K
clumps. The H antigen is strongly immImogenic antigens ot cdliiorms. It is heat labile . Bacilli
and induces antibody formation rapidly and in high inagglu finable with the O antiserum become
litres following infection or immunisation . The igglutinible after boiling or heating at 60 "C for
flagellar antigen is of a duul nature, oerurring in one hwr. It is also destmved liy N 11C l and O.Ji N
one of two phases. NaOH It is unaffected by alcohol of 0.2% formal.
p

O antigen: The somatic O antigen is a Originally observed in S typhi , the Vi

phospholipid-protein- polysaccharide complex antigen with similar antigenic specificity is present
which forms an integral part of the cell wall. It is in 5. pzmtyj hi C and S. dtiblin, a£ Well as LII certain
’
identical with endotoxin . It can be extracted from strains of Citrobacrer ( the Billerup-Bettueidi
the bacterial cell by treatment with trichloracetic group) . The Vi antigen tends to be lust on serial
acid, as first shown by Boivin ( and therefore called subculture . " I h e Vi polysaccharide nets :LS a
the Boivin antigen) . Treatment with phenol splits virulence factor by inhibitingphagocytosis , resisting
off the protein moiety, removing the iniigenkity compilement activation and bacterial lysis by the
but retaining the toxicity of the complex . alternative pathway and peroxidase mediated killing,
Table 32.1 Biochemical characters o! typhoid and paratyphoid bacilli
Gfocvtt Xyfift d-Tartrafe AfULflCT

S, typhi A d A d
Sr purmphi A AG — —
S. paratyphi B AC AG — AG
5- paratyphi C AG AG AC

Copyrighted material
292 TRxtbook or
*
In human volunteer experiments, steal ns possessing
the VI antigen were found to cause clinical disease
more consistently than those lacking it.
The Vi antigen L ? poorly immunogenic and only
low rirres of antibody are produced following
infection . No Vi antibody s induced by the
]
phenol iscd vaccine though low times arc produced
by' the alcoholiscd vaccine, 'Hie protective efficacy
ot tile Vi antigen is demonstrated by the success of
the purified Vi vaccine tor typhoid now in routine
use. Detection of the Vj antibody is nor helpful for
the diagnosis of cases and hence the Vi antigen is
not employed in the Widal test . It has bccti stated
that the total absence of the VI antibody in a proven
case of typhoid fever indicates poor prognosis . The
antibody d] sappears early in convalescence- Its
persistence indicates the development of the carrier
state. The VI antigen affords a method of
epidemiological typing of the S. ryji/ij Strains based
on specific- Vi bacteriophagies.
ANNUL- ]lie variation*: The antigens of salmo-
nellae undergo phenotypic and genotypic variations.
H- { ) variation: This variation is associated
with the loss of flagella . When salmonellae are
grown on agar containing phenol ( 1:HG0) , flagella
are inhibited. This change is phenotypic and
temporary. Flagella reappear when the strain is
suhcultured on media without phenol . Rarely,
salmonellae may Lose their flagella by mutation.
A stable nonmorile mutant ofi. typhi a the 901-0
"
Strain which is widely employed tor the preparation
of O - agglutiraHe bacterial suspensions . Generally,
the loss of flagella is not torsi and there occurs
only a diminution in the number of flagella and
the quantity of the H antigen . Flagellated cells arc
found in small numbers in such cultures. To obtain
S population of motile cells rich in H antigen , from
*
such cultures, selection may he carried nut by using
Craigic ' s rube . This cunsisti of a wide tube
containing soft agar (0.2*Hi) at the centre of which
is embedded a short, narrow tube open at both ends,
in such a way that it projects above the agar. The
strain is inoculated carefully into the inner tube .
M exobiology
m
w
.
Fig 32 i Craigie s tube . Inoeulslion s made inside th :
inner lube and after incubation, subculture is isksn
from the surface ol Ihs medium n lha oultr lube
After incubation, subcultures withdrawn from the
top of the agar outside the central tube will yield a
population of motile cells { Fig, 32,1k I instead of
Craigic ' s tube , a U - tubc of soft agar may he
employed* inoculation being made into one limb
and subculture taken from the other .
Phase- variation: The flagellar antigens of most
salmonellae occur in one of two phases that is , [ lie
*
flagella may exhibit one or the other of two
alternative sets of antigens defined hy two separate
*
sets of genes in the bacterial genome. Phase 1
antigens arc cither specific for a SIH.-LIO or shared
by a few species only. E Icucc phase I is called rhe
'specific ' phase. Phase 2 antigens are widely shared
and hence phase 2 is called the 'nonspecific’ or
‘
group phase - Phase 1 antigens arc designated a, h,
1
cL , d , etc. , and after I, as ill , z2 , Ctt . Fhasr 2 antigens
art far fewer and urc termed 1 , 2 etc. In some
*
species, antigens belonging to phase 1 mav occur
.
as the phase 2 antigens ( for example, e n, zlS ) .
Strains thar possess both phases are called dipiuiyii.-.
Some, tike & typhi, occur in phase 1 only ami art
called morlOphahiu.
Copyrighted material
 1 Entorobaetenaeeae ill Salmonella *
A (.illume will contain. LL'IIS with the flagellar
antigens of both phases but generally One Or the
other phase wi]] predominate so that the culture is
agglutinated only by one of the phase antisera . For
serotyping of Salmonella isolates , it may be
necessary to identity the flagellar antigens of both
phases. A culture in phase 1 can be converted to
phase 2 by passing it through a Craigic's rube
containing specific phase 1 antiserum, and the
reverse conversion achieved by using phase 2
antiserum .
\ variation; Fresh isolates of S, typhi
generally carry a surface layer of Vi antigen that
completely musks the O antigen . Such bacilli arc
agglutinable with the Vi antiserum but not with
the O antiserum. This is called the V form. After a
number of subcultures, the V] antigen is completely
Lost. Such cultures arc inagglutiiiable with the Vi
antiserum but readily agglutinable with rhe O
antiserum . This is called the Wform. Intermediate
stages during the loss of the Vi antigen , when the
bacillus is aggluti nable wi r b both Vi and O antisera,
arc called ' V\V forms’,
Ocher Vi-conraining bacilli such as 5. paratyphi
C anti S . duhlin seldom have the O antigen
completely masked by the Vi antigen .
S R variation: The smooth -to- rmjgh variation
is associated with the change in the colony
3 . iinalum
I . ysopen lotion
S. ncvLinginn
LysogcnuMHl
S . Tniuneupohs -
Fig. 32.2 Phage conversion ol Salmonella serotypes
293
morphology and loss of the O antigen and of
virulence. The colony becomes large , rough and
irregular. Suspensions in saline arc
auto aggluti nable. Conversion into R forms occurs
by mutation. R forms may be common in laboratory
strains maintained by serial subcultivation. S - R
variation may be prevented to some extent by
maintaining cultures on Dorset 's egg media in the
cold , ot ideally by lyophilisition.
Mucoid colonies, associated with the
development of a new mucoid or LM’ antigen , have
been described with S , paratyphi B and some other
species.
Variations in O antigen: Changes in rhe
Structural formulae of the Q antigen may be 1nductd
by lysogcnisatEon with some converting phages,
resulting in the alteration of serotypes . Thus, $,
anatum is converted into S. newington by one
phage and the latter into S, jiajjmeapoLs by another
phage ( Fig, 32,2).
Classification and nomenclature: The
classification and nomenclature of salmnnellac have
undergone several modifications over the years.
Inclusion in the genus is based on common
biochemical properties. Classification within the
genus is on antigenic characterisation based on the
Kauffmajin-White scheme. This scheme depends
on the identification, by agglutination , of the
scrOiype 10: c, h: l , ft.
with ptli e 15
^
veretype 3. 15 : c . h : 1.6,
Wltll pllJjiJC 34
serotype 3. 15.34: 6» "h: I. 6
Copyrighted material
294 * Te* ttHH3 k ot Microt^ ology *

Table 32.2 Kiufrrsnn- WhLte scheme — iIllustrative samples

SetOgfcMtpt SerafVjfX- Antigen O Aotfgtn FI
2 A
j- B
S. paratyphi A
S. paratyphi B
S. Eyphinsnrium
1, 2, 12
1.4. 5 h 12
Phase i
2
b
L
—Phase 11

1.2
1.4, .5 , 12 1.2
S, cAesctr J , 5, 12 e, h E n, x
7 Cl
- S' paratyphi C
, &, 7 , ( Vi) c 1.5
S. choleric-sun 6s 7 c 1.5
S - C2 S' rrruejfcifwn 6, 8 d 1.2
-
9 D 4, 12 , ( Vi ) d -
S. trtKfitidis
Sr gpHmamm
1, 9, 12
1, 9, 12 - ——
10 - £1 S' amtum
,
1 10 e, h 1.6

structural formulae of the G and H antigen* ofthe
strains {Tabic 32,2).
Salmonellae at Initially classified into
ierob icil groups , based on the presence of
^
distinctive O antigen factots, which ire designated
inn > four subgcnera (Table 32.3):
ubgrjjtrs /, the largest and medically the most
^
important group contain* all the species which
commonly cause human and animal infections.
hah U contains mostly specie
; isolated
^ 3
enuf

1, 2 , 3, etc . Strains possessing factor 2 belong to from reptiles .

group A, factor 4 to group Bj, factor 9 to group D Subgeous Hi copra ins bacilli * formerly
and so on . Scrogroups were originally designated designated Arizona, originally isolated from lizards
by Lapin] letters, A to Zpand afterwards by numbers hut subsequently found in reptiles, birds, domestic
- currently npty f ,7. It would he more logics] To animals and human beings. Many of them arc
name the serogroups according to their prompt lactose fermenters.
characteristic O antigen factor numbers, rather than SuAgesus IV strains arc rarely encountered and
by letters. It Ins therefore been proposed to , may be considered atypical member* of subgCOUS ]|.
c
designate group A as 2 , B as 4, 1 is 7, C2 as S, ( ) Kwing proposed that only three species should
as 9 and so on. be recognised in the genus SitlinuneUs- S . choferae -
Within each group, differentiation ol serotypes StUff, H . f >pi) r and ji. errfcnndw - all other species
is by identification of phase 1 and 2 flagellar being considered serotypes ot S . enterfEHiis. TTiis
. -
latignu The Kauffmann Wlute scheme gave proposal is now not followed .
species each *erotypc . The species were
status to
Table 33.3 Siuchemicjl reactions ol Salmonella
named according to the disease caused ( £ cyphi), suboenera
the animal source [ S. gzlhnanum ) , the dirrtwtrtr
Test Siifejgfnerj
( S. ichottmulkri) , the name of the patient from
whom the first strain was isolated (£. rbompsorf ),
; u m IV
or the place of isolation ( f >. ptumn ) . This was Lictow
Dukitul
— — + —
—— ——
satisfactory so long as rhe serotype* were not tBO
d -Turtrate
4

4
— #

-- - —
many but now with some 2400 serotypes of
salmoncllae, giving individual names isuoi realistic.
On the basis of biochemical reactions ,
KaufFmann proposed that salmoncllae be classified
Milonare
Sol Lein
KCN — —— # 4'

-
+
+

Copyrighted material
 * EntiefDtiaciBriaD V
' '
n III: Salmonella
Modern tajconnm ical techniques, especially
DNA studies, have shown that all the members of
tin genus Salmuneifae and of the funner genus
Arisema are so closely related that they should all
be considered as belonging to a single species, in a
genetic , phylogenetic and evolutionary sense.
Variations in properties such as antigenic structure,
biochemical reactinns and host preferences
exhibited by d liferent strains can he considered
intraspecies divergences. A new species name
i. eHten i’ii has been coined to include all
' '
-—
salmonellae. S. enterica i classified into seven
subspecies based on DNA reussut latum tests.
These subspecies are named enrerica , naiamac,
a.r / zonae. didrizotae* houtcnac, bongori and indict
Subspecies ertrenca corresponds to the former
submenus T.
Such classification and nomenclature , while
being taxonomic ally correct , would be loo
complicated for use in clinical bacteriology. For
example, the taxonomically correct name for the
typhoid bacillus would he * Salrrumella enterjca,
subspecies enteric a , serotype typin . Therefore, the
'
old pracucc of referring to clinically imports or
salmoncllae serotypes by the species name continues
m clinical bacteriology.
Sometimes serotype* may have to be further
differentiated. Thus, S. gallinanun and S. puiloruiA
cannot be distinguished serologically but they can
be idenrified by biochemical reactions ( S , .
galiinanim is anaerogenic and ferments dtdciiol
unlike ft . pul/firum]. Important pathogens such as
S. lyphif S. pafxtyphi A and B, and S . typhimurium
can be further typed for cp^ icrniolog ^ cal purposes
by phage susceptibility, biochemical properties ,
bacterociti production and antibiogiam,
I Vti hogtnicityt Salmonella* are strict parasites
of animals or humans. S. typh\ t S , para pfu A and
^
usually, hut not invariably S . paratyphi B are
confined to human beings. Other salmoncllae are
parasitic in various animals - domestic animals,
—
rodents, repifles - and birds. Some species axe host
adapted S. abuttus-rqiir found only in horses, Sr
295
abortus-nvis in sheep and S.gulWnanjir; in pouitr v.
Others such as S. typhimurium, have a wide hcM
range affect ing animals, I 'i rds and humans. lniL L [ iofl
in animals may vary from an asymptamit ic
conditi on to fatal, and someti mes cpi ^ootic di seasc.
,\ typhimurrum and 5. cnKtiridis cause a fatal
septu emi .i in rats and mice. S- putfanim causes
'
'white diauhea in chicks and 5- galiinarvm fowl
'
typhoid.
Salmoncllae cause the following clinical
syndromes in human bemgsi ( I ) enteric fever
( 2) septicemia, with or without local suppurative
-
Ir ionsj and (3 ) gastroenteritis or food poisoning.
ENTERIC FEVER
The term enteric fever Includes Typhoid lever
caused by ft. Typh; and paratyphoid fever caused by
V p.ojrcphi A, B and C.
Typhoid fever was once prevalent aii er the H
world and was not well demarcated fr other
prolonged fevers. A detailed study of the disease
was presented by Bretnnneau (1 j who Jn m 11 tied
the intestinal lesions. The name typhoid wa > u cn
^
by Louis (1829) to distinguish it from typhus fewer.
Budd (1856 } pointed out that the dtsejic was
transmitted through the excreta of patients, Ebetth
(i 860) described the typhoid bacillus and Gafftcy
-
( 1884 ) i olated ii in pure culture. Its causative role
was confirmed by Mete bn i kntff and Besredka (1900 )
hy infecting apes experimentally, S- paratyphi A was
isolated by Gwyn (1898) , S, paratyphi B ( £.
.schoftmuMcn) by Achard and Rcnsaude (189fi ) and
S. paratyphi C ( S. frrahfcjtfjf ) by Uhlenhuth and
Hubcncr (1908 ) from cases resembling typhoid
fever.
Thc infection is acquired by ingesnon . In
human volunteer experiments, the ID50 was found
be about Iff to 10" bacilli . On reaching the gut ,
to
the bacilli attach themselves to microvilli of the
ileal mucosa and penetrate to the lamina propria
and submucosa - They are phagd rtosed there by
^
polymorphs and macrophages ^ The alulitv to resist
intracellular killing and to multiply within these
Copyrighted material
II JR 'ligR
296 i Twcttxnfc of Microbiology *
cells is a measure of theb virulence. 'I'hey enter the
mesenteric lymph nudes, where chey multiply and ,
via the thoracic duct , enter the bloodstream. A
transient bacteremia follows , during which the
h.icilh are seeded in the Liver , bladder spleen,
, relapse rate is higher in patterns treated early u i.ch
bone marrow, lymph nodes, lungs and kidneys,
where further multiplication takes place . Towards
the end of the incubation period, there occurs
massive bacteremia from these sites of
multiplication, heralding the onset of clinical
disease .
A & hilt ii a
"
good culture medium fur the
bacillus , it multiplies abundantly in the gall bladder
and is discharged continuously info the intestine
where it involves rhe Feyer’s patches and lymphoid
follicles uf the lie urn . These become inflamed,
undergo necrosis and slough off. leaving behind
the characteristic typhoid ulcers. Ulceration of the
bowel leads Co the two major complications of the
disease - intestinal perforation and hemorrhage,
DLLIMG the 5-4 weeks that normally constitute the
course of the disease , [ he intestinal lesions undergo
healing-
The incubation pcr: id is usually 7-14 days but
may range from 3-56 days and appears to be related
to rhe dose of infection . The clinical course may
vary from a mildundiflfercnt iated pYrw i a (ambulant
typhoid ) to a rapidly fatal disease. The onset is
usually gradual , with headache, malaise, anorexia ,
a coated tongue and abdominal discomfort mth
cither constipation nr diarrhea. The typical features
are a step-ladder pyrexia , with reladve bradycardia
and toxemia. A soft, palpable spleen is a constant
finding. J Jepatomegaly is also common. 'Rose spots
that fade on pressure appear on the skin during the
second or third week but are seldom noticeable in
dark skinned patients.
The most important complications are intestinal
perforation, hemorrhage and circulatory collapsc.
Some degree of bronchitis or bronchopneumonia
\ < always found. Some develop psychoses, deafness
or meningitis. Cholecystitis, arthritis , abscesses,
peri osteitis, nephritis, hemolytic anemia , venous
thromboses and peripheral neuritis are other
complications found . Osteomyelitis is a rare sequel.
Convalescence is slow. In about .5-10 per cent
of cases, relapse OCCUR during convalescence - The
chloramphenicol ( 15-50 per cent ).
S . paratyphi A and B cause paratyphoid fever
which resembles typhoid fever bui is generally
milder. 5. paratyphi C may also cause paratyphoid
fever but more often it leads to a fbmk septicemia
with suppuradve complications. Other salmonellae
have on occasion been repotted to cause enteric
fever. These have included S. dublint S. baricUy^ S.
sendai . 5. enfe .nr.'dis, £ typhimvrium, S- eastfcnume ,
S . saintpaul , S . oranienburg and S . panama .
Infection with Alkaligenes fiecalit also may
sometimes cause a similar clinical picture .
Epidemiology: Typhoid fever has been virtually
eliminated from the advanced countries during the
last several decades mainly as a result of
improvement in water supply and sanitation hut it
,
continues to be endemic in the poor nations of
the world . The control of paratyphoid fever has
not been so successful . The distribution of
paratyphoid bacilli shows marked geographical
differences. £ paratyphi A is prevalent 11 L India and
other Asian countries, Eastern Europe and South
America, £ paratyphi B in Wfestcrn Europe, Britain
and Notch America; and S. paratyphi C in Eastern
Europe and Guyana.
Enteric fever is endemic in all parts of India.
An incidence of 930 per 100,000 was recorded in
the late 199Gs in a 5-year community based study
of children in Delhi . Worldwide, 16 million cases
are estimated to occur annually with 600 ,000
deaths! The proportion of typhoid to paratyphoid
A is about 10: 1 . Paratyphoid B is rare and C very
rare . The disease occurs at all ages but is probably
most common in the 5-20 years age group. The
age incidence is related to the eadecnidty of the
disease and the Level of sanitation.
The source ol infection is a patient , or far mote
frequently, a carrier Patients who continue to shed
Copyrighted materia
 < EntoiotacteriKWB III: Salmonella *
typhniJ hacilli in fcccs far three weeks To three
month* after clinical cure are called conwafescMf
current Those who shed the bacilli for more than
three months but less than a year are called
' temporary' carrion' and those who shed the bacilli
for over a year are called 'chronic Cartiers ', About
-
2 4 per cent of patients become chronic carriers.
The development of ( he carrier State is more
common in women and in the older age groups
(over 40 years ). Some persons may become earners
following inapparent infection (syinptamleSs
eret < ] r ) . 1 he shedding ( bacilli is usually
intermittent. The hacilij persist in. the gall bladder
or kidney and arc clin iinatcd in the feces ( fecal
'
carrier ) or urine ( urinary carrier ), respectively.
Urinary eairiage is less frequent and iv generally
associated with some urinary lesion such as calculi
or schistosomiasis
Food handlers or cooks who become carrieth
ate parti iTularly dangerous, The best known of such
typhoid carriers was Mary Mai Ion {'Typhoid
Mary „ a New York cook who, over a pc ri ; >d of 15
years, caused at least seven outbreaks affecting over
200 persons.
Carriers occur with paratyphoid bacilli also.
While & paratyphi A occurs only in human beings,
S . pimrypbi B can infect animals such as dogs or
cow*, which may act as source* of human disease.
Typhoid fever occurs in two epidemiological
types. The first is endemic or residua] typhoid that
occurs throughout the year though seasonal
variations may sometimes be apparent. The second
is epidemic typhoid , which may occur in endemic
or nonendemic areas. Typhoid epidemics are water,
milk or foodborne .
Laboratory d inruns is : Bacteriological
diagnosis of enters fever consists of the isolation
of the bacilli from the pat i LLnt And the demonstration
of antibodies in his scrum , A positive blood culture
is diagnostic, while the same significance cannot
be attached to isolation from feces or urine .
Demonstration of antibodies is not conclusive
evidence of current infection - A thud method is
297
the demonstration of typhoid bacillus antigen in
hiood or urine.
Dkiid culture: Bacteremia occurs early in the
disease and blood cultures are positive in
approximately 90 pci cent of cases in the first week
of fever. The popular belief that blood culture for
diagnosis of typhoid fever is useful only in the first
week i * erroneous . Blood culture is positive in
approximately 75 per cent of cases in the second
week, 60 per cem in. the third week and 25 per
cent thereafter t i 111 the subsidence of pyrexia. Blood
cultures rapidly became negative an treatment with
antibiotics.
About 5-10 m! of blood is collected by
venepuncture and inoculated into a culture bottle
—
Containing 50 100 ml of 0.5 per cent bile hnotb.
Blood contains substances that inhibit the growth
of the bacilli and hence it is essential that the broth
be taken in sufficient quantity to provide at least
fourfold dilution of blood. The addition ofliquoid
( sodium polyanethul sulphunale ) counteracts the
bactericidal action of blood.
After incubation overnight at 37 BC , the bik
broth is subculturcd on MacConkey agar. Fak
nonlactose fermenting colonies that may appear on
this medium arc picked out for biochemical tests
and motility, Salmonellae will be motile , indole and
urease negative and ferment glucose, mannitol and
maltose but not lactose or sucrose. The typhoid
bacillus will be anaerogenic, while paratyphoid
bacilli will form acid and gas from sugar*.
Identifier ion of the isolate is by slide agglutination.
A loopful of the growth from an agar slope is
emulsified in two drops of saline on a slide. One
emulsion acta as a control to show that the strain is
nor aucoagglutinable. If S. lyphl is suspected ( that
is, when no gas is farmed from glucose ), :L loopful
of typhoid O antiserum (factor 9/group D ) is added
to one drop of bacterial emulsion on the slide, and
agglutination looked for after rocking the slide
gently. Prompt agglutination indicates that the
isolate belongs to Salmonella group D. Its identity
a$ $. ryphi is established by agglutination with the
Copyrighted materia
296
*
fffgpgap antiserum (ami-d scrum). Quite often , fresh
isolates of S, tvphi lire in rhe V k >nn and do not
agglutinate with the O antiserum. Such strains may
he tested for agglutination against anti-Vi scrum.
Alternatively, growth h ttl ed off in :L mull
*
^
amount of saline boiled tor 20 minutes and tested
for agglutination with the O unrihenLcn . Where the
isolate is a noatyphoid Salmonella (producing g.is
from sugars}, it is rested for agglutination with O
:md H antisera for groups A , B and C. For
i dentilration of unusual serotypes, the help of the
National Salmonella Reference Centre should be
sought . The National Salmonella Reference Centre
In India is located at The Central Research Institute,
,
KasauEi . The reference centre tor salmoncll ie of
.MUII.II origin is at rhe Indue Veterinary Research
Institute, Ir..irnagar.
If salmorcllae ate not obtained from the first
subculture from bile broth subcultures should be
*
repeated every other day rill growth is obtained.
Cultures should He deelured negative only after
incubation fur leu days. To etiinmate the risk of
introducing contamination during repeated
subcultures, mid also tnr economy and safety,
Castaneda's method of culture may be adopted, In
tIns, a double medium is used. The bottle of bile
.
broth bus ,m igur slant on one side . After
inoculation of blood, the bottle is incubated in the
upright position.For subculture, the bottle is merely
tiEted HO that rhe broth runs over the surface of the
agar, Ir is reincubated in the upright position- If
salmoncllac ire present, colonies will appear on
the slant.
An altemuri vt ro blrrod tuI ti i re is rhe dot < i 11 ture.
Here , 5 ml of blood is withdrawn from rhe patient
into a sterile test tube and allowed to dot. The
senam is. pipetted oft ami used for the Widal rest .
The clot is broken up with a sterile lass rod ami
^ culture .
added In a hottle of bile broth. The incorporation
of streptokinase ( 100 units per ml) in rhe broth
facilitates lysis of rhe clot. Clot cultures yield a
higher rate of isolation than blood cultures as the
bactericidal action of [ he serum is obviated .
Textbook of Microbiology *
Another advantage is that a sample of scrum also
becomes available. Even though agglutinins may
be absent in the early stages of the disease, a Widal
tesr provides a bascl i nc ri r re against wh ich the results
of tes.CS performed Idler may i;: - " evaluated.
Feoes culture: Salmondlac are shed in feces
throughout die course of the disease andl even in
convalescence with varying frequency. Hence fecal
*
cultures: are almost as valuable as blood cultures in
diagnosis. A positive fecal culliire , hiTwever* nwv
occur in carriers as well as in patients, d bc use ot
^
enrichment and selective media and repeated
sampling increase rhe rare of isolation Fecal culture
is particularly valuable In patients on antibiotic as
. the drug LWS nor eliminate the hacilEi from the
*
guc as rapidly as it does from the blood.
Fecal -samples arc plated directly on. MacConkev,
DCA and WUson-Blair media . The last is highly
selective and should be placed heavily. On
MarConkev anil DCA media, nalinorirllaje appear
-
as pair Color i L H. OO the Wilhon-Rlair medium,
5. rty Ju forms large black colonies, ivith a metallic
.
sheen . S. ffcirjfyphj A producer green colonies- due
tothe absence of H ,S production.
For enrichment, specimens Are inoculated into
one tube each of selenite and recratliionute broth
*
and incubated tnr 12-1 H Knurs before subculture
ontoplates.
Urine culture: SalmonetIae are sEied in the
urine irregularly ini lntre< ] Lientlv. Hence urine
culture is less useful than the culture of blood
or feces. Cultures are generally positive only in
the second and third weeks and then only in
ab^ut .
yer cent of cases Repeated sampling
improves the rate of isolation. Clean voided urine
samples are centrifuged and the deposit inoculated
into enrichment and selective media as for fecal
Other materials for Cullure: Isolation may
he obtained from several other sources but they are
not usually employed. Bone marrow culture is
valuable as it is positive in most cases even when
blood cultures are negative. Culture of hi Le obtained
Copyrighted materia
 * Entergbacleriaceae
by duodenal aspiration is usually positive and may
be employed lor the detection of carriers . Other
materials which may yield isolation at times are
nose spots, pus from suppurative lesions, CSF and
sputum. At autopsy, cultures may be obtained from
[ he gall bladder, livef , spleen acid mesenteric lymph
nodes.
\S itliil reaction: This is a tent for the
measurement of H and O agglutinins for typhoid
and paratyphoid bacilli in the patient 's sera. Two
types of Cubes are generally used for the tesfr"^
narrow tube with a conical bottom ( Drcycris
agglutination tulie ) for the ]]
agglutination , and a
short round bottomed tube ( KclLx tube ) for the O
ugglutination . Ecjual volumes ( ( 1.4 m 3 ) of serial
dilutions of the scrum (from 1/ 10 to 1 /640} and
the H and Q antigens art mixed in Dreyer 's and
Felix agglutination tuhes , respectively and incubated
in a water bath at 37 *C overnight. Some workers
recommend incubation at 50-55 C tor two hours ,
followed by overnight incubation at room
temperature. Control tubes containing the antigen
and normal saline arc set to check for
autoagglutination . The agglutination litres of [ he
serum are read. II agglutination Leads to the
formation of loose , cotton woolly chimps, while O
agglutination is seen as a disclike pattern at [ fie
bottom of [ he tube . In both , the sujiernatant fluid
is rendered clear.
The antigens used in the test are the H and O
antigens of 5. and [he H antigens of .Si parafyphr
A and H . The paratyphoid ( f antigens are not
employed as they CTOSS-icact with the typhoid O
antigen due In their sharing of factor 12. The II
agglutinable suspension is prepared by adding 0.1 %
formalin -
to a 24 hour hmth culture or saline
suspension of an agar culture- fr> r preparing the O
suspension , the bacillus is cultured on phenol agai
( 1:1100 ) and the growth Scraped off in a small
volume of saline. It is mixed with 20 times its
volume of absolute alcohol, heated at 40-50 aC for
20 minutes, centrifuged and the deposit resuspended
in salhie TO the appropriate density. Chloroform
III : Salmonella 299
may be added as a preservative. It is important to
use standard smooth strains for antigen prejtararicm .
The strains used usually are the 5. iyphr 901 , 'O'
and ' If strains . Karh batch of antigen should he
compared with a standard . Readymade Widal kits
.iI stained antigens available commercially are now
widely used ..
r
The results of the Widal tost should he
interpreted taking into account the following:
1 . The agglutination titre will depend on the stage
of the disc asc. Aggluti nins usually appear by the
end of the find week, so thnt blood taken earlier
may give a negative result. The litre increases
steadily rill the third or the fourth week, after
which if declines gradually.
2. Demonstration of a rise in titre of antibodies, by
testing two or more serum samples, is more
meaningful than a single test , If the first sample
is taken laic in the disease , a rise may not be
demonstrable. Instead , a fall in titre may he seen
in some cases.
3. The results of n single test should be interpreted
with caution . It is difficult toby down levels of
significance though it Is generally seated that
tit res of 1/10Q or more for O agglutinins and 1 /
200 or mom for H agglutinin? are significant.
IL is necessary co obtain information on the
distribution of agglutinin levels in ‘normal sera'
in different areas,
4 . Agglutinins may be present OIL account of prior
disease , in apparent infection or immunisation.
Therefore, the mere presence of agglutinin in
flic Widal test should not be taken as proof of
typhoid fever.
H agglutinins persist longer than O agglutinins .
Semm from an individual pmiOUiuwd with TAB
vaccine will generally have antibodies to -5’,
typhi, S. paratyphi A and B, while in case of
infection antibodies will be seen only against
the iniecting species.
5. Persons who have bad prior infection or
immunisation may develop an anamnestic
response during an unrelated fever. This may
Copyrighted material
300 ^ Textbook d UiorabiDbgy
bcditferciriated by repetition of the test after a
wreck. The anamnestic response shows only a
transient ri '-e, while in enter 1 , - fever the rise is
sustained.
6. Bacterial suspensions used as amigens should be
free from fimbria. False positive results may
otherwise occur .
7. Cases treated early with chloramphenicol may
show a poor agglutinin response.
Other serological met hods of diagnosis include
indirect hemagglutination , CIE and ELISA ,
i JnHar^ s r R A T i o N OF CIRCULATING
ANTIGEN
Typhoid bacillus an Ligetis are consistently present
in the blood In the early phase of the disease, and
also in the urine of patients. The antigen can be
demonstrated by sensitised Staphylococcal
coagglutination test. Staph surtus (Cowan I strain )
, media.
which contains protein A , i * stabilised with
formaldehyde and coated with 5- typhi anrihody.
When a 1% suspension of such sensitised
staphylococcal cells is mixed on a slide with serum
from patients in the first week of typhoid fever, the
typhoid antigen present in the scram combi nes wi rh
the antibody attached to staphylococcal cells
produc i ng vi sible agglut i nation wi:h i n two m L uutes.
The test is rapid, sensitive and specific but is not
positive after the first week of the disease.
Other laboratory tests: A white cell count
is useful Lcticopenia wirh a relative lymphocytosis
is seen . Eosinuphili are said to be absent bolt in. the
tropics., with a high incidence of helminthic
infestation, eosinophils are usually present .
D l x i i N O S l S O F l A u u i l- ltS
The detection of carriers is important for
epidemiological and public health purposes.
Laboratory tests are also useful in screening food
handlers and cooka to detect earner state.
The identification of fecal carriers i -. by isolation
of the bacillus from feces or from bile. The
fretpency and intensity' of bacillary shedding vary
widely and if is essential , therefore, to test repeated
sample^. . Cholagogue purgatives increase the chance
of isolation. For the detection of urinary carriers ,
repeated urine cultures should he carried out.
The Widal reaction is of no value in the
detection of carriers in endemic countries . The
demonstration of VI agglutinins has been claimed
to indicate the carrier state. While this is useful as
a screen ing test , confirmation should he made bv
culture.
The tracing of carriers in cities may be
accomplished by the 'sewer-swab1 technique. Gauze
pads left in sewers and drains are cultured , and by
tracing positive swabs, one may be led to the house
harbouring a carrier. Another technique of isolating
salmonellae from sewage is filtration through
millipore membranes and culturing die membranes
on highly selective media such as Wilson and Blair
B M :TBRROPH *GB TVPING
Intraspecies classification of S, typhi fot
epidemiillogical purposes was made pOStihle by
bacteriophage typing, first developed bv C raigic and
Yen (1937 ). They found that a bacteriophage acting
on the Vi antigen of the typhoid bacillus [Vi phage
II ) irn highly adaptable. The parent phage is called
phage A It could be made specific for a particular
strain of typhoid bacillus by serial propagation in
the strain . Such adaptation was obtained by
phenotypic or genotypic variation. At present, 97
Vi II phage types of S. rtp/ti are recognised . As
phage typing of S . typhi depends on the presence
of Vi antigens, a proportion of strains ( Vi negative )
will be untypable . The phage type is stable. Apart
from helping in tracing the source of epidemics,
phage typing also provider information on the
trends and patterns in the epidemiology of typhoid
at the local , national and international levels. Phage
typing is carried out at the National Phage Typing
Centres and is coordinated by the International
Reference Centre . The National Salmonella Phage
Typing Centre fur India is located at the Lady
Copyrighted materia
 H Enterabartariacflae III . Salmonella
Harding Medical Collegeh New Uclhi , Phage
types A and El are the most common and are
present throughout India. However* rhe relative
prevalence in different regions* in subiect to change
from time to limic.
The preponderance of one or two phage types
in a region limits the utility of phage typing as
.
an epidemiological tool Additional markers
have, therefore, been employed for the -subdivision
of strain H belonging It ) a phage type . These include
(1) Ni.CoU.cS complementary phage tvping of
type A strains into 10 Types; ( 2 ) Krisfenscns
biotyping based on fermentation of xylose and
arabinose; (3) production oftctrithiomte reductase;
( 4) bacteriocin production; and ( 5 ) antibiogram.
Currently , more discriminating genotyping
methods like phasmid finger printing, muhiLocu ?
enzyme electrophoresis , 1S-20Q profiling and
random amplified polymorphic DNA analysis haw
been employed lor epidemiological characterisation
in advanced centres .
Phage typing has been applied also tn S, paratyphi
. .
A and B, S typhimurium , 5 enteritidis and 5,
Dublin , Among the S. pa ^ iryphr A isolated from
India, phage types 1 and 2 are the most common *
Prophylaxis: Typhoid fever can he effectively
controlled by general measures , such as
improvements in sanitation and provision of
protected water supply. Many developed countries
have been able to eliminate the risk by rhese
measures, but occasional outbreaks do appear due
to unforeseen lapses.
Specific prophylaxis with hear killed typhoid
bacillus vaccine was developed and successfully field
tested by Almrorh Wright during the Hner war in
South Africa . The TAB vaccine which came into
general use later contained S. iyphit 1000 million
and S, pwtyphi A and B, 750 million each per ml
killed by bearing at 50-60 "C and preserved in
0,5 % phenol.
The use of polyvalent TAB vaccine was an
. . It was introduced in that form
accident of historyJr
in World War I, is British troops had to serve in
» 301
various parts of Europe, .Africa and Asia where
typhoid and paratyphoid fevers were endemic. No
controlled held trial has been conducted with the
TAB vaccine . In civilian practice , protection is
mainly required against typhoid fever. If paratyphoid
components are considered necessary either A or
H may be added, but not both, as only one of them
is found in any one area , 'nicrcfbrc, in India , instead
of the TAB vaccine , a divalent typhoid-paratyphoid
A vaccine {eliminating paratyphoid B which is very
rare in the country ) or the monovalent typhoid
vaccine is prefemed -
The vaccine is given in two doses of 0.5 mJ
subcutaneously at an internal of 4-6 weeks. Local
and general reactions lasting for one or two days
are |L ui [e frequent . Such reactions may be avoided
if the vaccine is administered in a dose of 0-1 ml
intradermally. In nonendcmic areas, vaccination is
recommended foe troops, medical and paramedical
personnel . In endemic areas vaccination is
recommended for ail children , in whom a single
dose might give adequate protection , which may
1001
t T
A
so:
601
\ 0
4G; Id
v
20J -
I I E I
i !3
1
4 6
FJg.&.3 Laboratory magnusis of typhoid lever The
approximate percentages or tests found positive
during diflerenl stages ol the disease { from 1 st to
5lh week I . A , Widal agglutination . 6. feces culture.
C . Blood culture,
Copyrighted material
302 * Textbook oJ Microbiology
he maintained for several years by the booster tflfect
a a
of repeated natural subelmicaJ infections.
Two new typhoid vaccines, one oral and the
other inject able , have been introduced after
successful field 1 rials. Vh.c live oral vaccine ( typhonl)
mutant ). On
- - —
if a stable imitanc of 5, typht strain Ty2 la, lacking
the enzyme UTVP gaJsLctote 4 ejiimtfvire (Gal F.
ingestion , it initiates infection but 'self
destructs" after four or five cell divisions , and
therefore cannot induce any illness. The vaccine LH
^
an enteric centred capsu containing 10’ viable
lyophilised mutant bacilli. The course ctinsists of
one capsule orally, taken an hour before food, with
a glass ol water or mi ]];, on days ], 3 and S . Wo
antibiotic should be taken during tins period .
The injectable vaccine ( typhiai-Vi) contains
purified Vi polysaccharide antigen ( 25 pg per dose )
from S, jyphj Strain Ty 2. It is given as a 'ingle
subcutaneous or intramuscular injection, which
causes only minimal local reaction.
Both these vaccines are recommended only in
chose over five tcatv of age, the same dose being
used IDT children and adults . In both case;
protection is stated to commence 2-3 weeks after
adminictation and lasts for at least three ye a rs , after
which a booster may be given . Roth the vaccines
are effective and only their relatively high cost stands
in the WuV . of their wider use.
J
Typhoid bacilli arc primarily intracellular
penuites, and cell mediated immunity rather than
humoral antibodies may he more relevant in
protection against die disease. Cell mediated
immunity develops during the course of thedisease.
Cellular i mm u n i tv to the typhoid bacillus is
common in population* in endemic :Lrrjie. Absence
of CM I ha* been claimed to indicate susceptibility.
The killed vaccines currently used do not stimulate
CMI.
Treatment : Specific antibacterial therapy for
enteric fever became available only in 1948 with
the introduction of chloramphenicol , which
continued as the sheer anchor against the disease
till the 1970s when resistance became common .
»
Though S . yphi i* susceptible in vitro to many
antibiotics such as streptomycin and tetracycline,
these are ineffective in vivo . AmpiciUin, amoxycillin,
furazolidone , and cotriiTioxuvmle were the other
drugs that had been found useful in the treatment
of typhoid ftver.
While antibacterial therapy has been so effective
in the treatment of cases , it has been disappointing
LII the treatment of carriers . A combination of
antihacterial therapy along with the vaccine has
been tried in the eradication of carrier state , litis
combination has also been used to prevent relapses .
Elimination of the carrier state muv require hemic
measures such a* cholecystectomy, pyelolithotomy
or nephrectomy.
Drug resistance: Though occasional resistant
strains had been identified in the laboratory,
resistance to chloramphenicol dial not pose any
problem in typhoid fever till 1972 , when resistant
strains emerged in Mexico ami in Kerala {India).
Tr Mexico, the resistant strain caused an explosive
epidemic, with high mortality. Traveller* who got
infected in Mexico bad, on occasion , conveyed the
mutant strain to Worth America and Eurofie but
Lt did not get established in these areas.
Chloramphenicol resistant typhoid fever has
become a problem in many countries in Asia,
In India, chloramphenicol resistant typhoid fever
appeared in epidemic form first in Calicut ( Kerala )
in early 1972 . Jr became endemic and was confined
to Kerala till 197fl . Subsequently such strains
carrying drug resistance plasmids appeared in many
Other parts of India . Though resistant to
chloramphenicol , such strains were initially
sensitive to amjncillin, amoxycillin , cotriUMBMik
and furazolidone, which were HucccssfiiUv used for
r
treatment . Rv late 11990* , typhoid bacillus Strains
resistant to many or all of these drugs began to
spread m most parts of India. Ar present, the drugs
useful in treat ment of such multi resistant typhoid
cases arc the later fluoroquinolones ( such as
ciprofloxacin , pefloxacin, oflaxacin ) and the third
generation cephalosporin:, (such as ceftazidime .
Copyrighted material
 1 Erlerobacteriacaae III : Salmon alia
ceftrioxune* cefotaxime}. Furazolidone is still active
against most isolates but its action is too slow for it
to be used alone in treatment. Rccendy many strains
have become res in taut to fluoroquinolones* but
several isolates of typhoid bacilli are now sensitive
chJonmpbenicol
to
SALMONELLA GASTROENTERITIS
Salmonella gastroenteritis ( more appropriately
enterocolitis) or rood poisoning is generally a
zoonotic disease rhe source or Infection being
'
.
anuxul products It: maybe caused by any salmonella
except SI typhi The first instance of salmonella food
poisoning to have been identified was in 18S 8,
when ( juftner in Germany isolated , L bacillus ( .S'.
enter! tidir ) from the meat of an emergency
,
slaughtered cow and from the cadaver of a fatal
- of salmonehue from the article of food confirms
ease of food poisoning caused by the meat. In 1898,
Durham in England and de Nobele in Belgium
isolated S - typhimurium from meat and from food
poisoning cases. Avery large immlier ( jfsalrtWnjellar
have since been identified f r o m cases of
gasuroentc] itis and food poisoning but i few
species account for the minority ot cuses . In most
parts of the world * S. typhimurium is the most
common species , SonKOtber common species hast
been S. atieritidis, S. huJtkr, 5. heidetba%,S.Mgottst }
S. vitchuwT S. sr/rerjherjf, S. Indiana, S. titwpotc
and 5- iinafmn.
E Inman infection results from the ingestion of
contaminated food . The most tinequent sources of
salmonella food poisoning are poultry* meat , milk
and milk products. Of great concern ore eggs .md
egg products , Salmonella *: can enter through the
shell if eggs arc left on contaminated chicken feed
or frees * and grow inside. Human carriers do occur
hut their role is minimal when considered in
, cases, antibiotic treatment is needed .
relation to the magnitude of infection from animals.
Even salads and other uncooked vege cables may
cause infection if contaminated through m HI Lire or
by handling. Food contamination may also result
from the droppings of rats , lizards or other small
> 303
animats . Gastroenteritis may occur without food
poisoning as in cross infectlor in hospitals.
Clinically, the disease develops after a short
incubation period of 2*1 hours or less. with diarrhea *
,
vomiting, abdominal pun and fever . It may vary in
severity from the passage of one or two loose stools
"
to an acme cholera -like disease. Ic usuallv
ir
subsides
in 2 4 days but in some cases a more prolonged
~
enteritis develops, with passage of mucus and pus
in tcces, resembling dysentery. In a few, typhnidal
or septicemic type of fever may develop ,
Laboratory diagnosis is made by isolating the
BihCK)Della from the feces. In outbreaks of food
poisoning, the causative article of food CM often
be identified by taking a proper history. Isolation
the diagnosis.
Control of salmonella food poisoning requires
rbc prevention of food contamination , Food may
become contaminated ar various levels* from natural
infection in the animal Of bird, to contamination of
f he prepared food . Ptoper cooking of food destroys
aalmnnrtlie.
While enteric fever is & major problem only in
the developing countries, salmonella food poitOoing
is largely a problem for the developed nations, Iliis
"
is due ro rho differences in food habits and living
conditions between them and also because food
production, packaging, storage and marketing have
become industries in the rieb countries while they
still remain agricultural in the developing world.
Treatment of u m omplicated , noninva ive
salmonellosis is symptomatic. Antibiotics should
-
not be used , Not only do they not hasten recovery
but they may actually increase the period of foc& E
shedding of the bac iili . But for the serious invasive
SALMONELLA SEPTICEMIA
Certain sahnonellae* 5. cfro/eraesLhs in particular,
may cause septicemic disease with focal suppurative
lesions,, such as osteomyelitis, Jeep abscesses,
Copyrighted material
304 * TsxUnoh of Wicroo dog * *

endocardins, pneumoniaand memngjitii, Antecedent
gastroentcrifis may or may not he present . The ease
fatality may be as Ki urh as 25 per cert.
Salmonellae maybe isolated from the blood or
from the pus from the suppurative lesion . Feces
culture may also sometimes be positive. Septicemic
salmonellosis should be treated with
diluramphcnitol < jr other appropriate antibiotics
as determined by sensitivity tests.
MULTI RESISTANT SALMON ELL AE
R factors conferring multiple drug re stance have
become widely disseminated among salmonellae .
The clinical significance of thus phenomenon was
first observed during the studies of human and
veterinary infections with drug resisranr
Srt} j?himunum phage type 29 in England in the
lVfitls. Human infections were initially gastrn
-
crteriti - due to spread ftorr- infected animafe, through
food. Subsequently some salmonellae appear to have
changed their ecology In some ways . From being
responsible for zoonotic infections only as in the past ,
some multiresisiant salmonellae have now become
important agents of hospital LTOSS injection . Such
nosocomial salmonellosis manifests particularly in
-
neonates as septicemia meningitis and suppurative
lemons, Diiintiea may not always be present.
In India * several hospital outbreaks of neonatal
septicemia caused by multi resistant salmonellae
have occurred in recent years. Mortality in neonates
is veTy high unless early treatment Is started with
antibiotics to which the infecting strain is sensitive.

Further Reading

Forsyth JILL. 1WH.

1

^ ^ ^ .
Chriarie AB. I 9 H7. Inf&xious Diseases. Epidemiology atid Cfinih Pri- ciire , 411 edsi. Friinhut h: Ch Lfchill-LfonEsftme,
itnd Paratyphoid . In Topicr and Wilson s Mktobiolffgy' and JWi refflJsra Jji&.'rint wh 4* cdri - vul -3.
'

LunJuri: Arnold.
Ivanort B c[ aL 1994. Vaccination ajjiinsi ryphoid fever: Fraeflt Bull Wlci Hkh Ota: 72^957-
Mindd BK.. 1994. SalfflHKfll cyphi iusil other KaLmuiiidcDyj. Gut 3- 5:726.
Mina SH cr JL 199ft . Midcaiim rnAtani Sdiunetli typhi: A kabnJ pfo koL JModMkriobioL 44:317.
^
WHO, 1W. C ?4 wifml nf Sal me HIE]la infer turns in
1 ^
jminuli, fluSt WIrf I f l t h ^ .
Orgn 12 S!M ,

vJ opyrighted materia
[
 CO
CO Vibrio

Vibrio* are Gram negative, rigid , curved rods that

arc actively motile by means of a polar flagellum.
Tiie name Vibrio' in derived bom the < haracteristic
vibratory motility (from vibrate, meaning to vibrate).
They are aspOTogenous and noncapsulated - Vibrios
are present in marine environments JUld surface
waters worldwide . The most important member of
the genus is Vibrio cholcrae, the causative agent of
cholera. It was first isolated by Koch from
cholera patients in Egypt, though it had been
observed earlier by Pacini ( 11354 ) and others .
VIBRIO CbOLERAE
Morphology : The cholera vibrio is a short ,
curved , cylindrical rod , abour 1.5 pm * 0.2 ~Q,4
pm in si/e, with rounded or slightly pointed ends.
The cell is typically comma shaped (hence the old
name V! comma ) but the curvature is often kret on
subculture. S -shaped or spiral forms may be seen
of 16-40 °C (optimum 37 °G ). tirowib is better in
an alkaline medium the range of pH beiru^ 6, 4 9.6
-
(optimum 8.2). Nad ( 0.5 1%) is required for
optimal growth though high concentrations (6%
—
and above) are inhibitory'.
It grows well on ordinary media. On nutrient
agar , after overnight growth , colonies are moist;
—
translucent, round discs, kbrnit 1 2 mm in diameter
with a bluish tinge in transmitted light.The growth
has a distinctive ( KIOUF. On MacCunkejra agar, the
colonics art colourless at first but become reddish
on prolonged incubation due to rhe late
fermentation of lactose . On blond agar, colonies
are initially surrounded by a zone of greening .
}
. /) * }i j j

. ir i
^
due to two nr more cells lying end To end .
Pleomorphism is frequent in old cultures. In stained
films of mucous flakes from acute cholera cases, ’
the vibrios are seen arranged in parallel rows ,
described by Koch as the 'fish in stream appearance.
1

It is activelv motile , with a single sheathed pol .ir

flagellum. The motility is of the darting type, and
when acute cholera stool nr a young culture is
examined under the microscope, the actively motile «r
_ *r

vibrios suggest a 'swarm of gnats . The vibrios stain

7 u r -- V 1

readily with aniline dyes and are Gram negative

imd numteid fast ( Fig- 33,1).
Fig , 33.1 A . Cholera vibrios. Right - Gram slain Left
Cultural characteristics: The cholera vibrio - Flagellar stain showing single polar flagellum.
tt strongly aerobic, growth being scanty and slow H . Electron micrograph Showing ElnuU polar -
anaerobically. It grows within a temperature range flagellum in iftOD ) (Source: AN Ghosh , HICEPI

Dvrraniei cl :C
3D6 * Textbook uT
which later becomes clear due ut hemndigCEti < ui .
In gckrin stab culture, mfiindibuliform ( funnel-
HHAYED) or nupLiorm ( tufntp'shapcd) liquefaction
nec- urn in three day* at 21 *C . In peptone water,
growth occurs m about six hours as a fine surface
pellicle, which on shaking brctki up into
membranous pieces . Tin bid it} and a powdery
1
deposit develop on continued Incubation.
A number i it special media hive been employed
"
for tile cultivation of cholera vibrios They may he
classified as follows:
Holding nr transport media: ( 1 ) Vrnkat -
nunarrRfifn &krighiifla ( VR) medium: A simple
modified form of this medium is prepared by
dissrilvirig 2i ) g oudt sea Halt and 5 g peptone in
Otic litre of distilled water and adjusting the pH to
K />-S.S. Jt is dispensed ID serewgapped bottles in
10-15 ml amounts . About 1-3 ml stool is to be
added to each bottle - In this medium vibrios do
(lot muhlplv but remain viable for Hcv-eial weeks.
(.2 ) Cary-Rlair medium:1his is a buffered solution
of Hodium chloride , sodium rhioglvcollate,
disodium phosphate and calcium chloride at pH
8,4 . It is .t suitable nanapmt medium for Salmonella
and Shigella an well as tor vibrios . (.1) Autoclaved
sea water also serves as ji holding medium.
Enrichment mediiu (1) Alkaline peptone water
at pH £.6; [ 2 ) Mfjn$ ur't tau. ro / holate tellurite
peptone water at pH . 2 . Both these arc good
^ on linen or thread , they survive for 1-3 days but
transport JIS well as enrichment media .
IJlilting nttdin: (1 ) Alkaline bile salt agar ( RSA)
pH S.2: This simple medilirci lias HEKKKJ ihe test of
rime and is still widely used - The colonics ate simitar
to those on nutrient agar. ( 2 ) Monsur 's gelatin
timiDcbalajte trypticase teflurrtsogir (GTDV) medium:
Cholera vibriiw product small, rr .inslucent colonies
with a greyrih black centre and a turbid halo. 'I'hc
colonics become 3-4 mm in size in 48 liourn. [3 )
rCIIS medium:Thin medium , « Jiitnini ug tiliosulfiltc ,
citrate, bile saits and sucrose, is available commcmoHy
and is v«y widely used at preset ]!. Cholera vibrios
produce large yellow convex colonies which may
become green O i l continued incubation.
Vibrio colonics maybe identified by the 'string
test’. A loopful of the growthis mined with a drop
of O. o’W sodium deoxychokte in saline on a slide.
If the test is positive, the suspension foses its
turbidity, becomes mucoid and forms a string' when
the loop is drawn slowly away from the flupeuuXL
Biuchemicul reaction At Carbohydrate
metabolism is fermentative, producing acid, but no
gas . Cholera vibrios ferment glucose, mannitol,
maltose , mannose , md sucrose bur nor inositol,
strabitiosc, or lactose, though lactose may be split
very lilowly. lndnh: is formed and nitrates are
.
reduced to nitrites These two properties contribute
lo the VI iultra red reaction ' which JH tested by
adding a few drops of concentrated sulphuric acid
to a 24- hour pepronc water culture. With cholera
vibrios, a reddish pink colour is developed due to
the formation of nicroso- indole . Catalase anti
caudate ten It are positive. Methyl red and urease
tests are negative. Vibrios dccarboxvlate lysine and
ornithine but do not utilise arginine. Gelatin is
liquefied. Vibrios elaborate several enzyme!;
including L -OILAGTNASE , cLiittte, chitinasc,
nucleotidase, decathoirylaEC, lipas<\ mucinasc and
neuraminidase (receptor destroying enzyme).
Resistance: Cholera vibrios are susceptible to
hear, drying ur.d acids, hut resist high alkalinity.
They arc destroyed at 55 UC in 15 minutes . Dried
die 1(1 about three hours on cover slips. Survival in
water is Influenced by its pHh temperature, salinity,
presence of organic pollution and other factors. In
geiLLTol , the El Tor vibrio survives longer rhan rhe
c lassical cholera vibrio. In the laboratory vibrios
survive for months in sterile sea water, and this has
been suggested us ;i method for the survival of vibrios
in nature hi grossly contaminated writer, such as the
,
1
Ganges water of India, the vibrios do not survive for
any length of rime* due to die apparently Jaige amounts
of vibriophflges present. They survive in clean tap
water for thirty dap. In untreated night soil, they may
,
sumvx for several days. Vibrios are susceptible to the
common disinfectants.
Copyrighted materia
 4 Vibnc »
On fruits, they survive for 1 - 5 days at room
temperature and for a week in the refrigerator. In
general , IOIHI materials left a.t room temperature do
not act as m important swnxofinftttinn for longer
than a day or two blit those stored in [ lie cold may
harbour vibrios for more than two weeks.
They are killed in a few minutes in the
gastric juice ot’ normal acidity bur they may survive
for 24 hours in achlorhydric gastric juice.
Clat &ificfltitm: Jn the past* many oxidase
positive, motile, curved rods were rather loosely
grouped as vibrios . Precise criteria have been laid
down for differentiating vibrios from related genera
'
( Table 33.1).
Hribcrg ( 1934) classified vibrios into six group
based on the forme ntution of mannose, sucrose and
atabincise . Two more groups were added later .
Cholera vibrios belong to Croup I (Table 33.2).
A serological classification WH introduced by
Gardner and Vcnkatraman (1935), Cholera vibrios
and biochemically similar vibrios , possessing , L
common fliLgellar ( H ) antigen were classified as
< Jroup A vibrios, and the rest as Group B vibrios
comprising a heterogeneous collection. Based on
IILL: major somatic (Q ) antigen, Group A vibrio*
we re classified subgroups' ( now called ( 1
into " vibrios gave rise to the seventh pandemic -of
berOgrOUpfi Or tCfOVUl ), 1 39 of which are currently
known ( Table 33.3) . All isolates from epidemic
cholera ( till 1992 ) belonged to serogroup 0 - 1 .
Therefore in the diagnostic laboratory group 0- 1
antiserum (commonly called ' cholera
nondifferentifll serum ) Lime to be used fur
1 ^
identifying pathogenic cholera vibrios ( which ate
referred- » ‘igghltfortblc vibrios' ) . Other vibrio
isolates which were not agglutinated by the 0- 1
antiserum. came to be called norragjf / urjNjMe or
!M AG wbflM. Frhcy were considered nonpathogenic
and hence also called nmcfeafeii vibrio* ( NCV ) ,
Both these terms are not strictly' appropriate .
Though SAG vibrios are not agglutlnablie by the
G-l antiserum , they arc readilyagglutinated by their
own antiserii. The term noncholera vibrio is uol
correct as some of them can cause a disease clinically'
307
indistinguishable from cholera . However, by and
large , N’ AC vibrios were nor pathogenic and
commonly isolated from envi momenta! sources and
healthy human intesrircs.
While all isolates from epidemic cholera
belonged to group 0 - 1 , not all tnemben ol the
group were capable ol causing L [ i 11 LL . iI dioleft, The
'
first such members which acquired prominence
were the vibrios isolated by CrOHschlich { 190?)
from sis llaj pilgrims who died at the Tor
i| uafajuiut station on the Si mu Pen insula. They had
died not from tholera but from dysentery or
gangrene of the colon. These came to be ealled the
lil Tor vibrios. They were identical to cholera
vibrios in all laboratory tents except that they were
hemolytic to sheep erythrocytes and gave a positive
\ bgcs-Proskaner reaction . As the El lor vibrios
were subsequently isolated from water sources and
normal human intestines , they were considered to
be nnnparhogenic , In 1937 , El Tor vibrios were
recognised as endemic in Celebes (Sulawesi ) ,
Indonesia, causing a choleraic disease ( paracbalera ).
I lowevcr , outside thfo endemic area. El lor vibrios
were considered nonpathogenic .
The situation dunged in 1961 when El Tor
cl Lid era . Besides increased virulence and
invasiveness, the pandemic El Tor strains showed
altered laboratory reactions . The new El Tor strains
were often nonhemolytic and V— P negative . Mew
differentiatm properties were defined such as chick
celt SLgglutIllation , polymyxin sensitivity arid phage
susceptibility tents ( Table 3.3.4) . It wan accepted
tbut El TOT vibrios were indeed cholera vibrios,
which contained two bfon jet , the old or 'classical '
^
cholera vibrios, and the El Tot vibrios.
Bused on minor surface antigenic characteristics
both classical and El Tor biotypes of cholera vibrios
were classified into three serotypes, Ogiwa , Inaba,
and Hikojima ( Table 33.5 ). The Ogawa And Inaba
Strains- are agglutinated by their own respective
specific sere only, while die Hikojima strains are
agglutinated hv both Ogswa and Iniba antisera .
Copyrighted material
 303 * Textbook ;: F Micre- biology

Tabie 33 1 Differentiation of vibrios Irom allied genera

(? xfda IBM F rffl^nrtwn
^ [/dlttuwvj c/i/TjirHKidit String test
( Hwgh-Lithvn Tfsr)
i

Oaddiikm FeimenitaiLon Lvsine Aqcinine Ornithine

Vibrio + ] + +
— —
4 +
Aeromonas *2 + V
Pseudomonas
PlcsLomcmas
*

+
+
—
+
V
+
V
+
V
+
—
~

Note : 1 * no gas produced; 2 - gas mayor may nor be produced; V = [motion virisble ,
There ts. no difference in pathogenic itv between
the three serotypes . Scrotypinj; is only of
efi ide miulogical signi fieanct .
The non-Gl vibrios ( the so tilled NAG vibrios)
have been classified into many serogroupt, currently
uplo 139. The latent Hemgrnup 0-139, identified
in 1992 causes epidemics of cholera, emphasising
that they tan no Longer be considered as nunchokn
vibrios.
Modern taxonomies! criteria , particularly , V. cholcrsc , In its most severe form, cholera is a
UNA studies , have led to the recognition that all
the cholera vibrios that belong to Gardner and
Venlsatraman 's group A and share simdar
biochemical properties and a common H antigen
arc so closely related that they constitute a single
species Vibrio cbolenc , which can be classified
into serogmups (or scrovars ) , bintypes and scrotypes.
Accordingly the present nomenclature will be
indicative of all these features, as lor example,
VT cho/crae scrovar 01, biotype El Tor, serotype
Ogawa,
Table 33 2 Heiberg grouping of vibrios
Group Fermentation of
T A
11
111
-A
IV -A
Further classification car he made hy phage
typing. Phage typing schemes have been
standardised for classical and El Tor bioiypet an an
-
well us for 0 139 vibrios. New molecular methods
like ri ho typing have added further rctmements to
strain typing.
CHOLERA
Cholera > an acute diarrheal disease caused by
dramatic and terrifying illness in which profuse
painless watery diarrhea and copious effortless
vomiting may lead to hypovolemic shock and death
m less than 24 hours. In treated canes , the disease
-
may last 4 6 dpys, during which period the patient
may pass a total volume of liquid stool equal iu
twice his body weight. All the clinical features of
severe cholera result from this massive loss of fluid
.
and electrolyte* The cholera stool is typically a
colnrlcw witerr fluid with flecks of mucus, said
to resemble water in which rice has been washed
mannose Sucrose AraWncic
A
A
A A
A A

-— -
V
VI —
-—
=
Vll A A
vm — A

Copyrighted materia
 * Vibrio M9

VIBRIO

Croup A Group B
Cholera vibrios Biochemically and
Biochemical M mllaiiricG; anrigenically
Common U aorigen hererojfe ntnui
( Vihrift fhalet it )-

O SUBGROUPS
( SEROGROUPS ) :
SEROVARS )
01 Mon -01 ( cujicA[]y up( G (M 3)

BIOTYPES ’
I
[Criteria Listed \
in Table 33.4 ) j
f
'
C1?aSri Es1,S
E3. Ttrf
l 1
f \
SEROTYPES Ogawa Inaba Hikciima

Tsfrle 33.3 Qinlrw and VmkttitMil'l tMUoPoit updatedi

{ hence called ' rice stools * ) - lr has a
water
characteristic inoffensive sweetish odour. In
composition it is a bicarbonatc - rich isotonic
electrolyte solution , with Little protein . Its
outpouring lead ' to diminution of extracellular fluid
, occurs only i rn humans and not in animals. A number
volumet hemocuncentia; hypokaleim . i , base -
dcficit acidosis and shock . The comm or
complications are muscular cramps , renal failure ,
pulmonary edema, cardiac arrhythmias and paralytic
ileus. The clinical severity of cholera varies widely*
from the rapidly fatal disease to a transient
asymptomatic colonisation of the intestine by the
vibrios* The incidence of mild and asymptomatic
infections is more with Kl Tor vibrios than with
the classical cholera vibrios .
The incubation period varies from less than
24 hours to about five davs, The clinical illness
^
may begin slowly with rnild diarrhea and vomiting
in 1=3 days m abruptly with sudden massive
diarrhea.
I \ i 11 iiiUt- 1 it' sias Natural infection with cholera
of animal models have been developed! which have
helped in understanding the pathogenic
mechanism* in cholera. The firsr of these wu the
rabbit ileal loop model of Dc and Chattcrjce ( 1953),
Injection of cholera culture or culture filtrate into
the ligated ileal loop caused fluid accumulation and
ballooning- Intestinal loops of many other animals
and also of chickens hart been shown to behave in
, a similar manner. Dutta and ITabbu (1955 ) showed
that a fatal diarrhea could be induced in infant
rabbits infected with vibrios perurally or
intrsintcstinally. Sack and Carpenter ( 1966 )

Copyrighted material
310 * Tgxlbaok GJ Microti ulOQy *

introduced the canine model infected with able 33.4 DttteraflMitlon between classical
cholera vfbrios through a stomach tube, after cholera and El Tor vibrio*
administration of sodium bicarbonate developed Twt CAurieii cAflipra El Tor
diarrhea and vomiting, usually ending in Liexrh.

—
v
In human infection, the vihrias enter orally fbmolpis - +

through contaminated water or food - Vibrios are —

Vcges Praskauer
Chick erythrocyte -
+*
+
highly susceptible to acids , and gastric acidity agfclun n (Ition
provides »n tfiectiit burner against small doses of IblymYxin B *
cholera vibrios . It has been shown that 1 D 4 sensitivity *
pathogenic vibrios administered to fasting Croup IV phagr +

nnnnochlorh vdric volun leers , without food or susceptibility

El Tor phage 5 -
buffer, did not product infection, while the same SUSCCpilOllJTy
dose given along with food or sodium bicarbonate * Strum isolated after 1961 give varinhlc results;
L juvrd clinical cholera in 100 pet cent of them. i 50 i.
' u, disc,
Achlorhydria predisposes to cholera m the field .
In the small iiitotine, vibrios arc enabled to cross Table 33.5 O serotypes of cholera vibrios
the protective layer of mucus and reach the epithelial
cells by chemotaxis , motility, mucim ^ c and Other Serotype O IflfjjfJM
proteolytic enzymes. A hemagglutinin protease -
(formerly known as 'cholera lectin ') cleaves mucus
Qgrgwa AB
Inabi AC
and tihroneotin. It also helps in releasing vibrios ABC
hound r : bowel mycosa , facilitating their s]>tead to
other parts of the intestine and also their fecal The toxin molecule, of approximately 84,000
shedding . Adhesion to the epithelial surface and MW consuls of one A and 5 B subunits. The ft
colonisation may he facilitated by special fimbria ( binding} unit * act neb rn the GM , ganglioside
such as tbc ' rosin cn - regulated pilos ' [TCP). receptors on the surface of jejunal epithelial cells .
Throughout the course oh infection, the vibrio* The A (active ) subunit, on being transported into
remain attached to the epithelium but do not the entenih tr into two fragments A.
dissociates
d .Linage or invade the cells. The changes induct'd and A .
^
iTie A
Gt ntfllOnly links the biologically
are biochemical rattier than hismJogicnl - ^
active A , to the B subunit - The Al fragment causes
Vibrios multiplying on the intestinal epithelium prolonged activation of cellular adeuvluu cyclate
produce a toxin (ehofengen, cholera emero-toxin , and lonunditioA D[ L-AMI '. leading to outpouring
'

cholera toxin, CT, or CTX) which is very similar
to the heat labile toxin ( LT ) of !' coif In structural,
chemical , biological nod antigenic properties ,
though CT is far more potent than 11 in biological
activity CT production is determined hy a
filamentous phage integrated with the bacterial
chromosome . It can also replicate ns a plasmid
which ] can be transmitted to n ^ ntoxigenk strains,
rendering [ hern toxigenic. Cl , TCP and Other
virulence factors arc regulated by the TodtR gene
product, ToxR protein.
into the small intestinal lumen , oflarge quantities
of water and electrolytes and the consequent warcry
diarrhea. The fluid secreted is isotonic with plasma
but contain * much more of potassium and
bicarbonate. The rflgjn also inhibits intestinal
absorption of idmm and chloride. All clinical
manifestations and complications in cholera result
from the massive water and electrolyte depletion
thus caused.
CT also exhibits other biological effects which
can be used for its detection and estimation. These

Copyrighted material
 i Vibrio *
indudc activation nnipolysis in rat tCttiCulU' tissue,
elongation of Chinese hamster ovary (CHO ) ccll-s
in culture and histological changes in adrenal
Tumour (Y ) cell culture and vero ull | , JT also
increases skin capillary permeability; and so has been
called the Lpemieiab Llity factor ( PF). It ran be
"
demons; truled by the skin blueing test ' when CT is
"
injected intradcnnally In rabbits or guinea pigi mid
pontaminc sky bl « c injected intravenously
afterwards* the site oftoxin Injection becomes blue.
CT can also be e^- limited by ELISA . Cl ] H
antigenic and induces production ol neutralising
antitoxins. CT can be toxnided ,
Cholera vibrios also possess the
lipopotysaaiharrde O antigen (I.-PS, endotoxin an
^
in Gram negative intestinal bacilli- This apparently
plays ro role in the pathogenesis of cholera but is
responsible for the immunity induced by killed
vaccines. It maV cause the fal .d illness produced
experimentally by peritonea] inoculation in mice.
Epidemiology: Cholera can occur in many
-
forms sporadic* endemic* epidemic or pandemic.
India * more specifically elite large deltaic area of
the [ hinges and Brahmaputra in Bengal , is its
homeland, where it has heen known from very
ancient Times. Till early in the nineteenth century',
cholera was virtually confined to India, periodically
causing large epidemics in different pans of the
country.
From tS17 to 1523 cholera vibrios had spread
from Bengal , in sis separate pandemic waves ,
Involving most parts of the world. It was largely
due to the threat of pandemic cholera char
international health organisations came into being.
After the cm! of the 6th pandemic in 1523* till
1961 tine disease remained confined to its endemic
ureas, except far an isolated epidemic in Egypt in
1947 ,
Theseventh pandemic originated from Sulawesi
(Celel >es) * Indonesia, in 1961 when the El Tot
vibrios which hid been smouldering there tor many
decades suddenly became more virulent. After
spreading to Hongkong anil the Philippines , it
311
spread steadily westwards , invading India in 1964 .
By 196 b , it had spread throughout the Indian
subcontinent and West ASM . In the 1970s the
pandemic extended to Africa and parts of Southern
Europe.
During the course of the pandemic the vibrios
,
had invaded affluent countries also. In the 1970s
small outbreaks had occurred in [.Queensland ,
Australia and the Gulf Cout in the USA tmm
special environmental foci in the coastal waters.
However, they remained Localised and were SOOT
con [ rolled , in contrast to the outbreaks in the poor
,
narsons, which developed into prolonged and
extensive epidemics.
Ln January', 1991 , the pandemic reached Itru,
thus encircling the glnhc in thirty years rime [ tig
33 2) . For the first time in the century* cholera had
invaded South America . I he epidemic spread
rapidly through mauv Central and South American
countries.* and by mid -1992 over halt a m illion cases
and 5000 deaths had been reported. By 1994 most
parrs of Central and South America had been
involved . . ud rendered endemic. a
The seventh pandemic of cholera lias heen
different from nil the others It is the first to have
*
originated from outside the Indian subcontinent h
,
is alio the first to have been caused by the El Tar
r
biotype, in contrast to all the earlier ones causerl by
the classical cholera vibrios. The severity of illness
was much less, with a large proportion of mild and
asymptomatic ink'd ions. Mortality was low and the
carrier rate high. El Tor vibrios tended to remain
endemic in many new geographic areas , causing
pcri*> div epidemic hursts. The El 'for vibrio has
proved to he much hardier than the classical vibrios ,
capable of surviving in the environment: much
longer . A peculiar phenomenon has been rhe
replacement nt the classical hintype by the El Tor
vibrios following the pandemic spread. Thus, in
India the classical vibrio is hardly ever encountered
after the El Tor epidemic took root , though in
Bangladesh , the classical vibrio had staged a
rundudL
Copyrighted material
312 4 Tiaytbook of Microbiology *

An event of gneat WftnifkartCt wa* the sudden may initiate the next tandemit of choiera , The new
emergence of non-0l V! fMfiatf ( former NAG strain continued spreading, eastward'; to tile South
vibrio) as the enust of epidemic cholera, In October East Asian countries, and westwards to Pakistan,
1992, a new non-01 vibrio was isolated from China and some parts of Europe. Bui surprising)), 1

*
cholera outtrrea.lt in Madras { Chennai). Similar
outbreaks soon followed in different parts of India.
By January, 1993, the tiew strain had become
epidemic in Bangladesh also. In Lite affected Ateis ,
this strain replaced the EL1 lor vibrios as the
"
.
epidemic and environmental serovar Jt also showed
a tendency to be more invasive, causing bacreremic
illness in some. The new epidemic strain was
bv 1994 the FI Tor strain regained its dominance
and the threat ot an 0-139 pandemic diminished.
-
Both 0 -1 ElTarud0 139 strains began tacoaast
in endemic areas.
Cholera is an exclusively human disease .
Infection originates from die patient or the carrier.
Carriers may be incubatory convalescent, healthy
or chronic. Incubatory carriers shed vibrios only
r '

Jr

designated Serovar 0- 139 ( or 0- 139 Bengal) .
Unlike the 0 -1 cholera vibrio, the 0- 139 vibrio is
capsulated. As it possessed novel surface antigens,
the 0- 1 atnun vaccines could not protect agajnst
0“ 139 infection. There was nn natural antibody
against the strain in any human population then. It
was therefore considered likely that the 0-139 strain
during the brief incubation period of 1 > days .
-
Convalescents may LXCICLC them for 1 3 week The
*
healthy or contact carrier who has had subclinical
infecrion usually sheds the vibrios tor les- s than 1(1
days. The chronic carrier continues to he active tor
months or years^ rhe longest duration recorded
-
hc mg 1(1 years . Chronic Curriers were rure in

ft
*
to

966

961 19B 1
*
1971

1934
h 19B1
7%
1991

' A' Explosive initial epidemics

Extent of pandemic spread

.
Fig 43.2 Spread . -
^
( ) Unique onvironmenfal reservoirs

El Tor Cholera 1961 lW, Fltwnll) WtflUlted $p«lal environ rtfttntal esmoltfi of toxigenic
El Tor Cholera vibrio 01 are also indicaled. (Source; WHO ] .

Copyrighted material
 A Vibrio
classical cholera but with El Tor infection they sue
seen more often . Persistent infection of the gall
bladder accounts for chronic carriage .
Infection it acquired through fecally
contaminated water 01 food . Direct person- to -
person spread by contact may not be common but
hand contamination of stored drinking water has
been shown to be an important method of domestic
spread of infection . Large scale movement of
persons, as occurs during fairs and festivals , has
traditionally beet ] associated with the spread of
cholera .
The persistence of the vibrio during the
intenepitleiTiLc periiHls was a matter of controversy.
In the endemic areas it may be maintained by
continuous transmission of subchnical or mild
infection . It is now known that the natural habitat
of cholera vibrios is the saline waters of coastal
seas and brackish estuaries, where they can persist
for long periods , particularly in association with
small crustaceans such as copepods , crabs or
plankton.
A significant difference in susceptibility to cholera
has been reported in relation to blood groups, group
O persons being the most susceptible and group AB
313
*
As cholera vibrios may die in a few hours at
tropical temperatures , it is necessary to preserve
the specimen at 4 °C or in some appropriate
holding medium . Stool samples may be preserved
in VR fluid or Caty-Blair medium for long periods.
If the specimen can reach the laboratory in a few
hours, it may be transported in enrichment media
such as alkaline peptone water or Monsur ' s
medium, thus saving the time required fbr isolation .
If transport media are not available, strips of
blotting paper may be soaked in the watery stool
and sent to the laboratory packed in plastic
envelopes . Whenever possible, specimens should
be plated at the bedside and the inoculated plates
sent to the laboratory.
Diagnosis by direct microscopic examination of
cholera stool is not recommended as the results
arc rot rclrahlc - For rapid diagnosis, the
—*
- 0 ' r
f

the least. The reason for this is not known. L

Laboratory diagnosis: Stool, collected in the

acute stage of the disease before the administration
,

of antibiotics , is the most useful specimen for

laboratory diagnosis. Isolation of cholera vibrios
from such stools is a simple matter as they are
present in very large numbers, lty-— IQ* vibrios per -

mb The specimen is best collected by introducing i

into the rectum a lubricated catheter and Ertting

the liquid stool flow directly into a scrcwcipped . .£
—
U nuui a#u#nvic Gci

container. Rectal swabs maybe used, provided they Hrch IVI1

are made with good quality cotton wool, absorbing

about 0,1-0.2 ml of fluid . They arc useful in
GSWW* iW
^3

collecting specimens- from convalescents who- no

longer have watery diarrhea. In such cases, the swabs
should be moistened with transport mediu m before
sampling. Collection of stools from pans is not -
Fig. 33-3 Spread of vibrio cholerae 0 139 epidemic in
recommended. VomltUS is not useful. -
Asia ( 1992 1994) ( Source : WHO)

Copyrighted material
 314 « Textbook
characteristic motilirv of the vibrio and its inhibition
iB
by apti serum can be demonstrated under rite dark
m
field or contrast mLcroj&uofje , using cholera
stool from acute C& KS,. or mosre reliably alter
eiaridimmt for six hours Dcriionsfratinri of vibrios
, and arabinose , hemolysis , VT, polymyxin B
in HTOOLS bv. direct inununolliLoresceiice has been
attempted liut nonspecific fluorescence is common mid
the technique is too complicated fur use in die Held ,
On arrival in the laboratory the spe \ Imcns sent
! Manorial Infinite of Cholera and Enteric disease
in enrichment media should be intubated for 6-8
hours including trans r time. The specimens sent
in holding media should be inoculated into
enrichment media* to be Incubated for 6-8 hours
before being streaked on a selective and a
-
nonsclcct: LC mediu nn . It i s also dc ' table to do d ircct
plating befure CTbrichiTunYt. The pilling cileri i used
vary in. different labors lories but the media
employed usually are bile salt agar , Maction key
agar for nunselective and TCB 5 agar fur selective
plates. The plates should not be older than 3-5
dap and should be dried well beforc streaking [t
is possible to identify vibrio colonies on nonseledivc
media after incuhatn m lor 4-5 hours by examina!ioU
under a stereoscope with oblique illumination .
Generally* the plates arc exan tied after nwernight
incubation at 37 Colonies suggestive of vibrios
should be picked w ith a straight wire and tested by
slide agglufina : ii ' ii w : rh cholera O subgroup J
scrum (cholera ' nondiflerenri - d' serum ) , If positive*
agglutination may be repeated using speciftu Ogjwa
and 1 naba sera for sen ttvp i ng. Hikoj im a sir J 11 is w 111
agglutinate equally well with Ogawa and 1 naba sera.
If agglurination is negative with one colony it is , diagnosis of cases though : t may be helpful in
essentLiil to repeat the test with at least five more
colonies* as ‘agglul i lable and nun -01 vibrius may
coexist in the name specimen. J: slide agglutination
i -i positive* the isolate is tested (or ehi ik red cell
agglutination. This is employed for presumptive
differentiation between El Tor and classical cholera
vibnoS. A report cat ] be sen! al ill is stage * usually
the day after the specimen is received , If no vibrios
are isolated * a second cycle of en richment and
plating may succeed in some cases.
gl Microbpoloqy »
The isolate may then be subjected to detailed
study* if desired, including theoxidase test * utilisation
of aniinoacids * lysine , arginine and ornithine,
fermentation of sugar, including sucrose , mlunoSc
scnsiirvity and susceptibility to cholera phage IV*
The strain may be sent to the International
Relerence Centre fur vibrio phage IV ] dilg at the
( NIC ED) at Kolkabi .
Isolates of vibrios that are not agglutinated by
the O subgroup 1 serum should not be ignored as
non -01 vibrios arc known to produce eholcra-likc
disease. An antiserum to the H antigen which is
shared by all cholera vinos has been (bund to be a
useful reagent . Any vibrio which is agglutinated
by this H am JSCRUM , hut not by O -T serum LS
'
considered to be unn -Ol chulera vibrio. Specific
antisctum against 0-139 is available. In the fully
equipped laboratory* diagnostic tests in cholera and
, other diarrheal diseases should consist of a batterv 'J
of tesrs designed to isolate other known pathogens
also.
FCKI isolation ul vibrio# from Carri e rs* essen: iiallvrf
"
the Bune techniques are to be followed, except that
more than one cycle of enrichment may be
necessary. As vibrio excretion is intermittent *
,
Jr "
repeated stool cxarc : nation will y: dd better results.
Examination of stools after a purgarive f magnesium
—
sulphate 15 30 g or Mannitol 30 g ) * or of Kile
after duodenal intubation is of special value .
Serological examination is of litrle use in the
assessing the prevalence of cholera I n an area. 'The
tests avi dabte are agglutination using live or killed
1
vibrio suspensions , indirect hemagglutination ,
vibrint idal test and antitoxin assay. Of these, the
complement dependent vibriocidal antibody test is
the most useful.
1
Fur examination of water samples fur vibrios *
clit ' hmentor filtration methods may be employed ,
'
In the former , 900 ml of water arc added to 100 ml
tenfold concentrated peptone water at pH 9.2,
u opyrighted
y materia
 * V brio »
incubated at 37 ‘-'CJ! for 6-8 hours and a second
ennchincnt done before plating on selective media.
For the filtration technique the water to be tested
should be filtered through the Mi11ipore menil e
filter, which is then placed directly on rhe surface
of a selective medium and incubated . Colonics
ap}>ear after overnight incuturion . Sewage should
he diluted in saline , filtered through gauze and
treated US for water .
Immunity: Jn cholera, the vibrios remain
uuitfioed to the mteseme, where they multiply and
elaborate the enterutoxii ] which is. responsible for
the disease . Immunity therefore, mzy be directed
against the bacterium or against the toxin —
antibacterial or antitoxic. Natural infection confers
some amount of immunity but it docs not seem to
last for more chan 6^12 months and rcinfcctinns
are known after tills period.
Immunisation with killed vactines induces only
antibacterial immunity. The protector effect of these
vaccines , especially puimed somatic antigens used
a* vuciines , though shor [ - Lived , proves that
antibacterial immuniry can protect agamsi intecrion.
The protection appears to be serotype specific hut
not hiorype specific .
Immunity may be local , in the intestine, or
SVStcmk . The appearance of local antibodies in the
'
nrestinc has been known for a long rime. These
are known as ‘coproamibodies' as they appear in
the feces . Tliey consist of IgG , IgM and IgA .
Prophylaxis The prevention of cholera requires
essentially general measures such as provision of
protected water supply and improvement of
environmental sanitation - As these arc noT easily
_ :
attainable , vaccination con nucs fo be the most
v. i dely used method of prevc nrion in enden iic areas.
Cholera vaccines wete introduced bi krran
, strains, with their toxin genes deleted. While the
within a vear of the discovery of the vibrio. The
r /
origin id vaccinffl were live sus- pen -sions of vibrius.
As they gav* rise to adverse reactions, they were
-
replaced by killed vaccines The vaccines used
iradiri -DnaHy are killed suspensions containing 3000
nil][ion K cholcrac per mtf , gym posed of equal
MS
numbers of Ogawa and Inaba serotypes* given by
subcutaneous or ini ram uvular injccidoo. Many
laboratories employ classical cholera and El Tor
vibrio in equal numbers in rhe vaccine . Strain
0* 139 vaccine has diso beco prepared. The
concentration of the vaccine has been increased to
12,000 million per ml ,, in order to improve the
anrigcnic stimulus,
Several eoncrolled field trials in endemic areas
with wiooa types of injected vaccines have shown
that the protection atfbrded hv them dod not exceed
-
5Q tifl per cent; the duration of protection m only
3 6 months the rate ®f protection in endemic ITeii
“ '
increases with age; i single dose of vaccine i $
ineffective in children below five years of age while
two doses at 1-4 week intervals mto ptoreeiire; a
single dose confers good protection in adults- due
to its acting m a booster on top of prior naturd
immunisation ; cell - free somatic antigen
preparations are as- effective as whole cell vaccine;
chcrc is good cross- protection between classical and
El Tor vibrios ; rhe crotfs- protection between Ogawa
and Inaba serotypes is doubtful and requires further
study , pending which ViCcirte Containing the
homologous- serotype is to be employed. .Aluminium
hydroxide and phosphate adjuvant vaccines
produced berrer inumuilly, particularly in young
.
children* TOKOid vaccln ee have not been been
successful. Injectable vaccines do not provide any
local immunity. in the mtcttiiul mucosa. They are
at f*
also unacceptably feactogenic. I lenee attention has
been directed to oral vaccines .
Two tvpe-s of oral vaccines have been tried
recently; MEW oral whole cell vaccines with and
without the inclusion of the B subumr of CT„ and
/eve ora/ vsiconeii with classical , El Tor and 0- 13^
results have been promising, problems- remain to
be solved before they are cleared fcf genets! m
An Ideal cholera vaccine is yet to be found .
Cholera vaccination was- a compulsory requirement
for International travel , but now very few countries
'
insist on chis.
Copyrighted material
316 i Texltiook of Miaobiology
Trout morn : The treatment of cholera consists
essentially of the prompt and adequate replacement
of lost fluid and electrolytes . Oral administration
of fluid containing glucose and electrolytes, either
alone or supplemented by intravenous fluids is a
highly successful and freely available method of
treal mg cholera . Cereal hafied preparations are-
equally effective and usually more acceptable .
AmibacCen .il therapy is of secondary importance .
Oral tetracycline was recommended for reducing
the period of vibrio excretion and the need for
parenteral fluids. Initially cholera vibrio* were
in Liifbnnly suscept i ble to ail ant i him ics acti ve agai nst
Gram negative bacilli , but since 1979, multiple
drug resistant strains have become increasingly
common.
VIBRIO MIMICUS
So named because it closely resembles cholera
.
vibrios m bioclientical features, V jujinfcu^ can be
differentiated by its failure to ferment sucrose. Like
V, cholerae, it grows best at low salt concentrations
( 0..5-l % ). Tt has been responsible for many sporadic
eases of diarrheal disease on the Gulf Coast of the
USA, Infection is acquired from eating seafood,
,
-
especially oysters i :ic di easc is self 1 incited.
Clinical manifestations resemble those caused by
- It is killed at aC in 1,5 minutes. It docs not
V , panthacmolyticus.
HALOPHIUC VIBRIOS
Vibrios that have a high requirement of sodium
chloride arc known as halophilic vibrios. Their
natural habitat is sea water and marine life. Some
halophilic vibi ios have been shown to cause human
— .
disease V pnrahicniultrticuii V. a mofyr / cijs and
V. vulnificus. ^ Not all strains of V , parahacmolyticus are
VIBRIO PAR AH \ EMOLI TICUB
V. patahaeinofyticus is an enternpathogcnlc
halophilic vibrio originally i -^olited in 1951 in Japan
as the Causative agent of an outbreak of food
poisoning due to sea fish - Gastroenteric due to
this vibri ' i has since been identified in several
»
countries and it is now considered an important
cause of food poisoning throughout the world. It
inhabits the coastal seas, where ir is found in fishes,
arthropods such as shrimps and crahs, and molluscs
such as oysters . In Calcutta , it has also been found
in small pond fishes.
In morphology, it resembles the cholera
vibrio, except that it is capsulatcd , shows b .|x>lar
'
sta:iling and has a tendency to pleomorphism,
espec ially when grown on 3% salt agar and in old
cultures . Unlike other vibrios, it produces
pen trie hous flagella when grown on solid
media. Pillar flagella are formed in liquid
cultures .
Ir grows only in media containing NaCl. It can
tolerate salt concentration upto 8 per cent but not
10 per cent. The optimum salt concentration is 2-
4 per cent. On 1 CBS agar, the colonies an; green
with an opaque, raised centre and flat translucent
periphery. The siring test is positive .
It is oxidase, catalase, niirate, indole and citrate
positive. Glucose, maltose, mannitol, mannose and
arah mose ate fermented producing acid only.
'
Lactose, sucrose, salicin, xylose, adonitol, inositol
and sorbitol are HOE fermented .
grow at 4 "C but can survive refrigeration and
freeing. Dtving destroys it. It dies in distil led water
or vinegar in a few minutes.
"
I hrcc antigenic components have been
recognised somatic O, capsular K and flagellar
H antigens . Scrotyping Is based on O and K
antigens, 12 O groups have been recognised and
-
59 di ; inct K antigens-
pathogenic for human beings, Jt has been found
that strains isolated from environmental sources
(such as water, fish, crabs or oysters } are nearly
always nonhemolytic when grown on a special high
salt blood agar { Wagauuma agar) , whi c strains
'
from human patients arc almost always hcmoiytjc.
This is called the Kanagawa phenomenon and is
due to a heat stable hemnl >sin. The significance of
Copyrighted materia
 4 Vibrio
Table 13.6 Some characteristics- d V, parabaemotylicm and V. aIgtnclytlcvs
VL pvrnhKcmolyticuF
Indole
V.P.
Nirrafc rcductLan
—
4 +
Urease
Sy cru e Imne ill aciufl
^
—-
Swanning »
Growth in Wt> NaCl
7% NaCl
-
+
ltffi NaCl - Hr
thin hevTmlv & is is not known hut it IH U -SL'J is i
r
laboratory test for pathogen icitv; Kan ugawa posi t ivc
Strains hiring considered pathogenic ioi human
beings and negative a trains nonpathogcnic . No
enterotoxin has been identified - The vibrio is
believed to cause en.teritii by invasion of the
intestinal epithelium. V. parjhacmohtLu^ causes
food poisoning ass wilted with marine food. It also
causes acute diarrhea, unutoditcd with food
poisonirg. .Abdominal pain, diarrhea , vomiting and
fever arc the usual signs. Feces contains cellular
exudate and often also blond . Dehydration is of
moderate degree and recovery occurs in 1-d days .
Cases ate more common in summer, and in adults
than in children . In Calcutta, V partihacmo/yTrcuff
-
could tic isolated from 5 10 per cent of diarrhea
cases admitted ro the Infectious Diseases Hospital .
.
V puahMcmofyticusis common in sea fish in some
other parts of India but human cases are much less
frequent.
VlBHIfl Al lilMil YTICJI *
This halophilic vibrio resembles V piTth^ cmo-
Jjtkut in many respects and was tormerlv considered
a biotype of rhe latter , It has a higher sail tolerance,
Is VP positive and ferments sucrose ( Tahle .33.6).
It i« frequently found in sea fish. Its status as a
human pathogen is uncertain - It has been associated
with infections of eyes, cars ml wuunde in human
beings exposed to sea water.
317
V, i
^
'
j jToiyrrCuJ
+
=
*
V i n i t m V l I.M F I C I s
V vulnificus, previously known as L* vibrio or
JJentvAcir vulnifici , is a marine vibrio of medical
importance . It if VP negative and ferments lactose
but not sucrose . IT has a salt tolerance of less than
eight per cent . It causes two Types of illness. The
first is wound infection following contact of open
wounds with seawater. The second type occurs in
compromised hosts particularly those with liver
disease. Following ingestion of the vibrio, usually
in oysters, it penetrates the gut mucosa without
causing gastrointestinal manifestations and cliters
the bloodstream, rapidly leading to septicemia with
high mortality.
AKHOMUNAM P l. hKltlMUN AS
AM>
'
Besides the genu*. Vibrio, the family Vibrionaceac
also contains the genera Aeromorias and
Pfei;it> jTionas, some members of which have been
associated with human lesions.
AtromooMt hydtophiliL, originally isolated from
frogs, in which it causes the "red icy disease', has
been reported from many cases ot diarrhea and from
some pyogenic lesions in human beings .
PlesmmoBMM thigdfaidcs also has been reported
from diarrhea ] disease . Both these arc oxidase
positive , polar flugclluted, Crum negative rods and
may be mistaken for vibrios . They may be
differentiated horn vibrios by bioohem ical test* such
as utilisation of aminoacids.
Copyrighted material
318 * Texlooo* ol MicrobiolDBV *

further Reading,
Rjiruu. D mi WB G ret:no ugh 1992, CiWffa. Maw York:Plenum.
Collier L ec al teds ) - 199H. Toptcy and Williams MknMology andMicrobial Infections S'1 , edn. LoreJon : Arnold Vok . 2
*
and 3 L

Cofatelt RR iirhJ Hut ] 1994 . Environment } reservoir of Vibrio choleric Annair Acad So 740:44.
* ,

FaruK] SM ec al. 200.1!. Kcnecgecu.i: and evoJuii^n of V, eholerjc 0- 139. Proc Nit ALSIJ Sci (USA } 100: 004.
Honda T nni.iT lida L 99 j. The (whitgen iciiy of Vainnio /idriharjiwdfrTkus. Rev Med Microbiol 4:106.
Kjper JIB el a.1. 1995, Chcdera. Oifi MicrohM Ret. S:4H.
1

.
Lacey SWr 1995 , Chdten CM Infect /3UL 20:1409,
MctaL-anus JJ .Tii -ij.:! |L S^dcd-T 1994. ChnJE-r-j. v-jeciiics. Sere-aiL-e 265: 0 7.
^
Naif GB CT al. 199 b. Vrrtno ohtu1*rae G 139 -\Rengd. RevM td Aficra6io/ 7:43.
,

Copyrighted material
 Pseudomonas
Pseudomonas are a large group of aerobic ,
nonsporing Gram negative bacilli , motile by polar
flagella , They are ubiquitous, mostly saprophytic,
being found in water , soil or other moist
environments Some of them are pathogenic to
plants* insects arid reptiles . A few cause human
infection , typically opportunistic .
based on molecular analysis, pseudomonads have
been reclassified and many former Pseudomonas
species reallocated to new genera such as
13urkhoIderiiit Stenotrophomonas and others.
PSEUDOMONAS AERUGINOSA
Ps- pyucyanca: BaaHus pyocysncus
Morphology: It in a HLENDER Gram negative
. and pyomelanin ( brown ) in various combinations.
bacillus, 1.5-3 pm * 0 5 pm, actively motile by a
, Some strains may be nonpigmented. It is Tint known
polar flagellum. Occasional strains have two or
three flagella . Clinical isolates ace often pillared . It
is noncapsulated hut many strains have a mucoid
slime layer . Mucoid strains, particularly isolates
from cystic fibrosis patients have an abundance of
extracellular polysaccharides composed of alginate
polymers. This forms a loose capsule (glycocalyx )
in which microcolonies of the bacillus ate
enmeshed and protected front host defences.
Cultural characteristics; It is an obligate
aerobe, but can grow anaerobically if nitrate is
available - Growth occurs at a wide range of
temperatures, 6-42 "C, the optimum being 3? “ C-
lt grows well on ordinary media , producing large,
opaque , irregular colonies, with a distinctive, musty,
mawkish or earthy smell. Iridescent patches with
A metallic sheen are seen in cultures on nutrient
agar. C rystals are seen beneath the patches. It glows
on MacCohkey and DCA media , forming non-
lactose- fermenting colonies. Many strains arc
hemolytic on biood agar . In broth, it forms a dense
turbidity with a surface pellicle.
Pi. &cfuginoi& produces a number of pigments,
the best known being pyocyanin and fluotescin .
Pyocyanin is a bluish green phcnazinc pigment
soluble in wuer and chloroform, Fluorescin
( pyoverdin ) is a greenish yellow pigment soluble
in water but not in chloroform , In old cultures it
may be oxidised to a yellowish brown pigment .
PyocvanLi} is produced only by Pi . aerujfrrtfMa but
fluorescin may be produced by many other species
also. Other pigment produced are pyarubin {red)
whether the pigments have any role in
pathogenesis. Some of the pigments particularly
pyocyanin . Inhibit the growth of many other
bacteria and may therefore contribute to Ps.
aeruginosa emerging as the dominant bacterium in
mixed injections.
Biochemical reactions : The metabolism is
OMidatiue and nonfermentative . Peptone water sugars
are unsuitable for detecting acid production, since
this i:- weak and gets neutralised by alkali produced
from peptone. An ammonium salts medium in
which the sugar is the only carbon source is the
best . Glucose is Utilised oxidatively, forming acid
only Indole , MR , VP and H ,S tests sue negative .
Nitrates arc reduced to nitrites and further to
gaseous nitrogen . Catalase , oxidase and arginine
dibydrolase tests arc positive .
ClyssilloilLont As Ps . aeruginosa has become
Copyrighted material
330 i Taxttnofc of Microbiology
a very important cause of hospital infections , ICS
classification is essential hir epidemiological
purposes . Serotyping , bacteriocin ( pyocin ,
acruginusin ) typing and bactenopli . ige typing have
been used but are not entirely sati ^factory.
Restriction endonuclease typing with pulsed-field
gel electrophoresis is the most reliable method
avaUable-
Rm ^ unce: The bacillus Is not particularly heat
resistantj being killed at 55 nC in one hour but
, agents responsible for iatrogenic meningitis
exhibits a high degree of re-.istsuicc to chemical
agents. It is resistant to the common antiseptics and
disinfectant* such an quaternary ammonium
compounds, chloroxylenol and hexachlorcphane
and may even grow profusely in battles of such
antiseptic lotions kept for use in hospitals- Indeed,
selective media hive been devised for ft, aeruginosa
incorporating dettol or cetrimide. It is sensitive to
acids, beta ghiraraldehyde, silver salts and strong
phenolic Jisirifectants. Its suscept i billty to silver has
been applied clinically in the us-e of a Liver
sulphonair.i. de compounds as topical cream in
bairns.
Psr aentgr /ioM possesses a considerable degree
of natural resistance to antibiotics. Examples of
din i tally effect ice antibiotics axe amlnoglycoKi des
(gentamicin, amikacin ), cephalosporins (edotsedme,
ceftazidime , cefopemone) , fluoroquinolones
(ciprofloxacin , ofloxacin , pcfloxacln ), penicillins
( piperacillin , ticarcillin , azlocillin ) . For localised
infetions, topical colistin, polymyxin B or 1% acetic
acid may be useful.
Palhogenrctt ) : ' Blue pus was known as a
1
surgic al enrity long before Gcasvd (1SS3) isolated
/V. aeruginosa from such cases. Both the specific
names of the bacillus refer to its capacity to cause
“ blue
pufi' the term aeruginosa , meaning verdigris
T Equipment such as respirators and endoscopes,
which is bluish green in colour and pyocyana ,
being a literal translation of ' blue pus . 1
The pathogenic importance of the bacillus was
not adequately recognised till recently, when It haa
established itself as one of the most troublesome
agents causing nosocomial infections . In the
community outside the hospital, the most common
infection caused by Pi. aeruginosa is suppurauve
otitis, which is chronic though not disabling. In
the hospital, it may cause localised or generalised
infections. Localised lesions are cn mm only
infections of wounds and bedsores, eye infections
and urinary infections following catheterisation. Fs .
aeruginosa is the most common and most serious
cause of infection in bums . It is also one of the
following lumbar puncture. It frequently causes
post - tracheostomy pulmonary infection. Septicemia
and endocarditis may occur in patients who are
debilitated due to concomitant infection, malignancy
or immunosuppressive therapy. Ecthyma gangreno-
sum and many other types of skin lesions have
been described occurring either alone or as
part of generalised infection , mainly in patients
with leukemia and other types of malignancy.
Infection of the nail bed is not uncommon
following excessive exposure of hands to detergents
and water.
ft. afitigjjTUiia 1 ns been described as one ot the
.
agents responsible for infantile Hi urfiea and sepsis .
Strains isolated from outbreaks of diarrhea may
form a heat labile enterotoxi n and give a pnsirive
rabbit ileal loop reaction, ft , acrugincuu has been
reported to cause a self-limited febrile illness
{ Shanghai Aver) resembling typhoid fever In some
tropical areas.
The pre-eminent role of Pi. aeruginosa in
hosp i tal i nfccti on is. due ti > i rs resi stance to common
antibioticsand antiseptics, and its ability to establish
itself widely in hospitals. Being an extremely
adaptable organism it can sut vive and multiply even
with minimal nutrients, if moisture is available.
articles such as bed pans and medicines such as
loLions, ointments and eye drops and even stocks of
distilled water or plants and flowers may be
frequently contaminated - ft aeruginosa is present
on the skin of the axilla and perineum in some
persons. Fecal carriage is not common but may be
-
jr"*
1
opyrig hted _ n-
n atari
L I IL L
 < Pseudomonas
frequent following oral antibiotic; treatment or
hospitalisation .
The mechanisms of pathogenesis are not
clearly understood . Several toxic extracellular
products have been identified in the culture
liltnttSn such as exotoxins A and S. Enotoxin
A acts as in NADase , resembling the
diphtheria toxin . Good antibody response to
exotoxin A is considered a favourable sign in
severe infections xvitli ft. aeruginosa, Orher
toxic products include proteases , elastises ,
hemolysins and enterotoxin. The slime layer
acts as a capsule in enhancing virulence.
Laboratory diagnosis The bacterium grows
readily on most media,The identification of
pigmented strains of the bacillus from clinical
specimens is easy. But about 10 per cent of isolates
may be nonpigmented . Prompt oxidase reaction and
arginine hydrolysis help in their identification . It
mi; be necessary to use selective media such as
-
ci irimide agar for isolation from feces or other
samples with mixed flora . As Ps. a&Vginot& is a
frequent contaminant, isolation of the bacillus from
a specimen should not always be taken as proof of
its etiological role. Repeated Isolations help to
confirm the diagnosi -s.
Control; Prevention of ft aerugiaota cross
infection in hospitals requires constant vigilance
- septicemia . It is inherently resistant to mnst
and strict attention to asepsis. Antibiotic treat merit
is not jdwavs- satisfactory. Animals with eicjWri-
mentally infected hums have been protected by prior
immunisation with the homologous strains.
Immunotherapy in human hunts cases with
antiserum to Ps, aeruginosa may be useful .
Pseudomonas vaccines are being tried in Cystic
flbtosis patients who arc highly vulnerable to
pseudomonas infection .
Specific antibacterial therapy constitutes only
one aspect of the management of serious
pseudomonas infections . Treatment of the
underlying diseases, correction of granulopenia and
appropriate supfjortive therapy need attention .
Occasional opportunist infections may be
> 321
caused by a few other species , such as Ps.
fhioicsrejis, ft puiidi and ft. sfurzeri.
STENOTRGPHO MONAS M ALTO PHI LA
-
( VO n Ml M I rY PSBUDOMONAS
MALTOPHILA )
This i* a saprophyte and opportunistic pathogen ,
causing wound infection , utinaiy tract infection and
septicemia . It is usually oxidase negative and
acidifies maltose in addition to glucose , lactose and
sucrose. Infections usually respond to cotrimoxazole
and chloramphenicol .
BURKH OLDEST A CEPACIA ( FORMERLY
PSEL OOMONA S Ch' PA t . fA J
I’ his is a plant pathogen causing onion rot ( ccpu,
I .arin for onion}. It is increasingly being recognised
as an opportunist environmental pathogen ,
particularly in those with cystic fibrosis or chronic
granulomatous disease, in whom it causes fatal
necrotising pneumonia , It is nutritionally very
versatile. It can grow in many common disinfectants
and can ever use penicillin G as a sole source of
carbon I It n oxidase positive and acidifies mannitol,
sorbitol and sucrose. It can cause urinary, respiratory
and wound in (ectlnns, peritonitis, endocarditis and
antibiotics .
GLANDERS
BLRKHOLDBRIA MALLEI ( FORHELY
PSEUDOMONAS MALLBI )
Tile bacillus had also been classified variously
as Locfficrdh , Pfeiffcrdh , Milteomym ,
Actinubacillus and Acjjrefrtiiacfer. It is the
causative agent of glanders ( mxfledjs, in Latin}, a
disease primarily of equine animals - horses,
mules and asses - but capable of heing
transmitted to other animals and to human
beings, The bacillus was discovered by Loeffkr
and Schutz (1882J .
.
ft. malfe; is a slender, nonmotile Gram negative
bacillus, 2-5 pm * 0.5 pm staining irregularly and
Copyrighted materia
322 ^ TexlOnok ql MiorObiulOQv *
often i> iving a beaded appearance . Il is an aerobe
and facultative anaerobe, growing on ordinary media
under a wide range of temperature. Coleriits which
are HIIUII and [ranslusecnt initially become yellowish
anil opaque J >[] ageing. potato, a chnwAAttic
jmhert Koacy - lilce growth appears , becoming
greenish lellow resembling Pi. aeii iaou , IL IS
^
quire inactive biochemically, attacking only gluc-efe- v .
The rafur .il disease in equities occurs nl two
forms — glanders and farcy. In glanders , the
respiratory system LS involved , with rhe t orimsition
'
of firm , round nodules and a pmluse catarrhal
discharge Irmii the nose. Farryr follows infection
through the skin and is an involvement of the lymph
vessel? and nodes, which stand out as hard cords
beneath the skin.
Guinea pig£ arc susceptible and intnpaxtoneal
injection into male guinea pigs induces chc fifrans
reaction. This consists of swelling of the regies ,
inflammation of mnica vaginalis and ulceration of
the scrotal skin . The Straus reaction is not
diagnostic of glanders, as ir may also be produced
"
by inoculation of Other bacteria such as Brucella
species , Preisz- NocarJ bacillus, Actinobadllus
ligmcmi and Pa . pseudomallui .
Human infection is usually occupational , found
in ostler?, grooms and veterinarians. It may be acute
or chronic and is protean in character, with
locali &arion in the respiratory tract , shin or
subcutaneous tissues. In acute glanders, there is
fever, mucopurulent nasal discharge and severe
proftration. The fatality rate is high. While htiman
infection is acquired only rarely from infected
animals, Laboratory cultures are highly infectious
and PSL mailer is one of the most dangerous bacteria
to work with.
Animals suffering from glanders develop a
delayed hypersensitivity to rhe bacterial protein.
This is the basis of the miUtiii test used for
diagnosing glanders. This is analogous to the
tuberculin test and may be performed by tile
subcutaneous, intracutfmttius or conjunctival
methods .
MELIOIDOSIS
UllILKHOl MHHI 4 PSttUOOMAE . I . R. C
( F 1 > HMERLT PsEtDOMO\A S
PshUPOHA S.i.E l )
Also known as Whitmore's bacillus, Actinobacillus
whitmori, Mutiromjfver pseudomnllci , LoeiEefdk
pseudomallei ) .
This is the causative agent of meliuidus- is, J
glands -like disease, epizootic in rodents in
southeast Asia, India and North Australia. (The
name is derived from JTTCJJS , a disease of asseS
[glanders ] , and ados meaning resemblance ). The
disease was first described in human beings by
Whitmore and Kmhnaswami (1912) in Rangoon .
Whitmore ( 1913) isolated the bacillus. It resembles
Pi , mallei but differs in being motile, Liquefying
gelatin and forming acid from several sugars . Two
thcrmolabile exotojcins, nnc lethal and the other
necrotisirg iuvt been identified in culture tikrJTo .
The human disease mav take different forms . It
Jr
rnav be an acute septicemia , a Subacute Cyphoid -
likc disease , or pneumonia and hemoptysis
resemhlingtulierLuldsis. In clrrotliC form , there mac
be multiple caseous or suppurative fiici , with absces- s
formation in the skin and subcutaneous tissues,
bones and internal organs. Acute melioidosis has a
high case faijJity rate. Serological evidence indicares
that inappareut infection is common in endemic
areas. Long latency and reactivation may occur as
rhe bacillus can survive intracellularlv in the
reticuloendothelial system. The bacillus has been
isolated from water and soil in endemic areas, Ir is
a soil saprophyte that cause? infection in rodent ?
and humans accidentally. Human infection occurs
commonly through skin abrasions or by
inhalation.
Diagnosis may be made by demonstration of the
bacillus in exudates by microscopy (small irregularly
staining Gram negative bacilli , showing typical
bipolar 'safety pin appearance with methylene blue
stain ), isolation hv culture from sputum, pus, blood
oi unne , or bv serology { F1LJ 5 A for IgM and lgG
Copyrighted materia
 « PHudomonHi > 323

antiljody,, indirect hemagglurinarupn ) . A PCR test cobrunomolt , tetracycline, amoocyc 11 Un davulamtc f

has also been dnrclopaL or chloramphenicol . Prolonged treatment , for many
Ceftazidime is the drug of choice , along with months may be necessary.

Further Reeding
.
Biltch AL and Smith RP ( tds ). 1994. Pseudomonas teruginoa infections NrwYcrk] Marcel Dekker.

Fick RE (cd ) , 1992. ftcwforncwflf

- -
Dimr DAE . 1991. Meljoadosis- Ciin .UHTH.' MI ?! R. r . > 2
The C wrtwnjst, ftfJkigrnwjs ant? Djseaw- Boca RatuniCRC-
^
'
'

G - lligan I Mi 199 ? Fseudominaa and Burkholdcua In Mnrr . iy I’ K rf : il Annual of CJjnrcid Afjrrrfripfc y,

,
\Vi 'ltLiiylnn: AmL iii :ui
' '
Tvl 5 trd11 - i dn y.
yn ' i' l ; t l
'
^
^
Gould LM and K Ri ^ 1985, P - eudomunis lefnpinosa - Clinical minifeftutions and inanapemenr . Lancer 2:1224,
1

Lwluwamw A and S. Eonnwonkitti 1989. Melkiiilwis. Rev fnfprtDir - 11:413-

-
MonAon AJ Jr. and RPAflfcrwl 1984, lipidemiulugy of infections due lu Pfceudori masaenrpinMa. Rev in fret Dis, drSuppl
5 -627 -
PatamasiKun Per al , 1982, McLiuidn*i*. / ftJj> rr 1 DO:175-

Copyrighted material
 K?
CO
Yersinia, Pasteurella, Francisella
The plague bacillus and many other Gram negative*
shore bacit!] that are primary pathogens of rodents
were grouped together in the genus Pasteuretla.
Based on cultural and biochemical differences* this
.
-
gnou p has been divided ] i no [] Ltee genera Yet surra *
PasteurcH i and Francisella. The genus Yersinia ,
containing the radical ly important species Y.patis
{ the causative agent ot plague ) * Y. pyeudo
'
fizbtn. rdnsr:i ( a primary pathogen of rodents) and
Y.cntcrocoHticM ( which causes enteric and systemic
diseases in animals and human beings) was so
named after Alexandic Yerstn * who discovered rhe
^
plague bacillus. The genus tjsfnni is now assigned
to the family Enterobjctcriaceae . The genus
PasreuneiJa contains several related bacteria causing
hemorrhagic septicemia in different species of
animals and occasionally producing local and
systemic infection * in human beings, grouped under
it common species named f ? rntdrochfa. One of these *
P. Aviacptkt is the chicken cholera bacillus used
by Pasteur for the development of the first attenuated
bacterial vaccine . Hence the name PdsCeurella. The
genus Fnndtttla , co mailing of F. rrWarensis* is
named after Francis for bis pioneering studies on
tularemia, caused by this bacillus .
Y K H S I M A PtiSTis ( K O k A t K i i i .Y
P A H i ' l - I I k l - l . l A PKS 'I IS )
The plague bacillus was discovered independently
and simultaneous I v hv Yersin ANLI Kitasato ( 1 S 44 )
in Hong Kong at the beginning of the last
pandemic of the disease.
Morphology: Yi peVtiY is a short , plump, ovoid,
Gram negative bacillus, about 1.5 im * 0.7 pm in
^
sire , with rounded ends and convex sides* arranged
singly, in short chains Dr in small groups. In smears
stained with Giernsa or methylene blue, it shows
bipolar staining ( safety pin appearance ) with [ he
two ends densely stained and rhe central area clear
{Fig. .55.1). Plegmorphism is very conmon and in
club -shaped, filamentous and glam forms.
—
old cultures, involution forms are seen coccoid ,
Pleomorphiiun i & ohanicterirally enhanced in media
containing 3% NaCl.
The bacillus is surrounded by a slime layer
(envelope or capsule ). Jt is nonmotile * nnrtsporing
and non acid fast .
Cultural rharaderistics: The plague
bacillus LH aerobic and facultatively anaerobic ,
timwth occurs over a wule range of pi I (pi I 5-9, 6,
optimum pi I 7.2) and temperature ( range 2~45 “ C ) .
The optimum temperature for growth ( unlike most
pathogens) is 27 ‘G but the envelope develops best
at 37 ^C-
1 T is not nutritionally exacting and grows on
ordinary media. On nutrient agar, colonies arc small,
delicate, transparent discs, becoming opatjue on
continued incubation. Colonics on blood agar or
other heolui containing media are dark brawn due
to the absorption of the hemin pigment . Colourless
colonics arc formed on MacGonkjey's agar, In broth ,
a flocmlent growth occurs at the bottom anti along
the sides i > t the tube, with little or no turbidity. A
delicate pellicle may form later. If grown in a flask
of broth with oil or ghee (clarified butter ) floated
on tup (ghee broth ) a characteristic growth occurs
which bangs down into the broth hum the surface,
resembling stalactites ( stxbctiCe growth ) ( Fig* 35.2).
Copyrighted materia
 * Yersi.- na .
1: nmii ^ niJii . jl rcueliotlNi Glucose * maltose and
nmnriito] but not lactose* sucrose or rhamnosc arc
k'nnenfet! with the rmrl rtirFfl afudd hut no
^
fndolc U not produced , IT is MR positive und VP
and citrate negative catalase positive and acseulin
positive and mxtdnse iud urease negative . Gelatin is
not Lt[ uefied * Based on the fermentation of glycerol
and reduction r,fnitrate * Devignat hsf iListirL ULshed
ihnrophysiological varieties of Y , /wstjs. This typing
'
^ 1 or FT) best formed in cultures incubated at
appeaih rn he o1 epldemioLogicd significance
because of the different geographical distribution
of the types (Table 35.1 ),
Ik' si ^ . nu L- : The plague bacillus is easily
destroyed by exposure to heat, sura light , drying and
chemical disinfectant. IT is destroyed by heat at
,
55 JC nr by phenol in 15 minutes. It remains
vi able for tong peri ods i n cold, moist ctjvi ran ments .
It can survive for several months* and cwn multiply ,
in the soil ot rodent burrows. All strains; are lysed
.
by a 5-pccitic Jintipl Tguc bacteriophage at 22 “ t .
I
©0
Fl < j.35.1 Smt^ ar Iram gland punei-tinl inert plague
A Cast;
showing Y pesiis with Bipolar staining {safely pin
appearand ), a few fed blond cell and leucocytes
Pasleurella , Ftaivci Etll .a 325
^ *
Aniigcnsir Inxios nnd other virulence
laclors: PI ag\ic bacilli arc antigcnically
homogeneous and serotypes da not exist . The
antigenic structure is complex. At least 20 antigens
have been detected by gd diffusion and biochemical
analysis. Many ui them have been claimed to be
virulence factors. They include the following:
1, A heat labile protein envelope antigen ( Fraction
37 aG, Tr inhibits phagocytosis and LK generally
present only in virulent strains. This plasmid
encoded antigen has been considered! a virulence
determinant but (KTaiianal strains deficient in
Fraction 1 antigen have been isolated from fatal
human cases. The antibody to this antigen is
protective in mice,
2, Two antigens designated V and W and always
produced together have been considered to be
the virulence (actors as they inhibit phagocytosis
and intracellular killing of the bacillus .
Production of V and W antigens is plasmid
mediated.
Fig. 3 s. 2 Y. in glut broth Luriufe. i1 nlaclit &
. growth.
Copyrighted material
336 * TB* Ebook of M crobioiogy *
3. Virulent strains produce A biiCierutcirt {l Sticin ll,
^
coagukse and tlbrinolysin . l ^csticLn l inhibits
strains of Y . pseudotubcrcuiosis , Y cntcro
coEtia and E. coli.
4. Tilt term 'plague toxins' refers TO at least TWO
classes of toxiTis found in culture filtrates or
cell lysates. T he first is the endotoxin , i
lipopolvsaccharide similar to the endotoxins of
nmeric bacilli . The second class of toxins is
protein in naturef possessing some properties of
both exotoxins anil endotoxins. They are
thcrmolahilc and may He tMflided Hut do nor
diffuse freely into the medium and arc released
only bv the lysis of the cell . They are called
murine wxint rhev arc active in rat* and mice
but nor an guinea pigs, rabbits and primates. On
, occurred during the reign of Emperor Justinian {AD
injegrion into experimental animal plague toxins
^
produce local edema and necrosis with systemic
effects on the peripheral vascular system and liver.
The role of plague toxins in natural disease in
,
human brings is not known.
5. Virulence -also appears to be associated with an
unidentified surface component which absorbs
hemin and basic aromatic dyes in culture media
to iorm coloured colonics.
Table 35.1 Biotypes o\ Yersinia paslii
Variety Glycerol
fermentation
Y. pans vac nriomlis
K pCSfjV Wtfa JJJ ffL/ Ciil + +
V, petti* w. mdifVtt / ji + -
6, Virulence has also been associated with the ability
for purine synthesis .
r
PLAGUE
Plague is INL ancient scourge of mankind. The
disease wan familiar to the ancient civilisations of
Ada. The B/wgavaiJid Bunina urged householder;
to dec when rat foils were noticed .
Central Asia or the Himalayas is believed to
hive been the original home of plague, from where
ir han, in wave- after wave, spread for and widch
causing epidemics and pandemics, exacting a toll
of human life surpassing Any other disease . The
identity of the Biblical plague of the Philistines
(1320 Be ) is in doubt but the pandemic that
342 ) wan undoubtedly bubonic plague ( believed to
hive been caused by Y . pads, van iiiriqua) and
caused a hundred million deaths. In the fourteenth
century, jwndenuc plague known as the ' black death '
is believed to have killed a quarter of all mankind ,
( rar. maJicvoJis believed responsibleJ . The name
' black death’
may have been derived front the
extensive cutaneous hemorrhages and gangrene
often seen in fotal cases of plague.
Menft GugnpAxcd distribution
reduction
* Primacy kri in India,
Myanmar, and China.
Causative agent of 1894
pandemic. Responsible for
wild in Western USA*
South America, South Africa .
Transbaikalia., Mongolia,
Manchuria, perhaps
icsponsible For Justinian
plague.
Southeast Russia.
Copyrighted materia
 * Ystrinla Paateuralla, FrandsaHa *
Historians of plague identify 41 epidemics
before the birth of Christ and 109 epidemics in
ihe next 15 centuries. There are records of 45
pandemics between AD 1500 and 1720, The disease
was quiescent in the eighteenth and nineteenth
centuries and confined to endemic foci . The last
pandemic started in Hong Kong in 1S94 and spread
throughout tine world , (caused by y.pcsfss van
of /cntaJis ) . Indi :t was one of the countries worst
hit by this pandemii Plague reached Bombay in
P
1B96 and spread all over rhe country during the
next few years1 caur- ing more than 10 million deaths
by 191S. It gradually receded thereafter , though
occasional cases continued to occur M -J endemic foci
till 1967. Mu furtlier plague cases were seen in India
til! 1994 , when in August a mmfatal outbreak of
bubonic plague was reported from Maharashtra
( Beed district ) . In September pneumonic plague
was reported in Surat and adjoining areas of Gujuirar
and Maharashtra , causing much panic and
consternation, A few cases were reported from
different pans of north India also, probably caused
hy the exodus from affected areas. During the
outbreak which subsided in two months, there were
over fitNK) suspected plague canes and 60 deaths. In
February 2002, plague struck again causing ashore
outbreak near Simla, claiming 4 lives.
Plague survives in several scattered natural foci
in many countries ( Fig. 35.3 ) in wild rodent*,
occasionally causing infection in human contacts.
In India at least four foci of plague arc known . One
is the region near Kn|ar at the tri junction of Tamil
Nadu, Andhra and Karnataka. The second is the
Bccd-Latur belt in Maharashtra from where the
Surat epidemic emanated - I he third is in Rhoru in
Himachal Pradesh where the 2002 outbreak took
place * and the fourth is a small pocket in
Uttaranchal .
In human beings, plague occur* in three major
forms: bubonic, pneumonic and septicemic. In
bubonic plague, after an incubation period of 2-5
.
days The lymph nodes draining the site of entry of
the bacillus become Infected. In some, the infection
327
remains localised at the site of flea bite, with only
minor constitutional symptoms ( pcstis minor). As
the plague bacillus usually enter* through flea bites
on the legs, the inguinal nodes are involved acid
hence the name 'bubonic’ : bubon meaning groin ) .
The gland* become enlarged and suppurate. The
hadlli enter the bloodstream and produce
septicemia - Sometimes there are hemorrhage:- in. to
the skin and mucosa. Disseminated intravascular
-
coagulation 1 common and may lead to gangrene
of rhe sk: n , fingers and penis. The case fatalitv in
untreated cases may be 30-90 per cent.
Pneumonic plague may be seen sometimes
during epidemics ofbubortic plague. Rarely, pm nary
pneumonic plague may occur in epidemic form , an
happened In Manchur ::i during 1910-1912 , cau ^ i ng
some 6(1,(M )0 deaths. Pneumonic plague is Spread
bv droplet infect inn . The bacilli spread through
the lungs producing hemorrhagic pneumonia.
Cyanosis is very prominent. The bloody mucoid
sputum that is coughed out contains bacilli in
enormous numbers . Pneumonic plague is highly
infectious and in untreated patients , almost
invariably fatal.
Septicemic plague i -. usually the terminal event
in the bubonic or pneumonic plague but may
somen i ncs occur pr i mari ly- Menmgi ri L involvement
may occur rarely. Human carriers have not been
recorded hut ahymptumatic oropharyngeal infection
has been observed in some contact*.
Epidemiology? Plague is a zoonoric disease .
The plague bacillus is naturally parasitic in rodent*.
Infection 1* Transmitted among them hy tat fleas.
The fleas acquire the infection hy feeding on
infected rodents. In the flea, the bacilli multiply in
the stomach to such an extent that they block the
pruventficulus. The interval between the ingestion
of infected blood and blocking in [ he provenifKtllus
is the extrinsic incubation period, which is usually
about two weeks in Xenopsylla cheo/ns. When such
a 'blocked fie? bites .mot her rodent it cannor suck
1
,
in blood because the bacterial mass blocks the
passage mechanically The blond, mixed with the
Copyrighted materia
 I

328 4 Textbook oS Microbiology

YJZRSINIA. PA5TEIMELLA, hRAM"ISELLA

"

l* CA^l
'7
.
'
*

*
- :
jpr
#

*3
I >

-
\ -
H

1
1 4
(?
P
/
Fig. 35.3 Natural foci ef plague, known and suspected jKnown areas - dark; Suspecled - shadedl

bacteria is regurgitated into the bite, transmitting
the infection. Infection may also be transferred by
contamination c »f the bite wound with the feces of
infected flenH . When a diseased r:tt ( lies ( rat fall ),
the fleas leave the carcass and in the absence of
another rut , may hire human beings , Causing
bubonic plague .
Several species of fleas may act as vectors, the
most important being Xcnnpsvlh chtopi* , X . astia
and C’erutnphyfhifr fa^qintus . X . ohttopis , the
predominant species in north Jndia is a more
efficient vector than the south Indian species X .
Utk. This has contributed to the more extensive
nature of plague outbreaks in the north a* compared
to south India . Plague epidemics generally occur
the twentieth century, helped to clarify the
epidemiology of plague. It ivss (bund thar plague
produced epizootics First in Rattus oorvegicua
( sewer rut ). When their number dwindled, the
disease passed to the domestic rat, R, It was
from the domestic ru that the infection Spread to
human beings.
Two natural cycles of plague CKJSI, the domestic
and the wild . The tenni htrban or domestic plague'
refers In plague that is intimately associated with
human beings And rodents living with them ,
possessing a definite potential for producing
epidemics. "Wild nr sylvatic plague ' occurs in nature
and in wild rodents, independent ot human beings.
I “ he rodents involved vary in di ffenmt regions, Over
"

in LMC cool humid seasons that favour the

, TOO Species and Subspecies are involved. In Western
multiplication of fleas, leading to a high Lflea index’ USA prairie dogs , ground squirrels* wood rats and
fmean number of’ fleas per rat ) , In the hot, dry mice arc found infected. In the endemic areas of
weather , fleas do not thrive and the transmission of the USA, cases of human plague have occurred
infection is interrupted . following contact with wild animals, and even with
The studies of the various governmental Plsgtw domestic carnivores, particularly pet cats. In java,
Commissions In Bombay, during the early years of [ he held lit is the reservoir . In India , [ he gerhil

Copyrighted material
 * Yersinia. Pasteurelist , FranciseHa
{ Tatcra indies) and the bandicoot are infected .
Human infection may occur during skiruling And
handling of carcases of infected wild animals ,
Carnivores, including cats and dogs can fret infected
by eating infected rodents or through their fleas.
Clinical plague isseldom seen in dogs, but may desktop
in call. Human infection frfntii[ili.dat ion < H RSpinloty
droplets from infected cans has been repotted .
In enzootic foci, plague may persist for long
periods. Infected fleas may survive lor ewer a year.
The bacilli can remain alive and even multiply in
the soil of abandoned rodent burrows. They can
infect new rodents rhat may reoccupy such burrows.
This Itay account for the 1* nag period of (JuitHCOtC
acid subsequent re - emergence charActerivtic of
plague. Attenuated strains of plague bacilli have
been isolated from natural foci. They may regain
virulence when plague becomes active. Eradication
of plague is an unlikely prospect as it is a disease of
—
the earth of rodents that live in burrows and of
the fleas that live oa them . Only when hu man be i ugs'
or domestic animals trespass on these natural t < >ci
do human infectious set in .
In the 1990s , there Iras been a n -emergence of
plague in countries where it had ceased to be
noticed for many years . This has happened in the
-
China in Asia, Malawi and Zimbabwe in Africa,
the erstwhile USSR in Europe and in the USA.
—
developing and the developed countries India and
Plague bacillus strains carrying plasmid h -nrne
resistance to mi il ri pic a ntihiotirs wr re re|iorted hunt
Madagascar jn 19^5, These have the potential to
spread and pose a great threat.
Laboratory diagnosis: The laboratory should
lie able to diagnose plague not only in Immins, but
in rodents also , as timely detection ul infection in
rats may help to prevent epidemic spread .
A rat which died of plague may carry infected
ileus and should be handled with care. Pouring
kerosene oil over the carcass is a simple method of
eliminating the fleas. In the laboratory, the carcass
should be dipped In 1% lysol to destroy
ectoparasites.
129
During epizootics, it is easy to diagnose plague
itl ran. Buboes are usually present NL the CffViCll
region . Tticv arc hard and can bo moved uitder the
skin t ) n section , rhe bubo may show congestion,
,
hemorrhagic points or grey necrosis . Smcare from
the bubo stained with methylene blue show the
bipolar stained bacilli . The fluorescent antibody
technique may be of use in identifying plague bacilli
in the impression films of the tissues. Bacilli in
bubo show considerable pleortiorphism. The liver
is mottled, with red, yellow or grey stippling. The
spleen is enlarged, and moulded over the stomach,
with granules or nodules on the surface . A
characteristic feature is pleural effusion which may
he clear, abundant and straw coloured nr, less often ,
bloodstained , Bacilli may he demonstrated
microscopically in spleen smear* and heart bfood.
Cultures tuny be made from the buboes , spleen ,
heart blood acid particularly, from bone marrow in
decomposed carcasses.
In badly puoiikd carcasses , microscopy and
culture may not be successful. The putrificd tissue
rubbed on the shaven abdomen of a guinea pig can
infect the animal. Diagnosis may also be established
fhiimcscenf staining.
-
by demonstrating the F I antigen by immno-
In human bubonic plague , the bacilli may be
readily demonstrated in buboes by microscopy,
culture or animal inoculation . Blond cultures ate
often positive .
In pneumonic plague , the bacilli can be
demonstrated in the sputum by micmscopv, culture
or animal inoculation.
Serological tests are sometimes useful in
diagnosis . Antibodies to the F-I antigen may be
detected bv passive hemsgglutination. Rise in titre
of antibodies in paired sera m titre of 128 or above
in a single scram sample can be considered positive.
IgG and IgM ELISA test -s have been developed.
PCk is a rapid and sensitive method for presumptive
diagnosis of plague in clinical material and fleas,
1'rophyLftxis:; In the prevention of domestic
plague, general measures such as control of fleas
Copyrighted material
Hidden page
 H Yersinia, Pas1auf & lta. Ranchsella 351

Table 35.2 Same differentiating Features among Yersinia and Pasieurella

Y . ptiff ? y. jMtudofuiidrcutoiJJ Y . Cflterocoliiica P muhoridjl

Motility at 22 ®
C +
*
Growth on MacCfonl^ jfs agar f
Acid from sucrose *
* + *
Add from nulbK t +
Indole + +
Oxidase s-
Urease 1

— + r
*
Ofnithine decarboxylase
- - * 4-

Y, enterocolitis^ h.i - been isolated from a wide
range of domestic and wild animals and , in recent
years, LS increasingly being reported from human
clinkaJ material. It produces Three types of disease
and sheep . It may some times occur
cats, rats, cattle
ns icorrmiensal in the human respiratory tract also.
Human infection is rare hut may occur following
nnimdl bites or trauma I'he clinical tnInifctUMni
,

in human beings. The first Type occurs in young
children ns self - limited gastroenteritis or
enterocolitis which may be either inflammatory or
noninflammatory. The second is mesenteric adenitis
and inflammatory terminal ileitis in older children
that may mi mb appendicitis . The third category LH
i systemic disease typically in adults , often
characterised by bacteremia, meningitis, arthralgia
or erythema nodosum . Persons belonging to HI . A
- B 27 group are prone to develop reactive arthritis.
PASTKI KEIXA M I i . m c m . v
( formerly FastcurclU Scptica )
A group of related bacteria isolated from
'
hemorrhagic septicemia in a variety of animals and
birds had, in the paat, been named according TO
their species of origin— P. hftvincptLii ,
HVWeplica, etc. Though rhev show some degree of
host specificity, they arc so alike in other respects
that rhev are now considered strams of a single
species designated P. muhocidx .
P. multcifhia is a non motile . Gram negative
bacillus generally resembling YendrUa but differing
In being osidase positive , producing indole and
failing TO grow on MacConkcyr’s agar.
The bacillus is often carried in the upper
respiratory tract of a variety of animals such as dogs,
may be local suppuration following animal bites
{ wound infection , cellulitis, abscess, osteomyelitis),
meningitis following bead injury, respiratory tract
infect LOO {pneumonia , bronchitis , sinusms ) or
appendicitis and appendicial ahseefis.
The bacillus is sensitive rn tetracycline and
streptomycin And most strains to penicillin SLS well.
FkAMCIStiLLA TIFLAKKNSIS
(TASTEL' flELJ.A TVLAREN5i$t BflULkLLA
TULARESSIS )
This is the causative agent of tularemia, a disease
of rahhitj anil other rodents, originally described
mTulare county, California. Infection is transmitted
hy ticks and several other arthropod vectors. Human
infection may occur by direct contact with infected
rodents such a * rabhits or through rich hires. It can
also be acquired hv ingestion of contaminated meat
or water and in halation of infective aerosols,
It is a minute , capsulaTed , non motile Gram
negative bacillus, about 0.5-0.7 pm * 0.2 pm in
sire. It resemhle-s mycoplasma in being filterable
and in multiplying by filament formation and
budding, besides binary fission- In infected animals,
It acts as an intracellular parasite, being found in
Urge masses inside the liver and spleen cells- Jt has
fastidious growth requirements and special media

Copyrighted material
332 Textbook of Microbiol
*
^
such AS Francis blood dcxrrosc cystine agar have ro like respiratory infection. The disease may also he
l:c employed for its isolation . Minute ttartSp.Lrent waterborne, as a result of water pollution by the
colonies appear after incubation for 3-5 days- \ LTL 1 .I ot infected rodents. The bacillus is highly
'

Srrainsof S. fu/arenfrjs have been subdivided i nto infectious and laboratory infection has been quite
'

biotypes based on their virulence and common. Diagnosis may be made by culture or by
epidemiolugical beliiviour. Highly virulent strains inoculation into guinea pigs or mice. Agglutinating
arc found only in N. America, while strains of low aniiliodLcE mav. be demonstrated in sera from
Jr

-
virulence arc seen in Europe and A i ^ also.
In human beings, tularemia may present as a
patients
An attenuated vaccine is available- which cin
local ulceration with lymphadenitis, a typhoid like be administered by scarification to pamnt who sure
fever with glandular enlargement or an influenza subject TO hiph risk of infection.

Further Rmding
Brubaker BR . 1991 . Factors ptfomoring infecriMs. caused by j-^rsiniae. Clin Microbiol Rev: 4:309.
Butler X 1994 . Yersinia infecnons. Clin injfikf Dis. 19:655,
Campbell and JM Hughes. 1995, Plague in India. Ann Inf Marat 122155.
Cover TL and RC Aber 1939. Yersinia entcr&calihca. New Engl J Med 321:16*
Gage KL 1993. Plague . In Topley amJ WJfson- l Microbiology and Mhcmbml Infections , 9 , K «3n. London: Ainold.
IWBtzcr R . 1954. Pb uc WHO Monograph scrie-s No. 22.
^

u opyrighted
y materi 3
 CO Haemophilus
CO i
i
i
Thegemis Haemophilus contain snull^ nonmotile ,
nonsporing, oxidase positive, pleomorphic, Gram
negative bacilli thut ire parasitic on hurmn being*
or animal *. They arc characterised by their
requirement of One or both of two accessory gn^wth
factors ( X and V } present in blood ( ffoemapWirj;,
meaning blood Loving ).
Pfeiffer (1892} observed that a small, Cram
negative bacillus was "constantly present ' in the
sputum of patient* from the influenza pandemic of
18 B9-92 and mistakenly proposed this as the
causative agent of human influenza . Tbia amt to
be known as the ’ influenza bacillus’ ( Pfeiffer's
bacillus),. liter renamed i iaanophilus influenzae
The causal relationship between this bacillus and
human in flucnza could not he Fubs-tanliated and Wit
finally disproved when Smith , Andrcwes and
Laidlaw (1933) isolated the influenza virus .
^.M O P H I I U S h l l I 1-. \ Z ,M-
I\
'
fInfluenza bacillus , Pfeiffer 's bacillus }
Morphology: / /. influenzae is a email (1.0 urn
* 0.1 pm ) , Cram negative , nonmorile, nonsporing
bacillus , exhibiting considerable plcomorphUm
( Fig, 36,1) . In spurum , it usually occurs as clusters
of CQceobiejllary forms, while in the CSF from
meningitis cases, long, bacillary snd filamentous
forms predominate. Cells from young cultures
( 18-24 hours) arc usually coccobacdiary, while
older cultures are distinctly pleomorphic. Strains
isolated from acute infections are often capsulatcd.
'
Hie bacilli are relatively difficult to Stain.,
Staining for 5-15 minutes with I oeffltr 's methylene
blur or dilute carbol tuchsin gives good resulfs.
Cultural cheracterittioE The bacillu* Lai;
fastidious growth requirements . The accessory
growth factors, named X and V , present in blond
are essential for growth . The heat stable X factor
is hem in or other porphyrin* requited for the
synthesis of cytochrome ,md other heme enzymes
such as catalase ,ind peroxidase involved in acrohic
respiration . The X factor is not required for
anaerobic growth . The V factor was so named
because it was originally thought to be a bacterial
vitamin. It is heat labile being destroyed at 120 °C
in a few minutes. It i* present in red blood cells
and in many other animal and plant cells lit is ,
. synthesised by some fungi and bacteria ( forexample ,
Staph , aunnts) in. excess of their requirements and
released into the surrounding medium. The V factor
i K a cocnzymo, nicotinamide adenine di nucleotide
( NAI 3) nr NAD phosphate ( NADP) which act*
as [i hydrogen acceptor in the metabolism of the
cell .
It is aerobic but grows anaerobically also. The
.
optimum temperature is .17 “ Q It does not grow
below 20' C. Some strains require 10% COr It
grows on biota! agar hut growth in scanty, as the V
factor Ls not freely available, being imprisoned inside
the itd blood cells. Growth is , therefore, better if a
source of the V factor is also provided . When Staph.
UEECH IS streaked across a plate of blood agjr on
which a specimen containing / /. rrrrtuenjtae has
been inoculated , after overnight incubation , the
colonics of IL influenzae wi 11 be large and well
developed alongside the streak of staphylococcus,
and smaller farther away. This phenomenon is called
satcllitism .tod demonstrates the dependence of
Copyrighted material
334 s TexItKHiK 01 Microbiology

II . infi'uM ^ jfon the V factor* which is available in

high concentrations near the staphylococcal growth
and only in & i n :. i 11 LT qu anti [its away (tom it , This is
a routine test in clinical bacteriology for the V *
iJcntificatioji of II . influenzae ( Fig 36.2 ). It is, \
however * not very specific as it will iiiso be positive
with other V factors requiring hrmophill an well
as with occasional strain of ncisHcriae and
diphtheroids.
*
-O C
<r\
When blood agar is heated to S0-90 *C or
boiled for A few minutes ( boiled blood agar ), the V
factor is released from within the erythrocyte* and
lienee these media are nu|icrioi Oil plain blood agar
for grown ng / /. in fhienzae. C lear tna nnpa rent med i -.i i L i §

may be prepared by boiEitig and filtering a mixture K \

of blood and nutrient broth ( Lcvinrhal 's medium ) if

Oi by adding a peptic digest of hlond to nutrient

gat ( Fildeti agar), Fildes agar Is best for primary
A \

isolation of H . j' nfJuejiijae and gives a copious

growth. Captulated strains produce ttanslusccnt Fig . 3£.1 H , intiuwz$t in fluid showing
pieumorphlim
colonies with a distinctive iride^-etice < > n Levin thaJ 's
agar,
BiDnhcmkai re action si: C Income and scylose
are fermented with idd prndbctiiin hut nor lactone ,
H Lie rose and mannitol. Catalase and oxidase
1

reactions are positive. Nitrates are reduced to

nitriles , Eight tintypes have been identitied on the
basis of indole production, urease and ornithine •• *
decarboxylase activity. Biorypc i is most frequently
responsible for meningitis.
Kesiitance: H . in fluent at' is a delicate
bacterium , destroyed by beating (55aC for 30
* 9 t# • < •

minutes), refrigeration ( 0- 4 °C ) h drying and

disinfectants. In culture, the cells die within two or
three diyi due to liutolystH . Cultures may be
preserved for about a month on chocolate agar
• k

slopes in screw capped bottles, For longterm

preservation, the culture may be lyophiIised -

^ nligcnic properties: There are three major

surface antigens - the capsular polysaccharide* the
outer membrane pro lei OH ( OMP) and F|g. 36.2 SaleIIif sm tf. are large
near growth ol smaller away
lipOoligosaccharidc ( LOS). bom ft.

i Hin / riC|r m n aeriei

 * Maumcphlui *
The major antigenic determinant of capsulated
strains is the capsular polysaccharide based on
which H . jjiij'Juc'rjjiae strains have been classified
—
hv Pittman into six capsuLr types types a to f.
Typing was originally done by agglutination but
other methods such as Quellung reaction ,
|mn iptlaliun, caflgglutiufllion , Cl E and ELISA may
also he used . Capsular typing in of m e d i c a l
importance as about 95 per cent of H . influenzae
isolates from acute invasive infections such as
meningitis belong to type b. Diagnostic kits for the
identification of H . influenzae type b ( Hit ) arc
commercially available.
The type b capsular polysaccharide has a unique
chemical structure , containing the pentose sugars
iibos- e and rihitnt instead of the hexoses and
hexosamines JS in the other five serotvpes. The
capsular polyribosyl ribitol phosphate ( PRP) ati digen
of Hit induces IgG , IgM and IgA antibodies winch
are hactericidsd, opsonic and protective. Hib PRP
is therefore employed for immunisation . Hib
capsular anCigen shows cros-s reaction with the
capsular an r igens of some frram positive and ( i ram
negative bacteria.
H . influenzae strains lacking a capsule can not
he typed and are called ' nontypahle strains . Next
1
ro JIib- the nontypablc strains arc the most relevant
i the tropics , non -type b strains may be responsible
in clinical infections,
The outer membrane prorein antigens show
considerable variation. OMP antigens of Hib
htive been classified into at least Id subtypes.
H , influenzae lipooligosaochandcs are antigenicaily
complex . OMP and LOS subtyping may be of
eyidemn iloyical value.
hi. influenzae is the first free I K i ng organLin
whose complete genome has been sequenoed.
Pathogenicity: hi . influence is an exclusively
human pathogen. It .s not naturally pathogenic for
'
animals but intraperiioneat inoculation of large
doses is fatal in mice, guinea pigs and rabbits.
Diseases due to hi. influenzuemiybeconsidered
under two groups, invasive and non i nvasi ve diseases.
In the firet group, the bacillus acts as a primary
335
pathogen, causing acute invasive infections. The
bacilli spread through blood, being protected from
phagocytes by their capsule . Haem up Inins
meningitis is ihe mwt important infection in this
group , others being laryngocpiglottitii,
conjunctivitis, bacteremia, pneumonia , arthritis ,
endocarditis and pericarditis. These infections are
usually seen in children and arc caused bv the
r r
eapsulared strains, type b accounting for nnxsi cases.
In the second group, the bacillus spreads by local
invasion along mucosal surfaces and causes
secondary or superadded infections, usually of the
respiratory tract. These include otitis media, sinusitis
and exacerbations of chronic bronchitis and
-
bronchiectasis These are usually seen in adults and
are often caused by rhe noncapsulated strains.
Meningitis: Thi* is the most serious disease
produced by H . influenzae with case fatality rates
up to 90 per cent m the uncreated. The bacilli reach
the meninges from the nasopharynx through the
bloodstream , The disease is more common in
children between two months and three years of
age. Thi -; age incidence has been correlated with
the absence of bactericidal anti - PRJ 3 antihodies .
Older children develop immunity as a result of
subclinical infection. It has been reported that in
for meningitis more often than in the temperate
zones.
Laryn &oep gLottiti * ( croup): This is an
acute
'
inflammation of the epiglottis , with
obstructive laryngitis , seen in children above two
years . Untreated cases may be fatal within hours.
Tracheostomy is often necessary to relieve
respiratory obstruction caused by the grossly
enlarged uvula. Thi -. cond irion is always associated
with bacteremia and blood cultures are usually r
positive.
PIIULIIVHHIITV. Haemophilus pneumonia typically
occurs in infants and is. accompanied by empyema
,
and sometime a meningitis M well . In older
children and adults, the picture is of Lobar
pneumonia, While dxft are primary infections due
Copyrighted materia
Hidden page
 t H a &m Dphi I u s *
the upper respiratory tract . Infection is transmitted
by the respiratory route . Carriage in the upper
respiratory tract is common parliculariy in young
children but such strains arc usually noncapsulated
and not responsible for acute invasive infection,
Immuniiy in type specific. As the large majority
of serious infectionn are caused by type l> strains,
active immunisation wiih Hib PEtP vaccine is
indicated . Purified PRP ii immunogenic in older
children and adults. However , in Common with
other polysaccharide antigens, PRP is poorly
immunogenic In children below two years - Its
immunogen^ icy can be jnpnoved by coupling with
protein carriers like diphtheria and tetanus toxoids
or meningococcus outer membrane protein. Such
conjugate Hib PRP are available for use in young
children .
Young household contacts of patients. with
svatemic H . jnriuenjfiite infection have increased risk
If
of getting intccted - llifampicin given lor four diyS
prevents secondary infection in contacts and also
eradicates carrier stare.
HASMOPHJLUS AEOYPTIU 3
-
(AocA Wfeefa bacillus, formerly H, SQjprjcus) '
Even before Pfeiffer described the 'influenza
hacitluP, Koch ( 1 SS3) bad observed a small bacillus
in conjunctivitis cases in Egypt . It was first cultivated
by Weeks ( 18® 7 ) in Mew York and came to be
known as Kuch-Weeks bacillus . Recent DNA
studio have shown that the baci '.lus is identical
with nou-capsulated H . influenzae. Therefore, the
former H . aegrptimx has lost its separate species
status and is now comr . lered as a biotype ofri .
i .' M I
- I t is worldwide in distribution and causes
a highly contagious form of corjunci iv iris (' pink
eye' ) . Jr is especially common in the tropics and
subtropics and may occur in epidemic forms . It
responds to Irmai sulphunainideB or ge ft : 11 Lei i L .
It has also been identified as the causative agent
of Brazilian purpuric fever ( BPF }, in which
conjunctivitis proceeds to a fulminant septicemia
in in bin H and children with high fatality. First
337
rreognsed in Brazil in J 9?4, EPF is now endemic
in South America.
HAEMOPHILUS DUCREYI
Ducrey ( 1890 ) demonstrated this bacillus in
chancroid lesions and Liv. inoculation into tile skill
Jr
on the firearm , was able to transmit the lei- 1 on
through several generations.
Chancroid or soft sore is a venereal disease
characterised by tender n-onmdurated irregular ulcers
on the genitalia - 'Phis infection remains localised ,
spreading only to the regional lymph nodes which
arc enlarged and painful , Autoinoculation lesions
may be produced by contact . There is no immunity
following infection but a hypersensitivity results,
which can be demonstrated bv Imradermal Jl
inoculaiion of killed bai Ni - "
ff , ducreyi is a short, ov. : id bacillus ( 1 — 1.5 pm
a 0.6 mm ) with a tendency to occur in end to end
pairs tir short chains . It is Gram negative but often
may appear Gram positive and frequently shows
bipolar staining . The bacilli may be arranged in
small groups or whorls or in parallel chains giving
a school ill fish or 'rail road track' appearance.
Primacy isolation is difficult . It can be growtl
OTl fresh clotted rabbi [ blond. Smears made after
24-46 hours incubation show tangled chains of
harilli. It may also be grown on the chuinnJJanioic
membrane of the chick embryo. On chocolate agar,
enriched with isovitalex and fetal calf serum , and
containing vancomycin as a selective agent , H „
ducreyi forms small , gray translucent colonics alter
incubation at 35 °C under 10 per cent CO . and
high humidity in 2-E days.
The species is antigenit ally homogeneous and
'
cultures mav be i den rifled by agglutination with
the antiserum . Intradermal ! - looilation of the culture
into rabbits produces a local ulcerative lesion .
H . ducreyi is susceptible to sulphomunidcs and
many antibiotics. Cases resistant to sulphonamidrs
and tetracyclines haw been reported . Etythro'
myein , corrimoxazole, ciprofloxacin or ceftriaxone
may be used for treatment .
Copyrighted material
Hidden page
 co Bordetella
The genus BordtteUi is named after Jules
Bordet, who along with Gcngou identified lilt
itmal ] ovoid baeillus cawing whoopunt cough * in
the sputum of children suffering from the disease
(1900) and succeeded in cultivating ii in a
crjmplex medium (1906 ) . The bacillus is now
known as Bofdctclin pertuisii ( jWTUSSrs meaning
.
intense cough ) A related blcillut, Bore ).
ia was isolated from mild cases nt
paftpertvs&
whooping cough ( 1937) . Bt>rd , branchixepticn
orjginallv isolated from dogs with broncho-
pneumonia ( 11911) may occasionally mfect human
beings- , producing a condition resembling pcrtussis.
It has been suggested that BvfiL bronchiseptia
represents the ancestral form from which [ fie Other
UVHH species hiive evolved - The ( mirth member oJ
the genua is Bond avium which causes respiratory
, Gmwtb is slow. After incubation for
disease in tuikcys.
RoKiisiThii. A FHRTIJSSIS
( Bordet ( iengou bacillus:
formerly I hemophilus pertussis)
Morphology: Word- pertUKK is a small , ovoid
-
coccobacULus ( mean length 0- i pm) In primary
cultures, ceils are of uniturnn > ize and shape but on
subculture they may become longer and thread lik^
It is noncnoiile and nonsporing. It is capsuUred but
rends to lose the capsule or repeated cultivation.
The capsule can he demonstrated bv special stains
bu t docs not swell i n the|neHc i LLC of the ant i scrum .
-
In culture him the bacilli tend to hf arranged in
,
loose clumps, with dear spaces in between giving
a 'thumb print ' appearance , freshly isolated strains
of fiord . pemissjs have fimbriae .
'
It is Gram negative. Bipolar me Achromatic
granules may be demonstrated on staining with
tnluidine blue.
Cultural characlorisirios: It is aerobic No
growth occurs anaerobically It grown best at 35-
36 C
Complex media are necessary tor primary
—
isolation - The msilium in common use is the
Bordet-Gengou glycerine |iotat ( i - blood agar . Blood
is required not to provide additional nutritive factors
but raTher to neutralise inhibitory materials formed
during bacterial growth. Charcoal or ioit exchange
resiilS incorporated in culture media may serve the
same purpose . Charcoal blood agar U a useful
medium. I [ does not grow on simple media like
nutrient agar.
2
, -
hours colonics on Bordet Gengou medium are
-
small , dome .haped, nontb, opaque , vise]d. greyish
white , retractile and glistening, resembling ' bisected
pearls' or 'mercury drops'. Colonies are surrounded
by a hazy zone of hemolysis . Contluem growth
presents an 'aluminium paint appearance.
'
ItiuchuTnicul reactions: It is biochemically
inactive. It docs not ferment sugars , form indole,
L reduce nitrates , utilise citrate or split urea . It
produces oxidase and usually catalase also.
Resistance: It is a delicate organism , heing
killed readily by heat ( 55 QC (or 30 minutes.), drying
and disinfectants. Rut unlike H . ui/Pirenfirc LI retains
viability at low temperatures, (0-4 PC).
Outside the body, Word , pertussis in dried
droplets is said to survive for five diys on gl &s*,
three days on cloth and a few hours on paper.
Copyrighted material
 340 < Texlbaak of Microbiology
Antigenic constituents and ^ irul ^ nc ^
factors: Several antigenic fractions and putative
virulence factors have been described bur their role
in the pathogenesis of pertussis remains to be
clarified They include the following:
l .Aggiutinagcn -i -. 15 ofd : * tullie possess genus
specific and species specific surface agglutinogens
associated with the capsular K antigens or
fimbriae . Gy agglutinin absorption tests , 14
agglutinating factors have been identified Factor
7 is common to all three mammalian species of
bordetrlhe, Factor 1 ? is specific for Bord . of the three hemagglutinins produced by Bord
btOitchiscptics and Factor 14 for Bord.
parapertussis . Factors 1 to 6 arc found only in
Strain!of BOfdt pCtttiS$Bt all gfwhich carry' Factor
1 and one or more of the other factors . Bordetellae
are cIn -bs- IFLCD into Various types based or the
agglutinogens thev carry. AH strains causing
infections belong to types 1 , 2 and 3 , it is essential
chat pertussis vaccine strains should have factors
1 , 2 and 3 . Factor specific antibodies arc present
in the Hera oi convalescent and immunised
persons. Agglutinogens promote virulence by
helping bacteria to attach to respiratory epithelial
cells. They are useful in serotyping strains and in
epidemiological studies.
2. Pertussin toxin (PT}- This is present only it ]
.
Bord , pertussis It plays an important role In the
pathogenesis of whooping cough . PI’ is expressed
or the surface of the bacillus and secreted into
the surrounding medium. The toxin exhibits
diverse biological and biochemical activities,
which formerly had been believed to be caused
bv different substances that had been named
accordingly. Examples are the LYJNPHOCJ-TOSIS
producing Victor or LPF causing profound
lymphocytosis in pertussis patients as well as an
experimental animals; and two effect? seen only
it ] experimental anini . LLH , hut not in patients, such
as the histamine seif ft rifting fitefor or HSF
responsible for heightened sensitivity to histamine
In experimental animals, and the islet activating
prtftein nr 1AP induong excessive i nsul ] n secreti on
I
»
by the pancreatic islet cells. It is now known that
all these are manifestations of the pertussis toxin,
PT is a 117 ,000 molecular weight hexamtr
protein composed of six subunits with an A - 11
structure ( the A portion heing the enzymatically
active moiety and If the binding component) . It
con he toxoided. PI’ toxoid is the major component
or acellular pertussis vaccina. Antibody to PT
'
can protect mice agaln &t intraaanl, intruperitoneid
or intracerebral challenge .
3. Filamentous hemagglutinin (FHA)\ Thin is one
.
pejTu.sSj.s, rhe otbtrS being PT and a lipid factor.
Purified FHA appears as a filamentous structure
in the electron mienusuope and hence the name.
If is present on the badlluy surface and is readily
shed , ll adheres to rhe cilia of respiratory
epithelium anti to erythrocytes. Resides facilitating
adhesion of Bord- pertussis to respiratory
epithelium, FMA and PT hemagglutinins also
promote secondary infection bv coating other
bacteria such as hfocinuphilos influenzae am!
pneumococci and assisting their binding to
respi ratory epithelium. This phenomena has been
termed piracy of adhauu.
Antibody to FHA can protect mice against aerosol
challenge . FHA is used in acellular pertussis
vaccines along with PT toxoid,
4. AdenvJjtr cyctsse ( AC ): All mammalian
bordctcUae but not Bord - tvinm produce adenylate
cyclase. At least two types of AC arc known, only
one of which has the ability to enter target cells
and act as a toxin . This is known as AC toxin
( ACT ). It act > by Catalysing the production of
CAMP bv various types of cells-.
.
5 jffe. jr labile toxin (HLT ): It is a cytoplasmic
protein present in all bordetcllae. It is in activated
in 30 minutes at 56 °C . It is dermoneerotk: and
lethal in cuice. Its pathogenic role is not known,
ft - cjtofariri ( TCT ): It is a low molecular
weight peptidoglyean produced by oil bordetcllae.
It induces ciliary damage in hamster tracheal ring
cultures and inhibition of DNA synthesis in
Copyrighted material
 * Bordetella
epithelial cell cultures, Its rule in disease is nut
known.
7. LJ pop*1h ^Jediunde ( LPS ) or the heat ATa dj/er
"
toxin in present in at! hordetetlae and exhihirs
features of Gram negative bacterial endotoxins, It
is present in the whole cell pertussis vaccine but
is not considered to be a protective antigen.
8. Ptmctiii : Pertactin is an outer membrane
protein (0MP) antigen present in all virulent
strains ot B . pertussis . Mice i mm united with
pertactir resist npiratoiy challenge wirh rhe
bacillus. AnlilnK.lv EL» pertactin can he seen in the
blood of children after infection or immunization.
PenaCtiii is included in acellular jsertussis vaccines.
Variation: tford. perrus.sjs undergoes a smooth
'
to rough variation. All fresh isolates are in the
smooth form ( Phase 1 ) . On -iuh culture , they
undergo progressive loss of surface antigen* , and
pass through phases II and III , finally becoming
phase IV which is the rough, avirulcnt form,
A reversible change in the capsular antigen has
been described as VnodulttHu'- The bacillus may
occur in one of three potential 'modes', X, 1 and C,
each of which has a characteristic surface antigen .
X, I am ) C refer to the colour of the confluent
colonies on rhe Bordet-Gengou medium - X for
Xnuhic ( yellow), C for cyanic ( blue ) and 1 for
intermedia te. Modulation is influenced by the
'
nature oH the culture medium. On the flordet-
GcngOU medium, fresh isolates always occur in the
X mode.
Patlmgoiriciiyi fiord , pertussis is an obligate
human parasite but infect ion can he produced
experimentally in several species of animals, the
white mouse being most often employed. hitiarusal
inoculation in mice induces a characteristic patchy
interstitial pneumonia, histologically resembling the
human disease. Intraperitorteml inoculation of large
doses is fatal due to ttueonii. Intracerebral
inoculation causes a fatal infection. Immunised mice
are protected - This forms the basis for the
intracerebral mouse potency utay for pert uuL$
vaccines .
341
In hum ,m beings, alter an incubation period of
-
about 1 2 weeks, the disease takes a protracted
course comprising three stages - the catarrhal ,
paroxysmal anrl convalescent - each Lasting
approximately two week * . The onset is insidious,
wirh Low grade fever , catarrhal symptoms and a dry
irritating cough Clinical diagnosis in the catarrhal
stage is difficult. This is unfortunate as this is rhe
stage at which the disease can be arrested by
antibiotic treatment. This is also the stage of
maximum infectivity. As the catarrhal stage
advances to the paroxysmal stage, the cough
increases in intensity and comes on in distinctive
I
bouts. During the paroxysm , the patient is subjected
to violent spasms of continuous coughing, followed
hv a long inrush ot air into the almost empty lungs ,
wirh a characteristic whoop ( hence the name ). The
paroxysmal stage is followed by convalescence,
during which the frequency and severity of
coughing gradually decrease .
-
The disease usually lasts 6 8 weeks though in
some it may he very protracted . Complications may
be 1} due to pressure eifeers duri ng the violent bouts
of toughing (subconjunctival hemorrhage*
subcutaneous emphysema ) , 2 ) respiratory
( broncho - pneumonia , lung collapse ) , or 3)
neurological (convulsions , coma). Respiratory
corn plications are self limited, the atelectasis-
resolving spontaneously hut the neurologic a I
complications may resulr in permanent sequelae
such as epilepsy, paralysis, retardation, blindness or
deafness.
The inflation is limited to the respiratory tract
and the bacilli do not invade the bloodstream. In
the initial stages, the bacilli arc confined to rhe
nasopharynx, trachea and bronchi. Clumps ot bacilli
may be see n c nmes hed in the cilia of the respiratory
epithelium. As the disease progresses, inflammation
extends into the lungs , producing a diffuse
bronchopneumonia with desquamation of the
alveolar epithelium.
Blood changes in the disease are distinctive and
helpflit in diagnosis . A marked IcucocvTosis occurs,
Copyrighted material
Hidden page
 - i Bondetelte 343

Innimnufluonescenct: is useful in identifying the
bacillus in direct smem of clicuCAl specimens and
Ilf cultures . The differentiating features of
bordetelkt are listed in Table 37.1 .
Serologicil diagnosis is not helpful. Klsc in
antibody hire may be demonstrated in paired serum
samples by agglutination , gel precipitation or
complement fixation tests. As antibodies appear Late,
the second sample of serum should he collected
some weeks after the onset of the discasc.
r emunstration of spc-L-ifLU *ecTctOrv IgA antibody
^
in nasopharyngeal secretions by ELISA has been
proposed as ;L diagnostic method in culture negative
produces better and more sustained protection and
less reaction than the plain vaccines. Pertussis vaccine
is usually
a
administered in combination with
diphtheria and tetanus toxoid { triple vaccine ) . Not
only is this more convenient, but Bord. pertussis
also acts as an adjuvant for the toxoids, producing
belter antibody response.
In view of the high incidence and severity ot
the disease in the newborn, it is advisable to start
—
immunisation as early as possible . Three injections
at intervals of 4 6 weeks are to be given before the
age of six months, followed by a booster at the end
of the first vear of life,
- r
,

cases. Children under tour years who arc contacts of

Prophylaxis: Preventing the spread of infection patients should recent? a booster oven if they had
by isolation of eases is seldom practicable , as been previous ]! immunised. They should Also
1

infectivity is highest in the earliest Stage of the receive chcmnprnphvlaxi * with erythromycin .
disease when clinical diagnosis is nor easy. Non immunised contacts should receive
Specific immunisation with killed Boni. pertussis erythromycin prophylaxis for ten days after contact
vaccine has bee n found very effective. 1 r is of u rmosr
m with the patient has ceased . Pertussis vaccination
importance to use a smooth phase I strain tor may induce reactions ranging from local soreness
wane production. The method of inactivation and fever , to shock, convulsions and encephalopathy.
should be such rhat antigen ic potency is unaffected. Provocation poUomyclmv is , L rare complication.
Detoxication with 0.2% merchaobte during several Factors contributing to toxicity or pOitYlcdna]
months,' storage at 4 C has been recommended as encephalopathy have not hecn defined - The latter
.
a satisfactory procedure The alum absorbed vaccine compticition is estimited ro occur in one in 15 10 —
Table 17- 1 Differentiating feature* of Enrdetella species
Bord . pertuswj Band. parapertussis Bord. bnonehisepica Bord , arium
MdCilitj'
Gruwth on nutrient agar
—— —
+
+

*
4

—
Growrh on Bordet Geng-Qu 3-6 1-2 1 1
medium {days )
Urease
Nitrate Cu nitrife ——
*

—
4 f

*
- ——
Citrate uliliwitwjn V + V
Oxidase
Toxins:
+
— --
I 4

HLT and TCT +

——
4 4 4

ACT 4 4 4

PI 4
- -
V Variable

Copyrighted material
344 * TexltiooK ol Microbiology

million inject ion R . Eslinutcd NEUROLOGICJI
complications of natural disease have ranged tnont
1.5-14 per cent in hospitalised cases; $. third of
-
these recover, a third Live mirhc tiild , L third die
or have severe detects .
[ fsvvcrc complications such &s enccjihalopathy,
seizures, shook or hyperpyrexia develop following
the vaccine, subsequent doses of the vaccine are
Ire fitment: fiord pctmu^is is- susceptible
, tn
several antibiotics ( except penicillin ) blit
antimicrobial therapy i;; beneficial only if initiated
within the first ten days of the disease.
Erythromycin or one of rbe newer macrolides is
the drug of choice. Chloramphenicol and
-
cotrimanaoU an also useful .

contraindicated. Routine pertussis Vaccination is not BDRUETELL \ PARAPERTUSSIS

advisable after the age of seven years as adverse This is an infrequent cause* of whooping cough .
(tactions are likelv and the risk of severe diseahe is The disease i -s mild . The ]n.Ttus*is vaccine does not
low. Acellular vaccines containing the protective protect against Bord pmpertusas infection ,
components of the pertussis bacillus ( PT, F11A ,
. ), fm- t developed it ] Japan , are
agglutinugenK 1 r 2 , 1 BOELDETELLA BRQN < ; HISEPTIC \

now used in some other countries also : LS they cause
far fewer reactions, particularly lit older children.
Both whole celt and acellular vaccines llave a
protection rate of about % ]ter cent . With wltole
cell WfljflBt the protection dedincR to 50 per cent in
about five years and is absent after 12 years, The
( Bond , bronchiCam's)
Tli i T- is motile hy peri I rich ate flagella . It is
antigenicaUy related to Bond, pertussis and firucelb
abortus. It occurs naturally in the respiratory tract
of several apedes of animals. It has been found to
Liiose . L very small proportion ( O. t per «nt) of cases

duration of protection with acellular vaccines is not of whooping cough.

known. Even fully immunised subjects may develop
peituHhih but the disease will be very mild in them .

-I U II ' L T h e a d i n g
Cheny JDtral. l 9Siit. ftepn ^ i of the usk !»(t on hrtllHii, ftdrj jr K 1 : 93£J .
EiiiTflrLaJ . 1992. E rtUllii: idulls, infant and herds. Lancer , 359:526,
^
Fritdmin RJ .. 193?. FtrtuisluThe IUSL-JW sod new diagnostic irischmL'- . { Vin JWnTwfrkJ Krv t ; 165.
Gustaffson I . et aJ. 19%. A connclled tnal of acellular pertussis vaccines. .A’etv tng! JXted. 333:3+9.
Tinman M . 19 H 4. The concept of perruasic as a rotm mediated disease. Pediair Infccr Die. .1:4t > 7.
WHM AA and EL f lewlrtt 1986. Virulence factors ofAnfaklla pemissjs. Arm Re v Micnibiot +0.661 .
Hewlett EL 1997. Pertussis: Currenr rancepTF of pathogenesiF . Fkdiatric Infect UJS. J 16:5

Copyrighted material
 Brucella
The genus Efircelii consists of very small ,
nomnotile , aerobic, Gram negative Coceobacilh that
grow poorly on ordinary media anti have little or
no fermentative powers , ITcy arc strict parasites of
uni trials and may also infect humans.
Brucellosis is a zoonosis, primarily affecting
goats , sheep, cattle. bu It nines pipt and Other
, that they may be mistaken lor cocci . as was done
animals and transmitted to humans by contact wifh
infected animals or through their products . The
human d is ease was recognised along the
Mediterranean littoral from very early times and
has been known under various names such as
Mediterranean fever , Miltn fever and undul.inr fever .
A British army doctor, David Bruce { 138 b)
isolated a small microorganism from the spleen of
fatal cases in Malta and transmitted the disease to
monkeys experimentally- This was named BnincfJa
meJitenm ( Brucella after Bruce, tnctianixr after
Melita, the Roman name for Malta) . A Maltese
bacteriologist Zartmiit ( 19051 showed that Bf .
mcffticn.sMwas transmitted to humans by goat 's milk .
Following this, the disease was eliminated from
British soldiers by prohibiting them from using
gnat s milk and milk products, while the disense
remained undiminished in the civilian population ,
which continued to use them . Bang ( 1397 )
.
described Hr. aimrftis [ be cause of contagious
abortion In cattle , The third major species in the
genus, Br. mix was isolated by Tnum ( 19 M ) from
pigs in the USA. These three species cause human
bnicellorrs.
Other Species causing animal in lections include
fir: cam .?, isolated from cases of canine abortion ,
"
Br, avis from abortion in sheep and Hr, neotomac
from desert wood fats. Br. canis may occasionally
cause a mild human disease , but [ he other two are
not pathogenic for humans .
Morphology: Bruccllae arc cotcobacllli or short
—
rods 0.5-0-7 pm * T6 1.5 pm in size, arranged
singly nr in short chains. The cells are &o small
by Bruc e who called them jMtCAKDCOU ItKllteJU.
In older cultures, irregular forms appear. They arc
non motile, noncapsulated and nnmpnring. Thry are
Cram negative and nonaeid fa*t , Bipolar sraining
is not uncommon.
( ltd!Lind characteristics:. Bruce line are strict
aerobes and do not grow anaerobically. Br. abortus
tor growth . The optimum temperature is 37
—
is capnophilic, many strains requiring 5 109 CO .
*
( range 20-40 "C) and pH &, h 7.4. They may grow
“
on simple media, though growth is slow and scanty'.
Growth is improved by the addition of scrum or
liver extract . Liver infusion media were widely used
tor the cultivation ofbnacellae. The media employed
currently ire serum dextrose agar, scrum potato
jnfus]on agar, trypt lease soy agar, or tryptose agar.
The addition of bacitracin , polymyxin and
cycluheximiide to the above media makes them
selective.
In liquid media , growth is uniform, and a
powdery or viscous deposit in lormcd in ddctiljtUftt.
On solid media, colonics arc small , rnoiit,
translucent and glistening. Mucoid, smooth and
rough types of colonies appear , associared with
changes in antigenic structure and virulence.
Erytliritol has a specially stimulating effect on
the growth of bruccllac.
Copyrighted materia
34* * Texltwok ol Microbiology *
I i r tL 11 I -LT i i L - ; 11 functions;: No carbohydrates are
^ *
L
1
ordinarily fermented, though th rponn uxid^itivt
capacity. Btucdke ^
catalase positive . oxidase
ate
positive ( except tor Hr. rjeoJTnmaeand Br. Otis which
are negative ) and urease positive . NitrsUc^ arc
reduced Do nitrites. Citrate is not uuliscd . Indole is
not produced aid MR and VP tenth are negative .
K exist mice: Bmcelke ate destroyed by heat at
60 ' C in 10 minutes and by 1 % phenol in 15
minutes. They arc killed bv pasteurisation . I hey
mav survive in soil ind m .irmrc tor hever .il weeks .
1
They remain viable tor 10 days in refrigerated milk ,
one month in 111- cream , four months ii butter and
for varying periods in cheese depending on its pH .
'fhev may also survive for raanv weeks in meat .
They ate sensitive to direct sunlight and acid, and
fend tn die in buttermilk. Hr. mttitensis mac remain
alive for sis days in urine, six weeks in dust and ten
weeks in water.
Antigenic structures The somatic antigens of
hrueeDae cdnciLLii two main antigenic determinants ,
A ami M which are present in dificrent iimiunls in
the three major species. Hr. abortus contains about
2U times A as M : Ih. mclitaua about 2D times M
as A - Kr. sib* has an intermediate antigenic pattern.
Absorption of the minor antigenic component from
an antiserum wjH teave most ot LILL major afttilsody
component and such absorbed A and M
monospecific ser .i arc useful for species
identification hy the agglutination tc^t . The species
identification of brucella strains is not , however , so
straightforward and strains are often seen t h a t
behave biochemically as abortus and serologically
as meUtensis and vice versa . Species and biotype
identification depend:, on a varietv of other factors
beside* antigenic structure ( Table 3S . 1 ) .
Antigenic cross -reactions exi ^ c between
brucellac and V. c holeme and persons receiving
rhe cholera vaccine may develop brucella agglutinins
Lasting tor about three years . Antigenic cross -
re actio ns also exist with F . coii 0 : 11 b ; 0: 1.5 7 ,
Sal monel La serotypes group N ( 0:30 antigen
Kauffman anti White ) , JV nulrophila , Y .
enrerocoJinrs and F. HJLAIENSIS , A superficial L
antigen resembling Salmonella Vi antigen has been
described .
Bructllfl bflCttrinphflgtt Several bacterid
phnigC' that Iy^c the Brucella strains have been
isolated . All these phases are serologically similar.
The Thlist (Tb ) phage has been designated as the
reference pha e and at RTD Lyses only Br. abortus.
^
Hr. sms is lysed at 10,000 KTD, while Br mditensis
is not Lysed at all.
ClflSNtficnlidn; Brucellac may be classified into
different species, b^sed on CO . requirements , H ,S
production, sensitivity to dyes {basic fuchsin and
lliiomn ), agglutination fo monospecific sera , pliugc
Ivsis and oxidative metabolic tests with amino acids
r
and carbohydrates. The three major species are Br
me / rJcn.sb, ffr. u / jorrus , infecting primarily goat* or
sheep, cattle and swine, respectively. Many hiotypes
have been recognised, in these species.
Br. itlia strains that produce H ,S are known 4s
'American' strains and those that do not a ' Danish'
*
strains.
Pathogenicity: All three major species of
bruccllac are pathogenic to human beings . Br.
rncMtnsps is the must pathogenic , Hr. nhtyrtii. and f
Br . suis of Intermediate pathogenicity. The
incubation fjenod is usually about 10— 30 days, but
may *i > meti mes be very prolonged - Hu man i nfisrrinn
may be of three types :
1 . latent infection with only serological hut no
clinical evidence;
2 . acute or subacute brucellosis; and
3. chronic brucellosis .
Acute brucellosis is moctiy due to Br ineliiensia .
( It is usual!v known undulant ftwr, but this is
misleading as only some cases show the undulont
pattern . ) It is associated with prolonged bacteremia
and irregular fever . The symptomatology is varied,
consisting of muscular and articular pan is , asthmatic
attacks , nocturnal drenching sweats, exhaustion,
anorexia , constipation, nervous irritability and chills.
The usual turnplication* are articular, osseous,
visceral or neurological .
Copyrighted materia
Hidden page
Hidden page
 i Bmcellh
Laboratory methods for diagnosis include culture,
serology and hypersensitivity tests .
Blood culture is the most definitive method for
the diagnosis of brucellosis- Blood is inoculated
. ufo 1 hnttfe of trypr i case soy broth and. incubated
at 37 °C under 5-10% CO . As bacteria in blood
^
ire usually scanty, large volumes of blood (5 nil.)
should be inoculated . Subcultures are made on solid
-
media every 3 5 days, beginning on the fourth day ,
Growth mav often be delayed and cultures should
not he declared negative in less than 6-8 weeks .
-
BACTEC cultures may become po itive in 5 to 6
days.
The Castaneda method of blood culture has
several advantages and is recommended. Here , both
liquid and solid media are available in the same
bottle. The blood is inoculated into the broth and
the bottle incubated in the upright position. For
subculture , it is sufficient if the bottle is tilted so
that the broth flows over the surface of the agar
slant. It is again incubated in an upright position .
Colonies appear on the slant . This method
minimises materials and manipulation, reducing
chances of contamination and risk of infection to
laboratory workers .
Blood cultures arc positive only in about 3CH50
per cent of cases, even when repeated samples are
rested ifr. meljfensjs and Sr mis ire isolated more
,
'
readily than Br.abortus Bone mannow cultures y ield
a higher rate of isolation and remain positive long
after the blood culture has become negative ,
Cultures may also be obtained from lymph nodes,
cerebrospinal fluid , urine and abscesses, i: present,
and, on occasion , also from sputum, brrusl milk,
vaginal discharges and seminal fluid .
As cultures are often unsuccessful, serological
methods are important in diagnosis. Several
serological tests have been developed , including
agglutination , complement fixation and ELISA.
The standard agglutination tes- t ( SA 1 ' iS
performed most often . Th .* is a tube agglutination
-
test ill which L L: U.II volumes of serial dilutions of
the patient 's serum and the standardised antigen (a
34y
killed suspension of a standard stm H of Br- abortus]
'
are rinsed and incubated at 37 for 24 hours or
50 C for IS hours , A ti ’ re of 160 or more is
3
considered significant - Most patn - nfs with acute
brucellosis develop ri tries of 640 nr more by 3 4
weeks of illness. Trines tend to decline after the
—
acute phase of the illness.
Several sources of error have to he guarded
against . Sera often contain "blocking or
nonagglutinating' antibodies . The blocking effect
may somet mes be removed by prior hcaf ng of the
serum ar 55 QC for 30 minutes or by using 4% saline
as the di I uent for the test.The most reliable meth nd
for obviating the blocking effect and detecting the
'
incomplete' anti I >oi lies i - the an r i irlobnli n {Coombs)
n
test. As the prozone phenomenon to high titres
( upto 1/640) is very frequent in brucellosis d : s
csscnfiij that several scrum dilutions he tested - A
positive agglutination test may be produced by
cholera , tularemia nr yersinia infection , or
immunisation. Cholera induced agglutinins mav he
differentiated by the agglutinin absorption test and
also as they are removed by treatment with 2 -
mercapto-ethanol. In order that results from
different laboratory arc comparable , it is the
practice to express agglutinin titles in International
l 1 n : 75- This done by using a standard reference
-
serum for compa i'- on .
In brucellosis, borh IgM and IgG antibodies
appear in 7-10 days after the onset of clinical
infection , As thedisease progresses, IgMnnt bodies
decline, while rhe IgG antibodies persist oi increase
in ritre. In chronic infections, IgM may often be
absent and only IgG can be demonstrated . The
agglutination rest idenffies mainly the IgM
antibody, while both IgM and IgG fix complement .
The IgG and IgA antibodies may act as ' block : ng’
or 'nonagglcitmuling ' antibodies . It is thus evident
that the agglut inatk m test is usually posirivr in acute
infection but may be only weakly positive or even
negative in chronic cases . The results of the
agglutination tests therefore have lo be evaluated
carefully. While a high titre of agglutinins, and
Copyrighted material
350 * Texlbook pF Micrnbiaioriy

especially demonstration of a rise in tirre, ran be
taken .LK diagnostic, even i negative agglutination
test may not exclude the possibility of brucellosis.
The complement fixation test is more useful in
chronic cates detects IgG antibody also.
as it
ELISA is sensitive, specific and can detect IgM
and IgG antibody separately l f i s therefore useful
fur differentiation between the acute and chronic
phases of brucellosi s and also for screcr.ing large
numbers of sera.
Delayed hypersensitivity type skin tests with
brucella antigens (‘brucellins’) ate not useful in
diagnosing acute brucellosis. They parallel the
tuberculin test in indicating only prior sensiiisanon
with the amigertSp and may remain positive lor years.
Brucellin testing may lead to a rise in litre of antibodies.
The methods used for the laboratoiy diagnosis
of human brucellosis may also be employed for the
diagnosis ofar.imal infections. In addition , bnicellae
may be demonstrated microscopically in
pathological specimens by suitable staining or by
immunofluorescence- Several rapid methods have
been employed for the detection of brucellosis in
herds of cattle. These include the rapid plate
agglutination resi and the Rose Bengal card test.
For the detection of infected animals in dairies,
pooled milk samples may be tested for bacilli by
Culture and In: antibodies by several techniques. In
the milk ring test a sample of whole milk is mixed
well with a drop of the stained brucella antigen (a
concentrated suspension of killed Br,abortus stained
with hematoxylin ) and incubated in a water bath
-
at 70 T for 40 50 minu tes, If antibodies are present
in the milk, the bacilli are agglutinated and rise
with the cream to form 1 blue ring at the top .
leaving the milk unstained. If antibodies are absent,
no coloured ring is formed and the milk remains
uniformly blue . The whey agglutination test is
another useful method for detecting the antiisodics
in milk _
Prophylaxis: As the majority of human
infections ire acquired by consumption of
.
contaminated m iIk prevention con ^ i *t5 of checking
brucellosis in dairy animals. In many advanced
countries, this1 iis achieved by the detection of
infected animals, their ehmina Non by slaughter and
-
the development of certified brucella free berds-
P 3Ceuri >-ati < m of milk is an additional safeguard.
^ Vaccines have been developed for use in am mils.
Br. abortus strain vacrine is protective in, cattle.
No suitable vaccine is available for human use.
Treatment: The usual regimen is a combination
of doxytydine for 45 days with streptomycin IM
daily for the first 2 weeks in adults., and in
children cotrimoxazole along with rifampin n or
£ LL:iUrn win .

Further It Lading
Corbel MJ. 1997. Brucellosis, an overview. Emeig. ng Infccf Off. 3: No. 2.
Corbel MJ , 199?. Vaccine* ajjaiurt bacterial J Afled jVJjrrobrof 46:267.
Expen Committee on hi ucthusii . 1936. Stub Report, Technical Report Series bin . 74. ). GUKVIIWHO.
Young J .' l . 1995. An overview of human ttfueellasu. Clin lnitci Lhi. 21:233.

:>pyrianiea materia i
L
 Mycobacterium I: Tuberculosis
Mycobacteria are slender rods that sometimes
show branching filamentous forms resembling
fungal mycelium. In liquid cultures they form a
mold-like pellicle. Hence the name 'mycobacteria ,
meaning fungusdike bacteria . They do not stair
readily, but once stained , resist decolourisatlon with
dilute mineral acids . Mycobacteria are therefore
called 'acid fast bacilli’ or AFB . They are aerobic,
non motile, noncapsulated and noisporing. Growth
is generally slow. The genus includes obligate
parasites , opportunistic pathogens and saprophytes.
The Unit member of this genus fn be identified
was the lepra bacillus discovered by\ Lansen in 1963.
Koch ( 1882 ) isolated the mammalian tubercle
bacillus and proved its causative role in tuberculosis
by satisfying Koch 's postulates , Tuberculosis in
humans was subsequently shown to be caused by-
—
two types of the bacillus the human and bovine
types, designated Mveotacreifiim firberoifos!? and
Mr iwvjs, respectively. The term M . fubejTii /osj's
'
comple* includes, besides the hunuin and bovine types,
two other mammalian types also : M. ifrjcatium
causing human tuberculosis in
possessing properties intermediate hetween human
and bovine types, and M. microti ( the vole bacillus
pathogenic for votes and other small mammals but
not for humans).,
The second human pathogenic mycobacterium
is the lepra bacillus causing leprosy. Though this
was the mycobacterium first described , it is the least
understood because it has not been possible to
convincingly culture it in vitro so far .
The third group of mycobacterium it a mixed
group of isolates from diverse sources; from birds,
cold blooded and warm blooded animals, from skin
ulcers, and from soil, water and o-ther environmental
sources . They were initially called atypical
mycobareria and broadly categorised into hour
categories: photochromogens , scotochromogens,
non-photochromogens and rapid growens, based on
their growth rates and pigmentation . They are
opportunistic pathogens and can lead to many types
of diseases; they arc described in Chapter 40,
Saprophytic mycobacteria were isolated from a
number of sources. These included M . butyrieum
from butter, M. phlei from grass, M, stcrcoris from
dung and M. smegmarif from smegma . {Contrary
to common belief,. M . ftnegmam is seldom found
in smegma , though other rapidly growing
mycobacteria occur frequenly there, contaminating
urine cultures ) .
MYCOBACTERIUM TUBERCULOSIS
Morphology: Mr tuberculosis is a straight or
slightly curved md, about 3 pm x 0.3 pm , occulting
ringlv, in pains or as small clumps. The size depends
on conditions of growth, and long filamentous, cluh
shaped and branching forms may he sometimes seen.
M . fid-vis is usually straighber, shorter and stouter.
Tubercle bacilli have been described as Gram
positive, though strictly speaking this is not correct,
as after staining with basic dyes they resist
dccolourisatlon by alcohol even without the
mordanting effect of iodine. When stained with
carboi fuchsin by the Zichl-Neebcn method or by
fluorescent dyes Uuramine O, rhodamine), they
resiat decofourisatian by 20 per cent sulphuric acid
and absolute alcohol for 10 minutes (acid and
Copyrighted material
352 * TcirtHck DI M c - obio 'ogy
aleoho ] fast } . A -rid fastness has been ascribed
variously to the presence in [ he bacillus of an
uniaporufiiblc wa* ( mycoloic acid ) or to a
semi perm cable membrane around the cell . It is
i el ,mi to the integrity of the cell and appears to be
a property of the lipitl- rich waxy cell wall . Stsiioing
maybe on iform or granular. Beaded or barred forms
are frequently seen inM. rubmido - -srs', bur M . bovi $
stains more uniformly J
Eleetf on micrographs of tl 11n sec ^ons show that
the thick cell wall is Composed of three layers
enclosing a trilaminar plasma membrane .
SpheroplasHs are formed when jfmwi in the
presence of Ivsniymr. L- fhrms are also setO-
Culturfll characteristic^: The bacilli grow
slowly, the generation time in vitro being 14-15
honrs. Colonies appear in abraLt two weeks and may
sometimes- take up to eight weeks- Optimum
temperature : s 37 UC and growth docs nor occur
below 25 °C or above 40 't . Optimum pH is 6.4-
7.0. AI. ru6en. ulo.sjs i - an obligate aemhe, while
j\f. bavin is mieroaemphilic: on
primary isolation ,
becoming aerobic on subculture, A1 tirbercir/osis
grows luxui ianily it ] culture as compared to M. bovi*
which grows sparsely. They are therefore termed
eugenic and dvsgonic respo^ ri \ ely, The addition of
0.5 4 glycerol improves the growth of AJ.
^ tend to form long serpentine cords in liquid media,
lubcrCtllptis, hut lias no effect on or may even
impair the growth of AJ. fwvjs. Sodium pyruvate
helps the gmwih of both types. Human tubercle
bacilli do not grow in presence of F- nirrobenzoic
acid, unlike orher slow growing nonchrumugenii.
Tnhercle bacilli do not have exacting growth
requirements but arc highly susceptible even to
traces of toxic substances Like fatty achk in culture
media. The toxicitj is neutraiIscd bysemm albnmin
or charcoal , Koch originally grew the bacillus on
hear coagulated bovine serum. Several media, both
solid and liquid Have been described for the
cultivation of tubercle bacilli . The solid media
conrain egg (LowcnsteLn"-Jensen , Petragnini ,
Dorset), blood Harsh is), scrum ( LoeffW) or potato
(Pawlowsky ) . The solid medium most widely'
»
employed for non fine culture i> Lowcnstcirv-Jcnscn
i. l J) medium without starch, as recommended hy
the Interna n ' nal Union Against Tuberculosis
'
CIUAT ) . This consists of coagulated hens’ egg,
mineral salt solution, asparagine and malachite
greenh the last acting as a selective agent inhibiting
other bacteria. Among the several liquid media
described, Dubos ', Middlebrouk's, Proskauer and
Beck ' s , Sula’s and SautorV media are the more
common. 1 dquid. media are not generally employed
for routine cultivation , but arc used for strainviry
,
testing, chemical analyses and preparation of
amigens and vaccines.
On solid media , JW. ruberaiJosj s forms dry,
'
rough, raised, irregular colonies with a wrinkled
surface. They are creamy whi rch bccom ingyellowish
or buff coloured on further incubation. They are
tenacious and not easily emulsified . M. bovis
colonies, in comparison are flat, smooth, moist,
white and break up easily when touched.
In liquid media without dispersing agents rhe
growth begins at the bottom, creeps up the sides
and forms a prominent surface pellide which may
extend alon r the rides above the medium. Diffuse
^
growth is obtained in Duhos' medium containing
Tween- 00 ( sorKtun monooleate). Virulent strums
while aviruient strains grow in a more dispersed
manner. The cord factor itself is not a virulence
factor as ir is present in some aviruient strains as
well.
Resistance: Mycobacteria are not specially heat
resistant, being killed at 60 in 15— 70 minutes.
^
Survival i- influenced by the material in which
the bacteria are present. Cultures may be hilled by
exposure to direct sunlight for two hours, hut bacilli
in sputum may remal n alivc for 20— 30 hours. Bacilli
in droplet nticle i may refcf n viability for 3— 10 days
'
under Suitable conditions. Cultures remain viable
ar room temperature for 6— S months and may be
stored for up to two yearn at -20 "C .
Tubercle bacilli are relatively resistant to
chemical disinfectants, surviving exposure to 5%
Copyrighted materia
 4 Mycobacterium I . Tuberculosis *
phenol , 15% sulphuric acid, 3% nitric acid * 5 %
oxalic wid and 4% sodium hydroxide - They are
sensitive to formaldehyde and gluicraldthyde. 'fhey
arc destroyed by tincture of iodine in five minutes
and bv 80% ethanol in 2-10 minutes. Ethanol is a
I!
suitable d is in tecta nr tor skin , gloves and clinical
thermometers.
Biochemical reactions
Several biochemical tests have been described
for the identification of mycobacterial species.
Niacin lest: 1 hi man tubercle bacilli form niacin
when grown on an egg medium . When 10%
cyanogen bromide and 4% aniline in %% ethanol
are added to a suspension of the culture , a canary
yclkrw colour indicates n positive reaction . The lest
is positive with human type and negative with
bovine type of bacilli . The Test is useful in
identifying human strains as no other
mycobacterium is positive except fur M . rijnf &c
.
and a few strains of M chcbiKii ,
Aryl hllphatnsu lest: This test is positive only
with atypical inycobacieria.The bacilli are grown
in ; L medium containing 0.001 M fri potassium
phcnolphchalcin disulphate. 2 N . NiOH is added
drop by drop to the culture. A pink colour indicates
j positive reaction .
Neutral red test: Virulent strains of tubercle
hiK illi are able to bind neutral red in alkaline buffer
'
solution, while avirtilent strains are unable to do
so.
-
Catalase Peroxidase tests: These help in
differentiating tubercle bacilli from atypical
mycobacteria and provide an indication of the
xc ns- Ltivi Cy of tk ? strain In iwnismd , MtJKt .atypical
mycobacterial strains are strongly catalase positive ,
while tubercle bacilli are only weukly positive in
comparison . On the other hand, tubercle bacilli
are peroxidase positive , but not atypical
mycobacteria. Catalase and peroxidase activities are
lost when tubercle bacilli become INli resistant.
Catalase negative strains of tubercle bacilli are not
virulent fur guinea pigs.
A mixture of equal volumes of 30 vol. H ,0 ,
353
and G.2% catechol in distilled water U added ro
5 ml of the test culture and allowed to Stand for
few minures . Effervescence indicates catalase
production and browning indicates peroxidase
activity.
Amid tr sc lusts: The ability ro split amides has
heen used id differentiate mycobacteria. A useful
m
pattern is provided by testing five amides, viz , *
acetamide , kenzamide , carbamide, nicotinamide and
pyTKommidc. A 0, 00165 M solution of the amide
is incubated with rbe bacillary suspension at 37 *C
anti 0.1 ml of MnSQ +.4 H ,0., 1.0 ml of phenol
solution and 0.5 ml of hypochlorite solution arc
added . The tubes are placed in boiling warer fbr
20 minutes. A blue colour indicates a positive test.
Nitrate reduction lest This is positive with
M. tuberculosis and negative with M. bovia.
A ' I iconic prt > perlies: Manv antigens have
been identified in mycobacteria. Group specificity
is due to polysaccharide and type specificity to
protein antigens [ ’allowing infection by tubercle
,
bacilli, delayed hypersensilivty is developed to die
bacillary protein (tuberculin). Tuberculins from
human , bovine and murine bacilli appear to be
indistinguishable - Some degree of antigenic
relationship exists between tubercle bacilli and
atypical mycobacteria * as shown bv weak cross
reactions in skin tearing with different tubercLilins .
Time is also some antigenic relationship between
lepra and tuliercie bacilli.
By various serological tests ir has been
established that JW. fuberctiiosj s strains arc
'
antigenirally homogeneous and very similar to
M. bov7> and M. nricrob, bui distinct from other
species. Antibodies against polysaccharide* proton
, M id phosphatide anti gen s of tubercle bacilli have
been demonstrated in sera of pwients, bur they have
not been found useful in diagnosis or relevant in
miiy
Bacteriophage: Many mycobacteriophdges
have been isolated from soil, water and other
environmental sources as well As from lysogenic
strains. Many toycob*cteria infected with temperate
Copyrighted material
354 * Textbaofc ol Microbiology *
phagw are not truly lysogenic, instead of being
integrated with the bacterial chromosome, the
phage germane appears free, like a plasmid . This is
called p&tudoljwjgeny.
Tubercle bacilli have been classified into font
—
phage types A , B, C , and a type intermediate
between A and B, and therefore designated I ( for
'intermediate'). Phage type A is the commonest and
is present worldwide . Type B is common in Europe
and N . America. Type C is seen rarely. Type 1 is
common in India and neighbouring countries.
Phage 33 D isolated from an environmental
mycobacterium lyses all variants ofM. tubcjT[i/osj^ h
but not BCG .
Bftcleriocdns: AJ. fuberru/osf .t is divisible into
two types by means of bacceriocins produced by
rapidly growing mycobaetcria.
Molecular typing: DNA fingerprinting
provides a method tor di fife re flt i ati n g between
strains of tubercle bacilli. Restriction endonulease
treatment yields nucleic acid fragments of varying
lengths, the patterns af which arc strain Specific .
1
This 'restriction fragment length polymorphism
( RFI.P} enables strain typing for epidemiological
purposes. Fingerprinting can also be done with a
chromosomal insertion sequence, IS Slid, which
K present in most strains of tubercle bacilli.
As the entire genome of tubercle hacillus has
been sequenced, more molecular typing methods
may become available .
Host ranfjt: M. rubercufosrir causes natural
infection in humans, other primates, dogs and some
Other animals which have close contact with
humans. Experimentally it is highly infectious for
guinea pigs and hamsters , but virtually
nonpathogenie for rabbits , cats, goats, bovines and
, much less importance in public health . The mode
fowl. Mice are moderately susceptible and develop
progressive infection following intraperiruneal,
intravenous or intracerebral inoculation. Variation
in virulence among strains is frequent . Isolates from
cutaneous and urogenital tuberculosis are often of
low virulence for experimental animals .
Af - bends is more pathogenic for animals . It
produces tuberculosis in cattle, humans, other
primates , carnivores including dogs and cats ,
badgers, swine , parrots and some birds of prey.
Experimentally it is highly pathogenic for guinea
pigs and calves , moderately pathogenic for dogs,
cats, horses and rats , and nonpathogenie for fowl.
BCG, the tuberculous vaccine, is an attenuated strain
Of M fan .
*
Strains of tubercle hacilli isolated from parts of
Africa, that show properties intermediate between
human and bovine types haw been called African
strains* or Af. aBicaaiun . The name Mdsn type 'has
beer given to strains of tubercle bacilli originally
isolated from south India, which are of low virulence
tor guinea pigs, susceptible to hydrogen peroxide,
lsoruazid sensitive and usually of phage type L Such
strains have also been isolated from some other
Asian countries and from Asian expatriates abroad.
Though species names are sometimes used for
different varieties of tubercle bacilli infecting
humans, they are not considered independent
entities, but only variants of the M. fufwjrti/oris
complex.
M. microti is a natural pathogen of voles. It
does not cause natural infection in humans, but can
cause ulcers after experimental infection . Because
it is antigemcaliy identical with human Tubercle
bacilli , it was tried as a substitute for BCG for
human vaccination . It was given up because of
serious Inca! reactions.
rmhngcnesis: The source of infection is usually
an open case of pulmonary tuberculosis. Ir is
estimated that an open case of tuberculosis in India
may infect cm an average some 25 contacts before
death or cure . Other forms of tuberculosis are of
of infection is by direct inhalation of KWtdlittd
bacilli Contained in droplet nuclei of expectorated
sputum. Coughing, sneezing and speaking release
—
numerous droplets- as many an 3000 infectious
nuclei per cough. Dried bacilli in dust arc much
less infectious. Spread occurs most often among
household or other close and prolonged contacts
Copyrighted material
Hidden page
356 * Textbook
widespread diueminvtion of lesions
Instead tbejt LH
and either organs.
in rhe lungs
Epidemiology; Tuberculosis is an ancient
Evidence id spinal tubervulosis is present
in Home Egyptian mummies. Tubercle bacillus
I >NA has been detected by molecular uyih/n in a
—
mummy dated circa 15.S0 1080 Bt , Tuhcrculwis
J
has been for many centuries the most important of
human infections , in its global prevalence ,
devastating morbidity and massive mortality. It has
been called the 'white plague' and Jlhc captain of
all the men of death'.
It is estimated that a third of the world ’s
population, about two billion people are infected
with tubercle bacilli. Even year between eight and
1
nine million new cases of nibenculosiH appear and
three million persons die from the disease . The
large majority of the cases and deaths are from the
-
poor nation? . Jn din. is one or the worse affected
'
countries. More than 40 percent of the population
are infected and some 1 million suffer from
^
tuberculosis in the country, of whom over three
million .irehighly infectious upim Halt .i
million people die from the disease every year in
—
India one every minute.
Poverty and tuherculofiiH go togerher, With
improvements in standards of Living, tuberculosis
had declined rapidly in the affluent nation*;, though
it continued unabated in the poor countries. Rut
with the progress of the AIDS pandemic ,
luberculosis became a prnhlem for the rich rations
also, with cases and even outbreaks among the IUV
, tint do iLot CAflfUmnicatt with jirwjvu . Sputum is
infected. A dose relationship has emerged between
mbcncLilosis and HIV, Not only does HIV infection
reactivate a latent tuberculosis infection, but it also
makes the disease more serious and renders
treatment in effective. Tuberculos- is in turn may r
hasten The development of HIV infection into active
disease. A third complication tbit has made rhe
situation more grave is the emergence- anti spread
of multiple drug tUHisrance among rubende bacilli.
So serious is rhe global threat of tuberculosis
combined with mull [drug resistance and
ot Microbiology *
coitooinitattr HIV infection that the World 1 icalth
Organism on in 1993 took the unprecedented step
of declaring this disease a global emergency.
Human infection with M. hotels used to he
'
common worldwide . In many advanced countries ,
an in the UK it has been almost eliminated bv
J
regular tuberculin res ring of herds and slaughter of
affected cattle . The infection spreads- between
animals through aerosol i SLBJ bacilli in moist cough
sprays. A tew infected cow shed the bacilli in milk,
which is infecrious in humans when consumed raw.
The primary infection, mostly in children would
occur in rhe cervical and mesenteric lymph nodes ,
from where it could spread to bcane and joints and
other extrapulrnona.ry sites. Human infection. with
A/ HOVPS could be prevented by drinking only
,
pasteurised or heated milk . Person - to person
transmission of of A f. hovrs Is very rare .
-
'
B
Laboratory diagnosis: Laboratory diagnosis
of tuberculosis maybe established by demonstrating
-
the bacillus in the lesion by miCttttOpft isolating
ir m culture or by transmitting rhe infection to
experimental animals . DetihmstntLon
hypersensitivity to ruberculoprotein maybe helpful
iti some cases. Molecular diagnostic methods have
also been introduced .
IhJl . M ^ V A K V TUBBRCI ' l -OSIs
The specimen tested is the sputum . Bacillary
shedding in sputum is jhiindjnl in cases with
caseation , but relatively scanty in organised lesions
bc*t collated in the morning before any meal . It
sputum is scanty, a 24- hour sample may be tested.
Sputum sampling on three days Increases chances
of detection . Where sputum is not available ,
laryngeal Hwahs nr bronchial washings mav be
collected. In small children who tend to swallow
the sputum, gastric lavage cat ] be examined.
Microscopy: Direct nr concentration smears of
sputum arc examined . Sputum microscopy is the
most rcl i able si ngic method i n d i agnosi s and control
Of tuberculosis. New slides should be used for
Copyrighted materia
 * MyMfcact & nLim l : Tubereulatia *
smtsuhs and they should tioi be reused, as acid fasr
bacilli always be rtnuwcd
Itfly nut (rum
slides hy
cleaning. Smears should be prepared from the thick
purulent part of the sputum. Smears are dried, heat
fixed and stained by the Zichl-Ncclscn tec hnique.
The smear is covered with strong cartel futhsin
and gently heated to steaming for 5-7 minutes,
without letting the stain boil and become dry. The
slide then washed with water and decolourised
is
with 20% sulphuric acid till no more stain comes
off and then with 95% ethanol tor two minutes .
Decolourindon may be carried out as a single step
with acid alcohol {3% HC1 in 95% ethanol ). After
washing, the smear is coumtTsraincd with Loetflcr’s
Tnethvlene blue , 1% picric acid or 0.2% malachite
green fix one minute . Under the nil immersion
objective, acid fast bacilli arc seen as bright red
rods while the background is blue, yellow or green
depe ndi ng on the conn rerstai n used - At least \ (1 ,000
acid fast bacilli should be present pet ml of sputum
for them to he readily demonstrablc in direct smears.
A negative report should not be given till at least
300 fields have been examined , taking about 10
minutes. A positive report can be given only if two
or more typical bacilli have been seen. Smears arc
graded depending on the number of biL- illi seen:
(Table 39.1)
When several smears arc to be examined daily,
it is more convenient to use fluorescent microscopy.
Smears are stained with aunmme phenol or
Table 39.1 ZN smear evaluation and APS report
JVrJ. of SWJT in Report
AFB ( m! immersion field )
0 300 r AFB rvot seen
1-2 300 F Doubtful,
repoiT smear
357
auramine rhodamine fluorescent dyes and when
, examined under ultraviolet illumination, the bacilli
will appear as bright rods against a dark
background . Beeause of rhe contrast; the bacilli can
be seen ever under the high drv objective, cnahling
large areas of the smear to be screened rapidly.
Microscopic demonstration of acid fast bacilli
provides only presumptive evidence ot tuberculous
infection , as even saprophytic mycobacteria may
present a similar appearance - However most
saprophytic species srain uniformly without
appearing barred or beaded and arc usually only
acid fist without being alcohol fast. As saprophytic
mycobacteria may be present in tap watcrh rubber
tubes, cork or bark, they can gel into clinical
materials unless scrupulous care is taker in their
collection - Saprophytic bacilli are not ordinarily
present in respiratory secretions, hue they may be a
problem with gastric aspirates, feces and urogenital
specimens .
Cnncentration method! ! Several methods
have been described far the homogenisation and
concentration of sputum and other specimens. They
can be classified as methods useful far microscopy
only and those useful for culture .md animal
inoculation as well. To rhe former group belong
treatment with and form!nT sodium ciubonare or
hypochlorite, detergents like tcrgitol, flotation
methods using hydrocarbons, and [ he autoclave
method . These methods kill the hucilli without
altering their morphology or staining reaction.
Concentration methods that do not kill the baLLlli
and so can be used for culture and animal inoculation
include the following'
PetrofFs method: This simple method is
widely used. Sputum is incubated with an equal
volume of 4% sodium hydroxide solution at 37 C "

1- 9 100 F 1+ with frequent shaking till it becomes dear, on JR

1 9
i
-- y
10F
IF
i
3+
average feu 20 minutes. It is then centrifuged at
3000 rgm fur 20 minutes and the sediment
10 or more IF J neutralised with N / 10 HC1 and used for smear,
culture and animal inoculation. Excessive exposure
NB: F-od immersion field to alkali is deleterious and should be avoided .

Copyrighted material
359 i Texioook oA MicrotiiDiogy
A - IITIP ] cr method has been described
eliminating ccntiifugatii m and neutral is? t ion.
Sputum is treated with an approximately equal
volume of a sterile sol Lit:on containing 20 g
cetrimonium bromide and 40 g of NaOH per
!: rrc of d i -- tilled water The contents are mi xed w: i h
. treatment. Sensitivity tests for tubercle bacilli are
a co[coo swab and let stand for five minutes . About
0.2 ml of the inoculum i - smeared firmly with the
.
B
swab over the entire surfaceof acid buffered medium
( IUAT- LJ medium with 20.5 g KH FO, per litre ).
The same nwab is used I LIT inoculating^ a second
slope after stirring the contents ag. iin. The results
arc as good as wuh Fetroff 's method .
Instead of alkali , homogenisation can be
achieved by treatment with dilute acids ( 6%
sulphuiic acid, 3% hydrochloric acid or 5% oxalic
acid ), N acetyl cysteine with NaOH ., pancreallit ,
desogen , zephiran and cetrimide . Flocculation
rnethoik have also been described.
( ] LI I in ret Culture is a very sensitive diagnostic
technique fer tubercle hacilh , detecting as- few as
10 to 100 bacilli per ml . The concentrated material
is inoculated onto at least two bottles of IUAT- LJ
medium. If the specimen it positive by microscopy,
a direct drug sensitivity test may also he set up.
Cultures arc examined for growth after incubation
at 37 UC for four days { for rapid growing
mycobacteria, fungi and contaminant bacteria ) and
at least twice weekly thereafter. A negative report
is given if no growth Occurs after 9- 12 weeks . Any
growth seen :. s smeared and tested by ZN stain : ng .
For routine purposes , a slow growing
nonpLginemed , niacin positive acid fast bacillus is
taken as AJ. ruhejTufosj s. Confirmation is bv.
'
detailed biochemical studies. When the isolate Is
niacin negative, a battery uf tests may be needed for
identification , including growth at 25 °C and 45 C ,
animal pathogenicity and biochemical tests ( Tables
39.2, 39.5 ) .
The use of liquid media with radiometric
growth detection ( sueh as BACTEC-4tijO ) and the
identification of isolates by nucleic acid probes have
simplified culture methods greatly and enabled
results to be given in 2- 3 weeks . But these are
available only in advanced laboratnrics-
Semitivity testa: As drug resistance is an
important problem in tuberculosis, it is deniable
to have sensitivity of : solatcs tested as an aid to
ma nly of three types. The first is the absolute
concentration method in which a number of media
containing serial concentrations of the drugs arc
inoculated and the minimum inhibitory
concentrations calculated . The second is the
jf
res i -- Cance rat i o method in wh i ch two sets of media
containing graded concentrations of the drugs arc
i noculatcd, one set wi ;h the test stm i n and the ot her
with a standard strain of known sensitivity, The
third is the proportion method which indicates the
average sensitivity of the strain, taking into account
[ he face thac any population will contain cells with
varving degrees of sensitivity to a drug .
Animal inoculation: The concentrated
material is inoculated intramuscularly into the thigh
of TWO healthy guinea pigs about 12 weeks old.
Suhcutane^ HiR insulation is not recommended as
it leads to a local ulcer wliivh may be infectious.
P
The animals arc weighed before inoculation and
at intervals thereafter . Progressive loss of weight is
an indication o| infection. Infected animals show a
positive tuberculin skin reaction . One animal is
killed after four weeks and autnpsied . it it shows
no evidence of tuberculous , the other is autopMcd
after eight weeks.
At autopsv a positive animal will show a
caseous legion at the site of inoculation .
Sometimes the local lesion may contain pus
under tension, which may spurt on incision. The
draining and internal Lymph nodes are enlarged
and caseous . The spleen is enlarged , with
uregular necrotic area -- , Tubercles are seen in
the peritoneum and some rimes in the lung , but
the kidneys arc unaffected . The autopsy lesions
have to be confirmed as tuberculous by acid fast
stain 11 ig of smears, to exclude Y. psetidotubtrculosin
am! other hacterial infections which may resemble
Copyrighted materia
 < Mycobacterium I : Tuberculosis » 359

Table 39.2 ; IdentiFicadon ol tubeicla bacilli and nested mytobacleria

Growth im L-1 medium

Rapid growth (within 7 days ) Slow growth

Growth on \1 acCoRki; v:
An ! supharase test
\ i .iL - in

+
M . fortuitum complex Tdluntc reduction Type of growth M. tuberculosis

+
M. M, pWir figment Scanty, smooth,
flar colonies

Rabbit [mthogcRLcirv
In light In dark No pigment

Group I Group JL Group 111

PhorochmmogerL Nonchromogen

—
B.tC ,
+
M. bans

the lesions of tuberculosis , but will be smear
Guinea pig inoculation, once *0 commonly used ,
is row seldom resorted to because it is cumbersome,
Costly ami less sensitive than culture, particularly
with catalase negative , 1 NH resistant strains isolated
based DNA amplification, "iTnc use of Rf’LP and
IS fingerprinting for epidemiological typing of
strains has been referred to . Demonstration of
mutation in specific drug sensitivity genes is a useful
indicator of drug resistance . Such tests for
rlfampicin resistance are already available ,

from south India. [ mmunodiagnoftic Serological tests arc not

Nucleic nuid technology: Polymerase chain useful in diagnosis, though antibodies to many
reaction ( PCII ) and ligasc chain reaction { LCR) bacillary antigens have been demonstrated in the
arc used as diagnostic techniques. I ranscriprion sera of patients . Detection ut antibody to
'

mediated amplification, targeting- rihosomal RNA mycobacterial lipo - arahinomannan has been
has been introduced at an improvement on PCR reported to he of some value.

Copyrighted materia
360 * Texitook oi Microbiology *
Type Niacin Nitrate reduction
HununCdaiuial ) + *
Asian type * *
Afcan r^TK' variable
Vole * }- variable
Bui- ini;
TCH = Th iophrnr -i Caibrocvl LL arid hydrazi dLL (5mgd )
Demonstration of hypersensitivity to
rubcrcuLopiatein ( tuberculin testing) is a starLiianL
procedure . Its scope and limitation* are discussed
below.
li \ T K A P C I . M D N -\ KY Tl I l K H i l t l . o M S
Microscopy , culture and occasionally animal
inoculation axe used tor diagnosis of extrapulmnniirv
tuberculosis also, though it is difficult to get
conclusive results as the bacilli arc present in fir
tewer numbers in these lesions than in pulmonarv
disease. CSb (inn tuberculous meningitis often
develops ; L spider web clot on standing, examination
.
of which maybe more successful th vn of the fluid .
I'he use of PCR and DNA probes may help detect
the bacilli speedily a ]uf enure often .
Bone marrow and liver biopsy specimens from
miliary tuberculosis and blood from those with
HIV coinfection arc useful for culture. Pus from
tuberculous abscesses often yields positive results
in smear and culture.
Pleural effusion and other exudates mav be
j
collected with citrate to prevent coagulation. If free
from other bacteria, they mav be used tor culture
afrer centrifugation. If other bacteria are present,
prior concentration Is necessity
Urinary excretion of bacilli in renal tuberculosis
in intermittent , licncc it is advisable to test 3 6
morning samples of urme . Each sample is
Centrifuged for 3UU0 rpm for 30 minute* and the
sediment used for culture after concentration .
Allergy find immunity: Infection with
tubercle bacillus induces cell mediated immunity
Table 39.3
Oxygen Growth in TCH' Phzge type
Preferen ce
Aerobic -
i A -B . C
Aerobic 1
MicrMetophilk A
Mkrtaftiophilit J
B
Mitroamphilif A
which in manifested JS delayed hypersensitivity
{allergy ) and resistance to infection ( immunity ). The
resultant of these two processes determines the
course of the infection .
The response of a tuberculous animal to
reinfection was originally described by Koch ,
Subcutaneous injection of virulent tubercle bacillus
in a normal guinea pig produces no immediate
—
response , hi it after 10 14 days a nodule develops at
the site, which breaks down to form an ulcer that
persists till the animal dies of progressive
tuberculosis . The draining lymph nodes are
enlarged and caseous. If on the other hand virulent
,
tubercle hat-ilk ]* is injected in a guinea pig which
had received a prior injection of the tubercle bacillus
4-6 WOeltfi earlier, an indurated lesion appear* ir
the sire in a day or two. TTiis undergoes necrosis in
another day or so to form a shallow ulcer that heals
rapidly without involving the draining nodes or
other tissues , This is known as the Koch
phenomenon and is a combination of hyper-
sensitivity and immunity. The Koch phenomenon
—
has three components a ‘local reaction, a < (bcaT
1
response manifested as congestion and even
hemorrhage around the tuberculous foci in rismesj
and a constitutional' or 'systemic' response of fever
which may sometimes He fatal.
- Allergy can he induced by infection with
virulent as well as uvimlent tubercle bacilli , but
not ordinarily by injection of killed bacilli or
bacterial proteins. Howevetf for demonstrating
allergy, live or killed bacilli or ruberculopiotein can
1
be employed.
Copyrighted material
Hidden page
\bl * Texioook oi Mi^biaiogy *
of early detection and treilment of eases ,
chcmoprophyla ^ h and immunoprophylaxis.
Immunoprophylaxis is :T . intrude min] injection
of the live attenuated vaccine developed by Calmette
. I I I > I Guerin (1921), the Bacille Calmette Guerin
-
or BCG. Thi H a strain of iVf. hovix attenuated by
229 serial subcultures in a glycerine-bilc-potato
medium over u period of 12 years. Injection of BCG
in animals induces self limited infection , with
multiplication and dissemination of the bacillus ill
different organs and production of small tubercles .
Within a few weelcs the bacilli stop multiplying
although they survive in the tissues for long periods.
This gives fine to delayed hypersensitivity and
immunity. Following BCG vaccination, a tuberculin
negative recipient is converted to a positive reactor .
The : mm unity may last for 10-15 years and is
similar to the immunity following natural infec cion,
except that it does not carry' any risk of disease due
to reactivation, as in the latter case-
Several In' Id ITI.LIH hive Keen conducted to assess
rhe efficacy of the BCG vaccine. The results have
varied widely, from fiO per cent protection to a total
absence of protection . In south Indi .i., a trial
conducted at Madanapalle showed 60 per cent
efficacy while a later large trial in Chingalpattu
did not reveal any protection in adults, though a
15-year follow up showed some protection in young
persons ! he reasons for such w ide disparity are
not clear but have been attributed to several factors
such as the differences in the prevalence and
virulence of tubercle bacilli in various communities,
the type and potency of vaccines used and the
presence of ’atypical mycobacteria in the areas.
The BCG vaccine aroused much criticism and
it has been suggested that it may regain its virulence,
though there has rot been any evidence of it so far.
The Lubeck disaster in which several children
developed fatal tuberculosis following oral
immunisation earned the vaccine much notoriety'
ui rhe early days of the vaccine. This was due to a
-
mix up by which ivc virulent tubercle was given
"
instead of BCG, Stringent safety- measures have
since been enforced in [ lie manufacture of BCG
vaccine., The recognised complications of BGG
vaCcirte are the following:
Local Abscess , indolent ulcer, keloid, tuberculides,
confluent lesions, lupoid lesions, lupus vulgaris,
fltyior .'flJ Enlargement and suppuration of dram:] i ;-:
lymph nodes.
Gejertk Fever , mediasn . nal adenitis, erythema
nodosum , tendency to keloid formation after
wounding at other sites, and very rarely nonfatal
meningitis . The very few cases of progressive
tuberculosis reported are believed to have been in
i m munodefic lent subjects.
The consensus opinion is that BCG may not
protect from the risk of tuberculosis infection , but
gi '.^s protection to infants and young children
against the more serious types of the disease , such
as meningitis and disseminated tuberculosis. The
recominendalkin therefore is that in endemic
countries such as India , BCG vaccine be
jdinn iKtered to babies by mtraderma] injection on
'
the deltoid immediately after birth, or as early as
possible thereatter, before the age nf 12 months.
The WK , i nc need not be admi n i ^ tcred after the age
of two years, BCG should not be given to infants
and children with active HIV disease, though it
may be g: vcn with benefit to asymptomatic HIV
positives. Babies bom to mothers wilh ATB positive
s p u t u m should not be gr ^en BCG at birth , but only
after a course of preventive chemotherapy.
BCG induces a nonspecific stimulation of the
immune system providing some protection against
leprosy and Icukcmi .i - Multiple injection of BCG
has been tried as adjunctive therapy in some
malignancies. Some workers have reported that
BCG is superior to PPD for tuberculin testing.
Chemoprophylaxis or preventive chemotherapy
is the administration of antituberculous drugs
( usually only isoniazid ) to persons with latent
tuberculosis (asymptomatic tuberculin positive ) and
a high risk of developing active tuberculosis, or to
the uninfected exposed to high risk of infection . It
is particularly indicated in infants of mother; with
*“ *u
opyrighted materia
j
 * Mycobacterium \ : Tuberculosis
active tuberculosis and in children living with a
case of arrive tuboculow in the house . bonis / id S
mg/ kg daily tot 6-12 months is the usual course,
Trial* have shown that this reduces the risk of
developing active disease by 90 per ccnr. HIV
infected coni acts of active tuberculosis also benefit
from tills prophylaxis.
THERAPY
Cbcmothei ipv bus revolutionised the OU|Qj| fl1tilt
ot tuberculosis , It has been established that ^ infecting strain initially sensitive becomes resistant,
sanatorium regimens, bed rest, fresh air and rich
fbodf as well as operative interventions, such as
artificial ptieuntothnrut and thoracopluty arc not
essential for cure it domiciliary treatment with
effective antituberculous drugs is given in optimal
dose and duration .
Antituberculous drugs are of two typesf
bactericidal and bacteri static. Of the bactericidal
drugs , rifamplciii ( R ) and pyrazinatnidc ( Z) are
called sterilising drugs because they are able to
effectively kill the bacilli til the lesions. Ot the Other
bactericidal drugs, uoniaad (H) is effective only
against replicating bacilli and streptomycin ( S ) only
against extracellular bacilli and so are not bv
themselves able to sterilise the lesions. The
bactericidal drugs , along witli the hucteristatic drug
etbamhutot ( E ) constitute the flrnt line drugs in
-
antituberculous fbenpy. ' 1 V old practice of daily
adminis [ ration of drugs for two years or SO bus been
replaced by short course regimens of 6-7 months,
which are effective ind convenient A typical
example of such a schedule for a new smear po > iTivc
ease is a combination o f four drugs ( HRZD- ) given
three times a week during an in it ill intensive phase
-
of two months , followed by 4 5 months of
continuing phase with only two dings ( HR ) three
times a week.
The major problem in chemotherapy is drug
tc si stance , which in tubercle bacilli is due To
mutation, with an approximate rate of once in 10*
cell divisions. This could have been effectively
checked bv multiple drug therapy, which was
363
1 ntr< id need fo r this very purpose. ( 1 nfortu n ately this
was not implemented properly. Due tn a
combination of lapses in prescribing practices, drug
del ivery a nd patient compliance, res istance h as built
up in tubercle bacilli over the years, reducing the
efficacy
ar
of [ reatnienr.
Drug resistance map be 'primary' ( pretieatment,
initial ), when the patient is infected with a strain
of tubercle bacillus which is already resistant, or
acquired’ (secondary pout -treatment ) , when the
usually as a result of impiopet or inadequate
treatment. This is the more common type of
resistance . When Acquired resistant strains become
increasingly common in an area, the chance of new
patients presenting with primary resistance
increases. When an infecting strain acquires
resistance to one drug, the chance of its becoming
resistant to other drugs increases , unless the
treatment schedule contains an adequate number
ofdfective drug . At present the spectrum ofsirams
^
prevalent in the community conn resistance to all
available antituberculous drugs .
A very serious consequence of unchecked drug
resistance has been the emergence and spread of
multidrug resistant tuberculosis ( MDK -TH ) .
Though the term multidrug resistance means only
resistance to two nr more drugs, in the context of
tuberculosis , it specifically refers to resistance to
rifumpiuin And isonisaid , with or without reuis-tanue
to One or more other drugs. This is because R ami
H form the sheet anchor of short - term
chemotherapy and any strain resistant to both three
drugs is unLikelv to respond to treatment.
MDR -TB is a global problem , menacing the
|xxir and tOie rich nations alike . It may lie primary
or acquired - Its presence in those with concomitant
HI v infection make* it more dangerous. When first
line drugs become ineffective , second! line drugs
have to he tried. I.argc numbers of old and new
drugs ,trc being used - tiiiinoloncs , uniiuglyooeides,
macroltdcs, para amino salicylic acid, thtacetazonc,
cycloserine , capreonTycin and others. They are
Copyrighted material
Hidden page
 Mycobacterium II: Non-Tuberculous
Mycobacteria (NTM)
Mvcobicteria other than manmiilian tubercle
bacilli, which may ocusknal aiiKhuiniii disease
^
resembling tuhercuLosis„have been tilled 'atypical',
anonymous' or WJuified' mycobacteria, MIL
"
names 'environmental' or 'opportunistic
mycobacteria" are better suited as their natural
habitat appears to be ^niI or water ant) they cause
opporni nisric infections in human beings,The name
'mmtuberculouu mytcobacleriif (N1M) has gained
wide acceptance in receiptyears. They have also
been called paratuberele’, 'tuberculoid 1 and
' after M. inum complex.
'MOTT* (mycobacteria tuber rhan tubercle) bacilli.
Thcv ;ine distinct from the saprophytic rtiVCObaCteria
such as .U. phlei which are incapable of infecting
human beings or animals. While human infection
with them is common in some areas, disease is rare.
, pynzimiiudc hydrolase and 1.- fbooHidue aL- tiviricR.
They are unable ro cause progressive disease when
injected into guinea pigs.
Non - tuberculous mycobacteria have been
classitied into lour groups hy RuuVOit ( 1959) huicd
cm pigment production and rate of growth: Group
1 photochromogeitif Group II scotoehmmpgens,
Group Ill no nphocochro Imogens and Group IV
rapid growers. Though other methods of
, form pigmented colonies (yellow-onfige-red) even
classification have been described, Runyon's
dUtiffcttiOA has found universal acLeptailce.
Species identification depends on several additional
characteristic;; (Table dthl).
Cmup I phntochreImogens: 1 hese strains
form colonics that produce no pigment in the dark
bur when rhe young culture is exposed to light for
one hour in die presence of air , and reineubated for
-
2 4 2 8 hours , a yellow orange pigment appears .
They are slow growing, [ bough growth LS faster
rh;m that [ if tubercle bacilli. The important species
in rhis group are M. Aaiwasjj, M . mttnnam and
Mr strniae.
M . kastsii ciuset chronic pulmonary disease
resembling tuberculosis, usualI v affecting the upper
lobes, with eaviity form at ion ,md scarring ,
M . kannsu has beer isolated from tap water
samples around the world and this is believed to
be the main reason and source of infection. It is the
second most common NTM seen in lung diseases
M. nunnum which causes a warty skin lesion
( swimming pool Dr fish tank granuloma 1, closely
"
resembles JVf. ^^sif but can be differentiated bv
a
its poor growth at 37 aC,negative nitratase, positive
Several photochromogen LC mycobacteria were
isolated in llJ6J from monkeys exported from India.
They have been classified into two species: niacin
positiveM, sjNiijL' and niacin negative M. rafarieiuzL
They have subsequently been associated with
pulmonary disease in human beings.
Group II — fwOtochromogens: These strains
in the dark. They are widely distributed in rhe
enviremnient and sometimes contaminate cultures
.
of tubercle bacilli. M SLToiulaceum may cause
scrofula (cervical oriemris) in children .
M, gunJonae, [iften found in Cap water (hence
called ‘ the tap water sCOtochromogtn ' h is a
common contaminant in clinic ,i[ specimens and a
rare cause nf pulmonary disease, Ir differs From Af .
scrofulxceum in failing to hydrolyse urea,
nicotinamide and pyraainamide .
Copyrighted material
366 * TexIbtiOfe. of MiCfObiOMpgy

Tablte 40.1 DiHerenfliationi belween tubercle bacilli and so species of atypical mycobacteria

1
Tbr
'w
.
J

j
5
E
_
_£_
:r
! Si t

i i 1 I 5
I t :f

Z^=
P" '

3 i i £ 8
E
£
"
j

«r* c

Growth in 7 days 4 4 4 4
Growth sit 25 "C + * * ± 4 4 4 4

Growth at 3-7 + + + 4 t 4 4 4 4 4 +
Growth at 45 , lC 4 4
Pigment in dark 4 4

Pigment in light t 4 4 4

Urease 4 + 4- 4 4 4 4 4 4 4
Niacin 4 ±
Nitrate reduction 4 4 4 4 4

M. szulgni is scotochrocnogcctk when grown

'
at 21 GC and phoioohromogertic ac 25 °C.
Group I I I — n< inphotMhromogena: These
Htruins do not form piemen t even on exposure light.
Colonies may resemble those of tubercle bacilli .-
The medically important specie H arc j\ f. aWuiJlj
Mr jnmcefluZue; M. xeaopi and the skin pathogen
M. uicerans.
Aft living which causes nuniral fubcrciilnKi* in
birds and lymphadcnopatlby iit pigs is one of
the most common opportunist human pathogens,
A/, in trALcUuiurt is eomrnonlv known a-s the RuttevJ colonies .
r
pathogen ac ilie Rut le y State Hospital tor
Tuberculosis, Georgia , USA
Al avium and AJ . Intracd/uluv art so closely
similar t h a t they have been considered as one
group, the M avium complex [MACJ - They cause
lymphadenopathy, pulmonary lesions or dissemi-
nated disease, particularly in AIDS patients. They
are related to the animal pathogens M- psiititbtt-
ctilosis and M lepnemimum.
Af. rrraljniten.H: which may cause pulmonary
diueate i F a slow grower, taking 8 12 weeks: to form

bacillus' beeaiiie it was fm> r identified as a human M . .tejTopj, originally isolated from toads, may
Table *>0.2 Dltfersnlkation between V, trfeerens and W. nwrinum
Chmrwcter Af. ufrera /tr M , marinum
Distribution Tropics Temperate zone
Clinical Course Chronic progressive ulcer Self -limited ulcer
Bacilli in ulcer Abundant Scanty
Rate of growth
GrowTh ar 2$
Slower, 4 8 weeks
-
—
l ister, 1 2 weeks
4

Growth at 37 - +
Culture him Bacilli in ititfds No cord formation
Pigment in tighr -
Mouse footpad legion Edema, rarely ulcer
j —
Marked inflammation purulent
ulcer

Copyrighted materia
 * Mycobacterium II : S Dir - Tuberculosis Mycobacteria ( NTMj »
occasionally cause chronic lung disease in human
beings, AJ. xcnvpi and Af. arc ihermuphiles,
capable of growth At 45 °C.
Though usually classified as a nonpholo -
chromogcn , Af . xenopi may form seoto - Cutaneous lesions may occur in leprosy or
ebromogcnic yellow colonies, AJ. xerwpi has been
isolated from water taps, mostly hot water caps, in
hospitals. It has also been isolated from main water
supplies.
Group IN — r n p i d growers; This is a
heterogeneous group of mycobacteria capable oi
rapid growth, colonies appearing within seven days
of incubation nc 37 °C or 25 °C, Within the group,
photochromogenic , scorovhromogenie , and
nonchrotnogciuc species occur. Chromogcniic rapid
growers are mostly saproplivtcs (for CXMlpk, Af.
phiefi. The medically important species arc Af ,
ii>rtuiturn and Af, chdonac both of which can cause
chronic abscesses in human beings. Outbreak* o +
abscesses following injection of vaccines and other
preparations contaminated by these mycobacteria
have been reported on a number of occasions . The
bacilli arc found in the soil, and infection usually
follows some injury;
AJ , fbrtrrri m and AJ , dieiond do not produce
^ Initially, smears from the edge of the ulcer ^ how
any pigment . Pulmonary lesions caused by Af,
'
be distinguished radio logically
/ortm frrjTr cannot
from typical tuberculosis. No effective
chemotherapy is available. Af . smcgtnttii ,
commonly considered as saprophyte in smegma, i ?
seldom seen in thar location . It is a frequent isolate
from soft tissue lesions following trauma or surgery.
Some noncultivable or poorly growing
mycobacteria identified from the blood of AH b
patients have been characterised bv their IfiS RNA
^ the limb though ulceration is infrequent. A toxin
base sequences lliey grow sparsely in some liquid
, is produced by Af , ulccmns that causes
media. Tramples are Af genevente , Af. ConBtittitia
and At intamedium.
A rapid grower, Af, vatxze is reported [ < be an
immunomoJulalert capable ui inhibiting tissue
destroying hypersensitivity responses and
stimulating protective immune processes in
tuberculosis. Clinical trials of Af, vacat vaccine
167
as an adjuvant to chemotherapy in tuberculosis am
on.
S h i v PATTMUI 1: Ms
tuberculosis , either as localised disease or as parr
of a generalised infection. In a different class arc
two species of l NYOOBAELERIJ , Af. uk -erzfis and Af.
ntMTinuntf which arc exclusively shin pathogens,
causing chronic ulcers and granulomatous lesions
Oil the skin . Systemic invasion does not occur and
the regional lymph gland* arc not involved .
Cutaneous localisation is because they multiply
optimally at skin renuperaturc.
M. ulccrans: This wis originally isolated from
human skin lesions in Australia ( 1948) hut has
subsequently been recovered from similar lesions
from Uganda ( liuruli ulcer ) , Congo , Nigeria ,
Mexico, Malaysia and New Guinea, Ulcers art 1
usually seen on the legs or arms and arc believed ro
follow Infection through minor injuries;.. After an
incubation period oh a few weeks, indurated nodules
appear, which break down forming indolent ulcers
which slowly extend under the skin ,
large clumps of bacilli which are add list and
alcohol fast. Cater, the irnmunoreactivc phase sets
in and the bacilli disappear, " [‘he ulcers then heal
with disfiguring scans.
The bacillus grows on LoWClUttin-Jensen
-
medium slowly, in 4 8 weeks . The temperature
of incubation is critical; growth occurs between
.10 ^Cand . So
’
hut not at 25 °C or 17 °C . Inocu
lation into the foot pad of mice leads to edema of
-
inflammation and necrosis when injected into the
,
skin of guinea pigs. "J ' bis is the only known instance
of rosin produced by any mycobacterium species.
M , mannum: This LV a natural pathogen of
coldblooded animals, causing tuberculosis- in llsh
and amphibia. It may also occur as a saprophyte in
fresh or salt water. I Inman infection originates from
Copyrighted materia
365 s Texlftook d Microbiology *

Table 40.3 Atypical mycobacteria associated with human disease

Specitt Natural habitat Type tif in few 1on

AJ, nfricnnum Animals, soil Pulmonary

AJ, asiiiticum Primates. Pulmonary
AJ. Avium / rpfraLfJ/uj'jrp Soil,. seawater animals Pulmonary; systemic,gufioiiiAHtiiulp
tymphadenim
AJ. chdonac
AJ. CjfwJo/TdC, £.£
_ Soil* seawater, animals
Soil* seawater animals
Porcine heart valves,surgical wound, pulmonary
Cutaneous, surgical absccssus wound -
pulmonary - systemic
AJ. fitlax Water, soil Pulmonarv
AJ. /ojtiu.’jhj.m Water, soil animals Pulmonary surgical wound cutaneous* systemic*
bone and joint
AJ, harmophilum LJn known Cutaneoua, tubcutaneau
AJ, Jcarujdu WART, animals Pulmonary, systemic, skin joints, lymph nodes
AJ. mMlmocnsc Unknown Pulmotuty
AJ. f.rr;uirtLfjrj Aquarium water, bill Cutaneous (swimming pool granuloma ),
joints
Af. ttroftiiceuffl Soil,water,fomitea Lymphadenitis (usually cervical );
pulmonary disseminated
AJ. xhinmdes Unknown Pulmonary
AJ. smiuc primates, water Pulmonary
AJ. szulgai Unknown Pulmonary, lymphadenitis* cutaneous;,
subcutaneous bursitis
AJ. ulccmns Unknown Cutaneous
M- Kitin '
Soil, water Pulmonary, epididymirjs;

Contaminated swimming pools or fish tanki-. The M mirimim is not pathogenic for guinea pigs but
lesion, beginning .is a papule and breaking down in trade mud inoculation in rabbits leads to a
to form an indolent nuvlcar, usually follows superficial granulomatous lesion - Footpad
ibrasicins ]id therefore DLLWE on [ he prominence*
j inoculation in mice leads to a more severe lesion
—was first described from Sweden under the
elbows, knees, ankles nose, fingers or toes . It
name
than with M. idcemv, local inflammation being
followed by a purulent ulcer formation .
SWirnrning pool granuloma ' , and flit hfltiHus was
‘
Infection with Af. marinuni , hut not M.
named At bidnei ( fmm baJneum meaning barb) It . ulcaans, may cause a low - grade tuberculin reaction.
lias since been reported from other European M hiemophihim , fir* t described in 197S ,
countries and from North America - Its distribution causes skin lesions . It requires henlin for gtowth.
is in temperate areas in contrast to Af , tdeerans, Tt grows af .12 ¥ C! in 2-4 weeks but not ;U 17 DC -
which lias ,a tropical prevalence. EI uni an infection Tahle 40.3 shows rhe range of human infections
niav occur in epidemic form . The LLLLTIS are self - produced by ditferenr species -of atypical
limited and undergo spontaneous healing- mycohactcriiL.
HaciEli are scanty in smears from ulcers .
Growth occurs io uboul two weeks it TO ''C [range
1’icnlngy and epidemiology: As environ
mental bacteria are widely distributed io nature,
-
-
25 35 rC) and primary cultures do nof grow ^ t itifectiun with them is quite common, from soil,
37 <C but they do after adaptation. Colonies arc
'

nonpigmented in the dark; however, they lteoome

-
wafer and air. Ptraon - to person infection docs not
seem to occur. Infection is mainly asymptomatic,
intense orange yellow to red on exposure to lighr , though it may result in sensitisation , causing weak

Copyrighted materia
Hidden page
 Mycobacterium III: M. Leprae
Leprosy is a disease of great antiquity,having been
recognised from Vcdic rimes in India anil from
Biblical times in the Middle Last . [ r probably
originated in the tropics nod spread to the rest of
the world. Leprosy has always been held in
superstitious dread and the person suffering from
leprosy considered "unclean and a social outcasts .
Tire lejura bacillus was first observed by Hansen in
1A 69. Though this wan the first baeteriai pathogen
of humans to be described , it remains one of the
.
least understood This is because it I Las not been
possible to gfQW the bacillus in culture media.
MYCOBACTERIUM LEPRAE
.
Morphohrgy: M leprtc is a straight or slightly
curved rod, 1-9 urn « 0.2 -0.5 pm in size, showing
considetable miirphologic al vanstuns, Pollr bod ics
aud (ITLLUT intracellular elements may be present .
Clubbed forens, lateral buds nr branching may be
observed- It is Gram positive and stains more iciidilv
than rhe tubercle bacillus. It is acid fast, but less so
than tubercle bacillus . Hence 5% sulphuric acid
instead ot 2096 is employed tot decnlrmriRation after
staining with carbol fiich^m , It is the practice to
differentiate: between lir e and dead bacilli in stained
smears, assuming without Conclusive prr >of that the
former appear solid and uniformly stained, while
the lac ter are fragnienLed and granular. The
percentage of uniformly stained bacilli in tissues
{ morphological i n d v r ) provides ,t method ot '
assessing the progress of patients on chemotherapy
and is more meaningful than the old criterion of
bacteriological index { tlie number -of bacilli in
tissues) .
The bacilli are seen singly and in groups,
.
ii it race!lul Lib or lying fate outside the cells. TlLey
'
frequently appear as aggloincrates, the bacilli heing
-
bound together hy a lipid likc substance, the glia.
These masses are known as 'glohi '. The parallel
rows ol bacilli in the gtobi present a "cigar hundlt '
appeamticc . In tissue sections, the clumps of bacilli
resemble cigarette ends . The globi appear in
Virchow's lepra cclh' or Toacry cells' which are
"
large undifierentiated histiocytes.
Cultivation: It has not so far been possible no
Cultivate leprabfltcilli cither in bacteriological media
.
or LLI tissue culture There have been several reports
of successful cultivation but none ft as been
.
confirmed One of the best known of such reports
(1962 ) came from the Indian Cancer Research
Centre, Bombay, where an acid fast bacillus was
isolated from Leprosy parienti, employing human
fetal -spinal ganglion cell culture . This ICRC
bacillus has been adapted for growth r > n
Lowenstcin-Jcnscn medium.Its relation to the lepra
bacillus is uncertain.
There have been many attempts In transmit
.
leprosy to experimental animals However, the real
break through was rhe discoveryby Sbepard (I960)
:h.it lepra bacilli could multiply in the brntpatis of
mice kept at a low temperature ( 20 rC ) . This
observation liis been confirmed and lias become
rhe standard procedure tor experimental work with
rhe bacillus , hollowing inrradermal inoculation into
the footpads of mice, ;L granuloma develops at the
site I I L 1 — 6 months. If cell mediated immunity is
suppressed by ( hymectornv or tbe administration
of ancilympborrte senim , a generalised infection is
Copyrighted material
 « Mycobacterium III - M Lsprae
produced, simulating lepromacous leprosy; The
nine- banded armadillo { Dasypus novemcioctus) is
highly susceptible to Infection with lepra bacilli ,
Following inoculation into armadilloes , a
generalised infection occurs with extensive
mutt [ plication of the bacilli and production of
le"-- i ' ins typical of lepromatous leprosy. Some wild
armsud Hoes in captivity have beer observed ro he
naturally infected with a mycobacterium resembling
the lepra bacillus. " Natural disease' has also been
identified in chimpanzees and mangabey monkeys
from West Africa but ir is nor known whether they.
have any relevance to human infection.
In animal experiments, rhe generation time of
the lepra bacillus has beer found to be excepl i -unally
long, 12-13 days on the average but may vary from
-
£ 42 days, in comparison with about 14 hours in
the case of rhe tubercle bacillus and ahuut 2Q minutes
in the case of coliform bacilli
Af . leprae genome has been mapped and the
genes for its major protein antigens cloned and
scqucnced -
Resi stance: Lepra bacilli have hcen found to
remain viable in a warm hum .d environment for
'
9- 16 days and in moist soil for 4b days.‘iTacy surv ivc
exposure to direct sunlight for two hours and
ultraviolet light for 30 minutes .
LEPROSY
Leprosy is a chrome granulomatous disease of
humans primarily inyoking [ he skin, peripheral
nerves and nasal mucosa but capable of affecting
into four types
—
any tissue or organ . The disease may be classi:i.ed
Sep com a rou s , tuberculoid ,
d .' nwrphv us and imie terminate {Midi id
classification , 1953). The type of disease is a
reflection of the immune status of the host. It is
therefore not permanent and varies with
chemotherapy and alterations m host resistance .
'
-
Bacilli i olutcd from dilferrnt types of lepro&V do
not differ in virulence or other propcrties.
The two extreme or Lpo!ar’ forms of the disease
are the lepromatous and tuberculoid types. The
» 371
lepromatous type is seen where the host resistance
is Low. The bacilli are seen in large numbers or as
globl inside lepra cells or extracellular!)'- To is is
known as 'multibaci 1.1 ary disease . Superficial
1
nodular lesions ( lepromata ) develop which consist
of granulation tissue containing a dense collection
of vacuolated cells in different stages of
development from mononuclear cells to lepra cells .
The nodules ulcerate, become secondarily infected
and cause distortion and n :i:illation . Bacilh invade
the mucosa of the nose, mouth and upper respiratory
tract and are shed in large numbers in nasal and
oral secretions.The reticuloendothelial system, eves,
testes, kidneys and bones are also involved .
"
-
ILu iLcima L common. The lepromatous type LS
more infective than the other types. The prognosis
Is poor. Cell mediated immunity' is deficient and
the lepromin test is negative MI leproittaiOUS leprosy.
On the other hard, there exaggerated and
in an
broad humoral immune response . Antibodies in
high litres- are seen against mycohacCen.il as well
as several other an! igens . Autoan I ibodies ate
common. Most cases show biologitaJ false positive
reaction in standard serological tests for syphilis.
At the other end of the spectrum Ls tuberculoid
leprosy, which is seen in patients with a high degree
of resistance. The skin lesions are few and sharply
demarcated , consisting of macular anesthetic
patches. Neural involvement occurs early and may
be pronounced . leading to deformities, particularly
in the hands and feet. Bacilli are scanty in the
lesions and infectivity is minimal. This is known as
' paucihacillaiy disease*. Celt mediated immunity is
adequate and the lepromin test is pO* itnrc -
AiidmycDbactieriaJ and autoimmune antibodies are
. -
rare The prognosi is good .
The term borderline or dimorphous type refers
to lesions possessing characteristic! of both
tuberculoid and lepromatous types. It may shift to
the lepromatous or tuberculoid part of the spectrum
depending on chemotherapy or alterations in host
resistance .
The indetem’ irate type is the early unstable
Copyrighted material
Hidden page
 H Mycobacterium III: M. L &prao » 373

The deficiency in cell mediated immunity is
-
specific ro the lepra bacillus aittignu, 1 cpromafous
patients a« not more susceptible to viruses, parasites
acid Other pathUgertii against which CM! is
important Tuberculin reactivity may be suppressed
in untreated Icprnmatous patients hut it becomes
positive following treatment , unlike the lepromin
response which remains negative Lepromaious
^
patients hair large numbers. of C K * (suppressor)
lymphocytes in circulation , which can he
specifically activated by the lepra bacillus antigens .
The lymphocytes present in skin granulomas are
altruist exclusively CDS + cells, in contrast to
tuberculoid lesions which contain pcedocniniiitly
, chemotherapy Crops of tender, inflamed
CD 4+ cells . In lepromatous leprosy, there is
extensive polyclonal B cell activation with large
amounts of antibodies being produced, hnth
anti mycobacterial and autoimmune . The
ilbuminigkibuliti ratio is reversed . The antimyeo-
bacterial antihodics ate nor beneficial. On the other
hand, they may have an enhancing effect.
There is evidence of genetic effect in the pattern
of response to lepra bacillus infection , HLA DR2
is seen preponderantly in persons with the
- in leprosy was a skin test for delayed hypersensitivity,
Lulterculoid type uf reactiuith while HLA- MT1 and
-
HI J\ DQ]arc associated with leproma tons d i sease.
Though leprosy is a chronic disease, its course
may ho intenvperaed with acute evacerbations which
a re of an allergic rub Lre - Two types of such reactions
occur.
Type 1 or the ' reversal reaction’ or the 'lepra
reaction ' is seen mostly in borderline leprosy,
occurring spontaneously or more often during
cbe mother spy . It is a cell mediated immune
reaction , with an influx of lymphocytes into Lesions,
and a shift to tuberculoid morphology. The lesions
develop ervthenia and swelling, along with pain
and tenderness- A similar clinical picture is seen in
the ’downgrading reaction' which occurs usually
in untreated or pregnant patients , Here, biopsy of
the lesions shows a shift to the lepromatous pattern,
reflecting a decrease of CML
Type 2 reaction or Erythema Nodosum
Leprosum ( ENL) occurs in the LL and lfL types,
usually a tew months after institution of
subcutaneous nodules appear, with fever ,
lymphudenopathv and arthralgia. This i & an Arthur
type response to antigens released from dear! lepra
cells and is characterised by neutrophil infiltration
and IgC and complement deposition in the lesiors-
L E P H O M I N TEST
Till nece n[ I y, the only method fur studying imiriLiii ][ y
the lepromin test first described by Mitsuda in 1914.
The original aotiger ] (lepromin ) was, boiled ,
emulsified, lepromatous tissue rich in Lepra bacilli -
Tire response to the intradermil injection of
lepromin is typically hip ha si;: , -consisting of two
separate events - The first is the early reaction of
Fernandez, which consists of erythema and
-
induration developing in 24 4& hours and usually
remaining lor 3-5 davs. This is analogous to the

Tdbte 41 ,1 Characterisation Di
^ diHerem types or leprosy
Jradeferminafe

Tuberculoid Bdtdethne f. epro /tijroiji

Lepi ± bacilli in tissues * 4 4+4
Lepromin test 4 4 ± -
Mvcob.iv:teri al antibodies
. . ± + +4+
L>Tnphoeytk infiltration of lesiims ++ 4 4- -
Plasma cells in lymphoid tissue 4 4 4 + 4-

Copyrighted materia
 374 i TfeVfbOOfc d Microbiology
tuberculin reaction, This is usually poorly defined
and carries little significance, The second and more
meaningful l:- the late reaction ot Mitsui La, starting
in 1-2 weeks, reaching a peak In four weeks and
gradually subsiding in rbe next few weeks. The
reaction consists of an induiared skin nodulE , which
may ulcerate, Histologically, there is infiltration with
lymphocytes, epithelioid cells and giant cells. The
Mitnuda btc reaction docs not indicate pre-existing
DTI L tn.it i s a measure of the CMI induced by the
injected lepromin itself. Tt thus disti ilguishcs
between persons who can mount a CMI response
against the lepra bacillus antigens and those who
cannot .
The original crude Mitsuda antigens extracted
-
from skin le ions of IcprorruitoLLb patients ( integral
lepromins ) were standardised on the basis of tissue
content . Modern antigens are standardised
according to their lepra bacillus content ( 4 * 10T
lepra bacilli per ml ). Standard lepromins arc be mg
.
ITLI .IRL . I Liwrcj -
ingly from armadillo derived lepra
bacilli (lepromin-A ), reflating human derived
lepromin - H.
The lepromin test is not used to diagnose
leprosy, nor does it indicate prior contact with the
lepra bacillus. Healthy person ^ in noncndcmiL; areas
with no chance of contact with the bacillus may
give a positive lepromin test. The test is employed
for the following purposes!
1. To classify the Ichors of leprosy patients. The
lepromin test is positive in tuberculoid , negative
m leprOittatous and variable in dimorphous and
indeterminate types of dtica$C -
-
2.Tu assess the prognnsi . and response to treatment.
A pos i r ive reaction indicates good prognosis and
a negative one bad prognosis, Conversion to
lepromin positivity during treatment is evidence
of improvement.
3. lo assess the resistance ol individuals to leprosy.
It is dcurable to recruit only lepromin positive
persons for work in leprosaria as lepromin
negative pensons are mote prone to develop the
disease.
4. lb verify the identity of caiHlidatE lepra bacilli ,
Cultivablc add fast bacilli claimed to be lepra
bacilli should give match mg results when tested
ML parallel with standard lepromin.
I laboratory diagnosis: Bacteriological
diagnosis is easy in the leprcmnacous but may be
difficult in the tuherculoltl ease?. The diagnosis
consists of demonstration of acid fast bacilli in the
lesions. For routine examination, specimens are
collected from the nasal mucosa, skin lesions and
ear lobules. A blunt , narrow scalpel is introduced
into the rose and the internal septum scraped
sufficiently to remove a piece of mucus membrane,
which is transferred to a slide and teamed out into a
uni form smear. Samples from the skin should be
obtained from the edges of the lesion rather than
from the centre. The skin ti pinched up tight to
minimise bleeding and a cut about 5 mm long made
with a scalpel, deep enough to get into the infiltrated
layers. After wiping off blood or lymph that may
have exuded, the scalpel blade is turned transversely
to scrape the hides and bottom of the cut so as to
—
obtain a .tittle tissue pulp which is smeared
uniformly on a slide. About 5 6 different areas of
the skin should be sampled, including the skin over
the buttocks, forehead, chin, cheek and ears. The
smears are stained by the ZiehTIVeelsen technique
using ?% instead of 20% sulphuric arid for
decolourisation. Biopsy of the nodukr lesions and
thickened nerves, and lymph node puncture may
be necessary in some eases.
The smear; are graded, based cm the number of
bacilli as follows:
1-10 bacilli in 100 fields - 1+
-
1 10 bacilli in 10 fields
-
1 10 bacilli per field
2+
3+
--
10-100 bacilli per field
100-1000 bacilli per field
and glohi m EintCy Lucid
= 4+
5+
More than 1000 bacilli, clumps *= 6+ -
Copyrighted t a terial
 « Mycobacterium III: M . Leprae
The bacteriological ( bacterial ) index ( hi ) is
calculated by totalimg fbe number of pluses ( +s !
scored in all the smears and divided by the number
of smears, Thus, if eight smears examined have a
rota! ( ] f sixteen pluses , the RJ will be 2. For
calculating BI , a minimum of four skin lesions, a
nasal swab and both the car lobes have to be
examined.
The morpliological index ( MI ) is expressed . L >
the percentage of uniformly st .lined bacilli out of
the coral number of bacilli counted.
Mouse foor pad inoculation has hern reported
to be more sensitive than skin slit smears for
detect ion of lepra bacilli in tissues . Bur this is
unsuitable for routine diagnosis and feasible only
for drug potency or resistance testing and research
studies.
Detection of antibody against M . ieprae
phenohe glycollpid anh en has beer clumed to be
^
a specific diagnostic test. Attempts to develop
molecular diagnostic methods arc in progress.
Meanwhile, microscopic demonstration of lepra
baulli and histology remain the most useful
diiigriosric procedures.
Treatment: Dapsonc was the first effective
chemotherapeutic agent against leprosy. Its use as
a monotherapy for several years led to the
-
development of re istunt strains o1 Lepra bacilli . In
view of this, mulriple drag therapy ( MOT ) is now
recommended in leprosy, as in tuberculosis, The
currenr recommendations for patients with
pautibacillary lesinns ( T, II , BT) is the Concurrent
administration of rifampicln 600 mg once a month
and dnpsofld (K) mg duly for tix inourhs . For
multib a ciliary lesions ( BB , BL , l . l . i , the
recommendation is tifampicin 600 mg once a
month, dapsone 100 mg daily and clofazimine 50
mg daily for two years nr until slki. n smears are
negative. Ethionamide or prothjonamide may be
» 375
added to this negimen or substituted lor clofazimine,
A minimum follow' up of four years for
pauciba . illary and eight years for multibacdiary
cases would be necessary to detect any relapse .
An immunoihenapeu:ic vaccine ( Mycobacterium
W ) developed at the National Institute of
Immunology New Delhi is cl.umrd to enhance the
effect uf MDT.
Prophylaxis: Case finding and adequate therapy
have been the methods employed for prophylaxis.
Long - term chemoprophylaxis has given
encouraging results in child contacts of infectious
cases in India and the Philippines.
There is some degree of antigenic relationship
between the lepra and tubercle bacilli . It is an old
clinical observation that leprosy and tuberculosis
do not usually coexist. BCG vSc/ ine- was observed
to induce lepromin positivity and hence its use in
the prevention of leprosy was suggested by
Fernandez as- early as in 1939, Shepard found that
lepra baciili did not multiply in the footpads of
mice immunised with BCG, Controlled trials gave
divergent results, from high to no protection. Field
trills with different leprn vaccines ( BCG + lulled
^
lepra baciili ; 1CRC bacillus ) have nor given
conclusive results so far.
MYCOBACTERIUM LEPRAE MURIUM
This is the causative agent of rat leprosy. It was
first described by Stefansky in 1901 at Odes&a - It
has beer subsequently reported from several
Countries , Rat leprosy ii characterised by
subcutaneous indurations , lymphadenopathy,
emaciation, ulcerations and lots of hair. Acid fast
bacilli resembling lepra bacilli are found in the
lesions in large numbers . However, tbc disease
differs from human leprosy in rhat the nerves are
not affected. M. leprae and Af. itprjt murium are
not closely related by DNA studies.
Copyrighted material
Hidden page
 Spirochetes
Elongated, motile, usable bacteria twitted ipiraDy
along the long Ji\ is are Termed ‘Spirochetes ' ( from
Span, meaning coil .md tVjjjfe, meaning hair).They
are structurally more complex than other bacteria.
A characteristic feature is die presence of varying
no tubers oh enJoffagtHif which are polur flagella
wound along the helical protoplasmic cylinder, and
situated between the outer membrane and t.ie- 11 wall.
Endofogclte arc believed to be responsible for
motility hut the enact mechanisms of locomotion
i
are nor understood .
Spirochetes vary- widely in size, some being as
Icing a * 5(H) pm and others as short as 5 pm. They
are f ’rran negative , Many are free - living
saprophytes , while some are obligate parasites .
They muv be ,LC tabic, anaerobic or facultative .
Reproduction is by transverse fission .
Spirochetes belong TO the order Spirocherales,
comprising two families— S piroebetaccae
containing the genera Spirocticts , Cristapirs ,
Trepoaemt i\ud Eorrelit; and Leptncpinoeie
containing the genus Leptoajwta. Human jiathogens
arc found in the genera Treponema, honvli .i -.ind
Leptospira . Members of the genus jfunxhiea :mL
s;ij>ri 5|Thvrys found in water and lew » jjTn while
Cnstzspira are found in molluscs.
TREPONEMA
Treponemes ffcnrpoe, meaning to turn, and nema,
meaning thread ) are relatively short slender
spiroc hetes wi fh fi nc spj nils and poi riled nr mu nded
ends. Some of them arc pathogenic, while othcra
occur US commensals in rbc mouth , inrestines, and
genitalia . Pathogenic treponemes have n< n been
successfully cultivated in cell free media though
the commensals may be grown in artificial media.
Treponemes cause the following diseases in
humans:
L Venereal syphilis caused by T. pallidum
2 . Endemic syphilis caused hv X ptfSiiUtm
[T. cndcaiicum )
T . Yaws OJUSCLI bv F. pertenae
4, Pints caused by 7! canrcum
Ihcy art almost identical in their morphology,
antigenic structure and other features, though there
are differences in the clinical features and natural
history' of the diseases rhey produce. If has been
suggested that the pathogenic treponemes represent
only evolutionary variation! of a single species and
that the diseases caused by them, though deferent
clinically and epidemiologically, should he
considered pares of a continuous spectrum of
treponemaJVises- Accordingly, the species 71
pallidum is now considered to include three
subspecies— subspecies pallidum causing venereal
syphilis, endemicurn causing endemic syphilis and
pertenue causing yJws.
TRKPONEMA PAU.mirM
Treponema palEdum, the causative agent of syphilis
was discovered by Schauiiinn and! Hoffmann ( 190 )
in the chancres and! inguinal lymph nodes of
^
syphilitic patients. The name paii/ dum refers to its
pale staining.
Morphology: It is a thin., delicate spirochete
with (apering ends, about 10 |im long (range 4^14
pm} and 0.1-0.2 pm wide lit has about ten regular
,
spirals, which arc sharp and angular, at regular
Copyrighted materia
37 K * Texltraqk n1 MicTobiglpgy >

intervals yf about ] pin . It actively motile ,

IH There have been tnanv of cultivation «
exhibiting rotation around the long axis, backward 7 pallidum in cultures bir none has been
and forward movements, and flexion pj the whole substantiated . The : - i have been
body, During morion , secondary curves appear and nonpathogcnic trcponcmcf , . morphological
disappear in succession bur the primary spirals are and antigenic similarities with T, pailidum. The
unchanged { Kig. 42.1), bc? t known nf these is the H . r strain, which has
T. pjJiidfuTtl earn Lot he seen under rite light been widely used as; the in group specific
1

microscope in wer films hut ran be made out by treponemal tests for the 1
of syphilis. The
relative staining with Indian ink. Its morphology Keitei trepnneme grows well thioglycollatV
and motility on be seen under the dark ground or medium containing serum . The Reiter treponeme
phase contrast microscope. It does not take ordinary is now classified as 7? ; i/rnis

bacteria ] stains bur stains light rose red wirh
prolonged Giemsa staining. It can be Stained bv
silver impregnation method?- Fontana 's method is
useful for staining films and Levadiri's method for
tissue HCtlrUms.
KCMVLLIIICC : 31 pallidum ii i L delicate, being
readily inactivated by drying or i beat ( 41-42
in one hour ), Hence fomires are ! i importance
in the transmission of infection. S isvcpfit ) lit) of T.
paiiiduin to heat was the basiv fever therapy'
"

Ultrastmemrally, the cytoplasm of 7T pallidum for syphilis, ft is killed in \ ~3 At 0-4 *Ct so

is surrounded by a trilaminar cytoplasmic that transfoskui syphilis can be : : u by storing
membrane, cnctoscd by a celt wall containing blood for at leas!four day? bit hi . raiorbcfotl
pepcidoglycan which gives the cell rigidity and tn nsfosion , Stored frozen at -70 1 in 10% glycerol,
shape. External tn this is the lipid rich outer
membrane layer. Originating from each end of the
cell , three or tour cndoflagella wind round the
axis of the cell in the space between the ccU wait
—
or in liquid nitrogen [-130

o \vgunr disti lied water, soap,

bismuth , common . M . : . : ' ; ^
i
1 it remains viable
for 10 15 years. It is i iccivated by contact with
. . me rcurials ,

andantibiotics.
and outer membrane layer, to interdigitate at its Viili iiiL' sir'incuirtC The unt gttnk structure
centre. Unlike die flagella of other bacteria, these
^
cndofUgella do nut protrude outside,, but remain
within the outer membrane layer. w

Saprophytic spirochetes arc generally coarser

in ^ p|H? jrancc , lack the uniform spirals with regular
spacing, and show lashing motility.
1 r i i l t u L i l m u i: Patfsbgenic treponcmes do nor
grow in artificial culture media. Limited growth
of T- p?llirivm Jins been reported in cocultivirion ,

with tissue culture Cells. It IS possible to maintain

71 pallidum in motile and virulent form for 1 ( > 112
days in mm pint media under anaerobic condition*.
—
Virulent 7? jxflitkun strains have been maintained
For many decades by serial resdcular passage in
rabbits . Otic such strain ( Nichols strain ) isolated
fmm the hr.iin of a fatal ca ^c of general paralysis of
the insane in 1012 is still being propagated and .
Fig 42.1 Treponema pallidum - - ground
used for diagnostic and research purposes . illumination.

nnaiGNa
Hidden page
m * Textbook ol Microbiology
primary Icsfon heals , During this in he mil ( he patient
i & asymptomatic . The secondary Lesions arc due ro
wideypre .n] multiplication of llie fipiruchetea and
their dissemination through rhe blond . Roseolar
or papular skin rashes * mucous patches in the
oropharynx and condylomata at the mucocutaneous
junctions arc the characteristic lesions. Spirochetes
arc abundant in the lesions and consequently the
patient is most infectious during the second ire
stage , 'Hiere may also be ophthalmic * osseous and
meningeal involvement. Secondary lesions are
highly variable in distribution , intensity and
duration but they usually undergo spontaneous
healing, in some instances taking as long as four or
five years
j
. asymptomatic periods during the natural course of
After the secondary lesions disappear, there is a
period of quiescence known as ' Latent syphilis".
Diagnosis during this period is possible only by
serological tests. Jn many eases , this is followed by
natural cure but in others , after several rears ,
manifestation !; of tertiary syphilis appear. These
consist of cardiovascular lesions including
aneutySms , c humic gmauLOttttia (gURimtia ) :md
meningovascular manifestations. tertiary lesions
contain few spirochetes and may represent
manifestations ot delayed liypeirensiTivity. In ; L few
eases , neurological m anilestations such as tabes
dorsalis or general paralysis of the insane develop
several decades after the initial infection . These are
known as late tertiary' or quaternary syphilis.
In syphilis acquired nonvenerratly ( as
occupationally in doctors or nurses ), the natural
evolution is as in venereal syphilis, except that the
, the lesion and the scrum that exudes is collected
primary' chancre is extrageoital , usually <1 0 the
fingers . In the rare instances where syphilis is
transmitted! by blood tfUlifutioil * the primary
chancre docs not occur . In congenita! syphilis ,
where infection is transmitted from mother to fetus
transplacentally, the manifestations and course are
different . Transplacental transmission can rake place
at any stage ui pregnancy. A woman with early
syphilis can infect her fetus much mote commonly
-
( 75 95 per cent ) tlnm nnc with syphilis of osier
*
two years duration (.15 per cent ) . The Lesions of
congenital syphilis usually develop only after the
fourth month of gestation , the time when fetal
-
immune competence starts ajqK aring . This suggests
that the pathogenesis ncquireH immune response
from the fetus. Congenital syphilis can be prevented
if the mother is given adequate treatment before
the fourth month ot pregnancy.The ohstetrie history
in an untreated syphilitic woman is typically one
of abortions and stillbirths followed by live hirth &
m
of infants with stigmata of syphilis and finally of
healthv infants.
Laboratory diagnosis: As the manifestations
of syphilis are protean and as there are
the disease , Laboratory aid is essential for rhe
diagnosis of the disease . It is also Important in
assessing the cure alter treatment, Because of the
social and emotional overtones of the disease , the
diagnosis ot stplulis should impose a great sense
of responsibility' on the laboratory'. Laboratory
diagnosis consists of demonstration of the
fpifOCheteti under the micft > S £Ope and of ^rttihiklie*
in serum or L!SF.
Microscopy ; Diagnosis by microscopy is
applicable m primary ami secondary stages ami in
eases of congenital syphilis with superficial lesions.
Specimens should be collected with care JS the
lesions arc highly infectious. The lesion is cleaned
with a gauze soaked in warm saline and the margins
gentlv scraped so that the superficial epithelium is
abraded . Gentle pressure is applied to the base of
preventing admixture with blood . Wet film ate
prepared with the exudate and after applying thin
covurs- lips , examined under the dark ground
microscope . 7? paffidum is identified by its slender
spiral structure and slow movement. Differentiation
from saprophitic spirochetes commonly present in
the genital area needs experience .
Dark ground examination is useful but negative
results do not exclude the diagnosis of syphilis *
because of m low sensitivity. A treponemal
Copyrighted material
 < Sp*roc*iete&
concentration of lfl + per ml in the exudates h
required for the test to he positive.
The direct fluorescent antibody test for i . pallidum
( UFA -TP ) ia a better and. safer method for
microscopic diagnosis, Smears of the exudate are
fixed with acretone and sent lo the Laboratory; where
the DFA -TP test is done using fluorescent tagged
anti - T! im anti > erum . The use uf specific
monoclonal antibody has made the test more
reliable -
Serological tests: These tests form the
ma -. iistay of laboratory diagnosis A large number
of Tests have- been described ., of which only a few
am now employed -
Serological tests for syphilis may he classified
as follows :
1 . tests for anribodies reacting with cardiolipin
antigen ncagin tests; standard tests for syphilis;
STS
2. tests for antibodies reacting with group specific
trcpnnemal antigen
3. tests for specific anybodies to pathogenic
treponema. ( T pa/hdum ) .
r . Rcaginanybody tests These tests use the !ipnidal
or cardiolipin antigens and arc known as 'standard
tests for syphilis’ or STS . (The antibody reacting
with cardiolipin is known as mgin. This can he
misleading, as the 7gF antibody in atopy is also
called reagi :i , though there is no connection
between the two . )
The first of the reagh antibody tests was the
Wassermann complement fixation test ( 1906 ),
which ori i natly used a watciy extract of the liver
^
of a syphilitic ferns AS the antigen . This was Inter
substituted by an alcoholic extract of ox heart tissue,
to which Lecithin and cholesterol were added . This
crude extract was subsequently replaced by a
purified lipid extract -of beef heart (called
furdiolipin) , with added lecithin and cholesterol ,
as standardised by Pangborn ( 1945 ) . The
complement fixation test remained the principal
serological test for syphilis, tLIL i : was replaced by
the simpler flocculation tests which also use the
381
cardiolipin arnigen, The Wassermann test is no
longer in use now.
The first flocculation test used widely was rhe
tube flocculation rest of Kahn. The Kahn test lias
been replaced by the simpler and mote rapid VDRL
test , which ghres more quantirati .re results ( VDRL ,
1
for Veneral Disease Research Laboratory, (JSPHS,
New Turk, where the test was developed ) . In the
VDRL test, the inactivated serum ( that is, serum
heated at 56 UC for 30 minuta ) is mixed u rh
cardiolipin antigen on a special slide and located
for four minutes. Cardiolipin remains as uniform
crystals in normal serum hut forms visible dumps
on combining with reagin antibody, The reaction
is read under a low power micmscnpe . By testing
serial dilutions, foe antibody titre can be determined .
The results arc reported qualitatively as 'reactive',
'weak reactive' or 'nor rcacrivc’ . For quantitative
i*e|jcirtmgj the reciprocal of the end point is giver
as the litre, for example 'reactive 4 dilution or 1
Htitnc 4’.
VDRL test can be used for te &dng CSF also ,
but not | 1 na. CSF need not be heated prior to
foe test.
A number of mod locations of the VDRL test
' "
have been developed, of which the Rapid Plasma
Reagin ( RPR ) test is the most popular. The RPR
test uses VDRL antigen containing fine carbon
particles, which make foe result more clear cut and
evident to the naked eve . . RRR test can be done
with unheated scrum or plasma * but is not suitable
for testing CSF. Automated RPR test ( ART ) is
ava i labtc for large scale tests. An automated VDR\r
ELISA rest has been devebsped which can measure
IgC and lgM antibodies separately and is suitable
for large scale resting of seta .
As candioii ] iin antigen is present both in T paltdfon
and in mammalian tissues- , reagin anl iboiLies may
be induced by treponemal or host ti -isue antigens.
This aecnu nts fnr foe bi ilogic a I false positive ( BI V !
reactions, which constitute foe major di -iadvantage
of STS. BIT reaction are defined as positive
^
reactions obtained in cardiolipin tests , w . th ncgaf .ve
'
opyri <
B dr \
hted material
382 * Tfrultiook ol Microbiology *
results in specific treponemal tests, in the absence
—
ot past or present treponemal infections anti not
caused by technical faults . They represent
nontiepaneaul cardinKpin antibody responses,
BFP reactions may occur in about one percent
of normal sera . BFP antibody is usually IgM, while
tragi n antibody insvphilii. is mainly IJ J , Clinically ,
^
HI'"!3 reactions may be classified as acute nr chronic.
Acute BFP reactions last only for a few weeks or
months and are usu & Uv associated with acute
infections , injuries or inflammatory conditions .
Chronic BFP reactions persist lor longer than si*
months and arc typically seen in SJ,F, and other
.
coDagen diseases Leprosy, malaria, relapsing fever,
infections mononucleosis, hepatitis and tropical
cnsiuophilia examples of Other conditions
arc
associated with BFP reactions.
Rcagin antibody becomes detectable 7-10 days
after the appearance of primary chancre (or 3-5
weeks atrer acquiring fhc infection ), The sensitivity
in the primary stage i* 6(1-75 per cent with the
ntres being low , upto dghl In the secondary stage,
the sensitivity is 100 pet cent and tit res range from
16 to 128 or more, Prozone phenomenon may be a
problem in high litre sera and it is therefore
essential to tc ^ r sem in dilutions . Another stage of
syphilis in which such high t i m s a r c seen is
congenital syphilis. After the secondary stage, litres
diminish and about a third of patients with late
.
syphilis are seronegative The titTCS may rise in
patients developing cardiovascular, neurological or
gummatous lesions. In some cases of neurosypbilis,
rcagin tests may be negative wi th scrum but pontive
with ihe CSF. Reagin teste usually become Negative
6-18 months after effective tnc .itment ot syphilis,
depending on the stage at which treatment is given .
.
I lowcvcr if treatment is started laic, the rests may
remain positive in low ticres .
2, Gruujj- s[ v(.-itic nepontma} nesis: In order to avoid
BKP reactions, tests using cultivable trepememes as
antigens were developed: These employed the Reiter
trcponcmcs ( originally believed to be an adapted
strain of 7. paUSdirm ). The Test most commonh
employed in this group was the keircr protein
complement fixation ( RPCF) test , using a
UpopolyBicehimde-protem complex antigen derived
from the treponeme . Its sensitivity and specificity
were lower th:in those of Tests nsing T. p.-iUrdiim.
Though RPCF was. generally free from BFP
reactions, it still gave some false positive reactions.
RPCF and Other Reifer tfEpOnaiK tests are not n -mv
in general use.
3. Specific T puSbdutn rests. These rests use the
virulent: Nichols strain of T. pallidum maintained
by serial inoculation in rabbit testes.
The first in this group is the Treponema
pnHkiun ] immobilisation (TPI ) test introduced in
1949 . The test serum is incubated with complement
.
and T p illiilnm maintained in a complex medicn:
anactobicalllv. It ' antibodies are present , the
ireponemes arc immobilised , that is, rendered
ronmotile, whfcn examined under dark ground ,
illumination .
.
Tn its time TPI was the most specific test
available for diagnosis of syphilis and was considered
the gold standard in syphilis serology. 1 lowcvcr,
because of its extreme complexity, it ivas available
only in a tew laboratories . The T111 test ha ? now
been supplanted hy other tests such as FTA -AlSS
and TPI £A which arc quite as specific and much
simpler.
The fluorescent treponemal antibody (PTA) test
is an indirect imiminofluarcsccocc test using as
antigen, smears prepared on slides with Nichols
strain of 7 . paifuiurn . I ke slides can hu stored for
several months in deep fireree . The currently used
modification of the test is the FTA-absorptinn
( FTA - ABS) test in which the test scrum is
preabsorbed with a sonicate of the Reiter
trepooemes ( surhenf ) to eliminate group specific
-
reaction?, FTA ABS is K specific as die TPI test
and is now accepted as a standard reference test.
I luwever , us it can be done only in suitably equipped
laboratories , it is not available for routine testing.
The T. psiliiium hemagglutination Assay
( TH I A ) use tanned erythrocyte sensitised with
* *
Copyrighted material
 i Spirochete* *
T pallidum, ns antigen. Hit
a sonicated extract of
procedure now employed is a mkrci
hemaggluti nition rest ( MHA-TP) , which is
- patients response to treatment, indicating the stage
capable of being automated.
The test yen for TP HA ire absorbed with a
diluent COfltaLfting components id rile Reiter
frepoTicmc, rabbi t testis a nd sheep cryth nocytes- Sen
am screened in an initial dilution of 1:$0 but tines
of 5120 or more are common Ml the secondary stage.
TPHA is junt as specific as FTA AfiS nml almost
as sensitive, except in the primary stage. Jt is also
much simpler and more economical. No special
equipment is needed . Kits are available
commercially. These advantages have made TPHA
a standard confirmatory t« t -
Tablc 42.1 shows the relative sensitivities or
the serological tests in common use.
Enzyme immunoassays ( ElA ) have been
developed using T. pallidum antigens and are
available conn merci ally ( Bio - Enza Bead test; Captia
Syphil is - CJ tost) . A rapid aggluti nation Lest J i as been
developed , using latex particles coated with three
immunodominant proteins of T. pallidum , obtained
by recombinant technology'. It is claimed to He as
specific as TPHA* and more sensitive .
The practice tor uero logical sere en I rig for
syphilis varies in different countries. In the UK , a
combination ot VDRI , and TPHA tesla LS Lurid .
"
This is an efficient combination for the detection
or exclusion of syph i I is at all stages, except the early
primary stage. A repeat test 1- 1 months later will
bring even this no light . In the USA, screening is
by VDRL or RPR test alone. This may fail ro detect
about one per cent of secondary syphilis due to the
proaone effect and about 30 per cent of latent or
hue syphilis.
Table 42. T Frequency ol reactive serological tests
in untreated syphilis [percentage )
Siage VDRL / RPH FTA -ABS TPHA
Primary 70-80 H 5 -10 Q 65 -Si
Secondary 100 100 100
Latent/ late 60- 70 93- 100 95 -100
2E 1
Quantitative tests axe useful in monitoring the
of the disease and in detecting reinfection . Rcagin
Lears are preferred because they usually become
negative following treatment. If treatment is given
VIJIV early, the serum may not become jHiririvu at
all - Treatment in the primary jtage leads to
scrorcvcreal in about lour months; in the seconds ry
-
and early lament stages , it takes 12 IS months-: in
later stages, it may take five yean- or more. In some
cases low rltrc reactivity may persist indefinitely in
spite of effective treatment Specific ii*E >ui ]eT[]al tests
are of little value as indicators of clinical cure, as
they tend to remain positive in spite of treatment -
TPHA titres may foil rapidly following treatment
'
in secondary syphilis bn remain positive lor life in
low litres ,
TPIIA and FTA-ABS .ire helpful in excluding
or confirming the diagnosis of syphilis and for
identi lying BFP reactions. Though false positive
reactions were believed u > have been eliminated
with the introduction of these specific tests, it is
not truly so. Both TPHA and FTA - ABS can give
false positive results , though verv rarely. All
serologic ,il rests for syphilis may he positive in
nonvenureal trepone mitoses , and some in a lew-
other spirochcatal infections as well . In Lyme
disease, VDRL rest is negative, but FTA- ABS may
be pnsitivc.
A negative TPHA excludes the
virtually
diagnosis- of syphilis, past or present, except in the
very early sriges . In ncurosyphilis, a negative CSF
VDRL test may not be conclusive but a negative
ITJH .A test eliminates the possibility of
ncurosyphilis.
Detection of specific IgM antibody may he
helpful in sume situations. Being the initial type of
antibody to appear, IgM is detectable by the second
week of infection . IgM mti body production ceras
soon after elimination of inferrion by treatment.
Persistence of IgM antibody indicates continuing
active disease and the need for treatment. As IgM
does riiit cross the placenta its presence in neonatal
,
Copyrighted material
184 H Te tboo-k al Microbiology
*
confirms
^ ^ . syphilis and helps
scrum cisn iiir il
d] fferenrLa1 e ] tfrom surdpoSrlnvctv due W passively
transferred maternal antibody (lyphilo toxemia) ,
Many techniques have been developed tor the
selective detection ot IgM antibodies, These include
modifications of rhe FTA-AES, TPHA,E1A and
VDRL using whole sera or separated IgM
rests,
fractions. When such te>- ts are not avai 'able-, parallel
tests of maternal and neonatal sen may settle the
diagnosis of congenital syphilis „ in which the
neonatal serum ma y show LL IIL LICT Li tie of antibody
^
than the maternal scnim. Serial tearing is also useful
because the litre of passively transferred antibody
decreases rapidly, rhe VDRL test becoming
negative by three months.
Epidemiology; Venereal syphilis is worldwide
in distribution. During the five centuries that it: has
been recorded and studied, the disease has
undergone much variation in its natural history and
.
clinical features As originally described it was a
, antiseptics (potassium permanganate ] or antibiotics
highly virulent disease with tlnrid cutaneous
.
manifestations With the discovery of the dramatic
therapeutic response to pcnidllin, it was hoped that
it may even be possible to eradicate syphilis, as the
di >ease has no extra human reservoir. However, not
on I y has it not been pnss iblc to el [minatc the d i sease
but an mcrease has occurred in its incidence, due
ro the changing Liisroms, habits and values in
ancicty.
The advent of the AIDS pandemic has had an
impact on syphilis, in most places, fear of AIDS
and safer sex practices led to ;L tall in rhe inc idence
,
of syphilis and all ST t init i ,illy, but this trend did
m >r continue everywhere. Concurrent infection with
syphilis and HIV is common andmay lead to earlier
evolution of neurosyphilis.
Immunity: The immune mechanisms in syphilis
arc not adequately understood. Humoral immune
response against the Crepontmc docs not appeal to
be effective rhe disease progresses unhindered
for decades even in the presence of a vigorous
antibody response , Cell mediated immunity mav be
more relevant . T lymphocytes and macrophages are
*
predominant in early syphilitic lesions.
Specifically sensitised Thl cells secrete cytokines
favouring clearance of spirochetes bv activated
macrophages.
Reinfections do not appear to occur in a person
already having active infection , Tl was believed that
prut ? ] iminon or infection immunity, as seen in some
parasitic infections , holds good in syphilis also and
that a patient becomes susceptible to reinfection
-
only vvliL rt his original intention is cured. However*
it has been shown that some degree of immunity
to reinfection occurs in experimental animals and
persons whose infection has been completely
eliminated by treatment .
ProphyIjixiii As transmission is by direct
contact, it is possible to protect against syphilis hy
avoidance of sexual contact with an infected
individual. When this- is not complied with, the
use of physical barriers ( such ns condoms) ,
may minimise the risk . The use of prophylactic
penicillin carries the danger that it may suppress
the printin' Lesion without eliminating the infection
so rhar recognition ami treatment of the disease
.
may become more difficult No vaccine is available .
Treatment: Penicillin is uniformly effective in
syphilis hut it is necessary to give an adequate dose
and maintain the drug Level for a sufficiently long
period to establish cure . A single infection of 2 A
million units of he n/ athine penicillin G Is adequate
-
in L urly cases . For Late syphilis, this amount may be
repeated weekly for three weeks.In pa bents allergic
10 penicillin , doxvcvcline mavbc usedl. Ceftriaxone
is effective, particularly in ncumsyphllis Penicillin
treatment in syphilis sometimes induces a reaction,
the Jarisch- licrxheimer reaction, consisting of
fever, [il.Liaise and exacerbation of symptom!. It is
frequent, but harmless, in primary and secondary
syphilis , and can be managed with bed rest and
aspirin. It is rare in late syphilis but can be
dangerous in some cases of gummatous ,
Cardiovascular or ncurosyphihs It is believed to be
doc ro the liberation of trade products like tumor
Copyrighted material
Hidden page
336 * Tox ( book of Microbiology *
characterised by hype rpig mentation 0 i
hypopigmentation. TISSUER other than skin are
seldom affected .
calmative agent is T. cir .- ircuiti . Il is very
|Te
J
closely related to Tpaffidunjbut is nLit & nti geni cat] lv
identical so cross immunity between pinta And
philiv i -H only itanial .
^ morphology and man ) other features but differ in
NoM^T f l f K i l i M C TRt’ I'O M' M ]^
Several commensal tn:|>nneme £ occur on the buccal
aid genital mucosa and may cause confusion in the
di ;Lgno*i s of vy' hiliK by dark field examinant HI. I be v
^
are a heterogeneous group and have not been
adequately characterised, Best known among them
is the oral spirochete, 71 dcitticob, which can he
readily cultivated , Treponcidti also occur on the
surface of gastric and colonic epithelium in human
beings and animals.
During early attempts tt> grow the syphilis
spirochete in cultures, several treponcmes hjid been
grown anil mistakenly Called 7] jwlhtlunt — for
example, the Reiter and Kazan strains which hive
been identified as 7 ] phagcd&lis anil the avirulent
Nichols and Noguchii strains which have been
recognised as T. refringen^ .
Jn experimental work on T. pallidum in
rabbits , T, parmtuiscunkuii (formerly T. curuciJi),
which has a very similar appearance and causes
natural venereal infection in rabbits , mar pose
problem H.
BOftREUA
Borrcliae are Imge, motile, retractile spirochete
with irfcguLir* wide, open coils. They arc usually
-
5 30 pm long and 0.3 0.7 pm wide . They ate
^
readily stained by ordinary methods and arc Gram
negative . Several species of Uorrelia occur as
commensals on the buccal and genital mucosa.
Borreliae of medical importance are those causing
relapsing fever, B. vinctxrh which sometimes causes
fusospirochccosis and 13, burgdorferi , the causative
agpor of l .ymc disease .
L i k i A P S I\n F h \ l - l i
Relapsing fever (RF) has heert known since rhe
time of Hippocrates and has occurred in epidemic,
endemic or sporadic form throughout rhe world -
RF is an arthropod - bomc infection, two types of
which occur - lousebome and tickborne. T. he
horreli .Le c nosing them are indistinguishable- in
’
their arthropod hosts.
Hie causative agent <> t epidetnic or lousehorne
RF is H. recurrentis , first observed hv Obemuier
( 1 B7.4 ) in the blood of patients Lairing an epidemic
in Her I in . It it an rtHnurve human pathogen, being
transmitted from person to person through hody
lice (Adjcufut hum , mus corporis). No extrahuman
reservoir is known .
Horrr- li .ic causing endemic or tickbornc RF
—
normally live in their natural busts rodent* or
other mammals on which the vector ticks fccd -
13 uman infection is only an Accidental event ,
iiorrelliae have been assigned to various species
based on the Iccki that CArty them. Over ten species
of borreliae are known to infect human beings and
cause RF ( 13. duttemii, B. hcrsnuii, B. purhcri , etc . )
They are generally confined to certain geographic
areas. There is evidence from DNA homology
studies to indicate that ail of them may belong to a
single specie;;, with separate host adaptation. The
descriptions that follow apply to all of them, unless
stated otherwise.
Mi irpluNlo|jy; B . recurrent }* i* an irregular spiral
with one or both ends pointed. Jt is ft-20 pm long
and 11.2-0- 4 pm wide. It possesses 5-10 loose spiral
colls at intervals of about 2 mm. It stains well with
Giemn and Wterial trains and is Gram negative,
Cultural characteristics: Borrelia are
micTuaeropbiJic. Optimum temperature for growth
is 2 K-30T, Cultivation is difficult but has been
successful in complex media containing serous
fluids. Growth occurs on the chorioallantoic
membrane ofchkk embryos. For primary isolation ,
the best method is to inoculate mice ur rats
Copyrighted material
 « Spirocnmes
Lntrapcrtlcineally.When using experimental animus,
,
great care has to be eaten to ensure that the animals
are free from pre-existing borreliosis.
Vnliguiiic properties: Horrclia readily
undergoes antigenic variations in vivo and this is
believed to be the reason for the occurrence of
relapses in the disease. Antigenic variations have
been shown to be caused by DrvA rearrangements
in linear plasmids present in borretia. Ultimate
recovery after a number of relapses may be due to
the development of immunity to all the antigenic
variants . Agglutinating, complement fixing and lytic
antibodies develop during infection but their
demonstration is not possible as a. routine JiagniKtLC
rest due to the difficulty in preparing satisfactory'
antigens.
PathogeniciLy; Afrer an incubation period of
2-10 days relapsing fever Sets in as fever of sudden
onset. During this period, borreliae are ahundanr
in the patient 's blood. The fever subsides in
-
days. After an afebrile period of 4 10 days during
which borreliae arc not demonstrable in blood,
another bout of lever sets in.The borreliae reappear
in blood during the relapses of fever, The disease
ultimately subsides after 3-10 relapses.
Experimentally, rodents such as rats, mice and,
less readily, guinea pigs may be infected by
intrape riloneal iniection. Borreliae may survive in
the brains id infected animals after they haver
disappeared from the blood.
Epi (itimiulu | y: Louscbarnc relapsing fever
tends to occur as epidemics whenever poverty,
overcrowding and lack ot personal hygiene
encourage louse infestation. Epidemic relapsing
fever used IO be very common during wans and in
jails of former days but with improverneinls in
hygiene and the discovery of insecticides., it has
now become rare-. It survives in some areas, as in
pans of Africa and appears as outbreaks whenever
civil strike and famine encmarage large scale louse
infestation. The louscbnrnc disease presents a more
severe clinical picture than the tickbome variety
and is associated with jaundice, hemorrhages and
p
387
?v V.
I
:
-
*
I
Gi
JTwM 1
Fig. 42.2 Borrrito rEcurrentii in peripheral blood smear
in Hume outbreaks, a high rate of fatality. tr lift;,,
the horrrlia is, confined tn the hemotymph and 15
not shed in saliva or excreta . So the infection is
transmitted not by the bite of lice but by their being
curbed and rubbed into abraded *kiq, fi , rKvrrentis
is not transmitted transovariallv m lice .
Jr
Tickltome relapsing fever occurs an sporadic
cases in endemic areas . It is a ‘place disease' and is
frequently associated with certain dwellings or other
locations rhur are inhabited by infected ticks. The
disease is milder but relapses are more frequent
than in Lousebome fever. The borrelni persists in
the body of infected ticks throughout their life and
is also transmitted transovarially so that the ticks
act as reservoir mi well as vectors. The borrelia
^
invades all pam of the body of the rick and is shed
in its saliva and feces, So the infection is transmitted
to humans Through the hire of ticks or their
discharges. Several species of soft ticks hclonging
to the genus QiPithodorus act as vectors , different
spcciea being responsible in different regions. In
India , the vector species are O, cholozoiu ,O, cro&si,
O. hhorensa and the fowl tick, Aign persicus.
. These *oft ticks can live for ten years or more with
Copyrighted material
Hidden page
Hidden page
Hidden page
 4 Spirochetes
and truss absorption reactions usingtmnumf rabbit
sera or more recentlyJr
with monoclonal antibodies .
( icuetic methods , such as restriction endonuclease
analysis and 1 > NA pairing arc i ] *ed tor further
classification into serotypes.
Pathogenicity: In natural reservoir Hosts ,
Icptospiral infection is asymptomatic:. However,
when infect ion is transmitted to other animals ,
dlinica] disease may result. Human * are Inferred
when the leptospires in water contaminated by the
urine of carrier animals enwrs the body through
cuts or abrasions on the skin nr through intact
mucosa of mouth , nose or conjunctiva. The
incubation period is usually about JO davs ( range
.
2-26 days) . The clinic il picture varies from mild
undifferentiated pyrexia to severe or fata ] illness
with hepatorenal damage (Weds disease ). Ln severe
, by 1 mmunofUior'cscL- nce but this is of lit; k practical
cases, the onset La acute , with rigor, vomiciog,
head .ichcand intense injection of the eye*.The fever
is irregular and usually subsides in about ten days.
Jaundice occurs in about 10-20 pci cent ol ases '
I or similar medium. The bottles are incubated at
by the sccnnd or third day. I 'urpuric hemorrhages
sometimes occur on the shin and mucosa .
Albuminuria is a constant feature.
This typical presentation is unusual .
Leptospirosis is now classified into two clinical types
— jcfcrj e and nevuVrenV- Many eases present as
'
aseptic meningitis and in some , abdominal
symptoms predominate Clinical diagnosis I *
impossible in the majority of cases and unless a
high index of suspicion is maintained and laboratory'
assistance sought , leptospirosis will be missed in
all but a tew instance?.
Leptospircs are seen in the blood during the
acute phase of the disease but can seldom be
-
demonstrated after S 10 days. They persist in the
internal organs, and most abundantly In the kidneys,
HO that they may 1>e dtmonxtrated in the urine in
the Eater stages of the disease.
Serious cases of leptospirosis are caused most
often byserottpe ifterohaemorThiLgiiie , though they
may also be due to Copenhagen! and less often
bataviae, grippocyphosa , pyrogprres and some others.
* 391
Aseptic meningitis is common in canlcoht infection
and abdominal symptoms in grippotvphosa
infections However, clinical syndrome* are not
semtvpe specific and any type of ilines* can he
produced by any serotype.
Laboratory diagnosis : Diagnosis may He
made by demonstration of rbc 1 ept £« pircs
microscopically in blood or urine, by isolating them
in culture or by inoculation of guinea pigs or by
serological tests,
l , Examination of blood: A* leptospines disappear
from the blood after the first week , blond
examination is helpful only in the curly stages of
the disease , he fore antibiotics are given .
1 cptospircs may be demonstrated by examination
/
of the blood under the dark field microscope or
value.
Three or four drops of blood arc inoculated into
each of several bijou bottles containing EMJH
37 V . for two days and left thereafter at room
temperature in the dark for two weeks. Samples
from the cultures am: examined cvciy third day
for the presence of leptospires under dark ground
illumination. Primary isolation may be delayed
and may take many weeks to months. Chances of
isolation are increased by culturing blood daily at
the early stage of the disease, Leptospires may
sometimes be isolated from fhe CfiF also.
The blood from the patient is ll» inoculated
intraperitoncally into young guinea pigs. With
virulent serotypes like icterohaemorrhajdac , the
animals develop fever and die within 8— 12 dav *
with jaundice and hemorrhagic into the lungs and
serous cavities. With other serotypes such as
cantcola and poniona the animal may not become
111 and infection will have be hientified by
demonstration of the Icpmspiies in the peritoneal
fluid , by blood culture or by serology, from the
third day after inoculation, the peritoneal rluid is
examined daily under dark ground illumination
and when leptospires are defected, the blood
Copyrighted material
392 i Textbook of Micrctnoiagy »
withdrawn by cardiac puncture is inoculated into
culture media.
%
^ t urine Leptospira appear in
the urine in the second week of the disease and
intermittently thereafter for A— 6 weeks . The urine
should He examined imincdiafely after voiding as
leprospires readily undergo lysis in acid urine .
Centrifuged deposit of the urine may lie examined
under dark gnuind illumination . Direct culture of
urine is seldom successful because of
Lontamination but isolation is usually possible bv
inoculation into guinea pigs .
The identification of the isolates of kptnspircs
itf made by agglutination with type specific sera.
Due to the large number of serotypes and the high
degree of antigenic cross, reactions between them ,
identification of isolates is a complicated procedure
and is generally confirmed hy one of the WHO/
FAQ Reference I .aboratorics,
i . Scmhigii .il ttingnif is : Antibodies ap iear in
^ [
serum towards- the end of the first week of the
disease und increase till lllL- fourth week declining
thereafter . Agglutinins may, however, he
danemstrabk years utter the infection . Two types
of serological tests arc available , the broadly
reactive screening tests and the serotype specific
tests .
The broadly reactive or genus specific tests identify
Leptospira! infection without indicating the exact
infecting scrovar . The antigens for these tes.rs arc
prepared from the nonpathogenic L . biiSen Panic
1 strain. The rests employed .include sensitised
erythrocyte lysis (SET) , complement fixation ,
agglutination and indirect immunofluorescence.
ELISA Has been used to detect IgM and IgG
antibodies separately, in order to indicate die stage
of infection, A simple and rapid difi- stick assay
has b«n developed tnr the assay of leptospira-
specific IgM antibody in human sera.
The type specific tests identify the infecting
scrovar hy demonstrating specific antibodies .
Macroscopic and microscopic agglutination teals
arc used for this purpose . In the former ,
formal] nised suspensions of prevalent leptospira
serovars are tested for macroscopic agglutination
with serial dilutions of the test scrum . The
microscopic agglutination test (MAT) generally
uses live cultures of different serotypes and
agglutination is observed under the low power
dark field microscope . This tcsi is more specific
and is usuallv done only in reference laboratories.
Due to the presence of some degree of cross
reaction between different serovars, agglutinin
absorption tests may sometimes become necessary
for accurate diagnosis .
J. /)r: mwiv (if Jepiainimijs rn :INJJDJ /S: Infection
^
in rodents and ocher animals may be diagnosed
by serological rests or by culturing pieces of
kidneys.
5. F y j j t n n.j r p f J D of u-. j f L T for patliafienii
leptospin * ( f a shaved and scarified area of the
skin of a young guinea pig is immersed in water
for an hour, infection takes place through the
Abrasions.
k[Hdemiolr }£ s : Leptospirosis is considered to
be the most widespread of zoonoses, being regularly
present in all continents except Antarctica .
Pathogenic Leptospiras survive for long periods in
the convoluted tubules of the kidneys in natural
hosts,, multiply and are shed in the urine. Animal
carriers often excrete upto 100 million leptospires
per ml of urine. If the infected urine contaminates
the water or mud that is neutral or slightly alkaline ,
the leptospires survive for weeks . When people
come into contact with such water, the leprospires
enter the body through abraded skin of mucosa
.
ind initiate LO fee non. Certain occupational groups
such as agricultural workers in rice or cane fields,
miners ,aod sewer cleaners are more often exposed
to infection , and so leptospirosis is more common
JO them. Ijeptuspires mav be shed in the milk of
lac fating animals.However, they die rapidly in milk,
and human infection through milk is TIDI known .
They are not shed in saliva, and so animal bites are
not infectious. Arthropods arc not known to transmit
the infection.
Copyrighted material
 * Spirochete 393

Several animals act as carriers. Rats arc
particularly important Ifi they are ubiquitous and
carry the most pathogenic serotype ictcro
haermirrhagiiie . Field mice carry gripporyphosa
pigs pOtnOrta and dugs Canicula sem-t^fjes. However,
the HITH{ serotype may He carried Hv different
mammals and one mammal may tarty different
serotypes. While leptoapires are generally
nonpathogcnic in the reservoir animal , leptospirosis
is of veterinary Importance as infection of cattle
and pigs cause considerable economic loss . Infection
among animals is also transmitted by urinary
cuniammsiiurt of water and fodder. Human beings
arc an aberrant nr 'end ' host. There is no evidence
that human patients Infect others.
From being predominantly a rural disease of
agricultural worker leptospirosis has , in recent
^
times dtD become an urban problem in the
developing countries. This is perhaps doe to
vcrcrowdingh insanication , increasing rat
population and the habit of walking barefooted .
- Prophylaxis As leptospirosis results from
. contact of skin or mucosa with contaminated water ,
general measures of prevention such as rodent
control, disinfection of water and the wearing of
protective clothing contribute to its prevention.
Vaccination Has been attempted with some success
in dogs, cattle and pigs. Immunity following
vaccination or infection is serotype specific .
Vaccination has also been cried in persons at high
risk such a -i agricultural workers.
Therapy: l ^ ptospites are scnAltifUe ft ] penicillin and
tetracyclines, bur the treatment to be effective should
be started earlv m die course of the disease. PtuLclUjn
'
is given IV, 1-2 million units 6 hourly for 7 days m
serious cases . A mild Jarisch-HenKbeimar reaction
may occur in some, Doxycycliric 200 mg orally nn.ee
a week ! H effective in prophylaxis.

-
Table 42.2 Important leploapi al infections
Serotype ClimcaJ picture Animal Distribution
reservoir
lctcrohacrnorrhagLat Weirs disease
Can kola GanicoJa fever
.
Fever jaundice, titmorrhafle*
Influenza like, aseptic- ,
R*i
Dog
Worldwide
Worldwide
mejlingitU
Grippetyphosa Swamp or marsh Fever, prostration * ascpric- Field mice Europe., Africa*
fever meriinsitis S.E. Asia, USA

Pomona Swineherd's Fever Kg America,

dime Europe,. Middle
Fast., Indonesia,
Australia
Hebdomad is Seven day fever Fewer* lymphadenopathy Field mice Japan , Europe ,
USA
Forrbragg Pretibiai Fever, Fever* rash uvei tibia Not known Japan , S.E.
Fort Bragg fever Asia* USA

Pyrogcncs Febrile Fever Kg S. E , Asia ,

spirochetosis Europe, USA
Bataviae Indonesian Weil's Fever Rat S.E, Asia*
disease Africa* Europe
Hardjp Dairy firm fever Fever Cattic UK, USA * New
Zealand

Copyrighted material
Hidden page
 CO Mycoplasma

Mycoplasma arc it group of bacteria that arc
devoid of cell walls and so ire highly pleomorphic!
with no fixed shape or size. They lack even cell
wall precursors like muramie acid or
diiTninopirrctic acid- The cells arc bounded by a
soft trilaminar unit membrane containing sterols .
Because of their plasticity; they car pass through
bacterial filters and have often been rristateri tor
viruses . The first member of the group was the
organism causing bovine pleuropneumonia,isolated
by Notard and Roux (1R98). A simitar organism
was found to cause contagious agalactia in sheep.
When manv similar isolates were obtained from
Jr
LULU rials human beings, plants and environmental
] ,
sources, they came Co tie caller! yrJermipm'nmnnfHi'
like organisms'(PPLO). This unsatisfactory name
has heen feplat ed by the term Mjncnphcsma
from the fungus - likc form of the branching
filaments, pliama , denoting their plasticity of
shape).
MycopUsmis have been placed in tin- class
Mollicutes (literally meaning soft s k i n o r d e r
Mycoplasmaralcs , which contains the following
families and genera;
.
1 Family Afycopltsm ,which belong
ttou to
parasitic mycoplasma!^ requiring cholesterol or
other sterols as an essential growth factor,lliis
contains cwo general
a. CcmiH hcoptasma which utilise glucose or
arginine but do rot split urea, and
b. Genus Lfaeaphisrnrf which hydrolyse urea.
2.. Family ."ifho/cpffl.^mjrfjrcFie, mostly saprophytic
..
mycoplasrnis which do not require sterols as
growth facror
3. Family Spiropiaamttaeete, containing tin- Genus
SpiropLsilia, which parasitise arthropods and
plants. They are stem! dependenl. These are
helical in shape.
4 . Family djUAV liUdtltMcie, containing the
^
genus Ari.kWfiisma, which am strict anaerobes,
found in the rumen of cattle and sheep .
Mycoplasmas may be saprophytic, parasitic or
pathogenic . More than 100 species of mycoplasma
? re known rn cause disease in a variety of

Table 43.1 Mycoplasmas ul Humans

A. Parttide:
.
1 EiabUthed pathttfen;
M. prKuntwniii Causing pneumonia
2 . Presumed parhogen.c
MloffiinV and ( J.u/v?Jyticum .. sMK' iaLrjLi wbi 11 gt' miul infecriorih
.
.1 Non- puthogenie:
M, onafc, M. bwxak, M. Mbwium, AT- fruc* tmq in nmphafymc
M temienrarw, JEf. oenitaCun JVf. pBKMUH, IVf . pomatum,
M. spermitophiium in genual ^ mart
B. Saprophytic
AchoiepSdsrru .' jid/ jwu on skin and in mnirh.

Copyrighted materia
196 H Teat & ook &l WKfflbwNjOf *

MYCOPLASMA

. \ .*-/ ,
0
,r .
;
;*
. V/ ''
. cJ >r-
'
'
A
>*
-
T V
« /r ' ;.

iftf. ...
' «
v
p
i
. .V i

i
__ i /
/y
.
—
fhg . 45,1 Morphology of mycoplasma otlreme plcomofphism
mammalian, inject and plant hosts . Ahour sixteen Mycoplasma? may he cultivated in fluid or solid
species, belonging lo dlftt families are found in media. 1 hey are generally facultative anaerobes,
human beings (Table 43.1), growth being hotter aerobically. They grow within
Morphology: Mvco plasmas arc the smallest a temperature range of 22 -41 ‘C . tbe parasitic
"

tret'living microorganisms, and one of the most —

pleomorphic ( Fig. *1.1. 1), They occur as granules
and filaments of various sizes. The granules may
ho coccnid , balloon * disc, ring or star forms. 'Hie
filaments ate sltndcr, of varying lengths ami show
true branching. Multiplication is by binary fission,
but as genomic replication ami cell division arc
often mnuthreiMilt, budding tomiS and chains of
heads arc produced, A distinctive feature seen in
some species is a bulbous enlargement, wicli a
differentiated tip structure , by means of which the
organisms get attached to suitable host cells
carrying neuraminic acad receptor*. They rnav be
responsible for the hemadsorption shown by some
species.
Mycoplasma* Jo not possess spores , flagella or
fimbria. Serene specie? exhibit a gliding motility.
MvcopEasmas art Gram negative but arc better
stained bv Giemsa stain .
species growing optimally at 311 37 "C and the
saprophytes at lover temperacures . Media for
-
cultivating nuvopl .i ui .L arc enriched with JO per
cent horse nr hunun serum -and yeast extract.
JVniciliin and thallium acetate are abided as selective
agents. The high concentration of serum is
necessary as a source of cholesterol and other lipids .
Colonies appear after incubation for 2-6 days and
nrc about 10-600 pm i n size - The colony is typically
biphi& ie, with a 'fried egg' appearance , consisting
of a central opaque granular area of growth
extending into the depth of the medium, surrounded
by a flit, translucent peripheral nine ( Fig. 41.2).
C! ninnies maybe seen with a h .ind lens but art best
studied after staining by Dienes method. For this, a
block of agaj containing rite colony is cut and placed
on a *lide. It ih covered with ,L cover slip on which
h,w beer, dried ,in alcoholic solution of mcthslcnc
blue and a ?jure.

m aerial
Hidden page
39S * Tfijclboek
The mycoplasma may remain in the throat for two
. Appearance in a high proportion of case* with
or more months after recovery from the disease.
Eaton (11M 4 ) win the first to isolate the
causative agent ofthe disease in hamsters and cotton
rats. ] ie was able to transmit the infection later to
chick embryos bv amniotic inoculation . Because it
j" J
was filterable, it was considered To he .L virus ( Eaton
agem ) , hnc was subsequently shown to he a
mycoplasma and named M. pneumnnixe.
Laboratory diagnosis of mycoplurnal primary
atypical pneumonia may be established either bv
isolation of the mycoplasma or hy serological
methods, For iscjlation , thm ^ r swabs or respiratory
secretion are inoculated into mycoplasma medium
LuaiC : LLJIIEIJI glucose and phenol red . GtOWtll is s- U
on primary isolation and mav take 1 -3 weeks.
Growth is indicated he ILL - id production in fhc
medium. M . pmimonitie produces beta hemolysis
and agglutinates guinea pig erythrocytes, t lolonics
on agar adsorb erythrotylM. The hemadsorption is
enzymatic and occurs optimally it 37 °C , The cell
neceph > rs are destroyed hv neurarn Lnidase . It inhibit!
ciliary motility in hamster trachea organ cultures.
M. pneumoniae is unrelated to other human
mycoplasmas and may be identified hv growth
inhibition by specific antisera. As isolation is
difficult and delayedh PCR assay which is rapid
and specific is being used where tcasihlr.' ,
Serological diagnosis may be made by specific
tests using mycoplasmal antigens or hv nonspecific
methods. Among the former, immunofluDrescence,
hem agglutination inhibition and metabolic
inhibition ure the rrm-st sons iridic tests . Complement
rotation and indirect hemagglutination testa are less
sensitive: .
The nonspeeifiit: serological tests are
MG and cold agglutination tests. The
former is done by milting 5Cfi .it dilutions of the
pa lien r 'a unheated scrum and a heat killed
suspension uf Streptococcus MGt and observing
agglutination after overnight incubation . Lt 37 fL .
A title of 1:20 fir over is considered suggestive .
The cold agglutination test Is based on the
a ( Microbiology
primary atypical pneumonia , af maLrEJglubulin
antibodies that agglutinate human group O cells
:it ]( i-vv temperature. The patient 's blood sample
should rot be refrigerated before separation gf the
serum , as the agglutinins are readily absorbed by
ilic homologous eryihrocytes at low temperatures.
VtiT tli LI test , serial dilutions of the patients serum
are mixed with an equal volume of a 0,2% washed
human O group erythrocytes , and clumping
observed after Leaving at 4 ’ C overnight . The
dumping is dissociated at 37 °C. A litre of 1:32 or
overis suggestive but demonstration of rise in litre
inpaired serum samples is more reliable . The
indirect Coombs teat may also be positive in BOOK
cues.
URBAPLASMA ilRKALYTIfil M
Some strains of mycoplasma frequently isolated
from the urogenital tract of human beings and
animals form vtr> tiny colonies, generally 15 50
r
-
ptdi in EEZCL They were called T Strain or 7 form
mycoplasma? (T b * r tinyl/Fhcy are peculiar in their
ability to hydrolyse urea , which is an essential
growth factor in addition to cholesterol , 1 Etiman T
strain mvcnpla -smas have been reclassified as
l ' rexphy-mx urafytievw-
Genital infections arc caused bv M . hominis
. .
j
and U urealjrtkum Tliey an; transmitted by sexual
contact, and may cause urethritis, proctitis,
balanoposthiti? and Reiter's syndrome in men, and
acute saljihingiris, pelvic inflammatory disease,
cervicitis and vaginitis in women. They have also been
assoviited with infertility abortion postpartum fever,
^
churutfumkniilis and low biithiveiglil ot infants.
Mycoplvi & ma mid HIV infection:
Mycoplasma ? tend to cause more severe and
prolonged infections in the HIV infected and other
iiumunodeficicnt subjects .
Mycoplasma as cell culture
contaminant*: Continuous cell cultures
maintained in many laboratories have been found
to be contaminated with different species of
Copyrighted materia
 t Mycoplasma * 399

iDfDOpliinna.'Ilx contamination nay originate from
the worker or front animal seTa or trypsin Used m
cell culture. Contamination generally docs not
luoduce evtopathic effects but may interfere with
the growth of viruses in such full cultures uiid may
ill (id produce misleading results in serological, tests.
MynjplaWtV growing in cell cultures have often
hcen mistaken for viruses - Rradieatinn ot
myCOplasinas from infected Ceils is difficult ,
-
irc atmenl ate the drugs of choice tor the
treatment of mycoplasmal infections. Some
ureaplasmas are resistant to tetracycline ,
doxycyclinc , the newer macrolides and quinoloncs.
Mycoplasma * and [ . forms of bacteria :
hlcincherfitrr (1935 ) found pleuropneumonia -
like forms in a culture of Srnepfo6acj Jj' yff '
omaiubaois and termed them L forms, after 1.inter
Instilmer London, where the observation was made.
It was subsequently shown that many bacteria,
either spontaneously or induced by certain
substances Like penicillin, lost parr or all of their
cell wall and develop into 1 . forms. Such L forms
mav he ‘unstable', when they revert to their normal
morphology; or stable ^ when they Continue in the
cell wall deficient male permanently Cell wall
deficient forms ( L forms, protoplasts, flphtiOplatfs)
may not initiate disease hut may be important in
bacterial persistence during antibiotic therapy and
subsequent recurrence of the infection. It Hus been
suggested that mycoplasmal may represent stable
L forms of bacteria hut genetic , antigenic and
biochemical evidence are against the possihiliry.

Punhtr i t o i u h i L k
. .
Lin J 5 1985 Human, rnycoplafmaJ infections Serologic observations. Rev /nicer f .Ji 7 - 21 fy .
. ^^ . .
Symposium 1 3 The chaffing rule er mycoplasrnaE in nespiratury disease and AIDS CVifl /ritecf
'
17;f 5 uppl , l ) ,
.
Taylni- Kj iinsoTi [1 and J Bradbury 199S. Mywplanna distast * In Tbpfcyantf WiJwin'x .VIrcarpfcnj/qgy and Mkrvbiid
^
Jnfnrirjm, V' cdn. LondofcAri jld

Copyrighted materia
 Actinomycetes
Actinomycetcs arc traditionally considered to he
transitional forms between bacteria and fungi- [ .ike
fungi they tunm a mycelial network of branching
filaments but, tike bacteria , they arc thin, possess
cell waits containing marunlc it id, have
prokaryotic nuclei and ire susceptible ro
antibacterial antibiotic ;!- They are therefore true
bacteria bearing a superficial resemblance to hingi .
ActinomytCtics arc related to mycobacttti ^ and
curynebacteria, They are Gram positive , nonmotile,
nonspoting, nomapsubted filaments rh;it break up
into bacillary and coccoid elements. Most are free -
jKLiEicukrly in the soil.
Acfmomytctc* indttdc many genera of medical
interest SLLHTI as the anaerobic dCfinonytcJ,
ArMchaia, 9i£dobtcrerrumT Rothia and aerobic
.\’cjcarJja , Aetinonudvra , Dermatophilut, and
StTcpjomyces , The major pathogenic genus
Actinomyces is anaerobic or mjcroaerophilic and
nnnacid last, while JVOL- jnlia Speede* art; aerolite
and may be acid fast. Some species of StrtjrnojDytcs
may L inise diheane rarely, hut their importanee is an
' errujTP
the major source of antibiotics.
ANTINOMY I : K S
Bollinger yltf 77) IOUILJ a mould-like organism in
the lesion of 'lumpy jaw ' [ actinomycosis) in cattle .
The name actinomyces was coined by I larz to refer
to tbe ray I ike appearance of tire organism in the
granules that characterise the lesions ( nctinnmyces,
meaning ray fungus) , Wolff and Israel ( 1891)
. isolated an anaerobic bacillus from human lesions
and produced experiment infection in rabbit* and
^
guinea pigs. This was named Axtinoinj/as israeSi
It causes human actinomycosis. Art inti mycosis in
cattle is produced by A. hovrs-
Actinomycosis The disease is a chronic
granulomatous infection occurring in human beings
and animals . It is characterised by the development
of indurated swellings, mainly in the connective
tissue, ^suppuration and the discharge of 'sulphur
granules’.The lesion often points towards the skin ,
hading It) multiple sinuses.
Actinomycosis in humtin beings is an
endogenous infection. The actinomyces species are
normally present in the mouth , intestine and vagina
as commensal*. Trauma, foreign bodies or poor oral
hygiene may favour tissue invasion . A- jsraefir is
'
tbe most common causative agent - However, other
actiiiomyecies such as A. iiaeslundii , A, vtscastis,
A UiJojtftjfytJcum, A. jntyerr, A. getcnctiunci and
AifuMaclcnumpropionicum may sometimes he
responsible. Actinomycosis is usually a co-operative
dtsea -se , the actimmivces being accompanied by
utlicr associated bacteria which may enhance the
pathogenic effect. These Lnelude fljricltab;iL7
"
Centrum, Acrinobaallvs ictinaiBfvBcnicoiiutsat ,
Eikcnell# corrodcm, Haemophilus iphrophilut ,
b act cm ides , fusobicteria, staphylococci and
anaerobic streptococci .
Ac[ Loomycosi & io human beings occurs in (our
main clinical forms : ( 1 jeerrioofarialwith indurated
lesions on the cheek and submajdliaiy regions;
( 2 ) tiwnnic, with lesions in the lung that may
involve the pleura and pericardium and spread
outwards through the chest wall; ( 3) abdominal
where the lesion is usually aruLiiid the CifUifl, with
the involvement of the neighbouring tissues and
Copyrighted material
 < Atlincmyctiies * 401

the; abdominal wall. SoiQCCEnicS die uifeLtion spreads
to the liver vi ;i the portal win, {4) Pelvic. Many
cases of pelvic actinnnuTcosis hnvc been Kported
in association with rbc use of intrauterine devices.
Actinomyces have been incriminated in
inflammatory diseases of the gum; ( in svifis and
^^
periodontitis ) and with sublingual plaques lending
to root surface caries.. Actinomvcusis may also
present as mycetoma .
Laboratory iNa nosis; The diagnosis is made
^
by demonstrating nctinomycefcs in the lesion hy
microscopy and by isolation in culture - The
specimen CO be Collected is pUS. In pulmonary
disease , s- putum is collected. Sulphur grannies mav
methods. Ge! diffusion and immunofluorescence
can differentiate A isrselii from other actiiionycetc
species and from other filamentous- anaenrhes that
may produce granules in tissues.
F ^pidemiology: The disease occurs throughout
the world but its incidence i n the advanced con ntrics
has been declining probably as a result of the
widespread use of antibiotics. Actinomycosis i*
more common in rural areas and in agricultural
workers. Young male persons (10^30 veajlJ old ) are
most commonly affected. The reason for this
predisposition is not known. About 60 ]>cr cent of
the cases afe Cervicofacial and some 20 per cent
abdominal. I let vie latino mere* is seen mainly in
m r

be demonttrated in pus by shaking it up in a test women using intrauterine devices ,

tube with some saline . On standing , lEie granules 1refitment: The disease responds to prolonged
treatment with penic i Eli n or rctracycli ne. frcatnic nr
J

sediment and maybe withdrawn with a capillary

pipette. Granules may also he obtained by applying will have to be continued for several months and
gauze pads over the discharging sinuses, supplemented by surgery where necessary.
The granules are white or yellowish and range
in size from minute specks to about 5 mm . They NOCAHULA
arc examined microscopically under a cover slip. Mocardia resemble Aeriaiomyectcsmorphokigieally
They arc crushed between slides and stained by but are aerobic. All species are Gram positive and
Gram stain and examined. The granulev are * in tac£ h
barteria ] coionics ami will he found to consist of a
dense network of thin Gram positive filaments*
surrounded by a peripheral Hide Cif swollen
,

radiating club shaped structures, presenting a s« n

ray appearance . The 'club?/ are believed to he
antigen^ antibody complexes ( Fig, 44.1),

OkJ
Sulphur granules or pus containing
acrinomvcetcs arc washed and inoculated into
f

rhinglivollate liquid medium or streaked on brain-

heart infusion agar and incubated anaerobically at
37 LC . In thiogiycollatc A btms produces general
.
turbidity whereas A itmlii grows as fluffy ball* at
the bottom of the tube. Oil solid media A . URietii
prixluces small 'spidery colonies' in 4S-72 Sours
rltat become heaped up, white and irregular or
smooth , large colonics in 10 days. Other species
have different tvprs of colonies. Fig. 44.1 Sulphur granule . Section of tissue showing
T h e isolate is identified bv microscopy, an aiding mycotic clQlqny , I he clubs at I he pfrffphtfy
biochemical reactions and fluorescent antibody giving A $un r y appearance.
'

^
Hidden page
 Miscellaneous Bacteria
USTEFUA MONOCYTOGENES
Listeria jnonocrro ciics is si small, coccoid, dram
^
po$itiw ITIL LIIU -;, witli J t&ndetuy toocwrin chains.
Rough forma may he seen u long filament?- . If
when grown at 25 C boil at 37
L
'
.
exhibits a characteristic, slow Tumbling motility
is nonmutile.
This is because peritrichoiLS flagella are produced
by the bacillus optimally at 20-30 °C but only
sLacnily or not at all at 37 JC Jt is aerobic or
fnicroaemphil i c. t Jrnwf h LS improved whim cultures
arc incubated JT reduced oxygen Tension and with
5-10% COj. It grows best between 30 “ C and
37 °C , but slow growth occurs even at A "C ,
Colonies are hemolytic on blood agar . L. mono-
cyttigencs ferments glucose ^ maltose* L rhailimose
and alpha methyl D- mannosidc, producing acid
without gas , It is catalase positive. Tr grows in the
presence of 0.1% potassium tellurite, 10% salt and
,
at pi I 9.fi. Many sernvars have been recognised .
L, monocytogenes is widely distributed in
nature. It has been isolated from a wide range of
mammals, birds, fish, ticks and crustacca. It occurs
on a saprophyte in soil, water and sewage. Listeriosis
in human beings may present ill many forms. It
may cause meningitis Or meningoencephalitis ,
particularly in neonates and in the elderly Infection
of pregnant women may lend to ah onion or
stillbirth . Asymptomatic infection of the tccnale
genital tract may cause infertility. Listeriosis may
also present as abscesses, conjunctivitis, pharyngitis,
urethritis, pneumonia , infectious mononudeosis-
like syndrome, endoc.Ltdnis or septicemia.
MOM tinman infections arc caused by scrovar
l /2a nr l / 2h and 4b. Experimental inoculation m
rabbits causes a marked monocytOtof ( hence the
name moiMcytogeiMs ). Monocytosis Is a feature of
human Ettet»sis also, Inttilkbon into tlieeyes of
rabbits produces keratoconjunctivitis v - lntnn fe.sr).
Human Infection is believed to result from contact
with infected arim .ils, inhalation of contaminated
dust or ingestion of contaminated milk or food.
Outbreaks of foodbome listeriosis have been
known.
Laboratory diagnosis LE esc tf Mi shed by the
isolation of the bacillus from appropriate clinical
material such as cervical and vaginal accretions,
lochia , meconium , cord blood * blood and
cerebrospinal fluid. Greater SIH^ E &S in isolation is
achieved if the materials arc stored in tiyptuse
phosphate or thioglycollate broth at A and
subculture!; arc dune at weekly intervals lor 1 £ >
months ( cold enrichment) . Listeriosis in human
beings is being increasingly reported. Isolates ire
likely to be missed as nonputhogenic diphtheroids
unless properly Investigated . AmpicMILn *
cotrimttitzoLe and gentamicin arc effective.
Cephalosporins are iioi recommended .
Table 45 ,1 lists some distinguishing features
f nunspuring Grain posit i « bacilli found i n cl ] llical
specimens .
ERYSIPELOTHRIX RHUSIOPATHIAE
Erx^irvlothrix rhusiopathixe 'vi A slender* tionmotilc,
'
nonsporing, noncapsulated Gram positiye rod, witli
a tendency towards formation ot’ long filament .. It
LS microae.ophilie on primary isolation but on
-
lubcultuiv, grows as an aerobe or facultative
anaerobe. It grows on ordinary media . Black
Copyrighted materia
404 < Textbook
colonics are developed in tellurite ircdiL Itfenrents
glucose and lactose, producing acid without gas ;
sucrose and mannitol arc not fermented . Different
antigenic types have been recognised .
E rhi&iopEthiJi: is a natural p &rasiLc oi many
animals. Jr causes swine erysipelas and human
erysipeloid , E luman infection usually occurs on the
hand or fingers of persons handling animals fish
or animal products. The lesions are painful,
edematous and erythematous, usually involving the
local lytnphnodes and jisines. Occasional eases of
endocarditis have been reported . The bacrUos it
aenaitive t < > penicillin , erythromycin and broad
spectrum antibiotics.
ALCALIGENE 5 FAECALIS
The name JJacferjum faecalis tlkaligenes was
originally applied to an ill defined group of Oram
negative bacilli indited from human feces , which
did not ferment sugars bur produced an alkaline
reaction Ln litmus milk. The term Alc\iKgcncs
facL &il
* now refers to Gram negative , short ,
Ttonsporing bacilli , which Lire strict aerobes and do
not ferment sugars. They arc motile by means of
peritrichouj flagella . They ate usually oxidase
positive. Nitrate reduction is variable.
Ait, /pectEuia a saprophyte found in water and
soil contaminated with decaying organic matter .
They are also commensals Ln human and animal
intestines. They have been isolated from a variety
of clinical specimens such as urine , pus and blood.
Thty have been considered responsible for a
typhoid - like fever, urinary infections, infantile
gastroenteritis and suppuration in various pares ot
the body.
CHROMOBACTERIUM VIGLACEUM
Q lOOblUtrnun vrn /aneum is Gram negative ,
^ ] ,L
nonsporing bacillus, motile hy means of polar and
lateral flagella. They are facultative anaerobes
growing on ordinary media and producing violet
pigment soluble In ethanol and insoluble in water
and chloroform . They are oxidase negative and
of Microbiology *
saprophytic in water and soil . Human infections
have been recorded mainly in the tropics and consist
nt skin lesions with pvemia and multiple abscesses .
FUWGBACTERIUM MENINGOSEPTICUM
FJJVUBACTERIUM mcningosepdcum is a Gram
negative non mo rile tod , producing a yellowish
pigment. It is oxidase positive, proteolytic and
weakly termentalive. It is a ubiquitous saprophyte
i tottunistic infections. It has
capable ot causing o[|
been tcsponsible tor outbreaks of meoingitis io
newborn infants. Infection in adults leads to a mild
febrile ill lies?,
DGNOVANIA GRANUL0MAT1S
l CalymnritobactErnim granuhmatix)
Donovan 0 05 described the presence of
^ )
characteristic intracellular bodies in smears from
ulcerated lesions of a disease now known as
Douovanous. He considered the bodies to be
parasites . DonovanoELH is a venereal disease , first
described by McLeod in Indk in 1882 and seen
mainly in the tropics. The incubation period ranges
fn>m 1 It ) 12 weeks. It begins as a pamless papule
on the genitalia , which leads to a slowly
progressive, autoinocLiJable ulcers . The disease runs
a chronic course, Donovan 's intracellular bodies
have since been identified as bacteria and named
Dunovarna granuJeimurh - '
Diagnosis can he made hy demonstration of
Donovan bodies in. Wright-Giemsa stained
impression smears From the lesions. They appear
as rounded CQCObfKilli* 1-2 pm , within cystic space.;
1
in large mono nuclear cells . They show bipolar
condensation of chromatin, giving a closed safety
pin appearance in stained smears . Capsules are
usually seen as dense acidophilic areas around the
bacilli . They arc nonmotilc and Gram negative.
They can be grown on egg volk medium and on a
modified Lcvinthal agar. It is morphologically and
antigcnksUy related to Idcbsielke.
Pathogenic tv is Limited to human beings .
)
In trade rimiil inoculation of whole cultures or of an
Copyrighted material
Hidden page
406 * Textbook ol M rotiiokigy »
Relapses arc common n untreated CIHI. The
disease can .1 KI occur A* outbreaks, m the absence
of rat hitC - t his ci»u) itill]ih first observed in
Haverhill, USA , Is called Haverhill fever or
emhema arthricicuni rpfJemjamt , li is believed
to be caused also by consumption. of caw milk or
water contaminated hv rat ;;.m
Laboratory diagnosis iR by boiatu *r of the bacillus
-
I r i i i blood or oilier tjodi fluids , Smears of flic 01111
fluiL L may KIIOW pleomorphic Gram [legulive rods.
Agglu 1 : m :1 :m, CF and fluorescent antibody tests have
been used for serological diagnosis.
Spirillum minus is a short, actively motile
bacterium , 1 — —
. 5 * 0.2 0.5 )jm in ' .r.e , with two or
three regular spirals and 1-7 amphitrichous flagella.
It is Gram negative but 1* better visualised by
Ciiemsa or Fontana stains or by dark field
microscopy. It was first observed in a nit hy Carter
ill India. Japanese workers identified it as
the causative- agent of one tvpc of RRI', called 1
Soihtb.1 , It lias Trot been cultivated in laboratory'
media,
-
Spnillary RBF has ar. incuhatinn per . td of 1-4
wteks /lTic rat bite wound winch may have healed,
suppurates at the unset of fever, with regional lymph -
ailenopathy. The HLibsccjncnt course is sil uilar to the
streptobacillary type, Mortality rates of up to 10 per
dent I lave bun reported , mainly due to endocarditis.
Laboratory diagnosis is bv the mirtOKopic
examination of the blood and exudates from the
lesion , by intraperitoncal inoculation into guinea
pigs and mice and by dcmonstrai inn of the spirilla
in their blond ami per . tones! fluid . Biological false
pnMiivc reactions for syphilis serology occurs in a
proportion of RRF patients, more in tile spirillary
form .
Botli ty|jes ol RBF respond to penicillin and
tetracyd . ne. Oral penicillin or doxycyelinc After
rat bite is effective in prophylaxis ,
CAMPYLOBACTER
The genus IdJ7ipvj'ob!
, L tcr ( Crcek, meaning curved
rod ) contains slender spirally curved Gram negative
Hods, 0.2 -0.5 pm thick and 0.5^5 uiii long. They
are typii ally comma shape ;! hut mAY occur as lb Of
mnltispiral chains. Old cultures arc coccoid and
pleomorphic. They are nonsporing and motile with
A single unsheathed polar flagellum at one or both
pules . Growth occurs under m icroacroptl i lie
cunc . i Mns, 5% oxygen concentration being optimal.
'
Many pathogenic species are thermophilic, growing
well at 42 ° C . Campylobacters do not attack
carbohydrates but arc strongly oxidase positive.
Campylobacter* first gained prominence in the
1970s as a common cause of human diarrheal
-
di ease, affbi ting children and adults.They can, on
occasion, also cause systemic infections. They are
important veterinary pathogens. Campylobacters of
medical importance are the following:
h r: .
-
Cau ing diarrheal disease: C- jejuni , C- cofj, t ,
Causing extra - utcs’ - nal infection: t’. ferns
Canting abscesses: C. sputonim, C . conciscus
CAHPI LOBACTBH J h J l NJ
Med cally, this is the most important campylobacner
'
Species as it causes attacks of ni &Trhea worldwide.
The infection i* voonnti . :, the source being food of
animal oriu: nh especially raw milk . It is part of tire
normal intestinal flora of domestic animals and
b:’ ds, and is shed L11 thr. i feces- It can, be - wlated
frequently from stirface waters .
Infection occurs by ingestion. The jejunum and
ileum are the primary sites of colonisation, but it may
-pathogen
. -. i Lown the colon and
|ire L i hi It is an i
rectum.
and may Involve mesenteric lymph node
pwasive
*
and cause bacteremia. The incubation period is 1~7
days - The illness starts with fever, abdominal pain
and warcty diarrhea. Stool contains leucocytes and
blood. The disease is usually self-limited, though
Campylobacter shedding may continue for weeks after
recovery Fluid and electrolyte replacement is all tliat
is generally required. When needed, erythromycin is
the best antibiotic.
Copyrighted materia
 * Miscellaneous Bacteria
LtbulMy Jiagnosi -s depends on isolation of
the campylobactcr from feces. Direct microscopic
examination - phase contrast or dark licit ]
microscopy to detect the darting or tumbling
motility of the spiral rods, or demonstration of the
-
small curved rods in grained smears may be useful
for presumptive rapid diagnosis. Feces or rectal
swabs art pi ated on sc I ective medi a. In fuse of del av
in culturing, a Iran sport medium has to lie
employed' Campylobacters survive for 1-2 weeks
at A °C in Cary El Lair transport medium but
- -
glycerol s:i1 inc is not .arkfactnry. The pitting media
commonly used arc Sorrow s , Butzlefs or
'
Campy
HAP selective media . C , jejuni, as well as C roll
and C. l.dj- j , are thermophilic .and Jo not grow at
25 rG . Inoculated plates are incubated at 42 °C in
an atmosphere of S% oxygen , 10% carbon dioxide
and 8.5% nitrogen. Thermophilic tumpylnbactcrs
ca n grow well ar 37 °C also hot i ncuhation at high er
temperatures suppresses normal focal flora ro some
extent .
Colonies appear usually by 4? hours. They are
nonhemolytic, grey or colourless, moist, ami Jlat or
convex. Sugge ft Lve colonies are screened by Gram
staining, motility and oxidase rests , Confirmation
is. by further biochemical rests , including positive
catalase and nitrate reduction tests.
C . coli causes an infection clinically
indistinguishable from that due to C.JCfUTli ( ’. caii
is commonly found in healthy pigs . It is
differentiated from C. jejuni by the hippuratc
hydrolysis test which is positive only in the case of
C, jejuni,
C . lari also causes a similar diarrhea! disease, lr
i n o be distinguished from C - fejuni and C. Coli by
its resistance to nalidixic acid.
C . jejuni and C. coli can be serotyped for
epidemiological puriMiHes.
C. jejuni is the most common bacterial cause of
diarrheal disease in many developed countries -
more common than salmon elite or shigdlac . In
[ he developing countries, C. jejuni is endemic ,
asymptomatic infection being widely prevalent in
407
humane, as ivcll as domestic animals anil birds . In
this situation , clinical disease is infrequent and
usually confined to children, while older age groups
arc immune due to subclinical in factions.
The related genus Arcobacteria species (A
^
buti/erj', A . Crvittuphila also cause diarrheal
d ]>ease. They are capable of aerobic growth .
CAMPYLOBACTER F a r i a
This organism was isolated in 191 H by Theobald
Smit h from i nfeeiious al H inion 11 L cattle and named
Vibrio fetus , lr is a very important veterinary
pathogen . Human infection by C. telus may lead
to bacteremia, sepsis and meningitis.
HELICOBACTER
Spiral, campylobiCttr Hike- bacteria were observed
in close apposition ro the gastric mucosa in several
cases of gastritis and peptic ulcer, by Warren and
Marshall in Australia ill 1983.They were original I v
named C&tnpyl&bectet pylftri. As they differed in
many respects from campylobaercrs, they have been
redesignated as Hclicobactei pylori. It now appears
that helicobacters have caused human infection from
ancient times. By enzyme immunoassay ,
helirohacter antigens have hern detected in the
-
intesti n rs of pre Columbia n mu mmirs in (be 1 A.
Toddy, heUrnbacters colonise the stomachs of halt
the human population of the world !
I Icliicobacten inhibit die stomachs of different
animals, each with its own helieobacner species. / /.
pylori is adapted tti the luiman gastric mucosa. The
only animal it infects is the monkey A larger spiral
bacterium of uncertain taxonomy,'if. heiintMilf can
occasionally infect hmimns arui some animals like
cats and dogs also . 11. cifiardi and II . fcnnelliae
are associated with proctitis in the HIV mfevted.
HBLICO&ACTBk I - OR ]
H , pylori is a Gram negative spiral rod, motile by a
unipolar cuft of lophottiehous flagella. h grows on
chocolate agar or Campylobacter media under
microaerophulii.- conditions, with 5 -20% CO.. , and
Copyrighted material
 400 H Textbook d M crobiology
pH 6-7. At 37 ;' C , no Ionics like 2 -7 Jays Iu
develop. Coccoi . l forms appear in old cultures. It
produce* oxidase, catalase , phosphatase and E E \
A distinctive feature is the production of abundant
unease , and this property lias been Used as a rapid
diagnostic test in gastric biopsy samples. It does
not metabolise carbohydrates
.
or reduce nitrate.
11. priori is global , with a prevalence of 30-60
per cent— more in the developing than in the
developed countries. The sole source of //. pylori
is the human gastric mucus. The exact mechanism
of transmission is not clear, but it is likely to be
oral -oral or tcc^- l— tiral. Rwerty, overcrowding and
poor hygiene favour transmission, Wjtl : inprovements
in lifestyle, tlx prevalence of cluldhood infections has
declined in the developed countries.
After an incubation period of a few days, H .
pylori causes, in some persons, a mild acute gastritis
which mav last for about two weeks. The infection
jf
may he transient in some, but in most , it per- 1 sis
for years or decades. Such colonisation is usually
asymptomatic, though chronic superficial gastritis
may be demonstrable histologically. The bacteria
are present only in the overlying mucus and do not
hvade the mucosa , Gastric antrum is the commonest
ate of colonisation , though any part of the stomach
may he involved. The infection i.s strictly confined
to the gastric mucosa , in the stomach , as well as in
areas of gastric metaplasia and heterotopia in the
duodenum. The exact pathogenic mechanisms are
not clearly understood . Bacterid protease , toxins
nr ammonia released bv urease activity nr t
autoimmune responses to gastric antigens may all
contribute .
Peptic ulcer disease occurs in a proporrion of
the infected . Chronic atrophic gastritis may be seen
in the later stages. The infection is recognised as a
risk factor for gas- trie malignancid such an
adenocarcinoma and ’mucosa associated lymphoid
tissue' ( MAI. E ) lymphomas. Such MA[Tomas
appear to he antigen driven and are found to regress
after elimination of H . pylori by Treatment ,
Infection induces IgM , IgG , IgA and cellular
»
immune responses , but they do not seem to be
protective .
11. pylori shows considerable genetic diversity,
as evident in molecular typing . The complete
genome of the bacterium has been mapped .
Virulence has been associated with certain alleles
in genes, such as cag {cytotoxin associated gene )
and vac ( vacuolating cytotoxin gene ).
Diagnosric tests aie of two kinds, invasive and
non invasive . Invasive tests Involve cndoscop : c
biopsy of gastric mucosa, for examination by
microscopy, culture and urease tests . Microcopy' of
biopsy sections by silver staining or of Grant
stamed smear- is a useful method. Culture is more
sensitive, but requites expertise and takes 3-7 days.
A Li [ of the biopsy material put in a urease indicator
trediuni shows positive result in minutes .
Non invasive tests include serology ( ELISA ) and
the ' urease breath test’. In the latter, the subject
drinks a urea solution containing labelled carbon,
which can be detected in the breath . It is sensldve
and reliable, but needs isotope assay facilities.
SI . pylori is sensitive to several antibiotics and
to bismuth salts. The standard treatmenl is a
combination of bismuth subsalicylate, tetracycline
(or amoxycillin ) and metronidazole for two weeks.
An alternative schedule employs a proton pump
inhibitor like omeprazole and clarithromycin .
Treatment is indicated only for H pylori related
gastric or duodenal ulceration and not for
asymptomatic colonisaLLon . Drug resistance and
recurrences are frequent .
LEGIONELLA PNEUMOPHILA
The name Legionnaires * disease was given to an
apparently new illness which broke out among
members of the American Legion who attended a
convention in Philadelphia in 1976 . The disease
was characterised by fever , cough and chest pain .
Leading on to pneumonia and often ending fatally.
The causa rive agent has been called Legionella
pneumophila . Subsequent investigations have
revealed that the disease is neither new nor localised .
opyrighted materia
 « Miscellaneous Bacteria
Infection w ith L, pncLr/nopMa is now known to
cause protean manifestations . rwn disci net clinical
patterns bare- beenidentified and dc - ignafcd as
Legionnaires disease and Pontiac fever, together
'
known as /ejfjcviei/usjy.
Legionnaires ( listasc: may he either epidemic
or sporadic . The incubation period i -. 2-10 days .
'1'hc d - scasc presents with fever, nonproductive
cough and dyspnea, rapidly progressing, if untreated ,
to pneumonia . Diarrhea and encephalopathy are
common . Last fatality maybe 15-20 per cent , the
cause of death being progressive respiratory' failure
and shock . All age groups arc susceptible, though
more cases haw: occurred in the elderly
Ptyntiae fever is a milder, nor fatal influenza'
’
likeNllness u ith fever, chills, myalgia and headache .
Outbreaks with high attack rates may occur.
-
The discovery ot J . pneumophila led to the
isolation of many related hacteri ;:i, which have beer
placed in the genus LcgioncUa , under the family
T jcgLorctlaceae. Some 40 species of leg i nnellae have
been recognised, many of them with multiple
serogroups. The original isolate in tills genus is
designated / „ pneumophila senogrnup 1 (SGl ) ,
whieh accounts for nearly all severe infections .
Examples of other species char cause human
infection less often are L . mit-thiclei, L . bozirmAnii .
L . dusftti&ii and L. ur.u.ir.'Ji .
j
^ , noncapsulated hacilii , 2-
I ^eginnellac are thin
5 pm * 0.3-0.1 pmh coccobacillaTy in clinical
material and assuming longer forms in culture .
Most ate motile with polar or subpolar fligeda ,
They arc Gram negative but stain poorly,
particularly in smears from clinical spec mens ,
They srai - i better by silver impregnation , but arc
best visua! ]sed by d 11 eCt fluorescent mil. i body ( DFA )
staining witli monoclonal or polyclonal sera .
They hare fastidious rcqr. jirements and grow on
complex media such as buffered charcoal , yeast
extract ( liCYE) agar, with L- cystcinc and antibiotic
supplements , with 5% CO , at pH 6,9 , 35 =C and
90% humidity, Growth is slow and colonies take
3— 6 days to appear.
409
Lcgioncllae arc widely d i - tributcd in natural
water sources, sueh as stagnant waters, mud and
hot springs , where tbc nutrition a L and growth
requirements for these fastidious bacteria arc
provided by some types of algae . Legionellae survive
and multiply inside free - living amefee and other
protozoa . They also mulliply in some arlifici . il
aquatic environments, which serve as amplifiers.
Human iiife- clinn is typically hy inhalation of
aerosols produced by cooling towcrsh air
condiI innets and shower heads which act as
disseminatora . Acrosolised lcgioncllae can survive
for long and can be carried over long distances . No
animal reservoir exists, and infection is limited to
human benign. No carrierstate is established. Mari'
to -man transmission docs not occur.
The outcome of inhalation of lcgioncllae
depends- on the size of the infecting dose , virulence
of thestnun and resistance of the host. Known risk
factors arc smoking, alcohol , advanced age,
intercurrent illness, hospitalisation and immuno-
deficiency. Men are more often affected than
women , In the developed countries , legionedoi is
accounts for 1 -3 per cert of community acquired ,
and 1CKJ 0 per cent ofhosptaJ acquired pneumonias .
Its prevalence tn the developing countries is not
adequatelv known.
Following entry into the alveoli through
aerosols, legiondlae multiply in -side the monocytes
and macrophages , Dissemination occurs by
endobronchial , hematogenous , lymphatic and
contiguous spread. Because of then intracellular
location , humoral anybodies arc ineffective .
Cellular immunity is responsible for recovery,
Laboratory diagnosis is by the demonstration
of legionellac i n clinical spec ] me ns, such as sputu m ,
bronchial aspirate , artd lung biopsy, by direct
fluorescent amibodv test and culture , by the
idemificstion of leginne-lla antigen* in urine by Latev
agglutination or h’ LJSA , and hy the detection of
serum antibody by ELJSA or indirect
immunnflunresccnt assay.
For treatment , the n e w e r microti des,
Copyrighted material
Hidden page
 .
* Misce laneous Bacteria 411

oxidase positive., nonmotilc* Gram negative rock , genus MonEetki They are part of the normal oral
with a tendency to occur as coccobacilkiy and flora , K- fctngflg has been associated with mdlrramJjtis
diplococcaJ forms, was formerly grouped under the
, and Sdktuoa of bones, joints and tendons

h u r d l e r I t e a d i m*
Bcrgone- Bcrran E and KJ Towns* 19%, Arirwhrimcfw *pp. AE nosocomial pathogen*, Clin Mictvbiol Rtf 9=148 .
Blawr MJ. 1998. liclicobMcnrr priori and gastric discasee, BMJ , 316, 1507.
fi laser MJ. t 990. Epidemiology acid pachophyaiolog) of Camp. pylori infections. Rev Infcci Dis 12:( Suppl.1 ) .,99 ,
1

Ctdia BWr 1992. G?vdaotlk WjiiULlk Clin MkmbivI Res 5, 111

Fang GD ec al I 9H9. Disease due co the Legkmalbeceae., iWediejne ( Baltimore}, 4& l16
'

GrahamD CE al 1990. Infections caused by Moraxellae. Revlniect Dis 12423.

lonnidcs JPA -et d. 1995. AJorarelia catarrhiEs bacteremia, Clin Infect Dis 2:390.
.
Lrvcrt PN . 1992. Bacteria] ngirwiia Ret- Mcrif Mkrohro ) 115 .
MiAurAllot B arid MJ Bluer 1995, Gflm> prlottfirer jtjuni, Clin Infect Dis 20r1092,
MoffUOn VA and KF Wagner 1989. jfcrjigw injection*. Henr In leer JJh’s 11:776.
.
Parenti DM and DR SyndinJn 1985 Capoiocyruphagi specieE infections . / Infect Dis 151:140.
Peterunn WJB 1991. HeUttbeCKt pybri and pep+ k ulcer disease, New Engl J Med 324:1043,
Schuchat A ct aL 1991. Epidemiology of human listeriosis. OLD Microbiol Rev 4:169.
Shenrrtz. RJ and Ml Sullivan 1985. InfecrionE with AdnefcJaacter ira/coflcetjcue, / MSEC OPS 151:252«
Vcrghese A and SL Bedk 1991, MnrarcHa cittrrhaZjs. Inter Dis Clin N America 5:523.
VVormper GP and EJ Boctone 1983. Gareftobacterium /lom-trir-s. Rev Infect Dri 5:680.
Stout JE and VE Yo 1997. Legionella. New Eng J Med 337:682.

opy righted material

 Rickettsiaceae
Rickettsiae are smatl, < Iram negative bacilli adapted
to obligate intracellular parasitism , and transmitted
by arthropod sectors, They are primary parasites
of arthropods such .LH ]iceN ileus , ucks and mites, In
which they are found ill the aluuentarv Canal . In
vertebrates, including human ?, they infect the
vascular endothelium and reTiculuendnchelia] cells .
The family Rickettnaceac is named after Howard
Tayh > r Ricketts who discovered the spotted fever
rickettsia ( 190 b ) and died oi typhus fever contracted
during his studies.
—
The family current I v comprises [ It tee
genera Rickettsia. Ofienm. and Ehrlichia which
appear to have descended from a common ancestor.
Former members of the family, CoxieMa burnetii ,
_
which causes Q fever and ffodjiiimjen cjurnrjo;;
causing trench fever have been excluded because
the former is not primarily arthropod -home and
the latter not an obligate intracelhilar parasite, being
capable ofgrowing ip cell tree media, besides being
different in genetic properties.
GENUS RICKETTSIA
TTlC gCTl- US Rickettsia consists of the causative
—
agents of two groups of diseases typhus fevers and
spotted fevers.
Morphology: In smears from infected tissues,
rickettsiae appear as pleomorphic coccobacilli. 0, 3-
0.6 pm n Q. S 2|im in sire. They are mm mo tile
and noncapsulated TTicv arc Gram negative, though
rhev do not take the stain well, ’ ihev stain bluish
i
purple with Gicnisa and Castaneda stains and deep
red with MachiavcUo and Gimencz stains,
Under the electron microscope, rickettsiae are
seen to have i three Layered cell wall, a trilaminar
plasma membrane and an outer slime layer.
Cultivation: Rickettsiae ate unable to grow in
cell- tree media. Growth generally occurs in the
cytoplasm of infected cell? bur in the case of the
spotted fever richerts- iae , growth may take place in
the nucleus as well. Rickettsiae grow best in cells
that are not metabolising actively, The optimum
temperature for growth is 32-35 ZQ .
They are readily cultivated in the yolk sac of
developing chick embryos , as first shown by COM.
They also grow on mouse fibroblast, llel .a ,. HEp -
2 , Detroit 6 and other continuous cell line? but
tissue cultures are not satisfactory for primary
isolation. Laboratory animals such as guinea pigs
and mice- ate useful for the isolation ot rickettsiae
from patients. They may also be propagated in
arthropods.
Resistance; Rickettsiae are readily inactivated
by physical and chemical agents. They arc rapidly
destroyed ar 56 aC and at room temperature when
separated from host components, unless preserved
in skimmed milk nr a suspending medium
containing -sucrose, potassium phosphate and
glutamate (SPG medium),
Rickettsiae arc susceptible to tetracycline,
chloramphenicol and ciprofloxacin. Penicillin and
sulphon amides are ineffective but pata
aminobcnxoK acid ha ? an inhibitory action on
-
ticket [siae. Sulphonamides may actually enhance
the growth of rirkettsiae and worsen the condition
if adm imstcnrd to patients.
Antigenic structure: Rickettsiae have species
and group specific antigens. The iirimuiiodnniinflrii
Copyrighted materia
 i Rickaitsiacoae
surface protein antigens (SPA ) of R. piawaztkii
and Rr ( yphi have borh species specific and cross
reActive epitopes, Spotted fever ricketlsiae have
dominant outer membrane proteins (OMP) A and
B, the former being a species specific antigen tiering
as an adhesin for hnsr cells , and the latter showing
Limited cross reaction wjih SPA of typhus
nckcttsiac . The third surface antigen is an alkali
stable polysaccharide found in some rickettsiac and
in some strains of Proteus bacilli , This sharing of
antigens klnui rickettsiae and proteus is the basis
-
for the Weii Felijc reaction used for the diagnosis
of rickettsial infections bv the demonstration of
agglutinins fn Proteus strains OX 19, OX 2 and
OX K.
Fslthojg« tie»ls: Packet [ hi at are transinittcL] In
humans by arthropod vectors through their bite
or feces. On entry into ihc human body , the
fickeltsije mull ink locally and enter the blood.
Thev hcLome localised chiefly in the vascular
endothelial cells, which enlarge, degenerate and
cause thrombus formation, with partial nr complete
occlusion of the vascular lumen. The overall
pathological features of the rickettsial diseases are
similar and can be explained hy the damage to the
vascular endothelium .
The long survival of rickettriae in\arious organs
and Lymphatic tissues of infected men and animals
is a distinctive feature in pathogenesis and is of
importance in rhe epidemiology of some ticltenial
diseases.
TYPHUS FKVKU CIUJIJI*
This group of diseases consists of epidemic typhuit
rrcrudescent typhus [BrilI - Zinsser disease ) and
endemic typhus.
Epidemic typhus: ( Louubome typhus
Classical typhus, Gaol fever) hits been one of the
great scourges of mankind, occurring in devastating
epidemics during rimes ofwar and famine, so vividly
described by Hans Zinsser in his bonk, 'Rats , Liu
and History'. The disease has bun reported from
nil parts of the world hut Lins been particularly
413
common in Russia and Eastern Europe. Napoleon 's
retreat from Moscow was forced hy typhus fever
there were some 2? million cases in Russia, with
about three million deaths. Lenin is said to have
—
breaksng out among Ins truops. .1 during 1917 1V22,
remarked, in reference to the outbreaks of lousc-
homc typhus and relapsing fever rampant during
the Russian revolution, that "either socialism will
defeat ihc louse or rhe louse will defeat (socialism'!
Tn recent times, the main foci have been Eastern
Europe, Africa, South America and Ajia, In India ,
the endemic spot is Kashmir.
The causative agent of epidemic typhus is R,
pfpwTLzeJai named after von PtOWfik, who died
of typhus fever while investigating rhe disease.
HLIMANH are the only natural vertebrate hnsts.
Several animals - guinea pigs, mice, cotton rats
and gerbils - may be infected experimentally.
Natural infection in flying squirrels Lias been
reported from south - eastern USA . They may
possibly act as reservoir hosts, infection being
spread by the squincl louse and flea.
The human body louse fWi’culus hum anus
coiparjs is the vector. The head louse may also
transmit the infection but not the pubic louse. The
lice become infected by feeding on rickettsiacrnic
patients , The rideflsiae multiply in the gut of the
—
lice and appear in the feces in 3 5 days. Lice
succumb to the infection within 2-4 weeks,
remaining infective till they die. They can traumit
the infection after about J week of being infected.
The lethal nature of the infection in the louse
suggests that the association between ft- jwcwMcJbj
anti its vector IR relatively recent Odd not well
established. Lire may lie transferred from pCDOD to
person. Being sensitive tn temperature changes in the
. host, they leave the febrile patient or the cooling carcass
and parasitise other persons. Lice defecate while
tootling. Infection is transmitted when rhe contaminateJ
louse feces is nibbed through the minute abrasions
caused byscratching - Occasionally, infection may also
be transmitted by aerosols of dried louse feces through
inhalation or through the conjunctiva.
Copyrighted materia
Hidden page
Hidden page
416 s Textbook of Microbiology
ticks, which therefore act as borh vectors and
reservoirs, The infection may he transmitted to
vertebrate hosts by any of the larval stages or by
adult ticks. Ticks ane not harmed by# the rickettsiae
and remain infected fur LIJC . TILL* riubeirsiite lie died
in tick feces but transmission to human beings is
primarily by bite, as the rickettsiae also invade the
salivary glands of the riuk.s . All rickcttsiae of this
group pass through nsturnt c)<ks in domestic and
wiki animals or birds.
ffocky Mountain spatted fitter is the most
serious type of sported fewr and is the first to have
been described- It is prevalent in many ports of North
,md South America and is transmitted bv
/ Jcnnacvno > f zndtrsani 2 ml related specie!: of ricks .
Tick typhus in several parts of Europe, Africa
and Asia is caused by R . cotton , strains of which
isolated from the Mediterranean littoral, Kenya ,
, where die micrt>e cosyitem is favourable (mite
South Africa and India are indistinguishable. The
species is named after Conor, who provided the
first description of the Mediterranean disease ‘ fievre
.
boufonneusc ' (1910) The disease was first observed
iti India by MeglW (|Vl 7) in the foothills ot the
Himalayas.The investigation pf Kalra, Ram, Soman,
Helig and Naidd had established that the disease
is found in many pact of India. The rick
*
Rhiph -t -phxio.'; sungitinCUt i* the most important
vector. Hsemiphviaik.s fradji, Ajjtbfyvm mu and
Hytlomnti ticks cun also transmit the infection.
Uiekutlsinl pox: The mildest rickettsial disease
ot ' humans is a self- limited, nonfaral, vesicular
exanthem first observed in New York ( 1946). The
name is derived from rhe resemblance of the disease
.
to chickctipox It is also called vesicular or
varied!ifbmi rickctrsiosis. The causative agent is
R . ikon ( from akarr, meaning mite). The reservoir
of infection is the- domestic mouse, Mus mustmlus
and the vector is the mite , Lipaays»oides ( formerly
AJfodcrmirlpfajus) sajTgurneLr.!;, m which tTffBO'sariul
transmission Occurs. R . aA.ir: has ulso been isolated
from wild rodents in Korea. The disease hus also
been reported from Eastern Europe and Korea.
GENUS ORIENT!A
SCRtJB ..
TVI'HI S iCTllC l hK - BlUt M:
TYPHIS)
Scrub tvpbus is caused by Orient!a tsutsugamushi
i formerly /{. Cntf garau^hi, R . ancmalfs), It occurs
^
all along east Asia , from Korea to Indonesia, and
in the Pacific Islands including Australia. It was
first observed in Japan where ir was found to be
transmitted bv mite?:. The disease was. therefore
r
called rs1r.Ui4gwmr.ftbi ( from rs'Lrtsug- j, meaning
dangerous, and mus hi meaning insect Or mite) . Tt
is a place disease and is found only in areas with a
suitahie climate, ptenfv of moisture and scrub
.
vegetation The vectors Hire trombiculid mites
belonging to fhc genus Leptntmmbidtum— L
.
abidtrdti in Japan and L dciicnsh in India The .
mites inhabit sharply demarcated areas in the sod
fyJ.djrtift) . Human beings are infected when they
trespass into these mite- islands and are hitten hy
the mite larvae ( rJuggen). The mite feeds cm. the
wmm of warm blooded animals only once during
its cycle of development , and adult mites feed only
on plants. The microbes art transmitted
tnansovarially in mites. Various rodents and birds
act ILS reservoirs and also help ill spreading the
orientiae to fresh areas .
Scrub typhus, originally found in scrub jungles
Iiaa alsobeen identified in a vaneCy ofother habitats,
such as sandy beaches, mountain deserts and
•t
equatorial rain forests. The term ebiggor-bome
typhiai has therefore been suggested as a more apr
designation. Four factors arc essential for the
establishment of a mierefocus, of infection, viz.,
coexistence nod intimate relationship among O.
tsuttugtmushif doggers, rats and secondary or
Cransitioral forms of vegetation (known as the
sojFtottr -
re rod) .
The incubation period is -
1 3 iveeks . Patients
typically develop a characteristic eschar at the site
of the mite bite, with regional lymphadenopathy
.
and a macufopapular rash The disease sets in with
fever headache and conjunctival injection.
,
Copyrighted materia
 i Rickflttsiaceae
Encephalitis snd pneumonia may be seen in severe
eases. The disease is not a serious problem in civilian
practice but assumes IC .LC importance LII military
^
medicine, especially during jungle warfare, ?s was
-
recognised in the In do Burmese theatre in the
Second World War .
Considerable differences oust among different
strains of O, autsugamushi in mucigenic properties
and virulence, a factor that OntplicMn Htodiagnosis
and iinmunopropbylajciH . Three major antigenic
Kata ,
—
types have been recognised Karp, G illi a in and
GENUS EHRLICHIA
Ebcliihiac arc small , Gram negative , obligately
inrracclluar bacteria which ham im iffinitv towards
Jr
blood cells . In the cytoplasm of infected phagocytic
cells , they grow within phagosomes as mulberry^
tlke clusters called aiomla ( morula , meaning
mulberry). They lire tick - borni:. Similar organisms
under the names of dnaplasroa, C'oWn? and
NeorickemiiU had long been known to veterinary
scientists an etoLugio.1 agurtv of ticldjome infections
ot cattle and sheep. Three human infection ^ caused
by this group id organisms have been identified .
The first of these human diseases, reported from
Japan in 1954, was a case resembling glandular
fever who showed serological response against the
agent of canine ehrlichiosis. The etiological agent
lias been named Ehnfichia tenuttsu (from ' jennefinf ,
dxJlpfllKK word fetf glandular fever). It is endemic
in Japan and parts of South East Asia. It causes
lymphoid hyperplasia and atypical lymphocytosis.
No arthropod vector has been identified. Human
infection is suspected to be caused by ingestion of
fish carrying infected flukes.
The Second type of infection is 'human
mtmocvtic
J
ehrlichiosis’ caused by F . c/wffCCThtJS.
r
It is transmitted bv Amblvomma ticks. Deer and
J
rodents are believed n be reservoir hosts. Human
: disease LH associated with leucopema , thrombt)'-
cytopema and elevated liver enzymes. Multisystem
involvement and fatality may occur .
» 417
The third is "human granulocytic ehrlichiosis’
caused by an organism either identical with or
closely related Co the eqUiOC pathugen , £. epur
(probably £. phngwYtophila ) - lr is transmitted by
Lcodes ticks. Deer, cattle and sheep are the suspected
reservoir. Leucopenia and thrombocytopenia me
seen in ] i .U. icuts . < ocmra stained blood dims muv
show morula form ot the ehrlichia.
LJowcvcline is recommended for Ireiirmcnr of
ehrlichioses.
La hunt Dry diagnosis ; Rickettsial diseases
may be diagnosed in the Laboratory either by
isolation of the rickettsiac or by serology. As
riekerfSUK are bigblv infectious and haw caused
seven!ct- riousond fatal infect ions among laboratory
workers, their Isolation should be attempted with
utmost care and only LII Laboratories equipped with
appropriate safety provisions.
Rickettsiae may be isolated in male guinea pigs
or mice from patients in the earlv phane of tLac
disease. Blood dot ground in skimmed milk or any
suitable suspending medium is inoculated
iittrapcritooealb The ANIMALH have to he observed
tor 3 -4 weeks and their temperature recorded daily .
Their response to rickettsial infection varies. In
Rocky Mountain spotted fever, guinea pigs develop
fever, scrotal necrosis and may even die . With R .
iyph ) . R , conori and R jJatn. they develop fever
and tunica, reaction . R , prowazekii produces only
fever u lrhouranv *
testicular inflammation. Smew
from the peritoneum, tuuia and spleen ot infected
animals may be stained by Giemsa or Gimeoez
methods ro demonstrate the rickettsiac-
Though laboratory strains of rickettsiac grow
profusely in the yolk sac of chick embryos, this
method and tissue culture are not suitable for
primary isolation. Egg and animal inoculation
methods have been replaced by the faster and more
sensitive cell cultures. Rickettsiae grow well in 3
to 5 days on Vcnocell MRG 5 cel! cover slip
cultures and can be identified by immuno -
fluorescence using group and strain specific
monoclonal anti hod La.
Copyrighted material
418 * Textbook ot Microbiology
Seruhigical di :i gnus i £ may be bv the hcDfroplnlf
Wei I- Felix reaction or by specific tests using
riduttiial antipens. Tk Wcil-Fclbc reaction is an
1
agglutination lest in which sera are rested For
-
agglutinins to the O antigen* of certain n " LtiiotiLe
Proteus strains OX 19 f OX 2 and OX K. The test
was developed from ( he chance observation of Weil
And Felix (1916) that a Proteus strain isolated from
the urine ( lf ; L patient of epidemic typhus was
agglutinated by the patient 's serum as well as by
the sera of other typhus patients. Tlte basis of die
test is the sharing of an alkali stable carbohydrate
antigen by soinc rickettsime and by certain strains
of Proteus, P. vulgaris OX 19, and OX 2 and P.
tnifabilis OiX K , The test is usually done an a tube
agglutination, though rapid slide agglutination
methods have been employed for screening.
bera From epidemic and endemic typhus
agglutinate OX 19 and sometimes OX 1 also. The
test is negative or only weakly positive in llrill-
Zin ^scr disease. In Hckbome spotted fever both OX
19 and OX 2 arc agglutinated . OX K agglutinins
are found only in scrub typhus. The test is negative
46.2 ) . ^
in rickettsial pox , trench lever , and. f lever ( Table
The Weil-Feliit reaction is a simple and useful
teat for the diagnosis of some rickettsial diseases.
The antibody appears rapidly during rhe course ot
the disease, reaches peak titres of upto 1:1000 or
1: 5000 bv the second Week and declines rapidly
during convalescence. False pOfltiVC reaction may
occur in some cases of urinary or other infections
l1
-
Table 46.2 WHII FBIIX reaction In rieksttaial diseases
DiSCMSC
OX 19
Epidemic typhus + *
—
Brill Zinsser disease Usually negative or
Endemic typhus
Tkkbornc sporred fever
4
-
HI +
+
-
# ±
Scrub typhus - -
by Proteus and at times in typhoid fever and liver
diseases. I fence it is desirable to demonstrate a rise
in titre of antibodies Fur the diagnosis of rickettsial
infection.
The most Frequently used serological method
using rickettsial antigens is the complement fixation
test. This may be done using the group specific
soluble antigen , or the type specific washed
rickettsial antigen. The former lest is- in routine
use but rhe latter is neces& ary for differentiation
between epidemic and endemic typhus . Other
serological tests me We agglutination of rickettsial
suspensions, passive hemagglutination of red cells
sensitised by ESS ( erythrocyte sensitising
substance) , toxin neutralisation , immuno
fluorescence . Hid radioisorope precipitation.
-
Immunoprophylaxis: Rickettsial diseases may
be prevented by general measures such as control
of vectors and animal reservoirs. Immunisation is
useful in special situations. Killed ami live vaccines
have been prepared against epidemic typhus . "Hie
earliest of these was phenoliscd intestinal contents
of hce infected pet rectum with R . ptvwfs kii
^
( Weigl 's vaccine ). This was too complicated for
mass production Castaneda developed a
,
formal!nised mouse lung vaccine . Effective
vaccination became posvihlc only after Cox
developed the inactivated yolk sac vaccine, A live
vaccine using rhe attenuated strain This been found
to be highly immunogenic hut a proportion of
vactinees develop mild disease. The Coot type
vaccine has also been prepared against Rocky
Aprfurimtiofl pattern with
OX 2 OXK
-
^ ^trve
r =
+ +
-
-
+++
Copyrighted materia
 * Ricketlsiacoae
MLivnttin spotted fever. However, there is nu
satisfactory vaccine available against any of the
rickettsial diseases,
GENUS COXIELU: Q FEVER
Derrick ( 1935 ) investgating an outbreak nt
typhoid-like leVtr in abattoir workers in Brisbane,
Australia, transmitted rile infection to guinea pigs
by inoculation of blood from patients. As the
etiological agent of the disease wan unknown, it
was referred to as 'Query ' or fever. As Burnet
identified the causative agent an a rickcttsia , it was
named R, burnetii. At about the same time, the
same agent was isolated independently by Cos in
the USA from ticks, and was called R , diaporica,
the name referring to its ability to pass through
]Hires of filters impermeable to other rickettsiae .
The Australian and American strains were
subsequently shown to be identical. As the Q fqver_
agent differed from other tickettsiae in many
features {smaller size, greater (WttUKC It ) beat and
drying, major transmit on route being inhalation
erf ingestionf (dependent uf arthropod vectors ), it
has been separated from rirkettsiac into a special
genus and rennmed Coxiclh burnetii. It has been
^
assigned to the group Vofo/Meferhtalongwith other
genera like Legionella and RrancisdU
_
Q fcvcr is distributed worldwide, as a zoonosis
solidly established in domestic livestock. Wild
animals such as the bandicoot may be the primary
reservoir. It is transmitted among them , and to cattle,
sheep and poultry by ixndid ticks . Transnvarial
transmission occurs in ticks . Cordelia are abundant
in rick feces and survive in dried feces for long
periods . They arc shed in the milk of infected
animals . They are particularly abundant in their
products of conception and con turn in ate the
environment parturition ..
lit
Human infection TTULV occur occupationallv
through handling wool or hides, meat nr other
animal ]>roducT £ contaminated with the organism .
Drinking infected milk can transmit the infection,
Coxiella may enter through abraded skin, uUIOOSa ,
419
Lungs or intestinal tract . Person- to- perion
transmission is a rarliy, Ticks do not seem to He
important in human infection.
Cur. burnetii is widely prevalent m birds and
animils in India , as shown by serological surveys,
hut human disease has been identified only rarely.
The human disease is ail acute systemic infection
characterised by an interstitial pneumonia The
clinical picture is very variable and asymptomatic
infections very common . In chromic Q^ feve, tilt
coxiella spreads through almost all organs and may
cause hepatitis , meningoencephalitis or
endocarditis . SpctlltaiXOtll recovery is usual . The
efiodella may remain latent in the tissues of patients
tbr 2-3 years .
Co* , burnerjj Li an obligate iutracellular
pathogen, i > rim.arLIv infecting the monocytc
macrophage cells. It occurs as rods OJA).4 pm x
-
0.4~l ,0 pm or as rphdej ( ]..SA >. 4 pm in cliameleT .
'
It is filterable . Generally regarded as Gram
negative, A . it is better stained with Gimme? and
other rickertsiac strains
In dried feces or wool it survives for a year or
more at 4 "C and in meat .it least for one month . It
is not completely inactivated at AO "C or by l 'ft
phenol in one hour . In milk it may survive
pasteurisation by the holding method, but ltic tlash
method is effective. It grows well in the Volk SAC of
chick embryos and in various cell cultures.
Cm, burnetii shows phase variation. Fresh
isolate - arc in Phase I . It becomes Phase II on
repeated passage in yolk sac, but reverts to Phase i
bv passaging in guinea pigs . Phase 1 cells are
autoagglutirtahlc and are phagocyiOScd in the
absence of antibody. Phase [ activity is due to a
periodate-sensitive trichloracetic acid- soluble
surface carbohydrate. Phase 1 is a more powerful
immunogen than Phase II and elicit* good antibody
response to both 1 and II antigens. Phase 11 antigen
is more suitable for complement fixation tests . Q _
fever sera do not cross - re aft with rickettsial or
proteus bacillus antigens.
Lab oratory diagnosis is by serology, by
Copyrighted material
420
complement nation or indirect immuno -
rluoresceiiLL: assay. Isolation of the conic! la from
blood , sputum or other dimeal specimens is
possible, but is run recommended due to the baiard
of laboratory infection -
r
Vaccines have been prepared from formal in
killed whole cells, trichloracetic acid extracts and
attenuated strains , hut they are not for guiLLTiil LLHe.
Treatment is with doxvcrcline. In endocarditis
I Jt
prolonged treatmentwith enmhi nations of
tetracycline , cotrimoxazolc nr ritampicin may be
'
required.
BARTONELLA
hartnnellac are tinv Gram negative bacilli Listi .LLIV ,
transmitted Hv arthropods, which invade
mammaEian endothelial cells and blood cells,
Homan pathogenic strains are B . btaffifbaais,
B, quintMfix and fi . hensdx. The genus contains
species causing a number of tickbome fevers or
animals. Eilmtificution and classification of members
ot bartoncllac , rickettsiae , chlamydiae and related
'
bacteria often depend cm sophisticated molecular
methods tike lb? SNA analysis. Is
BARTON H I. LA B : i 1.1 JFORMIM
In 1870 , when the railway line from T . ima roOrovy
in Ptru was hting built, an outbreak of fever IdUcd
(huusaiid ^ oi workmen. The disease was called
Omyir /era; which was seen in the mountainous
parts ot Peru , Columbia and Ecuador in South
America. Some of the survivors developed nodular
ulcerating & kin lesions, called verruga peruansL The
common etiology ol ihese two conditions was
established tragically in 1385 by the Peruvian
medical student Daniel Carrion . He inoculated
himself with material from verruga and developed
, The chronic intention and late relapses help to
Oroya lever from which he died , Oroya fever is
therefore also known as Carrions disease.
Oroya fever presents as lever and progressive
anemia due to bacteria] invasion ot crvthmcvtcs. m J
Mortality is high in untreated cases . A late sequel
* T^ jrtbo & k of Micr 60iolM] y *
in survivors or in fh < ne with asymptomatic infection
is vmugtf peruana. B . buciUifbnms is seen inside
erythrocytea and in the skin leaiutis . li is a
pleotnorphoie Gram negative rod, which is motile
bv u tuft ol polar flagella . It can be cultivated in
semi solid. agar with rahbir or human blond .
BARTONBU - A ( ROCHAMMABA )
QL: II\ TANA
During the E'irst World War, osier u million cases
of a disease known as trench fever or Bvc -day fever
occurred among soldiers fighting in the Trenches
in Europe . 'Hie disease was not fatal hue because of
ii ^ sloiv course and prolonged convalescence , it
caused very considerable loss ot manpower.
Trench fever i * ? n exclusively human disease
and no animal reservoir is known. It is transmitted
by LLIR body louse . The feces of lice becomes
infectious 5-10 days after an infectious meal . The
lice arc unbanned and remain infective throughout
'
tbeir lives. Vertical transmission does not occur in
lice.The causative agent was identified as a tidoetisU
and nacneJ R . quinfana Ilnur tjuintun.- i , meaning
fifth , referring To ' five -day fever’, a synonym for
trench fever ) . As it wag found ro differ from
rickettsiae in a number of respects, including its
ability ro grow in cell - free culture media such as
blood igar, it was separated into a new genus
Rochalimaca. ( nfter da Rocha Lima , an cirlv
Investigator ot rickettsial discuses ), In a subsequent
taxonomicaE s h i f t , i t h a s heen icclailLflcd ns
Bartonella and named B . tfuiawA,
rhe disease Jicqucndy leads to ; L chronic or
latent infection. Recrudescence mavr occur as in
Hrill -yinsscr disease ansi relapses have been
reported as king as 20 years after tlie primurv disease.
maintain the bartonclla in the absence of animal
reservoirs.
french fever was thought ro have vanished With
the world wars. But isolation of li . qtimraria from
Tunisia and Mexico recently suggests chat the
Copyrighted materia
 i R : d k en si ace a e » 421

clitica ?iHc may be more widely distributed than is
realised . Trench fever cases have been identified in
some homeless persons living :Ln unsanitary"
conditions in the USA,
agent. It can be demonstrated in lymph node biopsy
smears and sections by Warthin-Srany staining.
J3. henselae has been also linked with two other
conditions, seen more commonly ifi HIV infected
and other immunodeficienr persons. These are
*

BARTONELLA HENSELAE fwc/J/ajy angiomatosis, in which vascular nodules

A febrile illness with lymphadenopathy following or Tumours appear on the skin, mucosa and other
a cat scratch had been known for long under the location ? and hacittary peliosix Involving the liver
!

name "cat smteh disease * but its etiology remained and spleen.
elusive. R- hCMCkc has been isolated from tin? blood Angiomatosis may also be due to B, quintans in
of patients , in blood media after prolonged some eases,- Another organism Atijpta fili* had aJso
incubation and is now considered as its etiological been proposed as a cause of cat scratch disease .

further LICJILI IUP!

-
B

Baca O G and D Paretsky If S3. Qjtvei and Cobefr faroecfe

1
-
Ad a I KA . |W 5 BanoiKlb: N*w iptdtt ami new diaca.tc. fici. Med
Rev
6:15 S.
47:137,
BuL[iii 1' ci aL. Iff 5- Human grinulncyvc chrLichiosis-. Lancer 346:782.
I'lshbean [IB ef al , 1994. Human EhHkhiout, Ann jni Mod 120173$,
KwhlarJE ct nb 1994, Wi ^JjuJrrn ±ej Aensejfjc infecrirm. J Am Med Assoc 271:531.
Muffin M mi D KwUk 19OT. QJkmr Cihs Microbial Rev 12 518. =
Haoult D ;uid V Roux 1997, Rkktdmss as paradigms rtf new nr emerging infectious diseases. CKoMaotial jKor 1Q ;694,
|

ScMttMG, lW8 A hifBOfy of taftoodlpm [CliriQn disease). AmJ Trap Med 17;503.
r
^
Walker DH andlJS Dnimiar I 996 Envcrgence of Ehrlichiosis at a human health problem. Lmcnjii r Tn / rtT Dtf Nol.
r
^ ^
Wnhlmd TTE, WTO, A himrleal mwiM: vf riekirnsial diseases with a discussion of the involved prnhJcTTus. ,/ inf Dm
127:585.
ZbUKT H . 1935, tfatfc HLM arici J iisiofw London: George, Routledpr -and Sons Ltd..
*

Copyrighted material
 Chlamydiae
Chlamydiae arc obligate intracellular bacterial
piiJHMCfH <> t limuajiy , LLiUmuls and birds* With tfupkiti
for squamous epithelial cells and maerpphages of
the respirator)' and gascroinEegtinai tracts. Due to
their fikerability and failure tn grow in cell-free
media ^ they were considered to be viruses. Based
on the human disease thev were then known to
ir
cause, they were called psittaeo ^ is -
lymphogranulom a - trachoma ( PLT} viruses, or
noncommittally as TIT agents ' . However, they
differ from viruses in many respects. They possess
both DNA and RNAt have cell avails anti
ribosomes , replicate by binary fission without an
‘eclipse phase ' , and are Susceptible to the usual
antibiotics and chemotherapeutic agents . They arc
.
therefore accepted is bacteria. Unlike other bacteria,
thev d ( ] not have peptidugLycan cell walls, Thev
lack enzymes of rhe electron transport chain and
so require ATP and outrtenr resources from host
cells . They have therefore been called energy
ptnsitts,
hi recognition of the pioneering studies of Sir
Samuel liodson on psittacosis, the name Bcdsunix
was proposed for this group. However, they are
now officially classified ns- bacteria belonging ro
the genus Chlamydia , in the family Chlamydiaceac ,
under the order Uhhimydi tlcs ..
The genus Chlxmydia contains touf species :
— £ aacbtmiMtis , C. pstemci, t\ pneumoniae, which
can affect humans; and [ he fourth species , C.
pecarum created recently to include some Strains
effecting ruminant?. Species differentiation is based
on growth characters, nucleic acid profile, antigens ,
plasmids arid nature of the inclusion hndy.
C. rmc-homarfs strains form compact indsuns
wLtk t !ie glycogen matrix , are sensitive to
sulphonamidcs, and BA natural parasites of humans,
usuallv causing locdlHd infections nf the eyes and
genitals . C . pejfTad strains form diffuse vacuolated
i delusions wi thoot th e glvc ^ igcn matrix, a re ncsi stSITl t
tosulphonamidcs and arc natural parasites of birds
and animals, capable of causing pneumonia and
generalised infections in humans . C. pneumoniae
is ,in exclusive human pathogen with no animal or
avian host. It is a common cause of acute respiratory
disease worldwide .
Morphology and growth cycle:
Chlamydiae occur in two forms , the cJcmc/lfary
tody and the reticulate body (formerly also called
[ be 'initial body ) The elementary bnrly is the
'
extracellular, infective form . It is spherical particle,
200-300 nm in diameter, with a rigid trilaminar
cell wall similar to the celt walls of C.Sram negative
bacteria, and an electron dense nucleoid . The
reticulate body is the intracellular growing and
replicative form, 500-1000 nm in size. Its cell wall
is fragile and pliable , Leading to pleomorpbism.
Infection is initiated by the attachment of the
elementary body to the surface of a susceptible
epithelial cell , followed bv its codocytosu ( Fig.
47 . Ij. Inside [ he host cell , the elementary body Lies
wirhm the endosontr, IJC Lng separated from the host
cell cytoplasm hy the crtdosomsl membrane
throughout its active growth cyde. By about eight
hours, the elementary body within the endosome
undergoes sphcrnplast - hkc transformation to the
large retieulare body, which begins to divide bv
—
binary fission by 12 hours . By 20 24 hours, the
Copyrighted material
 H Chlamydias 423

@
© © © c9
2 3 U 5

I
1
c o5> co
a
dy
<W
7 G

. -
L Elenwntir)' body t EBi: 2 E $ aJtwhssx to* Lril rcceplnr; J. Eii crtim cell fey cwkscyiMis. By ft ImirsL 4. EB reoffuiKd
'

. .
inut MIIIIIAI.^ ( RBJ: 5. Hast cell growtih in?%j by 12 twine R.R wnkqspMnj finim fis . By 24 tanmv nndunion body wish RR .
j!xl (tevdnpinif Eft ; 7 . By hnun. uicliiAiofi ftuly tonmjiinj;. imfcoHUb EB. Nuclrus pus&tard to periphery: ft, Py 4H hnun .
.
dejim iiiJ lysis v4 L'CII releuMng bft

Fig. 47.1 Reproductive cycle of Chlamydia

pEeomorphLC progeny show central condensation
and are converted to elementary bodies. Binary
fission continues till about 40 hours . The
developing chlamydial microcolony within the host
cell is called the inclusion body,;. The mature
•f —
inclusion bodv contains 100 500 demetuarv . bndics
which arc ullirn &tcly retewd 1 i"m itr ^- l cell.
t
! : ho
/ contribute to the pathogenesis of chlamydial
In C . prittaci infection -, the host cell i > seventh'
damaged and release of the elementaly bodies
occurs wi t hi i : 48 hours by host cell lysis. With £
trachomatis* the mature inclusion appears rn hr
During the derive intracellular growth of the
organism, the chlamydia specific lipopoly’
sac . h .iriiilcK accumulate on illc hofft cell surface ,
This highly material induces
antigen i *
inflammatory and immunological responses which
diseases.
Chlannvdiac ran he propagated in the mouse,
clhck cmhrvo or in cell culture though they show
individual variations in susceptibility.
1

Kesistnnee: Chlamydiac arc hear labile, heing

exocytoscd in 72 6 houss, the f y i - c cell being h'lc
^ inactivated within minutes at 56 :'C . They arc m

with a scar ( Fig- 47.1 ). susceptible to ethanol , ether mid low concentrations

Copyrighted material
424
Table 47.1 Human diseases caused by Chlamydiae
Species Serotype *
a trachomatis A, B, Bi, C
C tr*chom*ti* .
D F . RCL H, 1J, K
C fradbomaris l . l . Id, U
C. psittsd Many serotypti
Cl pncuimmiRC Only one serotype
- |--
" J HTULLH 111 ri J.1111 [ v n jSMitLitrd ivilh I he cliscjM!
of phenol and formalin. Jnfectmty is maintained
for several days ai 4 "C . They can be preserved
ftazen at -70 C or lyophilised.
"
Antigenic properties;: Chlamydiae possess
three. major kineb < if antigens. The first is the heat
stable , genus specific a citizen common to ill
chliirmhie - Thiii LH a lipupdysaechstridt resembling
the IPS of enteric Gram negative bacilli. This is
present in all itages of the developmental cycle and
car he identified by the complement fixation tesr .
The second type of antigens are tile speciej fpccifK
protein antigens pnesenr at rhe envelope surface .
- and ( 4 ) demonstration of antibodies or
These iire present in all strains of a chlamydial
species . They help in classifying ehlamydiat into
—
the species Dadnuntr
^
paevtaoniaeand
pGCOrtltti. Tim third kind of antigens help in
inrraspecies typing* as they are found only in some
members of a species. They are located on the major
u u r e i membrane proteins ( MOMP ) anti c m be
demonstrated by microimmunofluoi'C WClCC, by
micro- IF* chlamydiae have been classified into
many serological variant* ( licrovars , semrypes ).
C- rfircfiomarjs is classified into two broad
brnv.irs ( biological variants ) which cause trachoma,
inclusion conjunctivitis ( TRIG } and
lymphogranuloma venereum ( LGV ) , respectively.
The TRIG biovar has been cllassLtied into 12
serovars A , H , Ha and C causing Minding
trachoma in endemic areas, and serovara D to K
associated witb the less serious occukr infection,
inclusion conjunctivitis and with various genital
infections, Scnwars 1.1, L 2 and L3 cause LGV and
* Textbook oi Microbiology *
Disease
Endemic blinding trachoma
Inclusion eofijimcriviris fneonitil and adult )
Genital chlamdiasis
Infant pneumonia
Lymphogranuloma venereum
Psittacosis
Acute respiratory disease
hemorrhagic proctitis.
The serological classification of C p&ttKi in
complex, many Serotypes having been identified -
C . pneumoniae has not been subclassifted as only
one serotype is known ( Table 47.1 ) .
Laboratory diagnosis : Four approaches are
available for laboratory diagnosis of chlamydial
infections: ( 1 ) microscopic demonstration of
inclusion or elementary bodies ; ( 2 ) isolation of
chlamydia; ( 2) demonstration of chLamydinl antigen
hypersen si tivi tv.
Chlamydial elementary bodies and inclusions
are large enough ro he seen under the light
microscope . Chlamydia are Gram negative but arc
Stained better bv Gienisa , Castaneda, Mjchiavcllrs
or Giminea stains . Microscopic examination of
^ ficmsa Gained conjunciival scrapings for the
inclusion bodies is useful in the diagnosis of ocular
infections particularly ir neonatal inclusion
conjunctivitis . Because of the glycogen mitri* uf
C. tnchomariJinclusions, they can be stained with
Lugol 's iodine . Iodine staining of conjunctival
scrapings has been used as a rapid and simple
screening method for trachoma and inclusion
conjunctivitis. However, irs sensitivity is poor as
iodine staining occurs only in certain stages of
development of the inclusions. It is, however, useful
in the rapid screening for chlamydial inclusions ir
cell cultures inoculated with clinical samples . Iodine
staining is not applicable in C , ptittxd because its
inclusions do not contain glycogen .
Copyrighted material
Hidden page
426 * Textbook ol Microbiology
or [ype-ipioc nuicrtf -IF. The CF rest is used
mainly in invasive chlamydial Infections
psittacosis and I ,CIV. A fourfold rise in titres is
— mechanically. If may also be carried by dust, in
diugrtostic- AH low titrv, grnup- specific .intibody
-
ITLJV be present m tliL sera of many persons due to
exposure to other chlamydias, a single CF antibody
test is not diagnostic of psittacosis nr I ,GV unless
the rirn: is high - 1 :64 or greater. CP rest is of
tittle value LII TR 1 C infections. i ] L which iniero-IF
is more useful. Micro- IF tan test IgG and IgM
nntibodv sepa rifely. Titres of 1 or gtiea ter a re u &n ,il
in infecred persons , Enzyme immunoassays also art
xvailubfe.The initial antibody response is IgM ,
which is replaced by IgG after about a month .
Recurrent infection with the same serotype induces
only IgG response . As llow tirre antibodies are
frequently seenin healthy individuals,, the diagnose it
criteriiL lor serology are Seroconversion, fourfold
rise in IgG t it re or presence of JgM antibody. High
title antibodies are usually Seen only ill infant
pneumonia , ntpuipris and LGV,
Demi nutrition of hvpersensi tiviry bv shin ten ting
Fnci 's test ) w .ts widely used formerly for diagnosis
^
of LG V but has been given up because false positive
results arc very frequent.
CHLAMYDIA TRACHOMATIS
C , aachotnatis is a leading cause of ocular and
genital infections worldwide .
Trnthomn: Trachoma is a chronic keratocon -
junctivitis characterised by follicular hypertrophy ,
papillary hyperplasia , panrus Formation. and in rhe
[arc stages, cicatrisation. 'Hie name trachoma is
derived from [ be Grech rraAJzus ( rough ) referring
to the roughness of the conjunctiva in the- disease.
Though Halhcrstaedter and ProwiL7.rk in 190"
transmitted the infection to orangutans and
demonstrated in conjunctival smears the
characteristic inclusion body that bw their names,
cultivation of the chlamydia became possible only
half a century later, when Fang and colleagues
(1957) grew it in the yolk sac of eggs.
Infection is Iran 5 m it ted from eye - to-cyc by
fmgtre or fondles . FtieS may Transmit the infection
which case infection may he facilitated by minor
abrasions caused by dust particles. The incubation
period is Variable and influenced by the dose of
infection. Onset is insidious.
Trichoma has been classified into several StagCS -
Thc earliest is trachoma dubium, where the disease
is jutf a suspicion. Protrachoma is the stage of
Conjunctival lesion before follicles become visible
The inclusion bodies arc nor usually demonstrable
in these early stages . Established trachoma
progresses through stages 1 — IV. Infectivity is
maximum in the early cases . Stage IV is
non infectious.
Laboratory diagnosis : The characteristic
IOLIUMDIIV ( HfJbcn&cdter Avuacdt or HPbodiet )
maybe demonstrated in conjunctival scrapings, after
Staining hy
Gierma, Castaneda or Machiavcllo
methods. Because they possess a glycogen matrix
they may be stained with iodine which enhances
the sensitivity' of smear diagnosis.
The chlamydia may he grown in the yolk sac of
6- ft days old eggs. Tile material is treated with
streptomycin or polvmyain B before inoculation .
The eggs are incubated af .15 ; C in 3 humid
atmosphere. Blind passages may be necessary for
isolation. This method is seldom used now as it is
rime consuming, cumbersome and relatively
insensitive,
I issue culture using Stationary phase cells
( ncmreplieating cells) is the method of choice for
isolation, McCoy cells rendered nonreplicating by
irradiation nr anrirnctaboiitcs arc used. IIcLa or
HL «11* treated with DEAE dextran may also be
used. The inoculum has to be driven into the cells
bv centrifugation upco 15 ,000 g to get a good
growth.
TrciHmcnt ; Local application and oral
administration of erythromycin and tetracycline or
other suitable antibiotics should be continued for
several weeks. A sirglc-dosc azitromycin treatment
has been used with good results.
Copyrighted material
Hidden page
42S * Te- n tbook ot Microbiology
LYMPIItHiKANl t.OM\ VBNEREUM
' ['
hi ' sexually transmitted disease, dunmtrisfd by
vUppurariw in uinul adcriritls , has been known in
^
the tropic* fhr a long time tnielt- r various names:
lymphogranuloma inguinale, porademns, climatic
tjftropical bubo . [ r is caused by the LCV serOvar;
of Chfoin, trachomatis , Ll , L2 and LJ most
commonly L2. LG V morns arc more invasive than
— cultures ( Lvgranum ). was inoculated irtradermally
P
the: other i m mu notypes- of Cfrfam , traduffilltij,
Their preferred site of multiplication is the regional
Ivmph node* , in contrast to TR.IC serovaiv which
grow in epithelial cells.
fhe primary lesion is a small painless
papulovesicular lesion appearing on the external
genitalia ( or rarely extragemral sires ) after an
incubation period of three days to five weeks. The
secondary developing about two week * later
results from lymphatic spread io ihc draining
lymph nodes . In men tbe inguinal Ivrnpb nudes .Lfc
involved most often and in women the intrapclvic
.
and pararectal rtoda Women and. homosexual men
may develop hemorrhagic proctitis with regional
lymphadenitis . The nodes enlarge , suppurate „
become adherent ro the skin and break down TO
form sinuses discharging pus . Mel as lull C
complications may so me rimes occur, with
involvement of joints, eyes, and meninges, The
rvrriiirv stage is chronic, lasting kn several vCiTi,
representing tilt Hqudacof scarring ami lymptntk
blockage* Lite sequel at' are more distressing in
women leading to rectal strictures and elephantiasis
( if the rtilva (esihiomene ) .
Laboratory tUagrwsii: The primary legion
usually goes unnoticed and the disease is seen
commonly first in the stage of inguinal adenitis
( bubo) . Smears of material aspirated from the buboa
may show fhe e lenten fare bodies ( Miyag mv.i 's
granulocorpusclcs) . The sensitivity of microscopic
diagnosis is very low. Isolation of rbe chlamvdia bv
intracerebral inoculation into mice and info yolk
sac of eggs has been replaced by cell cultures. LGV
-
patients develop h igh title - cJ L irvulati ng an tiln ulies ,
'
with litres of 1:64 or more in CF rest and 1:512 or
more in micro- IF. Serological diagnosis LS
therefore feasible
A a iittrftdeful resL originally described by Frei
in 1925 was commonly used formerly Tbe crude
chlamydial antigen originally obtained From the
bubo pus, and later from mouse brain or yolk sac
in the forearm, with a control on the other arm.
Induration of 7 mm or more in 2-5 days was
considered positive . .[ Jue to rbe frequent <>ccurrence
of false positive reactions, Frei’s test is now not in
use. Treatment is with tetracycline , which should
be given (or ar least three weeks.
CHLAMYDIA PSITTACI
PStTTA * Q 9 J S
Psittacosis is J disease of parrots ( pwfWCOS means
parrot ) and ocher psittacinc birds, transmissible to
human beings. A similar disease acquired by
nnnpsittucine birds wus called ornithosis- ! vfnillnw
meaning birds) but the distinction is now no longer
employed, both conditions being called psittacosis .
Infection in binds is usually subclmical leading
to a carrier state . Overt disuse may be ]>ieci|uraced
by caging or overcrowing and is manifested as
diarrhea, mu copurl ueiii respiratory discharge and
emaciation. Chlamydia are shed in the droppings
OT nasal discharge and aerosols are liberated.
Ho mam in lections are mostly OOCupacibfiaJ, as HI
poultry workers, pigeon farmers, petshop owners,
bird fanciers and veterinarians . In lection is by
inhalation. Rare cases of infertinr by parrot bites
have been repotted. Consumption nf poultry
products does not lead to infection - Case to case
transmission in, h u m a n* i * rare but has been
recorded. The high injectivity of psittacosis is
indicated by the frequency of laboratory infection ^ ,
Strains from parrots and turkeys are more virulent
than those trnm other avian sourer*.
I hc incubation period ts about ter days. Clinical
disease varies from a mild influenza - like syndrome
J
to a fatal pneumonia. Though pneurncmig is the
Copyrighted material
 1 Chlamydlw * 429

usual clinical manitestation , psittacosis is a called C. pneumoniae .

lepbumu and may lead to meningoencephalitis,
endocarditis,, pericarditis, arthritis or a typhoid -like
syndrome.
.S
Laboratory dJagnotii: The chlamydia can
be -soiatcd from blond (iurmg the early stages ol
i the world . Its clinical spectrum includes
the disease and from sputum later on. Infected cells,
including Jveular macrophages from patients, and
mouse bruin , yolk sac and cel! cultures show
—
inclusion bodies ( Lc ^ in.ftiaJ ^Coic L. illic or LCL
hodteiO . These differ from C. trachama '. > nuiluHtJn
in being more diffuse and irregular, not stained by
iodine and nor inhibited by sulphadtazinc or
tycloheri ne. Tt LH generally difficult to recover the
chlamydia from patients treated wirh antibiotics -
Isolation should he attempted only in laboratories
where special containment facilities are available ,
ns laboratory infection is a Serious hazard .
Serological diagnosis may be made by the group
.
specific CK test or type spc ific micro IF.
It appears to he a common cause tif respiratory
LI ^ casc in older children and adults worldwide .
Antibodies have been demonstrated in the sera of
about 50 percent of adults from different parts of
pharyngitis, sinus- iris, bronchilis and pneumonia ,
which resembles Myvop IFJTIJ pneumonia. It has
^
also heeo associated with aduit onset asthma. 1 he
incubation period is 1-3 weeks. Outbreak* have
been reported in closed communities. Primary
infections occur in young children. Reinfections
are common. Serum antibodies do not appear to
he protective.
I ) iagjiQsis i ' ;IY antigen detection by F.IA, Llirect
immunofluorescence or molecular methods , as
isolation of the organism is very difficult ,
Serodiagno& is is by CF, ELISA or micro- IF.
Treatment ii bv one of [ he new microlide
-
n

antibiotics like clarithromycin or azithromycin.

CHLAMYDIA PNEUMONIAE
Grays ton and colleagues ( 1986 ) isolated a
ctikmydial strain from acute respiratory disease in
adults in I '.uwan and designated it as C.
strain TWAR (from Taiwan Acute Respiratory) . Jt
Considerable interest lias been aroused by recent
reports linking C. pneumoniae with atherosclerosis
and iu clkucdl effects like comnan; carotid and
cerebral arterial disease . Apart from scroepidemio -
logical evidence., the finding of the chlamydial
antigens in the isolation ol the organism from
1

possessed the group -specific antigen in common coronary artery atheromatous plaques , and the
with C. psjttaej and C, trachonwti' but could he
distmjjuishcd from both of them by species- specific
- experimental induction id atherftffia in rabbits
infected with the chlamydia strengthen the
,

antigens , DNA hybridisation and restriction .

endonuclease analysis . I" his appears to be an
exclusively human chlamvdia transmitted from
niff" IB
human to human without anv avian or animal host -
.iff
It grows poorly in. cell cultures. Because of these
property it has been classilied as a separate Species
U»cintlDD Early results © f anti ^ chlamydial
intervention studies in experimental animals and
human volunteers also arc consistent with a
causative role of C+ pneumonia*? in vascular
atheromatous disease , However, more wM > rlk will
have no be done before the issue is Finally settled.

FurLher lie -idling

Beatty W[ et at. 1994- PeraisierU CBd ^ rhydi ^ir . MktfojfefoJ Rev S£i:& K6-
J

KnuMpinen M and. P Sajklui IW5. ftwumanid AM fo Chlamydia pneumoniae. Cfa Infixt Dtf 21 Suppl . 3, S-244, "

Peding RW in4 RC Bnitihani 19%. ChLimydiae *s pathogens Emerging Infect Di* 2:4,
.

Wefiefiack 11 cc al. 1W4. C . mchumazis infection*. Intfecr Dis Clin N Am 8:797 ,

py^ righted material

1 1
 General Properties of Viruses

Unicellular Ttntronrganisnis may he classified in Viruses occupy the twilight zone that separates
-
descending order of complexity as i uJfcaiyorejj, such ihe 'living" I TO 11 the ‘nonliving’. The demonstration
]

as protuzua mid fungi „ and ptoLtryoicy , such as
bacteria , mycoplasmas, rickeltRiae and chidmydiac -
Viruses do not fail strictly into the category of
unicellular microorganisms as thev tlo not possess
a cellular organisation . Even the simplest of
microorganisms are cells enclosed within a cell wall ,
containing both types of nucleic acid ( DNlA and
bv Stanley [19J5) that viruses could be crystallised
like chemicals , and the extraction by Gcirer and
Schramm ( 1956 ) of 'infectious nudeie acid' from a
virus that could infect host cells and yield complete
V i r u s progeny made it appear that viruses were only
'
living chemicals'. Recent advances In molecLLljir
biology seem to make rhe distinction between 'life: 1

KNA ) , synthesising rheir own macromoJecnlar
constituents and multiplying by binary fission .
Viruses, on the other hand , do not have a cellular
organisation and contain Only one type of duel tie
acid, cither DNA or- RN
a -
'A lmt never
.. . - both
- - . Thev
'
/ ability to cause a very large number of human diseases.
are obligate intracellular parasites. They lack the
enzymes necessary fnr protein and nucleic acid
synthesis and are dependent for replication on the
synthetic machinery of host cells. They multiply
by a complex process und no L by bii wry fission . TJ Ley
are unaffected by antibacterial antihiotiw . The majioi
differences between viruses and microorganisms arc
shown in Tabic 48.1 . In split' of these basic
differences. Viruses are generally tor side red
micrrjtjiganisms ir medical microbiology.
aiui 'nonlife' little more than a semantic exercise.
As the smallest Thing units', viruses offer the hest
models for understanding the chemistry of life'.
The medical importance of viruses lies in their
Viral diseases range from minor ailments such as the
common cold no territying diseases such as rabies or
AIDS. They may be sporadic like mumps, endemic
like infectious hepatitis , epidemic Ute dengue fever or
pandemic Like influenza. They may be localised to
circumscribed areas ( as some arbovirus diseases ) or
worldwide (as I Ierpcs simpdjex ] . Tlte control of bacte-
rial infection with anHbiotics has enhanced the role
of viral infections in human d isease, Viruses can cause
cancer in animals and birds, as well as in humans.

Table 4fi.i Propeiies of prokaryotes and viruses

CciJuljr Growth on Binary Both DXA Ribomis ScmitivifY to Sfnakiwitjr to
p

omnEAtion irtMirtwre ilmm ii /ic/ RNA \intihjctcrijj imerfemn

media zntihiotics

--—
Bacteria + 4 * + 4-
Mycoplasma* 4 4 * + * 4-
Rickettsiae 4 4 * + 4 4
Chlamvdiac 4 - 4- * 4 4

Viruses - - - - - - 4

Copyrighted material
 < General PrmcH« Q1 Viruses
MohCHOLOGt
Size The extracellular virus particle is
jrtfccrions
,
called he virion . Viruses are much smaller than
[
bacteria. It was their small size and 'filterability '
(ability to pass through filters that can hold hack
bacteria ) thar led to their recognition as a separate
class of infectious agents. Hence they were for a
time known as 'filterable viruses' - As they were too
small to be seen under the light muroscupe , they
were called 'ultrarnicmscnpic'. Some of the larger
viruses, such as poxviruses, can be seen under die
light microscope when suitably stained. The virus
particlei: seen in this manner are known as
' efemenfarv bores
J
'
Viruses widely in size . The largest among
vjrv
them ( for example poxviruses ) measuring about 300
nrn , are as large as the smallest bacteria
( mycoplasma ) . The smallest viruses ( for example
parvovirus ) measuring about 30 nm are nearly as
small us the largest proreiu molecules such as
hemocyai : i n.
The earliest method of estimating the size of
virus particles was by passing them through
collodion membrane filters of graded porosity
( gradocol membranes ). The average pore diameter
of the finest filler that permitted passage of the
virion gave an estimate of irs siac - With the
development of the ultraiccntrifugc. a second method
became available. From the rate of sedimentation
ol virus m the ultracentT i tuge, the particle size could
be calculated » dng Stokes' law, I'he th - nd and the
most (frcct method of measuring virus size is
electron jnieroseopy. Purified preparations of virions
mav be examined under the electron microscope
either unstaim.ed or stained. Lb this [rtethud , both
the shape and size of virions can be studied.
Structure and shape: The virion consists
essentially of a nucleic acid surrounded by a protein
coat , the capsid . The capsid with the enclosed
nucleic acid is known as the ilucl^vcap?. id . I be
fund ion of the capsid is to protect the nucleic acid
from inactivation by nucleases andother deleterious
» 43 L
agent* m die environment , The capsid is composed
of a large number of capsomers which form its
morphological units , The chemical units of the
capsid arc polypeptide molecules which arc
anranged symmetrically to form an impenetrable
shell around the nucleic aci .1 core ( Fig . 4fi. l ). One
of the major furcrions of the capsid is to introduce
viral genome into host cells by adsorbing readily
to cell surfaces.
Two kinds of svmmctrv are encountered in the
«F
B
capsid , icosahedrsl (cubical ) and helical . An
icosahedron is a polygon with 12 vertices or corners
and 20 facets nr sides. Each facet is in the shape oi
an equilateral triangle . Two types of capsnmers
constitute the icosahedral capsid. They ire the
pentagonal capsomcrs at the vertices ( pentors ) and
the hexagonal capsomers making up the facecs
i. hexons ). There are always 12 pentons hut the
number of hexons varies with the virus group, in
the nucleocapsid* with helical symmetry, the
capsomers and nucleic acid are wound together to
form a heEcal or spiral tube . The tube may be rigid ,
as in the tobacco mosaic virus but in die case of
animal viruses , the tubular nuclencap -- id is pliable
and may be coiled on itself , Not all viruses show
die typical iCosahedral Or helical symmetry. Some ,
'
ike the poxviruses, exhibit a complex symmetry.
Virions may be enveloped or noncnvelopcd
( naked ). The envelope or outer covering of viruses
is derived from the host cell membrane when the
progeny virus is released by budding. The envelope
is lipoprotein in nature . The lipid is largely of host
cell origin while the protein is virus coded . Protein
i- ubunirs 111 . Li be seen as projecting spikes on the
'
surface of the envelope . These structures are called
pepltlfiter* ( from jrcphin , mean 11 ILT enveli jpe J. A virus
may have more than one type of pcplomcr . The
influenza virus carries two kinds of peplomers -
the hemagglutinin which is a triangular spike and
the neuraminidase which is a mushroom shaped
structure. Envelopes confer chemical , antigenic and
biological properties on viruses . Enveloped viruses
are susceptible to the action of lipid solvents like
>pyrighted material
H dr w
432 Tfljdhook o ( Microbiology
i
*

Lilr.NtKAl. OK VIRUSES

PEPLOMER -
B ENVELOPED
ICOSAHEDRON
ENVELOPE ~
—H - CAPSOMERE — -
-- NUCLEIC AC ID — -
i CORE
CAPSID - l - -
I

A, NAKED ICOSAHEDRON

-PEPLOMER
HELIX
ENVELOPE vz
- - CAPSOMERE *
i

: L - NUCLEICACID,
CORE
• y ~ - CAPSID
C . NAKED RIGID HELIX

.
A - naked icnviliednil virus . c-i^nsi^lin|£ nl nn inner cure <il nufEdc ;iftil L'rx kiNcd h_v a LUJISILI. Ii: _ li i in;ui_- uf i-djtauibiCft.
'

ES Ji I iters In ini A HI puutfskitig; ifi CUV dupe.

C - niiJ^ri hf ILL'LJ ] vmih mrnpn^d nl npaortm Auund found Itif HULJCH: Add Lu form A IlifruJur riHUUdV. -

[) - envelope I i 31cMI virus in which Lhf Lu.huI.IT capsid. is pliable UIMJ is ciK'kiKcd wilhm an fin cl JH:
Fig, 46. t . Design And slfiiClure- nl virions

eilicr, chloroform und bile ^alts . Specific Ebolvirus filamentini* and 1

-
RLL L 1
an: . -
neuiritivarion of virus infecUvity depends on shaped, the tobacco rtVOssuC vir r 0 shaped.
antibndics c CJ 1 he sarface a tinge ns , UiologiciE Bacterialviiusethave a n r Kuphology il; .
properties sutb a* attachment t ( ] host cellsurfiace 45.2).
< > r licEiiagglLiimatum depend cm the envelope, Some Ghtmiuril proper ) h s: Viruses contain only
viruses pn ?- R £;sH additional ’.[ ructura] feanucs. Fur one type of nucleic acid, either single or double
cxitriple, fibrils protrude from the vertices of stranded DNA or KVA . In this respect, viruses are
adetifivirui particles. unique, for nowhere else in nature i - genetic
The overall sha ]>c of the vims particle varies in intorui.itKin solely carried bv RNA Viral nucleic
different groups of viruses. Most animal vim secure .uid - mrtv bn extracted bv I rvatment with detergent
-
roughly spherical. Some are irregular and or phenol and. in the case l "i it ISCH l
pleomorphic . The rabies virus is bullet shaped, example picornavirus, pj|Micnviru . rhe extracted

Copyrighted material
 * General Properties cri Vtases 4,13
*

i
i i l

uJHaici ihi

*
i

1 4
j

11

1G

$
9 7
S

Fi y. 44.2 Casparalive sizes end shapes cl different groups c! viruses, 1* Poxviruses 2. Rhabdovlrus
. .
3 Herpesvirus 4, fletnovLrus 5 Tog-ovims 6. Adenovirus 7 Parvovirus &, Plcomevirus 9 Bacteriophage .
10. Coronavkus 11 Orthomyxovirus 12- Paramyxovirus ,

Copyrighted material
 a
434 Te boofc of Microbiaogy *
4
^
nucleic a. id LK capable oi initiating : nter tion when
introduced into host cells,
Viruses also ciuilun protein which makes up
,
the capsid , Viral protein, besides protecting the
nucleic acid, also determines the antigenic specificity
of the virus. Enveloped viruses contain lipkls deri d
^
from the host cc]] membrane. Some viruses also
contain small amounts nfcarboh >drute. Most viruses
do not possess any enzymes for the synthesis of
viral components or for energy production but some
have other enzymes, for example, the neuraminidase
in the influenza virus. Retro virues have i unique
enzyme, RNA - dependent 'DNA polymerase or
' transcriptase’ which can transcribe RNA into
DNA.
Resistance! With few exceptions* viruses are
very Heat labile . There are individual variations but
i -n general , they are inactivated within seconds at
5 (> °C, minutes at 37 °C and days at 4 ’sC . T1tey are
stable at IcTyi" temperatures. For long term storage,
they arc kept frozen at -70 "C . A better method for
prolonged storage is lyophilisation or freeze drying
(drying the frozen virus under vacuum ). Lyopliilised
-
Vi [ US can be stored fur YL-. LT and reconstituted when
r
required by adding water. Some viruses ( such as
poliovirus) do rot stand freeze drying. Viruses vary
greatly in their resistance to acidity. For example,
enteroviruses ate very resistant to acid pH while
rhinoviruscs are very susceptible . Ail viruses are
disrupted under alkaline conditions.
V HUses arc inactivated bv sunlight, UV rays and
-
:<> ru mg raiLiations. They are, in general, mote
resistant than bacteria to chemical disinfectants,
probably because they lack enzymes. Phenolic
-
di i nfcctants are only weakly virucidal. Bacteria are
killed in SO per cent glyceroE saline but this acts as
a pieser val ive for many viruses (fhr example ilia,
rabies) . Molar concentrations of certain salts
(MgClj, Na SQJ also protect some '. miscs (for
^
example poliovirus) agn - nst heat inactivation. The
most active antiviral disinfectants ate oxidising
agents such as hydrogen peroxide * potassium
permanganate and hypochlorite;;. Organic iodine
compounds arc actively virucidal. Chlorination of
drinking water kills most viruses but its efficacy is
greatly influenced by the presence of organic matter.
Some viruses (such as hepatitis virus, polioviruses)
arc relatively resistant to chlorination .
Formaldehyde and beta propiolactonc are actively
virucidal and are commonly employed for the
prepara I ion of killed viral vaccines.
The action of Eiyid Solvents such as ether,
chloroform and hi He salts is selective, the enveloped
viruses being sensitive and the naked viruses
-
resr rant to them. The selective action is useful in
the identification and classification of viruses.
Antibiotics active ag .nnst bacteria are completely
ineffective against viruses. This property is made
use of in eliminating bacteria from clinical
specimens by antibiotic treatment before virus
isolation.
VIRAL HEMAGGLUTINATION
Viral hemagglutination was oritiinallv observed
with the influenza virus by Hirat (1941). A large
number of viruses have since been shown to
agglunnate erythrocytes from different species.
Hemagglutination bv the influenza virus is due to
the presence of hcmagglu tiini n spikes on the surface
of the Virus. The influenza virus also carries o i l its
surface another peplomcr, the enzyme
neuraminidase which acts on the receptor and
destroy- it , Neuraminidase :is, therefore, called the
' receptor destroying enzyme’ ( RDE ) . RDE is
produced by many microbes including cholera
vibrios, and i -. also present in many vertebrate cells.
Destruction of the receptor leads to the revered of
hemagglutination and the release of the virus from
the red «11 surface. This is known as elution ,
Hemagglutination is as a convenient method of
detection and assay of the influenza virus . When
red cells arc added to serial dilutions of a viral
Suspension , the highest dilution that produces
hemagglutination provides the hemagglutination
litre. The hemaggluti nation test can be carried out
in test tubes or special plastic trays. Red cells which
l
> pyrigmet i "i
ri
Hidden page
43« s Textbook ! Microbiology *

n
10 20 40 00 160 »0 6 JO 1260

•
4}
“’
^ ’V.

? s.
P
* •} #

*
4 HA units
A
End Pain 1 HA . unit

Fig .43.3 Viral hantgglulinillon, Virus continuing' fluid is dMsd In doubling dilutions and 0.5% jusperrslon
of chick red CEEI? added. Wrws virus Is presenl theta Is dltluse widespread even pallet or the to Mom at 1 he
wells In (he plastic plate. Where no virus is present the calls settle down tn a button lake aggregate wllh
sharp edges.
me rely the viral nuclei c acid and capsid protein retroviruses which arc synthesised partly in the
hut also of enzymes necessary in the various stages nucleus. Viral protein is synthesised only in the
ofviral synthesis, assembly and iclease. In addition, cytoplasm.
certain Regulator proteins are also synthesised
1
Biosynthesis consists essentially of rhe following
which serve to shut down the normal cellular steps:
metabolism and direct the stL|uetiii.aI production 1.Transcription ol messenger RKA (JTLRNA) from
of viral components. The site of viral synthesis The viral nucleic acid.
depends un the tv] *? of virus . In general* roost DNA 2Translation of the mRNA into 'earlv proteins'*
viruses synthesis: their nucleic Itid in die host Cell These fearly or sonstnieturaJ proteins' are
nucleus . Tbt Exceptions are the poxviruses, which enzymes \vhich initiate and maintain synthesis
synthesise all their components in the host cell of virus components. They may also induce
cytoplasm. Most RNA viruses synthesise all their shutdown of host protein and nucleic acid
components in rhe cytoplasm. Exceptions arc synthesis.
orthomyxnviruses and some paramyxnviruses and .1. Replication of viral nucleic acid-
Table 40.Z ChflracteiiSliCs Ol hemaggluiinalion by viruses
Virus Erythrocyte ipccics and other conditio
*
Influenza virus Fowl* human , guinea pig, others; Elution at 37 " £.’
Parainfluenza, mumps, F4DV Fowl , human* guinea pij', others; Elution at 37 "C;
Hemolysin prevent
Meulci Monkey, 37 *C
Togavini*— severd of Arbwimr GiXsii, pigeon , urtr Jay oltl chick: pH and
rc rrLfMiTiLr in L- u-rnri-e l
Rubella
^
|> i £conp traeday old chick; 4nC
Enrcrovirus * same CoXMckir and I CIIO
'
I Ium .rn; 4 "C and .37 1

RhiiKxvuuB, some scmtvpes sheep' 4 XT

Rabies Goose; 4 ’C, pi I t >.2
Reavirui I (urn an; .37 *<7

Copyrighted n i atonal
 * Gerter &l Properties
4. Synthesis of Llate' < u structural proteins , which
‘
arc the components of daughter virion capsids ,
The critical step in viral biosynthesis is the
transcription of mRNA from the viral TVLHZICIL
Once this is achieved , the host ceil resources cun
be utilised for Translating mRNA into viral
components. Depending on the structure of their
genorne, viruses use dliferent strategies for the
transcription of mRNA . Viruses hive been
categorised into six classes by Baltimore ( 1970)
bused on rhier replication mechanisms , is integrated into the host cell chromosome . This
dual 1: In rhe c ? sc ot fully double stranded
DNA viruses { such as adenn - , herpes - ,
papovaviruses), the DNA enters the host cell
nucleus and uses the host cell enzymes tor
transcription .'Hie extracted DNA from these viruses
is infectious. Wit ft hepadnivi ruses which have- a
partially double stranded DNA, the duplex is
completed by a viral DNA polymerase, inside the
-
host Lvropla -m . The m ,iCure DNA then moves into
the nucleus, to be transcribed by host transcriptases.
Extracted hepadnavirus DNA is not infectious ,
Pbxvi ruses which replicate in rhe cytoplasm form
mRNA usmg polymerases contained in rhe ifirion
itself lAoirirus DNA is not infectious.
Class 2: With single stranded DNA viruses (for
example parvovirus), the DNA molecule moves into
the host cell nucleus and isconverted into the duplex
form. Transcription is achieved by host enzymes
ChurI : In renvi ruses, the double stranded RNA
is transcribed to mRNA by viral polymerases . cell surface and their envelope is formed by rhe
Clas* 4: Depending on the method of mRNA
transcription , single stranded RNA viruses arc
classified into two categories. In the positive strand
, on the cell membrane endows the coll with rhe
{phj.s sfntnJ, positive sertSe) RNA viruses, the viral
RNA iirselt act as the mRNA . Viral RNA is
infectious bv itself and is translated directly into
viral proteins in the host cell cytoplasm ( fi>r example
picorna-, togaviruses).
Glass 5: Tlte nqptfve strand (znjntu sense) RNA
viruses ( for example rhahdo - , nrrhmnvxo- ,
parsmyxovirijac) the RNA is ‘antisense ' , with
polarity opposite to mRNA,They possess their own
Viruses * AM
RNA polymerases for mRNA transcription,
Extracted nucleic acids from these viruses arc not
infectious.
. CllSS 6: RctroviruLie exhibit a unique replicative
strategy. Their single stranded RNA genome is
converted into an RNAiDNA hybrid by the viral
reverse franrfcnpMM ( RNA directed D N A
'
polymerase) enzyme. Double stranded DNA is then
synthesised from the RNA ;DNA hybrid. The
double stranded DNA form of the virus ( pro virus)
integration may lead to transformation of the cell
and development of neoplasia .
Maturation: Assembly of daughter virions
follows flic synthesis of viral nucleic acid and
proteins. Virion assembly may take place in the
host cell nucleus or cytoplasm. Herpes and
adenoviruses nrc assembled in the nucleus* while
piedrna Hid poxviruses are assembled in the
cytoplasm- At this stage , the noncnvclopcd viruses
arc present .intraccllularly as fiiUy developed virions
but iu the case of enveloped viruses, only the
nucleocapsid is complete . Envelopes are derived
from the host cell membrane during die process of
budding. The host cell [nembrane which becomes
I lie envelope is modified hy Incorporation ol virus-
*pecific antigens - I lerpcs viruses assembled in the
'
nucleus acquire their envelope from the nuclear
membrane us diev are released into the cytoplasm
enclosed in a vesicle. Myxcmrum bud front the
modified cytoplasmic membrane of the host cell .
The incorporation of viral antigen (hemagglutinin)
property of hemadsorption.
Release: In the case ot bacterial viruses, the
release ot progeny virions taken place hy the lysis
of the infected lracEerium . However, in the case of
animal viruses, release usually occurs without cell
.
lysis Myxoviruses are released by a process of
budding from the cell membrane over a period of
rime . The host cell is unaffected and may even
divide, the daughter cells contiruling to release
Copyrighted material
Hidden page
Hidden page
440 * Texlboc* oF Microdoiogy
system generally consist ] ng of bicarbonate in
equilibrium with atmosphere containing about
S% carbon L!in KLIJC: . This is supplemented with
UptO S CflEf or fetal L ;ilf serum . Antibiotics are
^ '
added to prevent bacterial contaminants and
phenol red as indicator . Such media wiil enable
most cell types to multiply with a division rime
o t 2 4 -48 hours. The cell suspc nrion is dispensed
'
in bottles , tubes or Pfctri dishes. The cells adhere
to the glass surface and on incubation , divide to
form a confluent monolayer sheet of cells
covering due surface within about a week . Detection of virus growth in dell
Cell culture tubes ITIAV be incubated in a sloped
horizontal position , either as ‘stationary culture'
or may he rolled in special 'roller drums' to
provide belter Aeration. Some fastidious viruses
grow only in such roller cultures -
Uased on their origin, chromosomal characters
and the numher of generations through which they
can be maintained, cell cultures are classified into
three types (Table 48,3) :
1 . Primary cdl m /fufes. Time are normal cells
freshb taken from the body and cultured. They
1
arc capable of only limited growth in culture
and cannot be maintained in serial culture.
Comrnon examples of primary cell culture -s are
monkey kidney , human embryonic kidney,
human amnion and chick embryo cell cultures.
Primary cell cultures are usclul for the isolation
of viruses and their cultivation for vaccine
production.
2. Diploid cell SiraJ /lS ' These are cells of a single
type that retain the original diploid chromosome
number and karyotype during serial
subcultivation Ear a limited number of times.
After about fifty serial passugefe, they undergo
' senescence ' . Diploid strains developed from
human fibroblasts are susceptible to a wide range
of human viruses anti are very use Fill for the
isolation of some fastidious pathogens.They are
also employed for the prod net ion of viral
vaccines.
3 . CtMitJOunUi cell linen: These are cells of a single
*
type, usually derived from wncer cells, that arc
capable o t com i n uous set ill Cult i vati o n
.
indefinitely Standard cell lines derived from
human canceiy, such as lleLu, Hep-2 and Kfi
cell lines have been used in laboratories
throughout the world lor many years. These cell
lines may he maintained by serial suhcultivatinn
,
or stored in the cold (- 70 *C) for use when
necessary. Some cell lines arc How permitted to
he used tor vaccine manufacture* for dtamplt
Vcrocell tor rabies vaccine .
cultures: Virus growth ill cell cultures Can he
detected by the following methods :
1 . CvTopjffm effect : Many viruses cause
morphological changes in cultured cells in
which they grow. These changes car be readily
observed by microscopic examination of the
cultures. These changes are known W cytopathic
effects' ( CPE ) and the viruses causing CPE are
called ' cytopathogenic viruses The CPE 3
.
produced by different groups of viruses are
characteristic: and help in the presum price
identification of virus isolates. For example *
enteroviruses produce rapid CPE with ere nation
of celts and degeneration of the e ntire cel I sheet;
measles vims produces syncytium formation;
herpes vims causes discrete focal degeneration;
adenovirus produces large granular dumps
resembling bunches of grapes; and SV produces
^
prominent ectoplasmic vacuolation .
2 . Mt t \ boli . inhibition: In normal «11 cultures *
:
the medium turns acid due to cellular
metabolism. When viruses grow in cell culture
cell metabolism is inhibited ami there is no acid ^
production . This can lie made out by the colour
ot the indicator ( phenol red ) incorporated in
"
the medium .
3 . 1 Icnudiotption: When hemagglutinaring viruses,
(such as influenza and parainfluenza viruses)
grow in cell cultures , their presence can be
indicated by the addition ot guinea pig
erythrocyte* to the cultures. If rhe viruses are
Copyrighted material
 * General Properties of Viruses 441

multiplying in the culture* the crvthrueytcb will
adwjrh onto the surface of cclk. Thus ! H known
as hemadsorption '.
' virus particles in a suspension can he counted directly
-
4 . in tcrk - ft'rfin The growth of a non cytoparhogenje
virus in cell culture can he tested by the
subsequent challenge with a known
UYtopathngEnic virus. The growth of the first
will inhibit infection by the second virus by
interference.
5 . 7:. m.sifji'jrurron : Tumour forming (oncogenic)
viruses induce cell 'transformation ' and loss of
contact inhibition, so that growth appear? in a
piled-up fashion producing ' micrommoLirs'.
6. Irnmunofluorvsctncr: Cells from virus infected
cultures cm be stained by fluorescent conjugated
antiserum and examined under the UV
microscope for the presence of virus antigen .
This gives positive results earlier than other
methods and, therefore, finds wide application
enumeration are electron microscopy and
hemagglutination. Ry simple negative staining,, the
under the electron microscope . The virus suspension
can he mixed with a known concentration of latex
particles The ratio between the virus and latex
particles under the electron microscope gives on
indication of the virus count . With
herragglutiuating viruses * a convenient met find oi
quantitation is the determination of
hemagglutination times. Hemagglutination is not
a very sensitive indicator of the presence of small
amounts of virus particles. Thus approx i mutely 1 0 -
*
influcntst virions are required to produce
macroscopic agglutination of a convenient quantity
ot chicken erythrocytes (0.1 ml of 0.5 per cent
suspension ). However, because of its simplicity ,
hemagglutination is- n very convenient method of
virus assav,
a

in diagnostic virology.
Vi HA i ASSAY
The vims content of a specimen car be assayed in
two ways: either with reference to the total vims
particles or with reference to the infectious virions
only - Two methods employed for total particle
AHSAV til 1 NKI : C;TEVIT \
Two types of inicctivitv assays can be carried out -
quantitative and quanta! assays. Quantitative assays
measure the actual number of infectious particles
in the inoculum * while quanta! assays only indicate
the presence or absence ol infectious viruses. Using

Table 46.3 Some cell cultures in common use

a. Primary cell culture
]. Rhnus monkey kidney LXII culture
2 . Human amnion cell culture
.
3 Chick * mbr lihiubLm ccU culture
P

b - Diploid cell serai

1.WI 38
2.
-
HL -
8
Human embryonic lung «11 strain
Rhesus embryo cdl strain
c Continuouscell linns
1. He La Human cudnoma o( ctmx «11 Um
.
2 HFP - 2 Human epidulionaa of larynx cel] line
Human carcinoma nf nasopharynx cell line
1 KR
4. McCoy Human synovia] carcinoma cell line
5 . Detroit -* Sternal marrow cell line
6 . Chug C f l f U K Human conjunctiva ( Cl
Intestine (I), Liver ( L) and Kidney ( K ) celt lines
7. Vcro Vervet monkey kidney cdl line
Baby hamster kidney cell line

Copyrighted material
 AAl 4
-
T salbook of Microbiology *

GEN h h A l . HK ( JPERTIES oh VI Ft USES

CHO« lOAELANTO:G
MEMBRANE

t ' fl SAC

AMNIO TIC CAVITY

SHE LL
SHf -. L
MEV & HANE
A , UMO C j

LAVIT Y .
YO . K SAC & w
-
YA

ALBUMCM
.
Fiij. 4£ 4 Cross s«tlpn o1 J ten bay clu egg

serial JikuMn of virus suspensions and with the US a ntodiiicalioiL of the bacteriophage plaque assay.
.Liu of statist ic^J methods , reasonably accurate A viral suspension is added to a monolayer of
estimates nf injectivity can he obtained in quanta ] cultured cells in a. bottle or Petri dish, and after
SOWfl
J
. allowing time for absorption , the medium is
Quailtal ra of infect Lvity can he Lurried out removed and replaced with a solid agar gel , t< > ensure
in aniinnk, eggs or tissue culture. Examples of ' liar I TIL q >read of progeny virions is confined to

endpoints used for infect huy titration are the death
ot the animal, product ion of hema glLLtiiiin in
^
jliantoic fluid or the appearance of CfE in cell
.
cultures The titre is usually expressed us the 50
per cent infectious dos- c’ ( ITT,, ) per ml , which
-
indicates the highest dilution or rhr inoculum that
_
Mould produce an effect in Tijper cent of animals,
ggs or Cell Cultures inoculated . IIT LH calculated
ov the application of statistical methods, such as
that of Reed and Mucnch.
The quaniil .itive inactivity assay of viruses is
similar to rhe estimation of bacteria! viable counts
bv colony counting. Two methods arc available -
plaque assay in monolayer cell culture and pock
assay on chick embryo CAM. Plaque assay vm
introduced in animal virology by I >U lbCOCO ( 1952)
the immediate vicinity of infected cells. In this
system, each infectious viral particle gives rise to a
localised focus of infected cells that can lxe seen
'
with rhe nuked eye. bm l : foci arc known AS
" plaques’ and each plaque indicates an infectious
virus ( big . 48.5}. home viruses which ure
transmitted directly from cell to cell ( for example
herpesvirus) may form plaques even without an
agar overlay. Oncogenic viruses produce cell
transformation which can he seen as micro
tumours, Hcncc they can be enumerated by the
-
transformation assay.
Viruses thatJdrm pocks on CAM ( lorexample
vaccinia) can hr assayed by counting the number
of pork tbrmcd on CAM by appropriate inocula
*
of virus. This is known as pock assay.

Copyrighted material
 * Geno ; al Properties d Virus** *
V I R A L fii . .M- T I C S
Liter all other 'living beings', vinines obey The laws
of genetics. Several properties of viruses, such as.
virulence and antigenicity; that arc of great concern
to human beings in ihe context of infections it the
level of the cell, individual and community, are under
genetic control . Genetic studies, therefore, have
contributed to a better understimding of virus-host
interactions and the development of better viral
vaccines. Genetic mechanisms nidi as mutation and
selection in tbc past without
were utilised
recognising the biologic at mechanisms involved .
The development of the 'fixed ' rabies virus by
Pasteur is a CMC itl point -
The two main mechanisms for genetic
modificatioti in viruses are mutation and
recombination . In addition , viruses may exhihit
many nonhcritablc variations due to gene product
interactions.
Miitnbtmi Thf frequency of mutation in viruses
is about 10 A to 10 ’ , approximately the same as in
bacteria. Mutations, therefore, occur during every
viral infection. Most munitions arc lethal . A mutant
becomes evident only if the mutation confers some
readily observable property or survival advantage .
Mutation may occur spontaneously or may be
induced by mutagens, physical agents such as
irradiation or chemical agents such ns S
fluomuracil.
- obtained with the hemagglutinin of one parent and
Tilt; mutants may he of various Types. Some
mutations i >1 clinical ,in ; i laboratory Interest are
those affecting virulence, host range , antigenicity
and pock or [llaquc morphology. A class of mutants
that are of great importance in laboratory studies is
the conditional lethal mutant. These are mutants
which are able to grow under certain conditions
(called permissive conditions), but are lethal, that
is it cannot grow under certain oilier specified
-
condition ; ( called non permissive or rcstric five
conditions).There are different types of conditional
lethal mutants but the types most widely employed
7
in genetic studies are the ' temperature sensitive ( ts)
443
mutants . These can grow at a low (permissive )
tv in peri tore ( 2 B 31 uOJ hut not at ; t higher
( restrictive ) temperature (37 ,:C ) . The advantage
here is LII .M by using a HINGL $ selective test
( temperature sensitivity ), large numbers of mutants
with lesions in different genes may be obtained ,
The fit mu rants have not only contributed largely
to fundamental studies on viral genetics bnt also,
because of their low virulence, offer prospects of
better live viral vaccines.
Reoombiriaiioni G tic recombination may
occur when two different, but related, viruses infect
a cell simultaneously. The twin viruses exchange
segments of nucleic acid between them so that a
hybrid results, possessing genes from both parents.
Such recombinants breed true thereafter.
Recombinants may occur between (1) two active
( infectious) viruses; ( 2 ) one active and one inactive
virus; and (3) two inactive viruses.
When two different sE rains of the same vim?
(such as vaccinia or influenza) , possessing distinctive
markers (such as puck morphology or antigenic
pnrperl ies ) aregrown together , recombinants may
hr derived chat possess the distinctive properties of
both parent!;. Thus, if a human and an avian strain
of influenza virus ( whose hemagglutinin and
neuraminidase untigens are different and easily
identifiable) are grown together, a hybrid may be
the neuraminidase of the other 'fhis has been
,
demonstrated experimentally in vitro and in vivo.
This may be one of E he ways by which the pandemic
Strains of the inflmen ^:i vims originate in nfilure.
When a cell is 'infected ' with ait active virus
and a different but related inactivated virus, progeny
possessing one or more genetic traits nt the
inactivated virus may be produced . This
phenomenon is tailed OWJWtMtitn or marker
rescue. N’ew antigenic variants a\ the influe ru» virus
causing epidemics, often do not grow well in eggs
as compared to established laboratory strains.
When such an epidemic strain tlbr example strain
AJ iv grown in eggs along with a standard strain
Copyrighted material
Hidden page
Hidden page
446
( jj. VSSEIH: V tH > v \ \ |>
N IMKPJ< . T
"
( AN HR OK Vim
Till about little.- was known oJ the basic
] 950
properties of viruses . They were named
haphazardly, based on the diseases they caused or
o.n rhe place of their inlvian, They were grouped
according to assumed 'tropisms' nr affinity to
different systems or organs of the body.Thu human
* deoxyriboviruses arc those containing DNA .
viruses were classified as dcrmotnipic., that is those
producing skin lesions (upillpor, chiekcnpoitj
measles), neurnrropic, thal is thufie affecting the
nervous system ( poliomyelitis ,, rabies ) ,
ptieucintropic, that is those affecting the rapuntbty
1 (influenia, cortution cold ) and vifeero tropic,
trn.
that is those affect jpg visceral Organs (yellow lever ,
hepatitis)- Hawden { 1941) made the pioneering
suggestion that viral nomenclature and codification
should be based on the properties of viruses and
not upon host responses-. From the early 1950 s,
Mitirp rlcv] >
* faxihoOK at ^ ^
viruses began lit be classified into groups- based on
SK $ their physicochemical and Structural features.
Nomenclature and classification arc n o w rhe of haul
responsibility of (he International Committee on
Taxonomy nf Viruses.
Vituses .ire classified into two main divisidtii
depending on the type of nucleic acid they possess:
rihovi ruses are llirssc CQlit lining RNA and
Further classilicarui-n is basal nil other properties sncli
AS rhe srrandedness of nucleic acid , symmetry of
ruu-Jeneapsid, presence of envelope, size and shujic Lif
virion .i : d number ot rapsdtnca. Short tlc -< riptiors-
of the major groups of viruses arc given helow,
D N A VIRUSES
Moxviridne JnmiT : ' These arc Urge , brick
shaped or nvr>id viruses (300 * 240 * HW p ). with
complex structure , hiving a lipid containing an
roar, Line nr two lateral belies- and a enrr
outer
carrying i single linear molecule of double stranded
UNA . Multiplication and maturation take place in
the cytoplasm. The family is divided inm several
genera,
Herpcsviriilac fnmily: These arc medium
saved viruses L-Lintaining linear double stranded
DNA . The iweahcdraJ nuclcocapud (100 nm) has
UJ 2 eapsomers and is Surrounded hy a lipid
eoiuaiiliilg envelope . Multiplication lakes place in
111 L auckun and [liaturatinn by building through
1

the nuclear membrane . Only one genus ,

Herpcsvirw, has been characterised, hut several
member* of die family await classification.
Adnmv irulnc ; (dnsi | -H : These ate medium

>t sited (70- 90 nm) nonenveloped, icosahedral viruses

with 252 cupsomers. Members have heen classilied
into two genera : Mastadcn ovinia (mammalian
adenoviruses) and Aviadcnovirus ( adenoviruses of
birds)
Pnpnv nviridcuj I amill llicsc arc small (40-
55 nm ) nonenvelojjcd , double stiandeJ DNA
Fig tB.G Plaque formation tn monkey kidney cells VTRUSES with 72 Capsortiets. Two genera have bevn
by poliovirus recognised, ApTfprtjmryj aril Ri/vrtfnahru,

VI maerial
Hidden page
Hidden page
 Virus-Host Interactions: Viral Infections
Virus- host in tersedons may be considered at
clif -Ferenr levels - the cell, the individual arid the
community.
At tile cellular Level , vital infection ItdyonK i
bmad xpertnim at effects, ranging from m apparent
cellular damage to rapid cell destruction , Some
viruses, like poliovirus cause cell death ( cytoeidal
effect ) oi even lysis (cyrolysis ). Others may cause
cellular proliferation {as molluscum com agiosum )
or malignant transformation {..is oncogenic viruses).
In some instances the virus and ho*t cell enter into
3 peaceful coexistence , both replicating
independently without inv cellular injury , a
condition known as ‘steady state infection '. In tissue
culture viral infection may lead to readily observable
.
cellular changes ( cyiopathic effects) These may not
paraltel the changes produced in the infected
animal , as in the latter situation infection is
influenced by the various defence mechanisms of
the body.
Cellular injury may be due to a number of causes.
Early or nonstiuccura] viral proteins often cause a shut -
down of host protein and DNA synthesis. Large
amounts of viral ui . iciomalcculcv that accumulate ill
the infected cell may distort the cellular architecture
and exert a toxic effect. The permeability of plasma
membranes may be altered , releasing lysosomal
enzymes and leading to autolysis.
Many viruses produce alterations in the
cytoplasmic membrane of infected cells. Some (such
as respiratory syncytial virus) cause fusion of
adjacent cell membranes, leading to pofykaryocytosis
or syncytium formation. Virus coded antigens may
appear on the surface of infected cella. These
antigens may confer new properties on the cells.
For example, viral hemagglutinin appears on the
surface of cells infected with Influenza virus and
causes adsorption of erythrocytes to the cell surface
( hemadsorption ) , Virus coded antigens also appear
on the surface of cells transformed by oncogenic
viruses .
Certain viruses such as measles , mumps ,
adenoviruses, cytomegalovirus and varicella virus
cause damage to the chromosomes of host cells .
Chromatid gaps and breaks in cliromosotnc 17
occur frequently in cultured cells infected with
adenovirus types 12 and ill.
The most characteristic histological feature in
virus infected cells is the appearance of uhittaan
bodies. Inclusion bodies arc structures with distinct
size , shape, location and staining properties that
can he demonstrated in virus infected Cells under
the light microscope. They may be situated in the
cytoplasm (as with poxviruses) , nucleus
( herpesviruses ) or both ( measles virus ), lliey arc
generally acidophilic and can be seen as pink
structures when stained with Giemsa or cosin
methylene blue stains. Some viruses (for example
adenovirus) form basophilic inclusions .
Demonstration of inclusion bodies helps in the
diagnosis, of some viral infections. The presence of
intracytoplasmic eosinophilic inclusions ( Negri
bodies) in the brain cells of animals juttific ^ the
presumptive diagnosis of rabies. Vaccinia infected
cells show rather smaller multiple inclusions known
55 Guarnicri bodies. Large inclusions ( Bollinger
bodies ) are seen in fbwtpOOL Inclusion bodies in
molluscum contagiosum ( molluscum bodies ) are
Copyrighted material
450 * Tsidbook of MfemtMi3Jogy
very Large (2D~30|iin) and can he readily -seen under
tilt low power microscopCp inimuuletr inclusion
bodies were classified into two types bv Cowdrv
(1934), Cowdry type A indusionH are of variable
rise and granular appearn.net [as with herpesvims ,
yellowfenrvuui), while type R inclusions are more
circumscribed and often multiple (as with
adcnodrusp poliovirus ) - Indus ion bodies may he
crystalline aggregates of virions or made up of -vims
antigens present at the site ot virus synthesis. Some
inclusions represent degenerative changes produced
by viral infection which confer altered staining
properties cm the LCU .
PATHOGENESIS OF V I R A L INFECTION
Depending on the clinical outcome^ viral infections
Can be classified as inapparent ( subclinical ) or
apparent {clinical of Overt) infcctionsiP The latter
may be acute , subacute or chronic Some virus
. multiply and produce local disease . These are
infections are characterised Iby latency Latent
infections- are of clitTerent types. Recurrent herpes
Simplex and herfws zoster are examples of latent
infections in which clinical manifcsrattons appear
after prolonged periods ©f quiescence during
which the viruses remain hidden in the nerve-
mot ganglia. Another type of latent infertion is
persistent tolerant infection in which the virus is
readily demonstrable in the tissues of the host but
neither disease nor i mminie tttpQrttt develops. The
, Some of these nuch an rntavirus remain confined
host as immunologically tolcranr to the virus as a
result of congenital or neonatal infection - Disease
sets in when the tolerance is interrupted. The
classical example of persistent tolerant infection is
lymphocytic choriomeningitis of mice. Another
type of latent infection is seen in neurological
diseases such as scrapie in sheep and kuru in
human beings. Ibis is called slowly progressive or
slow infection as the incubation period is unusually
long. Vet another class of latent infection is infection
by oncogenic riinset. The human
immunodeficiency virus leads to a -special type of
latency, with an, initial asymptomatic period
followed by progressive immune damage causing
secondary diseases, ending fatally after many
,
vears
Viruses enter the body through the respiratory
and . LI i n i _ 111 ; LT y tracts, sldti , ntijunttivA and the
ger . ital tract . Many vimsos are transmitted vertically
from parent to prngiercy-
'Yhc respiratory tract offers the most important
portal ofentiy for viruses. A Large numlier of viruses
arc able to : ifcct the cells of this trace . Some of
them multiply Locally Lu initiate a silent local
infection which is followed by lymph ark or
hematogenous transport to other situations where
mote exter -Mve multiplication takes place before
system ic illness is ma : ike sled . Smallpox and
chiekenpox arc examples of such systemic diseases
in which the portal of entry is the respiratory tract .
Other viruses such as inlluenxa and rhi u-nvi rusts
are rc*t : xtcd to the respiratory tract where they
known as respiratory viiuses ,
The alimentary tract is rhe next most important
route of entry for viruses. However, only Home
viruses arc able to establish infection in the
intestines. All enveloped viruses are destroyed by
bile . Rhinoviruses arc inactivated by gastric
acidity. Gnh rntrruvi ruses, adrmovi ruses, renvi runes ,
hepatitis viruses and the viruses causing
gastroenteritis are able to sec up intestinal infection.
to the gut causing local disease . Others such as
poli ' wirus ) after i "iir - al multiplication locally, ate
transported to other <- ues (dr further multiplication
and subsequent spread tn the target organ -.
Of the viruses that enter through the skin ,
only a tew produce Local Lesions , Papilloma ,
vaccinia , cowpox , molluscum contagiosum and
Qrf are viruses that produce dermal led on -; at
the site oi entry. Skin lesions of exanthematous
viral diseases arc secondary ro systemic infection .
Viruses enter the skin through abrasions
( papillomavirus ) , insect bites (arboviruses) , animal
bites ( rahic ) or injections ( type B hepatitis ) ,
*
Systemic spread occurs through lymphatics or
Copyrighted material
 i Virus-Host Inieract .ons : V- ral Infections
blood. Rabies virus travels along the nerves to the
spinal cord or brain.
"
The conjunctiva also mav act as a portal of entry
for viruses . This may lead to local disease
(adenovirus) or tO HVHteinic spread ( measier ). Some
viruses may enter through the genital tract nr other
Kites of Hexufll contact ( HIV ).
Congenita! infection may occur at any stage
from tlir ; level ' )umeiit of the ovum up to birth. Jo
cut>C systemic infections . congenital infection
usually leads to fetal death and abortion. Rubella
and cytomegalovirus produce oiildecdopniL'tiL Dr
severe neonatal disease. Vertical transmission is the
natural mode of spread of many tumour viruses ,
flic uvian leukosis virus LH transmitted in uVb and
nuirinc mammary rumour vims through breast
milk .
SPREAD OP VIRUS IN LHIK BODY
The manner in which the infecting virus sjueads
from the poi nt of e ntry, multiplies in sites of election
mid causes lesions in target tissues was fiist studied
by Fenner ( 1 48 ) using mousepox: as the
^
experimental model ( Fig. 49.1) . The mousepox
-
vims enters the - kin, where it multiplier initially
and proceeds along the lymphatics to the local
nudes. After usultiplication in the lymph nodes, the
vims, enters the bloodstream ( primary vireima) and
is transported to the spleen and liver which act as
the ‘central foci ' for viral multiplication After
attentive multiplication LO the centra! foci, there
occurs a massive spillover of rhe virus into the
bloodstream (secondary viremsa) . This heralds the
onser of elfe -Lcil symptoms ( the prodromal phase
in eruptive feverc). The virus reaches the target
organ ( skin in eruptive fevers ) through the
bloodstream. Multiplication in the target sites
produces the distinctive lesions . With minor
modifications , this model holds good tor most
systemic virus diseases . The reasons for the
difference in the foci of multiplication and tmget
organs in the case of different viruses are
obscure.
451
SltJMrirANtlH OF THK l t d '
ll A TI O N
PERIOD
Tin: incubation period represents the rime taken
for the virus ro spread from the sire of entry to the
org,ins of viral multiplication and [ HENIL to the
target organs fir the production of Icrioitt, Its
duration is therefore influenced by the relation
between the sites of entry, nmkiplieation and lesion.
Where die site of entry and Kite uf lesion are the
—
same, ill* incubation period is short one ro three
days, as in respiratory viral infections and in
gastroenteritis . In systemic diHessen where [111 VirtlS
enters through the respiratory or alimentary tract
,
—
Lind produces lesions In remote target sites, the
incubation period is longer 10- 20 days , as in
chickcnpox or polioolyelitii. There are, however,
exceptions to this rule . In arbovirus diseases, as in
yellow fever or dengue , the incubation period may
,
be shorter \. S 6 days), probably because the virus
in introduced directly into the bloodstream by the
insect vector*. The incubation period in type R
hepatitis may be 2 ~ f > month* and in slow viral
infections, many years. Papillomas and molluscvim
coniagiosum have Long incubation periods, probably
because the viruses myltipty slowly:
HOST Itiispoissns T O VIRUS
INEECTIOMK
The outcome of a virus infection is influenced by
rhe virulence of the infecting strain and the
resistance offered by the host. Mechanisms of host
resistance may be immunological or tMQfpeciflc.
The latter includes various genetic and
physiological factors such as interferon production,
body Temperature , nutrition and hormones.
Immunity in virus infections: Virion in
*
general are good antigens and induce both humoral
and cellular immune responses. The multiplication
of a vin '- in die body during infection induces int
1
only a t \ .unlit atively greater iinn JILL FMpGrje nut
also liberates and makes available to the immune
system the whole range of virus antigens, including
Copyrighted materia
4S2 * Textbook of Microbiology

TEXTBOOK CV Ml( KUfcUULOGY

Sbtfn inyKiQfi
Or ^ =
mdiLp lci irirjn
lymph d« -
«

14 mijijpficfltian

8 »
Primary
vifaerma
2
s
/ p Q&s
3 i Mollification
Mulirp in
CentfSi foo"
< and sptean
§ 4-
2
Secondary
5" '

viraemia

6 - Skn - txal rnuUipItcaiKyi

>* Antibody *n

> *um
§js
S vv lryj erf
^
& l foot - primary lesion

s<
"a

Early rash +

9^ L pBpulH

3 10
U»te rash-
11- .
JCBr itKXl

F: g 49.1 Pathogenesis of mousepox e model lor acute exanthemata of humans jailer Fanrter )

surface and iurcrra ] nn ri ci . s as wc II as the "

^
nonstmdufJ aiLligeils such iS Cifly proidtlS.
In mediating humoral antiviral immunity, the
important classes of antibodies are IgG, IgM
IgA . lj[C and IgM play a major n Au in blood and
IIHH 11 v spaces, while IgA is more important on
mucosal surfaces . Antibodies effect v i r u s
neutralLKilion bv several mechanisms . Thtv may
R P
prevent adsorption of rhe virus to cell receptors ,
cause enhanced virus degradation. of prevent release
of the progeny virus from, infected ceilsr
Complement acts in conjunction with antibodies
in causing surface damage io enveloped virions and
in producing cvtolysis ui virus infected Cells.
Not all antibodies are able to neutralise viral
R
btfectmty. Antibodies to internal antigens arc nun -
 v r.js- - H{)Si
neutralising. Antilsodik's to surface antigens vary in
then ncu. tr3ili -. iiig ability. For instance, two types of
'
surface antibodies appear following influenza
—
infection antihcm &ggluri n i n and antineur
ami ^ idase- 'l l- c termer can neutralise inactivity tmt
^
the latter cannot. Antircuraminidasc antibody can,
however, i nh i h: t the release of progcnal virions from
infected cells. Some antibodies can paradoxically
enhance viral infecrivity, Humoral antibodies may
Homelim.es actually contribute to pathogeH.esis .
Antibodies mav cause complement dependent injury
to cells or induce an immune complex type of r issue
injury. The enlianced severity of respiratory syncytial
viral infection in early infancy is believed to be due
to the presence of passively acquired maternal
antibodies. In older children who have no antibody,
the virus causes a milder disease . The pathogenesis
of some viral hemorrhagic fevers is immune
thrombocytopenia , Most extrahepatic lesions in
type R hepatitis are due to damage caused by
immune complexes.
Cell mediated immunity is ofcrilivaJ importance
in viral infcctions-The earliest indication of cell
mediated immuniiy in viral infection! was the
demonstration of delayed hypersensitivity following
vaccination in immune individuals. Similar skin
reactivity is also seen in mumps. The normal
resistance to vi ms infections shown by
again miglobulincmics is ascribed to their cell
mediated immunity; though it may also be due to
interferon or Other non immune mechanisms.
Individuals with deficient cellular immunity show
a heightened susceptibility to infection hv the
-
herpesh pox and me - ivies v: rusr The administrtU ion
of antilymphocytc serum inducts fatal infection in
mice Injected with a sublethaldose oftbeectromelii
virus. Cell mediated immunity is considered to play
a m.ijor role in recovery from viral infections in
which viremia is not important and in which
mfccted cells have viral specific antigens cm their
surface . In some virus infections cell mediated
immunity may contribute to tiHSue damage, as n
lymphocytic choriomeningitis ill mice.
Intsratucnai Vral Infections 453
Some viral infections cause a suppression of the
mmune response. Measles infection induces a
temporary depression of delayed hypersensitivity to
- tuberculin. Infection of adult mice with lymphocytic
choriomeningitis or leukemia viruses inhibits
anfibodv resjvrnHe to other antigens. HIV strikes at
the centre of the immune system by infecting the
CD4+ helper T cell.
In general, viral infections are followed by solid
immunity to reinfect ion, which may often be
1 ifelong. Apparent exceptions I i ke the common cold
and influenza arc not due to lack o! immunity hut
to reinfection being caused by antigenically different
vi ruse 5, L ive vi ms vact i ties also induce more durable
protection than bacterial vaccines.
N o n tinlimnological K e s p u n s c s
Jr Ph$gOCytO$!5: Polymorphonuclear leucocytes do
not play any significant role in the defence
against viral infect inns. To fact , more viral
diseases are characterised by a
polymorphonuclear leucopema. On the ocher
hand, macrophages phagpeytoHC viruses and are
important in clearing viruses from the
bloodstream.
2. Body temperafurr Fever may act as a natural
defence mechanism against viral infections as
most viruses are inhibited hv temperatures above
39 C . An exception is herpes simplex which is
usually reactivated by fever to produce lfever
blisters '. Herpes febtilia is a frequent
accompaniment of fevers caused by pneumococci ,
streptococci , influenza virus and tnalana
parasires, hut for some unknown reason, is very
rare in other fevers (typhoid, tuberculosis).
3* fformwffi Corticosteroid administration
enhances most viral infections. Coxsackie virus
B1 does not normally cause disease in adult mice
hut will induce .i fatal infection in. mice treated
with cortisone. Normally mild infections such
as varicella and vaccir. ia may be lethal in
patients on cortisone. Injudicious use of steroids
i n the treatment of herpe tic keratocon i unctivitis
opy righted material
Hidden page
 N Virus-Hggl Interactions : Viral IntEdiana *
epithelial cells following stimulation by viruses or
polynucleotides. It is i glycoprotein.
Gamma interferon (IFN-y), formerly known as
immune interferon * Is produced by T lymphocytes*
on Stimulation by ant inerts or mitogens. It a
glycoprotein , Tt is more concerned with
i inmunomoduktoiy and intiprolilcntivc function!
than with antiviral defence. It alsodiffers from alpha
and beta interferons in having a separate cell
receptor.
Interferons are inactivated by proteolytic
enzymes but not by nucleases or ]i [';iscs. Thev resist
heating at 56— 60 —
for 30 60 minutes and are
stable over a w ide range of pH ( 2-10) , except
.
gamma IFN, which is labile at pH 2 They have a
molecular weight of about 17,000* are nondialyaable
and nunsetlimentable ( IQOJOQO g). They are poorly
antigenic, so no routine serological tests arc
available for their detection and estimation.
Interferon assay is based on its biological activity
such as the ability to inhibit plaque formation by a
sensitive v irus. The potency of 1 FN is expressed as
International Units (1U ) per ml.
Many properties of interferon make it an ideal
candidate for use in the prophylaxis and treatment
of viral infections; it is nontouic * nonancigenic,
diffuses freely in the body and has a wide spectrum
of antiviral activity, The rmijor drawback initially
was its species specificity - interferon produced by
nonhuman cells was not clinically useful. This was
overcome to some extent by producing interferon
from buffy coat leucocytes from blood banks * with
NDV or Sendai vims as the inducer. Now, human
interferon is available in unlimited quantities
following its commercial production by cloning in
bacteria and yeast . Even so, its initial promise as an
antiviral agent has not been fulfilled . Local
application of high doses has shown some benefit
against upper respiratory infections * herpetic
kcratitis and gen ital warts. L. i mited success has also
been reported against generalised herpes infection
in immunocompromised hosts * and against hepatitis
Ji and C infections. Some encouraging results have
455
been reported in che use of interferon as an
anticancer agent * particularly in lymphomas but
there have been reports of toxic effects in cancer
patients given high doses of interferon.
Although interferon was first recognised as an
antiviTal gcnt ; iris now known lobe a more general
^
regulatory peptide belonging to the class of
cyn>£inr& The main biological effects of interferons
are the following:
1, An:mral effects : Induction of resistance to
infcctinps-
2 Anrimicmbid effects: Resistance to intracellular
,
infections , for example toxoplasma, chl . Lnnvdi . L ,
malaria ,
3, Cellular effects: Inhibition of cell growth and
proliferation: and ofDNA And pro!ein synthes is;
increased expression of MHC antigens on cell
surfaces,
4, Immunoregulatoiy effects: Enhanced cytotoxic
activity of NK, K and T cells; activation of
macrophage cytocidal activity modulation of
^
antibody formation; activation of suppressor T
cells; suppression of UTH.
LABORATORI DIAGNOSIS OF VIRAL
ll> t s a -: AS KM
Technical difficulties in virus isolation and
identification* the length of time required for these
procedures and the lack of specific therapy lor viral
infections have contributed to the sparse use of
diagnostic virology till recently. The situation has
changed in recent years, With the development of
rapid techniques for the diagnosis of many vims
infections and the availability of Specific drugs
against at least a few viruses, diagnostic virology is
fast becoming 1 routine procedure.
The demonstration of viral infection in selected
groups of persons ( screening ) is an important
procedure Ln the prevention of some diseases (such
as screening for HBV and HIV in blood donors).
Etiological diagnosis of viral in lections is useful in
many ways . It is of vital important in some cases * as
in rubella in pregnant women . It helps institution
Copyrighted material
m + Textbook o Microbiology
*
ut early specific therapy as in herpetic encephalitis the capability of the virus laboratory. The
and. lesions of the eve. It serves to define the etiology appropriate specimens should be collected Grom
of vague syndromes such as upper respiratory patients* preserved and transported to the laboratory
infection or aseptic meningitis. It is essential for in the proper manner along with pertinent clinical
the detection and prediction of epidemics and the and epidemiological information (Table 49.1 ),
identification of antigenic variation in viruses. It is In the laboratory the following methods are
invaluable in the prompt control of outbreaks . It commonly employed; microscopic demonstration
may lead to the discovery of new viral infections. of the virus or its inclusion body, demonstration of
Successful diagnosis at viral infections depend
, the virus antigen, isolation and identification of the
,

as much on the awareness of the physician as on virus, or detection of the specific antibody.

Table 49.1 Types cl specimens ta be sent for virus diagnosis

SyJfem Specimens required1

For isolation For direct cmniflifioD; For serology

Respiratory system Throat swab 4 Nasopharyngeal aspirarc (IF) Paired sera
nasopharyngeal throar washings ( EM)
i -HpirjJe*

Central nervous system Feces, blood Brain biopsy ( IF -Sc EM); Paired sera
(for arbovirus isolation ) CSF ( EM &L IF)
CSF, (brain biopsy,
throat swab, rectal swab )

Cardiovascular system Feces Nil Paired sera

Skin Macular / papular scrapings* Vesicular/ pustular fluid ( EMfldD) Paired sera
vesicular/ pusrubr fluid, Uktr scrapings ( EM) ,
.
ulcer scrapings, crust Feces, crusts, (EM Sc ID)
throat swab

Eye Conjunctival scraping* Conjunctival scrapings, as smears Paired sera

or swabs on miftostupe slides [JLM & IF)1
Liver Blood (for ydJow fever) Serum (feces) Serum
General;. congenital Throat swab
infections { products of conception ) Nil Single sera
( mother Scbaty )
General; PUD Heparinised blood (arbovirus Nil Paired sera
and arenavirus infections )
throat swabs,
feces ( fresh urine )
Specimens within brackets are not appropriate for rouritie diagnosis byr many be indicated in particular
'

circumstances
IF = Jrnmun0fluAKKflkCC; EM Electron Microscopy, ID Immunodiffosion; LM = Light Microscopy
Tor diagnosis of rabies only
( Adapted from W1 IO)

Copyrighted material
 f -
Vir U B t+osl Inleracliofis : Vital Inlad uns
Microscopy; The demonstration of virus
elementary bodies by examination of stained smears
is now seldom employed. The detection of virus by
electron micTORropv is being Lined increasingly- In
some diseases , it used to be the only diagnostic
method ( for example viral diarrhea ), Demonstration
of the inclusion body is a routine diagnostic method
tor rabies in dogs. The microscopic diagnosis ol
rabies has hecn rendered very sensitive by
fluorescent antibody techniques . 'Hie use of direct
and indirect fluorescent antibody techniques for the
examination uf material from lesions, as well as for
the early demonstration of viral antigen in tissue
cultures inoculated with Specimens has enlarged
the scope and greedy increased the speed of virus
diagnosis.
Demonstration of virus antigen: In ases
where virus antigen is abundant in the lesions, its
demonstratiori by serological methods such as
precipitation in gel or immunofluorescence offers
a rapid method of Lliagnosis , Highly sensitive
serological [ M T S such a s con nterimmuno -
eEcctrophoresis , radioimmunoassay and enzyme
linked immunosorbent assay have found wide
application in diagnostic virology for the detection
of viral intigeiu in clinical samples. Molecular
methods such as probes and polvinerase chain
reaction provide rapid , sensitive and specific
information about the presence of viruses in clinical
sample s. Thev are fait liecoin i ng routine diagnostic
methods in the affluent countries, hut in the poor
nations, their availability is limited .
Isolation of virus For virus isolation it is
imperative that fbe specimen be collected properly
and transported with Least delay to tire laboratory.
As niost viruses ire heat labile , refrige ration is
essential during transport . The methods used for
isolation depend on the virus sought. In general ,
they consist of inoculation into animals, egg* or
tissue culture, After the specimen is processed to
remove bacterial contaminants. The isolates are
identified hv neutralisation or Other Suitable
serological procedures. It hit to he emphasised that
457
the mere recovery of a virus from a patient th »cs
not justify the assumption that it is the causative
agent nf the patients illness . Many viruses ( For
example adenoviruses, enteroviruses) are frequently
found in normal individuals . "lire results of isolation
should always be interpreted in che light of the
clinical data. Demonstration of an immunological
response to the virus isolate in tlie patient during
the course of the disease reinforces the significance
of die isolation .
Scroingienl diogiUMids: The demonstration Lit
-
a ri - c in titre of antibodies to a virus during the
course of A disease is strong evidence that it is the
etiological agent. For this , it is essential to examine
paired *erfl, the 'acute' simple collected early in
the course of the disease and the 'convalescenr '
ample collected 10-14 days later Examination of
a single sample of serum for antibodies may not be
meaningtul except when IgM specific tests are
done. The serological techniques employed would
depend on the virus bui those in general Use are
neutralisation, complement fixation, El . ISA and
hem agglutination inhibition tests.
Mnlccultir diagnosis; The availability of
molecular methods has transformed the diagnosis
of viral diseases, enlarging the scope, sensitivity and
specificity of such tests .
IMMUNOPROPHYLAXIS OP VIHAI
D I S K \ * E -. S
Prolonged and effective immunity is a characteristic
of most viral infections. Viral vaccines also confer
solid protection and are, iq general, more effective
than bacterial vaccines. Viral vaccines mav be live
Jr
at killed (Table 49.2 ) . Live Vaccines are more
effective than killed vaccines . Tlie smallpox vaccine
lias been used at the sole tool for the global
eradication of the disease. The early live vaccines
were developed empirically from natural viruses (as
Jcnncr 's cowpc* vaccine) or by attenuation by serial
passage (as yellow fe ^ er vaccines). The basis of the
latter technique was ail unconscious selection of
avimlent mutants . With the development of more
Copyrighted material
45« + Textbook Of Microbiology *

Table 49.2 Viral vaccines In common u»

£> jsetfw Type ( j/vacci'ne Mode of prepara tion
Poliomyelitis Live A virulent strain* gmwn in monkey Iddney cell culrure
Killed Virulent strains grown in monkey kidney cell culture,
formalin - killed
Rabies Killed (Semple type) Fixed virus grown in sheep brain and inscrivared by
phenol or beta peopiokcroK
Killed Virus grown in cell culture and inactivated with beta
propiolactofie
Yellow Fever Live (17D) Attenuated virus grown an thick embryos and lyophilistd
Japanese Killed Virus grown in mouse brain and inactivated by formalin
encephalitis
Varicella Live Attenuated virus grown in chick embryo fibrublaSICulture
Mumps Live Attenuated vims grown in human diploid cell culture
Influenza Killed (subunit) Virus disintegrated wjih sodium deo hcl me
LIVE [itiehiuted ) ^
Virus aitenuiTed hy serial passage in eggs
Live ( mutant ) fs mutants which are aviruknt
Live ( recombinant) Recombinants with surface antigens of new strains and
growth characters of established strains
Measles Live Atrenu 4iT«l virus grown in tissue culture
Rubella. Live Attenuated virus grown in tissue culture
Hepatitis B Cloned subunit HBsAg doited in yeast
precise genetic techniques , live vaccines have been spectrum of immunoglobulins to the whole range
developed by plaque selection (Sabin vaccine for of vita ] antigens. They also induce cell mediated
poliomyelitis ) or from rs mu rants rjr by immunity. They provide moie effective and more
recombination ( as in hifluenaa ). Lasting immunity than hilled vaccines. They can , in
general , be prepared mure economically and
Killed vaccines have been firepan' ll bv administered more conveniently, especially for mass
inactivating viruses with Heat , phenol , formalin or Immunisation , Some of them can be given as
ben propioLanone , Ultraviolet irradiation is not combined vaccines {measles-inumps^rubella
satisfactory hecause of the risk of multiplicity vaccine) .
reactivation . The reactogenicity of killed vaccines They have ihe following disadvantages: There
has been attempted tu be reduced by the purification
'
is a risk, however remote, of reversion to virulence -
of the viruses . Adverse reactions may also be Thc vaccine maybe contaminated with potentially
reduced by the use of 'subunit vaccines' in which dangerous viruses or other infectious agents. The
the virus is split by determents or other chemicals virus may spread from the vaccines to contacts.
and only the relevant antigens incorporated in the While this is a seriuus danger in some HI turnons,
vaccine . Vaccine production by cloning the desired as when spread occurs to immunodeficietit or other
antigen in bacteria or yeast is becoming increasingly high risk contacts , in other cases, it may even he an
common , as in hepatitis B. advantage (as in poliomyelitis where the range of
Live vaccines have [he following advantages: A vaccination is attended by the natural spread of the
single dose is usually sufficient . They can be vaccine virus among children and adults ) .
administered by the route of natural inficcrinn so Interference by pre-existing viruses may sometimes
that local i m munity ifl induced - They i nduce U wide prevent a good immune response following live

Copyrighted materia
Hidden page
m H Itaxtboofc a\ Wic 'st dogy >

own thymidine kinase fl ISV, V- Z ) are for more
susceptible than tKobe which do not (CMV- EBV ),
The related drug Ginci ^iimr LS more active against
CMV.
The widely publicised drug Azidorhymidinc
( Zidovudine, AZT ) used against LLIV infection is
a rhymidint analogue which blocks the svnlhe -- is
of proviral DNA by inhibiting viral reverse
transcriptase. AZT is used widely m HTV infection ,
h . IE is toxic and costlv
.s .
A series ot Jideosynudetjsides DiiJ .drt £>.Sirie,
1
StJWJtJjnf , Lamivudine ) have been
svnthesised and found tn possess anti - Hl \ activity
by blocking reverse transcriptase . The second group
of drugs used m Hl \ infection is protease inhibitors
(SarjttjjMVjr; /JjfojiJt irh ftidjViiiVJ-r) ,
/Eifctfwmj { VitAZoh ) is a synthetic nucleoside
related to guanosme. It shows activity ags.inst many
DNA and RNA viru-ses. Administered as an
aerosol, it has been effective in the treatment of
respiratory syncytial viral infection and also in
mllurns. Intravenous ribavirin has been reported to
be effect! ve agai ost Lass* fever and other hemorrhagic
fevers.
i liiur A man rad: nr i Aifamantananune
hydrochloride, Symmetrel ) blocks host cell
penetration bv influenza A urus but nor Bor C. A
derivative rimantaibne is less tDxic and equally
effective, A second line of drugs against influenza
employs neuraminidase inhibititmr
Enviroxinc and related chemicals have shown
activity agtimsr rhinoviruses.
Foscarnct Triaadium phosphnnnfnrmate )
(
specifically inhibits DNA polymerase of the herpes
simplex virus and has some effect against hepauus
If and HIV also.
Suramin developed as an anti parasitic drug in
1916 was found to inhibit reverse transcriptase
activity and so was one of the first drugs ut-cd against
AIDS- Because of toxicity and inadequate efficacy
us use was discontinued.
Interferons: The discovery of interferons, with
activity against a wide range of viruses raised hopes
of their application as anriviral drags. However,
most trials have been unsuccessful. Eenefici .il effect
has been obtained in persistent infections such as
hepatitis B and C, laryngeal papilloma and against
CMV infecth ins in transplant recipients. High doses
of interferon lead to toxic effects .
In spite of intensive efforts, progress in the field
of antiviral chemotherapy has not been satisfactory
Various (actors contribute to this. Many compounds
show antiviral activity in tissue Culture but most of
them prove to be ineffective or toxic in animal tests.
The available drags have a narrow range of activity.
They are seldom able to eradicate the virus from
the host so recurrence is common. Viruses develop
resistance to the drugs and break through infection
takes place even duemg t r e a t m e n t . The AIDS
pandemic has been a catalyst in development of
antiretroviral drugs . It is hoped that better
understanding of the molecular and cellular biology
-
of viruses and of virus host interactions may lead
to the development of more effective antiviral agents.

Further Reading
Collier L andj Oxford 3000. Hvman Virology, London: Oxford University Prrse .
Fields DM et al . 19%, pyroJqgy 3 J tdn. Philadelphia: Lippmoolr- Raven .
Harper UK . 199 J . AfofeorJar Vlm/q T. Oxford: Bios .
^
Zuckerman AJ ft all 3000 . Ciinjcif Vjra/op; llh ed . Cbicheiterjohn Wiley.

Copyrighted materia
 L J Bacteriophages
*
Bacteriophages (commonly abbreviated as phages) are Phages occur widely in nature in close
viruses tliat infect bacteria. Twurt ( l 9 l5) described a association with hacterij. They be readily
can
degenerative change in staphylococcal colonics isolated from feces* sewage and other natural sources
isolated from calf lymph, which could be transmitted
, ofmbted bacterial growth. Early hopes that phages
serially by application of culture filtrates from the could be used in the treatment of bacterial infections
original growth. d’Herelfe (1917) observed that have not been fulfilled but these viruses have
filtrates of feces cultures front dysentery patients Contributed much to microbiology. As phages could
induced transmissible lysis of a broth culture of a be grown easily on bacterial cultures, they provided
dysentery bacillus. He suggested that the lytic agent
was a virus and gave it the name bacteriophage.
the only convenient model for the study of virus
host interactions at the cellular and molecular levels
-

\Z _ B

c
D

'
6

Flg.SQ . 1 Morphology oP bacteriophage . A. hevagonat head , B. DMA cote, C. demarcation between head and tailr
. F. tail litres . G- prangs ; Right, process of injection ol phage DNA into host cell
D. tail, E . baa ? plate

Copyrighted material
462 * Textbook d Microbiology

before The dcvrloprOCnt uf cell culture IftlutUJUtS
made similar studies with animal vim ^- es ] n.sn ^ iL )lc .
Phagts piay an important mV in the transmission
of genetic information between bacteria by the
process of transduction . The presence of phage
genome integrated! with bacterial chromosomes
confers on bacteria. certain properties by a process
known as phage convttsii JJI. Phages have been used
ns cloning vectors in generic manipulations.. 'I'hc
presence of high corcci' iirnitioiLH of phage particles ,
upiu 10s per ml in some natural waters suggests
that they may have ; L role in the control of bacterial
populations in such environments. The specificity
ot the host range ot phages is the basis of phage
typing methods by which bacteria can be identified
and typed.
bacterial genome , replicating synchronously with
it, causing no harm to the host cell ( Fig. 50.3),
! lie eyeduf Replication of a virulent phage can
be considered in the hdlowing stages adsorption,
penetration , synthesis of phage cornpon*nt 5 n
assembly, maturation arid release of progeny phage
particles .
Plixgt' particles Come into Contact with bacterial
cells by random collision . A phage attaches to the
surface of a susceptible bacterium by its tail.
Adsorption is a specific process and depends or
the presence of Complementary chemical groups
on the receptor sites of the bacterial surface and or
the terminal base plate of the phage. Under optimal
condition ?.. adsorption is- a very rapid process, being
complete within minutes (.iertaip oofcctoro, such
,

Morphology ; Certain hactLTHiphages thaL irtlkt as cations , are necessary for adsorption . The
£, coli , called the T even phages (T2, T 4, T6) h
have been studied in great detail and traditionally
serve as the prototypes in describing the properties
of bacteriophages.
T even phages have a complex and characteristic
morphology. They are tadpole shaped , with a
hexagonal head and u cylindrical mil . The liead
consists. of a rightly packed core of nucleic tcid
(double stranded DNA ) surrounded by ii protein
coat nr capsid . The SLAV of the head Varies hi
different phages from 2H n. m to 100 nm . The tail JS
composed of a hollow core, a contractile sheath
surrounding the core and a terminal base plate 1
which has Attached to it prongs , rail fibres or both
1
n - 1 Sl
.

( Fig. 50.1).
Though im > si bacteriophages have the
morphology and structure described aljuve, phages
that are spherical or tilamcntous and possess
single stranded DNA or RNA have also been
identified .
1 . iff eycks Phages exhibit two dif 'fercm types
of lifecycle. Irt tile virulent Or fyiic cycler ,
intracellular multiplication ot the phage culmin -Ate -s
in the Ivsis of rhe host bacterium and the release of
m .
Fig . 50 2 Bacteriophages [ t 200,040) Multiple intention
progeny virions , In the temperate or fysOgvnk tytJe PI a bacterial ceil ( lysis from Mlhouti ( Source;
the phage DNA becomes integrated with the HlCED )
 * Bacteriophages 463

ThXTflOoit OE M CR0BIOLOG1

1
FJ

C
Q O O

D
00 0/1
o.cAo
l
i "
E i K
od
M0 C ' i
o
F
i L_ J
L
G
*
Fig. 50.3 Lylte and lysogenic life of bacteriophage . A. adsorplian. B. Injection of phage DMA, C. elreularlsatlon cl
phage DMA . D replication ol phage DNA, E. production of phage components, F. assenbly of phage ,
G . release of progeny phage, H. integration of phage DMA with host chromosome, I. binary fission of lysogenic
bacterium , J . daughter bacteria Carrying prophage, K . eiCl$lQn ol prophage, L. same stage ± 6 C. ( A Ip C
injection; D ID G lytic ; H to J lysogenic cycle ; K tt> L induction. )
 b
Fiq
. 3 2 - layers &

50.4 11 athe rti J

of
£

ltp
"
a

£ 5
Z

cell re pcor
growth *1
H

_.
u
wal sites
or
TITflE OF INFECTIVE PHAGE
on may
r;
.
=
curve
cf T
PARTICLES
O. o
--
C
S

3
3
u
rr
-
a
surface situaed
be

struce difernt
b :-
- 5

aclsfiphag rn Q
W
§ 3 S
iJ
CL.
era
(

such
tn

—I
m NJ
ipg
4
z r rZr
.
~
2 s3* s3 Xz-Jr cr 2L r} .
J U5 ?
^ of
^- - - E the produces
5 IT
*
"

f 8 i
slJ- s.- g- l l!g *^J': Sg=
" S "£i o n

a
O LO
HO Rise Period 52 - ^
S TJ g E g s i g e
^s i 2. N i -l ^ P 111
l lis 3^§ "s~G- U T
'

S
S-
^
*
- L-
s §. ? ?K p ? S fl
'
3 !
S P p f H J f ai E hole 5 s
phage
1
hr
ora. <
« S
c
S
S
ti

•
E,
W “
Q 3

n
n P3
'
T3

Q
a

j .core ao
:
^ rc> a D "c - ' 8/
l

r*'
.. jS
1 &, §
*
T ? ; SI.45 B a. ?1
3 rt

f t
ir«
'
?G §
£ 5 S s- 8 & ?:|; s i «
O
- Pu
5
3 15

>‘ 2*
ITS 1 1 -
n A a '

a£ -*
jr “ $ *
B
> i r 1i The the
I T r.

— g
"3
® S
.
c f t
3 ^— 33 2
m 1

1f 1
complex bacterial
3 ^ 3
H- S
Burst SiZti
i 1. 3- P- f
E
S Ft
'

o
3

I s 3.
IA H, 5 **
;
.

E c «.
c '
-
Tj

^r
o 5 § Q Sr
I? S
Sr S 5 '
f I 5 n- K --“ T
E
wal
struc e entry
1
- IA ^ EC |P.
to
zr z
u
a G
s 0 zr r
=
Ill HI ri 3 s n

d
TJ X
|v.
3- S S? =
3
r.
9
3
"h F fl
for
3 £ s|
3
* i 2 the
3 S a * “O

= of
CD
S < i 1WI1
S- f H £
r. - -
3 --. the
Cd * f- 7
 < Bacienopnagat *
synthesised separately in the bacterial eel ] . The
UNA is condensed into a compact polyhedron and
’ packiged ' into the head and , finally, the tail
structures are added - Thi* assembly of the phage
components into the mature infective phage particle
is known as maturation.
Release of the mature progeny phages typically
occurs hy lysis of the bacterial cell . During rhe
replication of the phage, the bacterial cell wall is
weakened and if assumes a spherical shape . Phage
filliyma act on the weakened cell wall causing it
to burst or lyse resulting ill the release of mature
daughter phages.
The interval between the entry of the phage
nucleic acid into the bacterial cell and the
appearance of the first infectious intracellular phage
particle is known as the tcllpst phase. It represents
the time required tor the synthesis of the phage
component* and their assembly into mature phage
particles. The interval between the infection of a
bacterial cell and the first release of infectious phage
particles is known as the inenr/xnaci. I tin mediately
following the latent period., die number ol phage
particles released increases tor ;L tew minutes till
the maximum number of daughter phages is
attained. This period, during which the number of
infectious phages released rises , is known as the
rf >e - period. The average yield of progeny phages
per infected bacterial cell is known as the buTJf
size. This is estimated bv experiments in which
infection is established with ore phage per
bacterium and the release of infected phage particles
is estimated serially over a pencil of time. The
result* of such an experiment plotted on a graph is
known as the OIK -step growth curve ( Fig , 50, 4) ,
Lyngenio cycle Unlike virulcnT phages which
produce lysis of the host cell, temperate phage*
enter into a symbiotic relationship with their host
cells without destroying them . Following entry into
the host cell, the temperate phage nucleic acid
becomes integrated with die bacterial chromosome.
The integrated phage nucleic acid is known as the
prophet . The prophage behaves like a segment of
465
the host chromosome and replicates synchronously
with it. This phenomenon is called JyKjjejry md a
hacte ri urn that carries a prophage with i n i rs ge iiome
is called a fy*qgEajv bacterium. Lysogenisarion does
not upset the bacterial metabolism.
The prophage crullers certain new properties
Lin the lysogenic bacterium . This is known as
h yugetuc conversion or pJutgr conversion. Tiny is
due to the synthesis of new proteins dial are coded
tor hi the prophage UNA. An example is Toxin
'
production by The diphtheria bacillus . which is
,
determined by The presence in it of the prophage
herd. The elimination of the prophage abolishes
the toxigenicity of the bacillus.
During the multiplication of lysogenic bacteria,
the prophage may become 'excised ' Emm occasional
cell s.The exci sed prophage init iates lytic repliCation
and the daughter phage particles arc released ,
which in tec r other bacterial cells and render them
lysogenic. This Is KNOWIL as spontaneous induction
of prophage . While this is a rare event , all lysogenic
,
bacteria in a population can be induced to shift to
the Ivtie cvcle hy exposure to certain physical and
chemical agents . Such i mind rig agents include UV
rays, hydrogen peroxide and nitrogen mustard .
A lysogenic bacterium is resist , mt to reinfection
by the same or related phages. This is known as
fupericfecrirxD immunity.
Bacteriophages may act as carriers ol genes from
one bacterium to another . This is known as
fransdircrion . Two types of transduction are
recognised - In restricted transduction, only bacteria I
genes contiguous to the prophage are transmuted .
For example, rr JUSLIUCIION hy the pmpha c htaibdu
.
in E calr K 12 transfers only the gal gene ^ '
( determining fermentation nt galactose ) , which is
'
the bacterial gene contiguous to the prophage . On
the other hand, any bacterid gene inaylfe transferred
in generalise! trmfductioruTniwducrion has been
demonstrated in many genera of bacteria and
constitutes one of the most important mocha nis ms
of genetic exchange among bacteria in nature -
Plasmid mediated drug resistance in staphylococci
Copyrighted material
466 * Textbook o4 Microbiology *
in an example of a medically important property
that is transmitted bv transduction .
•iff
Phage particles cxhibil general ^ Tibili’ V of type
and a low rate of hcrirablc vari.irinp.
If bacterium simultaneously adsorbs two
jf
related but slightly different DNA phage panicles,
both can infect and reproduce- On lysis both types
are released. When this occurs many of the progeny
are observed to be recombinants.
PHAGE ASSAY
When a phagJC is applied on the lawn culture of a
susceptible bacterium, areas of clearing occur after
i niubati - wi. These zonc & of lysis are called plagues ,
rbr size, shape and nature of plagues are
characteristic for different phages. Since under
optimum conditions a single phage particle is
capable of producing orte plaque, pl ,n| UL .Lssay cart
be employed for titrating the number of viable
phages in a preparation. As plaques are analogous
to bacterial colonies, plaquing is also useful for the
pur fication of phages .
PHAGE TYPING
-
Tile specificity of phage bacterium interaction is
made use of in the identification and typing of
bacteria. Phages exhibit different degrees of host
specificity. Some phages possess wide host ranges,
covering many bacterial genera, while others have
a narrow range limited to certain str.iins of hactei la'
only. With some phages serial passage in a strain
-
of bacterium makes them spe Ific for that strain
and related strains ( adaptation of host range ).
Phages are available that lyse all mem bers of a
bacterial genus I for example genuR - Rpcri :1 c
bacteriophage for SaJmoneljfa ) , all members of a
species ( for example specific hucterinphagc (or ft.
anfhrycH , and all members of a biotype or
subspecies (for example Mdkeriee's phage IV which
lyses all strains of classical V. chaleme but not V.
choleric biotype El Tor ) . The most important
-
application of phage typing : s for intraspecics typing
of bacteria, as in the phage typing of if . fyphi and
staphylococci. Adapted phages, aedve only against
fresh isolates possessing the Vi antigen, are used
For phage typing of typhoid bacilli . Staphylococcal
phage typing is a pattern method using a set of
,
standard phages. A strain of Staphylococcus may
be lysed by a number of phages and the phage type
of a strain is designated by the numbers of the
different phages that lyse i:..
As lysis is influenced by the dose of infection,
phage preparations used for typing should be
standardised by titration , Titration is carried out by
appiving seri.d dilutions of the phage preparation
on a Lawn culture of a susceptible strain and
observing the lysis after incubation. The highest
dilution of the phage preparation that |List produces
confluent lysis i >. known as the routine test dose
( RTD) .
BACTHHIOCINS
Gratia ( 19.25 ) observed the production of a highly
specific antibiotic substance hyune &tr .u :i of E. call
which wasa< rive against another strain of the same
species . The name coin:in. was j: iven to such
substances produced by E. cali and other members
of the family Entcrobactericcac . With the
-
recognition that colicin likc substances arc
produced by several other bacteria also, the generic
name bacfeWodn was proposed for the group of
liighly specific antibiotic-like substances produced
by certai i L struins of bacter nwli Lch are active agai list
other R f r .L i r i R of the same or different species.
Bacteriocins are gtven specific names based on the
bacteri al species of origin, fui example colic ins from
E .cali , proems from Ps. jiynerrenea ( am rnosaX
mcgacins from B , mcgstcrium and diphthcricins ^
from C. diphrjfieriae.
-
B .LCIvnocin are proteins but some rn .LV have
Lssociated lipopolysiccharides derived from the cell
walls of bacteri . i producing them. B& cterincLns and
phages resemble each other in a number of respects.
Both adsorb on the surface of susceptible bacterial
cells on specific receptor sites some of which may
he the same for phages and bacteriocins, Under
Copyrighted materia
 * 6 ac1eriophages 467

( Col factors ) , (Jol factors arc cpi somes and can be

transmitted from cell m cell by conjugation or
transduction , Certain physical and chemical agents
(UV rays, nitrogen mustard } induce colicin
production by the cells harbouring Col factors.
A celt producing a bactcriocin is immune to it
but may be sensitive ro other bar ferine in s .
Bactcriocinshavc a very specific activity on bacteria,
being capable of billing some btJT not all strains of
a pecies. Tine Specificity it made UM: uf in typing
i-

certain specie^ such as Sil . iumiej', ProStua xp. Pi . r

aerujjjno.?. j . Harterioeins kill susceptible cells

without lysing them.

Fig . W -5 fisclerlocin typing - Gacleriocin produced by
producer strain nas Inhibited I no growth ai lest
i IK:
strains In Ihe centre.
rhc election microscope , some hactcriocins ,
especially proems , appear like the IAIL structures of
phages . ] hey may lie considered products of
F
While phage typing schemes are generally based
on the sensitivity of the test strains to the tytic action
of phages, bacteriocin typing schemes depend on
the ahi I iiy of bacteriociiw produced by the test strai n
to kill standard indicator strains of bacteria. The
usual method of bacteriod.il typing employs the
plate diffusion technique . The lest bacterium is
inoculated as a broad streak on the centre of a
culture medium , the bacterid growth is scraped
off and the remaining cells killed by exposure to
chloroform vapour. Standard indicator strains of

defective pbsgy genomes, able to code only h>r parti; bacteria are then streaked at light ingles to the
of phage particles. original inoculum . After incubation, the pattern oE'
The synthesis t > f baddunlins is determined by inhibition of the indicator strains represents the
rhe presence in bacteria of colicin-ngcnic factors bacteriocin type of the lest bacterium (Fig. 50.5).

Further Kending
Anderson IT. 1981. Reflection! on phage genetics. Ann RsvGenaia 15 :^ 05
"

Dav M. 199S. Baaenodns and bacteriophages, In TipTey & H ifemr 's .Hdi'cnjfctA y 4rtd Mitwhitl ( nfL-nion .-. , VruL. 2.
I.nndnn: Arnold-
' ^
MjdirwE CECctal («1E ). 1983, .JXfletenopA c T 4 , Washington D- C; American Soriery tor Mkrobtoiogy.
1

svrrahtet ia
Hidden page
 4 Poxviruses *
unique in thal it LH . LEI artificial vims jnd does not
' "
occur in nature is such. It his been studied m greater
detail than variohi , as it is safer to work with .
Vaccinia virus is being employed as a vector for
the development of recombinant vaccines. The
vaccinia genome can accommodate about 25 *000
foreign ha.se pairs sufficient tor introducing several
genes. Many genes have been inserted , including
those coding for the antigens of hepatitis B virus,
HIV, rabies, ami toi pharmacologically important
products such as neuropeptides. However the
vaccinia virus is not suitable as a vector for human
use due ro its pathogenic effects.
Vaccinia and variola viruses are so similar in
their properties that they car be considered together
Morphology : The virion is brick shaped fn
vertical section ir consists of a double lave red
,
ar
membrane which surrounds a biconcave 'nucleoid '
containing the DNA core On either side of the
* .
nucleoid is a lens shaped structure called the lateral
body (Fig, 51.1 ). The virion measures about 300 *
200 * 1G0 nm and so can be seen under the light
microscope . Variola virus was first demonstrated
microscopically hv Ruisr in 1BE 7. Paschrn in I 90fi
developed a staining technique for the virus particles
and demonstrated the elementary bodies ( Paschcn
bodies) in smears bom smallpox lesions .
Physical and chemical properties:
Poxviruses arc stable and if protected from sunlight
may remain viable tor months at room temperature .
In the cold or when freeze dried , they survive for
years. They are susceptible to ultraviolet light and
other irradiations. They are resistant To 5(M> glycerol
and 1 % phenol but arc readily inactivated by
formalin and oxidising disinfectants . The virion
consists essentially of DNA, protein and lipid .
Though enveloped, the vims is not inactivated by
ether. The virion contains a multiplicity' of enzymes.
The entire multi plication of the vims takes place
in the cytoplasm of the infected cell.
Antigenic structure: All poxviruses share a
common nuclcopmtein ( NP) antigen . By
immunodiffusion some twenty different antigens
469
LB
.
Flg.51.1 Structure of vaccinia virus The nucleic acrid is
contained within a dumb -beN shaped core ( C ).
FHiring Inio the concavities d Mia core are iwo lateral
bodies (LB) The virion is enclosed within a protein
shell which has an irregular surface .
have been identified . These include the L$ antigen
(a complex of two antigens, the heit labile L and
the heat stable S antigens ), agglutinogen , and
hcm .igglmiinir , which is responsible for rhe
agglutination of erythrocytes of those fowls which
ate also agglutinated uunsjiet ifu- jllv by tissue lipids.
Cultivation and host range: The variola
and vaccinia vimses can he differentiated hv rheir
growth characteristics and host range .
Chick embryo: Both viruses grow or the
CAM of 11-13 day old chick embryo producing
pocks in 48-72 hours. Variola pocks arc small, shiny,
white , convex, non - ueCTOtic , nun - hemorrhagic
lesions . Vaccinia pocks arc larger , irregular, flat,
greyish , necrotic lesions, some of which are
hemorrhagic ( Fig. 51.2). The viruses may also be
differentiated by their 'ceiling temperatures' , the
highest temperature above which pucks are nut
produced.1"he ceiling terrperatures art 41.(1 UC fqr
vaccinia, 38 "C for variola major and 37 -5 "0 for
variola minor.
Tissue cutaz/r; Variola and vaccinia viruses can he
grown in tissue cultures nf monkey kidney, HeLa
Copyrighted material
m * Toxibocfc of Micros: D - cgy *
-•
** j
7
F
Lv , _ c-
Fig. 51.1 Vanold and vaccinia pocks on CAW . Left
showing large , irregular pocks
and chick cmbiyo colls . Cfytopsthic effects are
produced by vaccinia in 24-4B hours and. more
slowly by variola . Eosinophilic inclusion bodies -
-
Guarmeri bodies can be demonstrated in stained
preparations. The inclusion bodies consist of
aggregations of virus panicles in a matrix. Vaccinia
but not variola virus produces plaques in thick
embryo tissue cultures-
Animiis: The vaccinia virus can infect a wide range
ui animain experimentally. Monkeys , calves , sheep
and rabbits can be infected by scarification leading
Co vesicular lesions. The variola virus produces
similar lesions only in monkevs. Scarification of
rabbit cornea with variola virus leads to keratitis
and sections of the cornea will show typical
Guarnicri hodics , Intranet instillation of the
variola virus in the monkey produces a self-limited
attack of smallpox with generalised skin lesluns.
SM A I T PCIX
Smallpox has been eradicated.The last natural case
of variola major detected was Saiban Bibi * a
Bangladeshi woman found with smallpox un ibe
Karimgani railway platform in Assam on 24 jVlav,
1975 - The Last Case < tf variola minor occurred in
Mcrea , Somalia, in October 1977 . The coming
. 'I
J
I
- variola , showing small, uniform pocks ; righl - vaccinia,
generations are unlikely to witness the disease but
its disappearance has been too recent for it ro be
inquired altogether . A brief account of smalljiox is
therefore being presented. ( For more details about
smallpox* the 3d edition of [ bis textbook may he
consulted -)
Smallpox was an exclusively human infection,
with no animal reservoir. There were no carriers
as the vims was eliminated completely from the
patient on recovery. 'Hie source of infection was a
patient in the early phase yf the disease* though
Infect ivity extended from the appearance of buccal
mucosa! lesions (enanThem s) to the disappearance
ot all the skin lesion (exanthems). Infection usually
occurred only in close contacts. Virus entered the
body by inhalation. After ini rial multiplication in
the local lymphoid tissues, the vims reached the
reticuloendothelial ceils , where further
mulriplication took place * leading to a severe
virertiLj with seeding of the mucosa and skin
heralding the clinical disease/ I'he incubation period
was around 12 dav$,
’ *
l he single crop of centrifugal exsnthenns passed
through macular, papular * vesicular and pustular
stages, before scabbing and healing by scar
-
formation in 2 4 weeks . The exanthems varied io
FIC r
Hidden page
Hidden page
Hidden page
 Herpesviruses
The herpesvirus family cuntMIW ( iVet x hundred
spcc i e s of enveloped DNA viruses that affect
humans nnd animals. ' [’ hey are L- h & ractcrised by
their ability to establish latent infecfifmj* enabling
rhe virus to persist indefinitely with in infected hosts
and to undergo periodic reactivation.
The herpesvirus cups id is icosahedral, composed
of 162 capsomers, and enclosing the core containing
the linear double straoded l ) N" A genome. The
nucLeocipsid i* surrounded by the lipid envelope
derived from che modified hose cell nuclear
membrane through wlti ^ li the naked virions bud
during replication . The envelope carries surface
spikes, about S nm long, Between the envelope and
the capsid is an amorphous structure called the
tegument , containing several proteins . The
enveloped virion measures about 200 nm and the
naked virion about 100 urn in diameter.
Herpesviruses replicate in the host cell nuclcus.
They form tfowdry type A intranuclear ( Lipschurc )
inclusion bodies . Like other enveloped viruses,
herpesviruses arc susceptible to fat solvents like
alcohol, ether, chloroform and bile salts . 1'hey arc
heat labile and have to be scored ar -70 "C.
The family Herpesviridae is divided into three
sub fa nil lies based on biological „ physical and
genetic properties :
Alphahcipcrvimscs , with a relatively short
replicative cycle ( 12 -1 S hours) , x variable host
range and a tendency to cause latent infection in
sensory ganglia . In culture the \ are rapidly
cytopathic and infectious viruses may be released
ir ^ m cells, for example herpes simplex virus ,
varied la - zosler virus .
ifefilierpesi'irtJies, which replicate slowly ( more
than 24 hours), have a narrow host range, grow
best in fibroblasts- with a tendency In produce
enlargement of infected cells (cyromegaly ) and cause
latent infection of s,divan' gland and other organs.
In culture ^ the cytopathic effect is slow and the
virus remains cell associated, for example
cytomegalovirus .
( 1 jnimLificfpe.svTruse.s, which have a narrow
host range, replicarc in lymphoblastoid cells, are
specific for either li orT lymphocytes and frequently
cause latent infection in lymphoid tissue , for
example le- pstcm-TJarr virus.
tight different type? of hepesvinuta are known
whose primary hosts are humas. They have been
officially designated ‘Human hcrpcsvini* types 1-
$ but their common names continue to lie in
general use, except for types 6, 7 and S (Table S 2.1 ).
The herpesvirus family has no common group
antigen and the different herpesvirus species do not
show any significant antigenic cross reaction, except
between Herpes simplex types 1 and 2 .
HERPES SIMPLEX
The her ]ieH simplex vims ( HSV ) occur* naturally
only in human , but it can produce experimental
infection in many laboratory animals. There are two
rypes of the herpes. sim|ilrx vims . HSV type 1
( Homan herpes vims type 1 or Hi IV type 1 ) is
usually isolated from lesions in and around the
niotith and is transmitted bv direct C ^ nt ? Ct or
droplet spread from cases or carriers. HbV type 2
[ ] I E I V type 2 } is responsible for the majority of
g e n i t a l herpes in feci ion * and in c o m m n r l v
Copyrighted materia
 * PoUYiruSiU* ¥ 475

Table 52,1Classification of human herpesviruses

Specie*
Subfamily Cytoptthology Silt of latent
Official name
, Common name infection

Human herpesvirus type 1 Herpes simplex virus rvpe 1 alpha qtioljflX neuft> rl
*
Human herpesvirus type 2 Herpes simplex virus type 2 alpha . cytolytic neuions
Human herpesvirus type 3 Varicella zoster virus alpha . tyrolytic neurons

Human herpesvirus type 4 —

Epstein Barr virus gamma ! vnrphoproILfera t i ve IjTnphold tissues

Human herpesvirus type 5 Cytonugikwirui beta cytomegalic secretory

glands
Kidneys, other
organs and
tissues

Human herpesvirus type 6 Human B cell lymphotropjc ben hinphopKiliimtive lymphoid iissues
virus*
Human herpesvirus type 7 R K yinis* beta lymphopFoliferatlve lymphoid tissues .

Human herpesvirus type B gamma

* These names are no Icmgct in use

iriitsmmed Verterc:LL!V. IiH racertrbfal inoculltiun in
J
rabbits anti mice leads to encephalitis and cornea ]
scJtrificicion produces kfnhKfflijuDCtmtii in rabbits.
The vims glQWE in a variety of primary and
enntiminus cell cultures ( monkey or rabbit kidney
human amnion, HeLa ) producing cytopaihic
changes, well defined foci with heaped up cells
aiul syncytia ] or giarr cell formation . Or chick
embryo CAM, small ( diirtWter less thart 0.5 rnfn )
-
white shiny QOfl CKCFOrfe pocks are produced (Fig.
52- 2 ) . The two types of the viruij cm? react
serologically. They can be differentiated by the
following features:
1 ) Antigenic differences can be made out using type
specific Tunnodtinill antihodic*.
2) On chick embryo CAM type 2 strains, form
5 ) Tvpe 2 strains are more neuruvirulent in
laboratory animab than type 1 .
6 ) Type 2 strains are more resistant to antiviral
agents like- 1UDR ami ivtarabine in culture .
7 ) Restriction endonuclease analysis of viral l ) NA
enables differentiation between the two rypes
,is well as between strains within the same type .
Pathogenesis: Herpes simplex is one of the
nurit common viral infectious in humans ,, about 60—
90 per cent of adults showing detectable antibody.
Prirtlny infection is usually acquired in early
childhood , between two and five years of ape .
Humans arc the only natural hosts ? nd the sources
of infection are saliva , skin lesions or respiratory
secretions.Asymptomatic carriera form the more
important source or infection , especially in genital
'

larger pocks resembling variola ,
d ) Types 1 siraiiw replicate well in chick embryo
fibroblast celts , while type 1 strains do so poorly .
4 ] The infectivity of tvpe 2 is more- temperature
sensitive than that ot type 1 .
infection with type 2 strains . Transmission occurs
by close contact and may be venereal in genual
herpes. The virus enters through defects in the skin
or mueous membranes and multiplies locally, with
-
cell 'To cctl spread . The vims. enterv cutaneous nerve

Copyrighted material
Hidden page
 * H&rpe
^viruMS
ateu with wide spread ulceral ion . A clinically
indi & tinguishabk picture is also produircd by
vaccinia virus infection, both designated Kaposi s
rMrkctlifoim eruption.
jMucoy .t }: The buccal mucosa is tlis site most
commonly ifFcctfd, GingivQttomatitis a nd
phiryngiris. are [ fie most frequent conditions in
primary infection and recurrent herpes labialis in
recurrent infection. The vesicles nuy ulcerate and
become secondarily infected.
t T/ltfut/pmc: HSV infection is the- most common
^
cause of corneal blindness in some developed
countries. Acute kcratoconjunctivitu may occur by
itself or by extension from Facial herpes - Follicular
conjunctivitis with vesicle formation o i l the Lids is
another manitestafi on . The cornea may he invotved ,
with typical branching dendritic ulcers .
[ )ehr i de me nt, topical antiviral dr\Lg$ an d i nterlc n trt
help in healing. Steroids are contraindicated as they
lead to deep stromal involvement and healing may
be delayed, with scarring and corneal blindness.
Chorioretinitis and acute necrorising rectnicis are
uncommon but serious manifestation.
Nervous system; HSV encephalitis though rjie , is
the most common sporadic acute viral encephalitis
in most parts of the world. HSV encephalitis has
an acute onset, with fever and focal neurological
symptoms. Brain biopsy was employed in diagnosis
ftir instituting early specific therapy. This is now
replaced hy demonstration of HSV DNA in CSF
by PCR , which is a very sensitive test in rhe acute
stage . HSV meningitis is a self - limiting disease ,
usually resolving in about a week, without sequelae .
The CSF shows lymphocytic pleocytosis and may
yield the vims in culture.
HSV can cause sacral autonomic dvsfuncrion B
and also rarely transverse myelitis or the Guillian-
liarre syndrome . HSV has been implicated in the
etiologv of Rel I 'H palsy.
Vi&Cttntl: HSV esophagitis may cause dysphagia,
substcrnal pain and weight loss. It may involve the
respiratory tract causing tracheobronchitis and
pneumonitis. HSV is an uncommon cause of
477
hepatitis . Erythema multi forme rrav be seen in
association with HSV infection. Disseminated HSV
infection UI .LV occur In patients with
immunodeficiency, malnutrition or Ibums-
GaiitAl: In rhe 1970s genital herpes became the
most rjpidlv increasing venereal disease, particularly
in the USA. In men, the lesions occur mainly on
the penis, or in the urethra causing urethritis . In
women , the cervix., vagina, vulva and perineum are
affected. When only the cervix is involved, the
infection may be asymptomatic. The primary
infection is usually more icriouc, accompanied by
systemic fen to res like fever and malaise , It is
followed by several recurrent episodes whieh arc
milder. The vesiculo- ulcerative lesions may be very
punfnl. Rectal and perineal lesions occur in
homosexuals . Both types of ) ESV tmy cause genital
lesions , though HSV 2 is responsible more
frequently and causes many more recurrences .
There have bee n several reports of ai L JMSociit M > n
between I CSV 2 ,uid carcinoma of the cervix uteri
but a causal reLiionshlp has not been established.
CH CIHUL' Ttmiphcetm] infection with HSV 1
^
Or 2 can trad to congenital mallurmalions , but this
is rare. Infection may occur during birtli , particularly
it the mother has genital lesions due to HSV 2. In
such cases, cesarian section may prevent infection.
Postnatal infection is more commonly due to
HSV i . Neonatal herpes may be confined to the
eyes , mouth or skin , but is more commontv a
disseminated disease having multi - organ
involvement, with oi without encephalitis. The
mortality rate is very high ,ind survivors may have
neurological impairment.
Laboratory diagnosis : The dlagnos is of herpes virus
infection may be made by microscopy, antigen or
DNA detecrion , virus isolation or serology.
AJrrfij.wnpr: The Tzanck smear is a rapid, fairly
sensitive and inexpensive diagnostic method. Smears
are prepared from the lesions, preferably Irum the
base ( if vesicle ^ and stained with 1% aqueous
solution of toluidinc blue '0 ' for 15 seconds .
Muldnudeared giant cells with faevred nuclei and
Copyrighted material
478 i T&xtbaak ol Microbiology
homogeneously nec1
. ground glass' chromatin
"
{ Tzanck cells ) constitute a positive smear .
Intranuclear type A jadwibn Undies may be seen
in Giemsa stained smears. The vims particle may
also be demonstrated under the electron
microscope . It is nor possible to different Late
between herpes simplex and varicella zoster by
micron copy. The herpesvirus antigen may be
demonstrated in smears or sect iotas from lesions be
the fluorescent antibiwJy technique. The fluOttStCiit
antibody test on hrain biopsv specimens provides
reliable £ nd speedy diagnosis in encephalitis. PCR
based DNA detection bis replaced brain biopsy.
.srjlatmji: Inoculation in mice and on chick
'
j
embryo LAM is insensitive and has been replaced
by tissue culture for virus isolation , Primary' human
embryonic kidney human amnion and many other
cells are susceptible, but human diploid fibroblasts
are preferred. Vesicle fluid, spinal fluid , saliva and
swabs may he used. Typical cytopalhic changes may
appear as early as in 24-48 hours but cultures should
be observed for two weeks before being declared
negative. Drug susceptibility too cm be tested in
cell cultures .
Differentiation between HSV types 1 and 2 mar
be made by a variety' of serological techniques, by
nucleic acid hybridisation or by restrict ion
endonuclease cleavage and electrophoretic analysis
of viral DMA or viral pnotiens.
Strategy: Serological methods are useful in the
diagnosis of primary infections. Antibodies develop
within .t tew days of infection and rise in title of
ami bodies may be demonstrated by ELISA ,
neutralisation or complement fixation tests . In
recurrent ot reinfection herpes, there mav he little
change in the antibody litre .
< jhanatherapy; Idoxyuridine uved topically in
eye and skin infections was one of the first clinically
r m"
successful antiviral agents. The introduction of
acyclovir and vidarabine enabled the effective
management ot deep and svstemic infections. Early
treatment with intravenous acyclovir lias improved
the outcome of encephalitis . Oral and topical use
»
may help iin less serious conditions . Valacidovir
anrl famciclovir arc more rftcctivc oral agents.
When resistance to these drugs develop, drugs like
toscamet which arc independent of viral thymidine
kinase action may be useful,
HERPESVIRUS 5IMIAE: B VIRUS
This vims was isolated by Sabin and Wright ( 19.14)
from rite brain of a laboratory worker who
developed fatal ascending myelitis after being bitten
by an apparently healthy monkey. It came to be
known as the ' R ' virus from the initials of this
patient . Many nimlircases have been reported since
then. Herpesvirus si m i ae i nfects old world monkeys
in the same manner that herpes simplex infects
humans, the infection usually being asymptomatic .
The typical lesions produced are vesicles on the
buccal mucosa which ulcerate shedding the virus
and inferring contacts, 'Though most human cases
have followed monkey hires, the infection in some
was acquired through che handling of monkey
tissues .
Herpesvirus simiae is similar to herpes simplex
virus in its properties . The [ Wo are antigenkallv
related but the herpes simplex virus antibody does
not protect against herpesvirus simiae infection . A
fcrfmoLised vaccine has been tried experimentally
in laboratory workers at risk.
The disease in humans is usually fatal, 'l'he rare
patients who survive have serious neurological
Sequelae . The official name for R virus i -.
Ccrcopfthcaac herpesvirus 1.
VARICELLA ZOSTER
As early as 1 RB9, Von frokay had suggested that
varicella { chickenpox ) anil herpes 7.nstcr a r e
dif feren t man i festatirms of the same vi rus infection .
Virotogieal and epidemiological observations have
proved this concept. The virus is. therefore culled
varicella aosCer virus ( VZV ). Chidcenpox follows
primary infection io a nonimmune individual, while
herpes zoster is a reactivation of the latent vims
when ( be immunity has fallen to lnelfective levels.
Copyrighted material
 •« Hwpwtruwa *
Thus ^ thicIccupM is Bcaft3gh/ hut not luster. Contact
wirh either zoster or chickcnpox may lead only to
chtclccnpox hut not zoster
VZV is similar to the herpes simplex vitus in
its morphology. It does not grow in experimental
animals or chick embryos. The virus was first
isolated by Weller in human embryonic tissue
culture . Ir can be BTOWH in cultures uf human
fibroblasts, human amnion or HeLa cells. The
cytopathic e r k .. i > are similar TO, but less marked
than those produced by the herpes simplex virus.
In cultures the virus remains cell associated and
does not appear free in the medium . By using highly
specilic .mlisera , iL is possible td distinguish between
herpes tirus types 1 , 2 and varicella zoster viruses.
Only one antigenic type of VZV is known.
Varicellfl (oliicluflpnx}! Chicken pox is one
of the mildest and most common of childhood
infections. The disease may, however, occur at any
age - Adult chickenpos, which is more serious, is
rather common in some tropical areas.
The source of infection is a ch ickenpax or herpes
zoster patient . Inactivity is maximum during the
ir trial stages of the disease when the vims is present
abundantly in the upper respiratory tract. The buccal
lesi i ms wl rich appear in the early stage of the di *ease
and the vesicular fluxl are rich in vims content.
Infecti s ity wanes as the disease progresses and the
'
scabs are virtually nonin fee ri < i.us- There arc no
animal reservoirs of vail -dla.
L the pei iiod of gestation when foe woman develops
The portal of entry of the virus d rbc respiratory
tract or conjunctiva . After an incubation ped ' d of
about two weeks ( 7— 23 days ) the lesions begin to
appear. The patient is Considered to be in lev Lx ms
during the two days before and five days after the
onset of the lesions . In children , there in little
prodromal illness and the disease is tir - r noticed
when skin lesions appear. Buccal lesions may not
be TII it iced . The rash appears usually on the trunk.
The evolution of the rash is so rapid that thcvariuois
stages - macule, papule, vesicle , pustule and scab -
cannot be readily followed in individual lesions.
The rash is centripetal in distribution, affecting
479
mainly the trunk and sparing the distal parts of the
limbs , and is very superficial without involving the
deeper layers of the skm , resembling a dew drop
,
lying cm. the skin . The rash appear* in crops during
the first three nr four days of the disease, so that
lesions of varying agp can be noticed or the same
patient. It matures very quickly, beginning to crust
within 4E hours.
When varicella occurs in the adult, systemic
symptoms may be severe , the rash very profuse and
the entire disease much more intense than in
children. The rash may become hemorrhagic and
occasionally bullous lesions appear. Fitted scars on
the skm may reninm after recovery . Varicella
pneumonia is more common in adults, and often
fatal in the elderly. Other complications like
myocarditis , nephritis , acute cerebellar ataxia ,
meningitis and encephalilis may ensue . Secondary
bacterial infections, usually due to staphylococci
or streptococci , may occur. Rey syndrome may
follow varicella ir some cases wiih a history of
administrai ion of salicylates, But in most cases *,
, jr
ckickenpox is an uneventful disease and recovery
the rule . One attack confers lasting immunity.
Chkkenpox in pregnancy can be dangerous for
both motherand bahy. The disease tends to be mure
severe in pregnant women, with enhanced risk of
complications like pneumom . L. The baby may
develop two types of complications, depending on
chicken pox . If maternal varicella occurs during
the first half of pregnancy, the fetal infection may
usually be asymptomatic . Some infants may develop
the fetal varicella syndrome, manifesting as
cicatrising skin lesions , hypoplasi . L of Limbs ,
chorioretinitis and CNS defects. Some babies may
not exhih i t any defects, but may carry latent VZV
infection. When maternal varicella occurs neat
delivery, babies may develop congenital ( neonatal )
varicella, wi tli in two weeks of b irth. If the mother's
rash began a week or more before delivery, she
would have developed . iiudnuLie - which would have
been passed , along with the virus , to the fetus
Copyrighted
1
- 1
material
490 * Tf *1(JOOk
tram placen rally. Such A biby, though infected,
usually '
clinical dincase. It the mother
escapes.
develops chicken pax lens the - i week (or wlrinn 2
, It Is not useful in treatment.
days) ot delivery. the b*by would have received
"
from the mother only the viru* ami not the Anribtjdy,
so that iT develops neonatal varicella , This is usually
a serious disseminated disease , with high risk of
pneumonia and encephalitis. As treatment l ot such
conditions will have to be started curly To he of ILJIV
nee , the babies ate to be given VZV anti sc mm and
chemotherapy immediately after birth.
Laboratory diagnosis: Uiagnosis is usually
clinical . Multi nucleated giant cells and type A
intranuclear inclusion bodies miybtAccn in smears
prepared by scraping the base -of the early vesicles
( Tzanck srncanO and stained with toluididc biucL
Cietnsa or Papanicolou stain. Electron microscopy
"
of the vehicle fluid may demonstrate the virus with
typical herpes morphology. Virus Million can be
attempted from the buccal or cutaneous lesions in
the early stages by inoculating human amnion ,
human fibroblast , HeLi or Veto cell*, The virus
antigen can be detected in scrapings Jrom skin
lesions :w immunodtiorescencc, and in vesicle fluid
by cotin rcrimmunoclcctrophorcsis wirh zoster
immune serum . ELISA and PCR techniques ate
also in use,
lJmphyln \ is nnd iroatmcnt: A live varicella
vaccine was developed hv Ttkahishi in Japan in
1974 hv attenuating a strain of varicella virus (Oka
strain, so named after the patient! by serial passage
itl [issue culture . Given subcutaneously, it Induced
good antibody response, hut it was very labile and
bad to be stored frozen . A modified lyophilised
form of the vaccine is now available, which can be
stored between 2 UC and H 'C It i > recommended
for children 1-12 years old as a single subcutaneous
—
dose , and for those older as 2 doses 6 10 weeks
jparl . It is safe and effective. Occasionally' children
may develop a tew vesicles which resolve cjuickly
It is not considered -sate in pregnancy Varicella
zoster Immunoglobulin ( VZIG ) prepared from
patients convalescing from herpes zoster provides
o' Microbiology »
passive protection ir immunocompromised children
exposed to infection , hut its availability is limited.
Specific treatment is indicated mainly in
iminunodetivient arid elderly subjects and those
with complications such as varicella pneumonia ,
encephalitis and disseminated zoster. Acydovjr and
famciclovir ire effective. Corticosteroid* are
contraindicated in varied I ; L as they enhance the risk
r
of pneumonia and disseminated disease.
HBHT &S ZOSTI - K (SHtNCLfiS, Zn ^ A )
Tlie name is derived from Horpcufi, meaning to
,
creep; zoster, meaning girdle .
While varicella is typically a disease of
childhood, herpes zoster is one of old age, being
common after the age of fifty years. 'Hus disease
may, however, occur ar any age and zoster has been
reported verv rarelv ever in the newborn .
Herpes zoster usually occurs in persons who
had chic ken pox several years earlier. The virus
remaining latent in the sensory ganglia, may leak
out it times hut is usually held in check hv the
residual immunity. Years niter rhe Initial infection ,
when the immunity has waned, the virus may be
reactivated, and triggered hv some precipitating
stimulus, travel along the sensory nerve to produce
zoster lesions on the area of the skin or mucosa
supplied by it . This reactivation is associated with
inflammation of the nerve, which KCUUdftS for the
neuritk pain that often precedes the skin lesions ,
The rash is typically unilateral and confined ro the
area supplied by a single sensory ganglion . The most
common sites arc the areas innervated by spinal
Cord segments 1)3 to L 2 and the trigeminal nerve,
particularly, its ophthalmic branch . The lesions are
identical in nature to varicella lesions, except tor
1 hen limited distribution. Tlie risk heals in about
two weeks. Pain and paresthesia ar the affected area
may persisr for weeks or months Other
complications are lower motor neuron paralysis
which sometimes ensues, meningoencephalitis and
generalised zoster where the lesions are scattered
Copyrighted material
Hidden page
462 * TemtJMk of Microbiology *
Primary
. infections in older clii.3d.rcn and adults
arc usually asymptomatic. However, a heteropEnle,
antibody negative, infect iotis moncmucleos] ; may -
be seen . I'll is is more common frlkvwi ng transfer ion
of CMV infected blood ( post- transfusion
moronudeo M ). -
In the immunocompromised host, CMV can
cause severe and even fatal infections, 'this occurs
in transplant recipients , cancer patients on
chemotherapy, and moft. particularly in the E [ IV
infected. CMV is an important pathogen in AIDS-
In AIDS patients, the already weakened immune
response is further damaged by the nonspecific
CM1- inhibiting effect of CMV. One of the
glycoproteins on the surface of CMV acts as a
receptor for the Fc portion of immunoglobuli n
molecules. This leads to masking of the virus by
attachment of irrelevant immunoglobulin molecules,
preventing access to specific ami - CMV antibody.
Laboratory diagnosis; Diagnosis may be
established by recovery of the virus from the urine,
saliva or other body fluids by inoculating human
fibroblast cultures . A simpler bur less reliable
technique is the demonstration of cytomegalic cells
in the centrifuged deposits from urine or saliva.
Demons [ ration of an ; ibodv is useful in [ he
diagnosis of primary infection but not in
reactivation. Serological tcchniqties in use include
CF> I HA, IF and EDSA, Antibody detection may
be necessary far screening blood or organ donors.
Epidemiology! CMV spreads slowly and
probably requires close contact for transmission. It
may spread through salivary or other secretions or
by sexual contact. A special method of transmission
- -
i j by blood transfusi m or organ transplants. The
virus has been detected in saliva, urine, cervical
secretions, semen, blood and milk . Congenitally
i nfectcd i nfants have viruria for uplo 4-5 years.They
.
aie highly infecti rus in early infancy. About one
per cent of neonates in the USA are infected with
CMV. In the developing countries, the rate may be
much higher . Upto SO per cent of adults show
CMV antibodies, indicating the high prevalence
of infection. Once infected , the person carries the
virus for life.
Prev«mbn and treatment; Prevention is
indicated only in high risk cases such as organ
transplants, immunodcficicnt persons and in
premature infants . Screening of blood and organ
donors and administration of CMV
immunoglobulins have been employed in
prevention. Acyclovir is useful in prophylaxis hut
not in treatment . Ganciclovir and foscarner have
been fiiund effective and arc used for the treatment
nf CMV disease in AIDS patients.
No vaccine is available . Experimentally, live
attenuated vaccines (Towne 125 and AD 169
strums ) and a purified CMV polypeptide vaccine
have been found to be immunogenic but not
effective in protecting immiinodefLcient subjects
from CMV LnfccfioTi -
-
EPSTEIN BARR VIRUS
In 1956, Burkitt described an unusual lymphoma
among chiidrcn in certain parts of Africa and
suggested on epidemiological grounds that the
tumour may be caused hy a mosquito borne virus.
This led m several attempts at isolating viruses from
such tumours. A number of different viruses,
apparently ’ passenger viiuscs", were isolated from
cultured lymphoma cells. One virus observed in
the cultured lymphoma cells by Epstein, Bair and
Achong in 1964 was a new type of herpesvirus,
named the EB vims, specifically affecting cells of
the B lymphocyte lineage. Only human and some
subhuman primate B cells have receptors [CD 21
molecules) for the virus, EBV infected B cells arc
transformed so that they become capable of
continuous growth in vitro.
Epidemiology: The Epstein-Barr ( EB) virus
is ubiquitous in all human populations. As with
other herpesviruses, infection with the EB virus
leads to latency periodic reactivation and lifelong
persistence. The EE vims antibodies are present in
about 95 per cent of adults. In the oveicmwdrd
developbig world, the EB vims infection occur? in
LJ opyrighted materia
 * Herpesviruses
infancy and childhood , when it is tisunlly
asymptomatic . In the affluent countries, primary
infection is often delayed till adolescence and early
adulthood, when it may lead to ini cer lout
'
mononucleosis .
The source of infection is usually the saliva of
r
infected persons who shed the virus in
oropharyngeal secretions for months following
primary' infection and intermittently thereafter.
The EB virus is not highly contagious and
droplets and aerosols ace not efficient in
Transmitting infection . Intimate oral contact, as in
hissing, appears to be the predominant mode of
transmission . This accounts for infectious
mononucleosis being called the ‘Idling disease'.
Infection may also follow blood or marrow , of immunodeficiencies, most typically in AIDS,
transfusion but these are rare event*.
f' H virus infection may lead to the following
clinical conditions;
1. Infectious mononucleosis.
2. EBV associated malignancies;
a . Burkin's lymphoma.
b . 1 ympbcimas in immurnN.tefLviert persons Huch
as AIDS patients and transplant recipients.
c . Nasopharyngeal carcinoma in persons of
Chinese origin .
Pnthogcnusis; " Hie virus enters the pharyngeal
epithelial cells through CR 2 (or CD 21) receptors,
which art the same as for the C’ dd component of
complement . It multi [dies locally, invades the
bloodstream and infects B lymphocytes i. n which
two types of changes are produced. In most cases,
the vims becomes Latent inside the Ivmphneytes,
which become transformed or fnrjntfrtaJist.ii ' so that
they become Capable of indefinite growth hi vitro.
They are polyclonal!} activated to produce many
'
kinds of immunoglobulins. The heterophde sheep
erythrocyte agglutinin seen characteristically in
infectious mononucleosis is an example of such
polyclonal activation . A second type of effect , shown
by a few infected II cells is Lytic iul ecUort , with cell
death and release of mature prngency virions.
The mononucleosis represents a polyclonal
4fl3
transformation of infected B lymphocytes, EB
virus antigens are expressed on the surface of
infected B cells, The atypical lymphocytes seer
in bDod smears in infectious mononucleosis
are T lymphocytes undergoing blast
transformation In response to such neoantigens.
Intermittent reac ovation of the latent EB virus
leads to clonal proliferation of infected B cells. In
immunocompetent subjects, this Is kept in check
bv activated "J " cells. In the immunodeficicnt , L5 cell
if
clones may replicate unchecked , resulting in
lymphomas. Hypercndemic malaria prevalent in
Africa is believed to be responsible tor the immune
impairment in children with Burkin 's Lymphoma.
The frequency of lymphomas sett ] in many types
may have d similar pathogenesis. Nearly half the
lymphomas seen in immuDodsficicnt subjects
contain EB virus DNA sequences.
Gercdc and environmental factors arc said to
be .important In tile nasopharyngeal carcinoma seen
in men i > t Chinese origin . EB virus DNA is
regularly found in the tumour cells. These patients
have high levels of EB virus antibodies. Genetic
Influence is best illustrated in the X - linked
lymphoprolifrrative (XLP or Duncan ) syndrome
associated with extreme susceptibility to EB virus
infection ,
I !N h KCTIOUH MONON ( C l.flGS LS
( GLANDULAR FIIVEH )
This is in acute self-limited illness usually seen In
non immune young adults following primary
infection with the EB virus . Tin: incubation period
is 4-8 week*. The disease is characterised hv fever,
J
sore throat, iy!U[ ihadenopathy and the presence of
abnormal lymphocytes in peripheral blood smears .
A mild transient rash may he present - Some patients
treated with adipicillin may develop a
miculopipular rash due to immune complex
reaction to the drug. There i * often associated
hepatitis which is usually ( ubclinicil and
demonstrahlconly by Liver function tests. A numher
Copyrighted materia
Hidden page
 < Hapatvliuvn 4 BS

Human Herpej Virus rih fn.- ^
A herpesvirus, first iosolitud in l 9flb from the
peripheral blood of patients with lympho
prol rerarive disease, was called the human B
^
lytnphotfopic vims , It has been renamed J JHV-6,
--
Thi i iubiiiuiros and spreads apparently chn ^.j rh saliva
in early infancy. Two vari .mts recognised, A and B,
Variant B is the cause ofmild but common childhood
illnrsK ' nsaittheiti subitum’ ( rtrtcolsi infantum of > Lxt I L
disease i . In older age groups, it has l >een associated
with infectious mononucleosis syndrome , focal
encephalic ^ and , in the i - nmu node fie lent, with
pneumonia and disseminated disease.
HHV 7 was isolated In 1990 from peripheral
CD 4 cells of a healthy person - Like HHV 6,
HHV 7 also appears to be widely distributed and
transmitted through saliva. Jr shares with HIV the
- same CD4 receptor on T cells and could therefore
- contribute to a frirther depletion ol CD4 1 Cells in
HIV infected persons. It has been said to cause
some cases of exanthetn subitum.
^ In 1994, L>N A sequences presumed to represent
a new herpesvirus were identified from tissues of
- Kaposi 's sarcoma from AIDS patients. This has
been named HHV fi. This has subsequently been
identified also in Kaposi's sarcoma in persons not
infected with HIV. Jt has been therefore referred
to some limes as Kaposi V sarcoma -assuoa fed
herpesvirus fKSHV ), but an etiological
relationship is yet to be proved .

Furthar iK- ndum

Curay I , and P Spear 19 K 6. InfectLana with Herpes Simple* viruses A'ew EngJ Med JIJ:6 W6 , 749 .
,

Ha M. 1991 . CyiamqpaJovjrciscj.' Biology and Infection, 2 u edn. New YbrlcPtenum Press.

Jaflie TIW and PE Fellerr 1999. I luman herpesvirus 3 and Kapusi 's sarcoma. New EngSJ Med 24 : 140.
Kaplan JE. 1989. Herpesvirus siiri .ie infection in monkey handlers. J Infeet Dis . JJ 7; 1090.
klij. Mii , i hi . i- i at . 1995. Immune regulation in Epstein - Barr virus disease . .V .- . .- •ten! fin 59:
LevyJA 1997 . Three new human herpesviruses. Lancet 349:558 .
Lusao P and RC Gallo 1994. Human herpes virus 6 in AIDS. Lancer 343: 5 S 3.
Onoraio 1 M er aL 1985. Epidemiology of cytomegalo'.- ini - infection . Rev Infect 1 1' - 7:479.
Rickmsn AB ?t al . 199 5 . TTlC Epstpiu- Bma virus as a ITICKIF] nf vinii - h < >jt i r. I- . r . I I . -. I I -- B::t Med Bull 41'.75 .
IL

Rowley AH e ( al - 1990 , Rapid dctectiim H *«pa simplex -. - i r -±ia UNA . Jjneer .135.
Strauss M - . er al . 1993. Epsnein-Barr virus infections . Ann Jnr Med 118:45.
WtlleeTH . 19 B 3. Varicella and Herpes- Zoster: changing concepts. New Eng } JlJed. J0913& 2 , 1434.

opy righted material

8 dr w
 CO Adenoviruses
in

Adenoviruses are a group of medium sized , Adenoviruses appear to have a spare capacity
rLonenYeioped > double stranded DNA viruses chat to cam DMA insert up to 7 kb and arc being
1

share a common complement fixing antigen. They
infect humans, animals and birds, showing strict
bast specific in'.
In 1L >3 , Rowe and associates grew surgically
^
removed human ad end id tissue in plasma eldt
cultures and nociced that the epithelial outgrowths
underwent spontaneous degeneration resembling
viral cytopatbic change. Thin wan neutralised bv
human sera . A viral agent was shown to be
responsible for this degeneration. Tins was the
investigated as potential vectors in gene tberapv.
Morphology: Adenoviruses arc 70-75 nm in
size . They have ,t characteristic morphology. The
capsid is composed of 152 eapsomcn arranged as
an icosahedron with 20 lri &rtgui])r facets and 12
vertices . Of the 252 capsomers , 24Q have six
neighbours and arc called hexnns, while the 12
capsomers at the vertices have Five neighbours and
arc called pentuns. Each pen top unit consists of a
petunn base anchored in the cap - id and a projection
1

prototype of the group of viruses subsequently or fibre consisting of a rod like portion with a knob
designated adenoviruses because they were attached at the distal end. Thus , rln - virion has the
originally isolated from the adenoids. I liUcman in appearance of’ a space vehicle .
195 *1 isolated a related virus from the throat
washings of military recruits with acute respiratory
illness .
Over SO serotypes? of adenoviruses have been
isolated from human sources . Most uf the recent
serotypes; were recovered from AIDS patients.
Adenovirus infections are common worldwide
mostly in children . Many infections arc
asymptomatic [’ he virus may persist in the host
,

for manv months- Adenoviruses cause infections of

r

the respiratory tract and eyes, and less often of rhe

intestine am! urinary tract .
In 1962 Huebner reported that adenovirus types
12 and 18 produced sarcoma when inoculated into
baby hamsters. Tbis led to the intense studv of
adenoviruses at the genetic and molecular lewis.
However, there is no evidence .it *11 r e l a t i n g
adenoviruses to natural malignancy in humans or
animals. Fig . 53-1 Morphology of dve navi re :

Copyrighted material
 * Adenaviru &frS m
Tabta 511 Classilicetlen of human adenoviruses

Group Sciutypc Hemagglutination Oncogenic potential

fSubgeruiij) ( Species )
Red cells Pxttem Tlr rtt 0Uttgifiiciry in Tin Tr* nefarm* tion of
cultured cells
A 12, IS, 31 Rat * High *
E 3 7
, , 11 , ,
14 16 21
,, "4,
.
35 Monkey * Weak +
C 1, 2.5,6 Rat £ Nil +
D a-10r 13, 15 , 17. 19, 20» 4 Nil
-
22 30, 32, 33, 3 39,
42- J 7
*-
E 4 Rar + Nit *
F 40, 41 Ra « £ NK *

Note;' + denores complmi: and ± uniil lutmijy lLLrirtaiiniii ; N'K = mu known

^ ^
KcsisTitnCtJ Adnoviruses arc relatively stable,
remaining viable for about a week at 37 aC, They
are readily inactivated at SO °C. They resist ether
and hide salts .
Growth and host ranje Adenoviruses arc
host Kpeofic . l so laboratorv
LHRJ > animals are not
susceptible to adenoviruses infecting humans.
Hurman adenoviruses grw only in tissue cultures
of tiu man origi it , such as human embry on ic kidney;
HcLa or HEP-2. CvEopathic changes may take
several days to develop and consist of cell rounding
and aggregation into grape likeclusters . 1 ntra nuclear
inclusions may be seen in stained preparations.
Classification: The family Adenoviradac
contains two genera : jVfavJjtJenu virus , the
adenovirus of mammals and Avudesmimx, that of
birds . In addition coat least 47 serotypes of human
origin, masradenoviruses include simian, bovine,
equine, ovine, canine, murine, porcine and cetacean
serotypes. Avia.de no viruses have been isolated from
fowls, gclc and turkeys. They infect only the
homologous species , with the exception of
oncogenic human adenoviruses (for example types ,
12 , 18, 31 ) that cause sarcomas when injected into
newborn hamsters.
All mammalian adenoviruses share a ainmiim
complement fixing antigen. The group antigens are
present mainly on hexons and can be detected by
-
iiminofltiorescerCe or EI . ISA. Type specific
antigens are located on pontons and fibres. Serotypes
are identified bv the neutralisation rest . Human
adenoviruses are classified into six groups (also called
subgroups or subgenera ) based on properties such as
hemagglutination , fibre length , DNA fragment
analyst and oncogenic potential ( Tahir 53,1)-
Patho enesis; Adenoviruses cause infections of
^
the respiratory tract , eye, bladder and intestine .
More than (ine typeofviru ^ may poultice the same
clinical syndrome and one type of vims may cause
clinically different diseases ( Table 53 - 2 ) . The
following syndromes have beer recognised:
Pharyngitis: Adenoviruses arc the major cause
of run bacterial pharyngitis- and tormllilU ,
presenting as febrile common cold . Types 1-7 are
commonly responsible.
IJNOIIMONIA: Adenovirus types 3 and 7 are
associated with pneumonia io adults resembling
primary atypical pneumonia. In infants and young
children types 7 may lead to more serious and even
fatal pneumonia .
Acute respiratory diseases ( A R l ) fc This
occurs usually AN outbreaks in military recruits.
Serotypes 4 , 7 and 21 are the agents commonly
hoisted.
Pb ary n £oco n j u nett va I .
fever This
syndrome of febrile pharyngitis and conjunctivitis

Copyrighted materia
4 SE H Ten [ book 01 W crobiology *
' rissuc culture ( use of trypsinised monkey kidney
Fig . S 3.2 Atfenovirus In stools Horn a chile! w 4 h
diarrhea in ZDCUJOD ; Virus panicles show
,
chi racier i Stic he rag 0 nil shape. N eg alive slain with
3 per cam ammonium molybdate. of [ seal sedlmenl
(Courtesy : Prof . M Mathen. Cbnslinn Medical Collage.
Vallora.)
seen in civil ian population is usually Associated with
tcrOtypet 3, 7 ami 14 .
Epidemic) ker & Uicimjunoti vilia CEKOi
This is a serious condition which may appear as
ait epidemics, usually caused by [ype & and less often
by types 19 and 37.
Acute litlLioular conjunctivitis: This is 1
nonpuruknt infUmrratkm nf the conjunctiva with
enlargement of rhe submucous lymphoid follicles
and of the preauriujlar lymph nodes. Types 4
and 11 arc commonly responsible . Adenoviral and
chlamydial conjuncrisdris arc clinically similar .
Dili [Then: Adenoviruses un often be i solaced
from feces but their relation to intestinal disease
has not been conclusively established . However*
Tabfte 53.2 Common syndromes associated 'with adenoviruses infection
Syndrome
Respiratory disease in children
Sore throat, (febrile cold, pneumonia
ARD in military recruits
FoJIicuJar ( swimming pool ) conjunctivitis
Epidemic keralocu-njuncfivitb (shipyard eve )
Diarrhea
some lasfidicniH adenoviruses , which can lie
demonstrated abundantly in feco by electron
microscopy but fail to grow in conventional tissue
cultures, can cause diarrheal disease in children ( for
example types 4Qh 41 ) / l "hcy have been designated
as coterie type adenoviruses. Special techniques of
cells or transformed human embryonic kidney cells )
have been developed for their cultivation . They can
also be idenrifted bv stool ELISA.
Jf
A cure hemorrhagic OFfiris in children and
generalised exanthem arc two other syndromes
which have been reported . Adenoviruses types 11
and 21 arc fc &ponstblc lor the former.
Adenoviruses have been isolated from
mesenteric lymph node* in cases of mesenteric
; idei ] j'fis and Aaoustueepdon in children.
1 . uhirrLLlnry diu innrifi : Diagnosis Can be
^
csiablisl Led by isolai ion of the virus from the Throat ,
eye, urine or feces. The materials are inoculated in
tissue cultures. Preliminary identification is possible
by noting theevtopithic effects and by complement
fixation tests with adeiUrt' ifUS arttiscruHi , Bv Jr
hcitkaggliidnaikon with rat and monkey erythrocytes,
the isolate can be classified intes subgroups. Typing
is done by neutralisation tests.
For serological diagnosis , rise in citre of
antibodies should be demonstrated in paired sera.
Examination of a single sample of serum is
inconclusive as adenovirus antibodies are so
common in the population .
Electron microscopy For fecal virus and
immunofluorescence for viral antigen detection in
Principal se w types
.
1, 2 5, 6
3S 4 , 7, 14, 21
*1, 7, 21
3* 7
B, 19, 37
40, 41
. P*
 * Adi IOVKU fct!fi- * 469

nasopharyngeal and ocular infections are useful.
I ' rrtphylaKie: Specific prevention required
only for flic control of outbreaks i :i closed
communities, ^ in military recruits. Killed and live
vaccines have been used in them for prevention of
ARD, with some success. No vaccine is available
for general use.
ADENO-ASSOCIATED VIRUSES ( AAV /
Electron microscopy of adenovirus preparations
have revealed small icnsahedral viral particles, 20~-
25 nm in diameter They are unable to replicate
independently as they lack enough DNA , They can
multiply only in cells simultaneously infected with
adenoviruses and are called adcno associated
viruses {AAV ) or adcnosatellite virases. They have
-
been clarified as the genus depenefonn ^ {referring
to their dependence on adenoviruses) under the
family Parvoviridae. They can be detected by
electron microscopy and complement fixation or
immunofluorescence wirh specific anri -icra. Types
1, 2 and 3 are of human origin and cause natural
infection while type 4 is of simian origin . Thrir
pathogenic role is uncertain.

Further Reading.
J3 . SC 1.1990 Adencmruiiex. In C_ .
De Jong J L aL 19K 3 _ FiuiieliftLis jtLertnrifu
x JVIjndpl ft jl. / Practice of In fc-CTJCHH IA IEWKE V sdn . New Ynrfc Wiley.
'

J UIIL . . jurat

&fs foam. hummus Infant H AKA.J M $d L IJTJJ 11:2 IS.

ft
^
Ginberg led ). 1934. The Adenoviruses. New Yoflc Plenum.
] IS
KeenJ iirle RA « *1. 19B3- KentaccmjwrtvvTtii asKKj? T*rt with. nhlenijiTirvi! type 37» J Infm Dk 147:191
^
Kemp MC ct aJ. 19B3. The changing ecLuLftgy af epidemk kcriiociwijimcciviTLi.J InfcLt Dis 14S:24.
T

[ ju C.199L . WencKvinLK ?. ] n TW 6CK-JA vfHvmvn

’ * 2^ cdn. BdlhcMoiby Year Bfflrftt .

opy righted material

8 dr w
 IT) Picornaviruses
The familv Picomavindac comprises a large dumber
of very small ( pko, mealing small ) RNA viruses,
They art- nonenveloped viruses, 27-30 nm in size,
resistant to ether and other lipid solvents, Two
groups of picamsviruies are of medical importance ,
the cntcrcvaufet that parasitise the enteric craft
and the rfujwvinisffs that infect rhe nanal mucosa.
Two other pieornavirus genera of veterinary
importance are *phr/tovi'rusej causing the foot and
mouth disease of cattle, and cardrcrniuse* of mice,
including the encephalomyocarditis virus .
ENTEROVIRUSES
Enteroviruses of medical importance include:
Pbfitmmi types 1 _ 3n Coxsackievirus A types 1 -24,
Coxsackievirus 13 types 1 -6 , Eehovim; typos 1-34
and Enterovirus rypej 6 H - 71 .
Paralytic disease of children ( m /autii/c partJysjs)
has been recognised from very early nines. I lowever,
it wan only towards the end of the nineteenth
century that poliomyelitis { polios = grey; myelitis
= i nflunmaftion oft he spinal cord } w as eh aractensed
as a separate clinical entity capable of causing
LfljectiuilS ill which paralytic cas- cs are tar
outnumbered by silent in apparent infections,
Landstciner arid Popper (1909 ) reported
experimental transmission of the disease to monkeys
hy inoculation of spinal cord and fecal extract
fdtrates from fatal cases of poliomyelitis. The
exjterimental study of the disease was restricted as
monkeys were the only laboratory animats
susceptible to the virus . Armstrong ( 1939 )
succeeded in adapting a poliovirus ( type 2 Lansing
strain ) ro cotton rats and mice hut few strums could
be sc ] adapted. Progress was also inhibited by the
dogma then held that polioviruses were strictly
nemolxopK, multiplying only in the neural tissues.
The demonstration by Endcn , Wtllcr and Robbins
( 1949 ) chat polioviruses could grow in cultures of
non-rte ur J ] cells from human embryos, producing
cytoparhk effects, was a major breakthrough . The
Nobel Prize was Awarded ro them in recognition
of rhe seminai importance of this discovery in the
development of virology as a whole.
A new type of virus was isolated by Dalldorf
and Sickles (194S) from the feces of children with
paralytic poliomyelitis , from whom type 1
poliovirus was also isolated . The virus caused
,
piaraivsis on inoculjtion into suckling mice. This-
was called the coxsackic virus, as the patients came
from the village nfCoxhackie in New York. Many
similar viruses have since been isolated from the
frees and throats of patients with different diseases
as well as tram healthy individuals. They have been
designated asconaddc viruses, classified into groups
A and U based on the pathological changes
produced in suckling mice.
The in traduction of tissue culture techni <|ues
in diagnostic virology led to the isolation of several
cytopathogeuie viruses from the feces of sick as
well as healthy persons. Thcv were called orphan
viruses as they could not be associated with any
particular clinical disease. They came to he known
by rhe descriptive Term ' enteric cytnpatbogenic
human orphan ( ECHO) viruses ' . Several orphan
viruses have also been isolated from annual feces.
The classification of enteroviruses as
coxsackieviruses and eehoviruses was not
Copyrighted material
Hidden page
4 n
* TaxjtbQOfc OF MI J : IJ LMIJ lij U y »
The vims grows readily in tissue cultures of
pi i mate 0 ri i.ii , Pri unary monkey kidney cultures are
^
'
used fur diagnostic cultures and vaccine production.
The infected cells round up and become refractilc
and pyknotic. Eosinophilic intranuclear inclusion
bodies may be demonstrated i n sfs i ued prepare ions.
Well- formed plaques develop in infected
monolayers with agar overlay.
Pathotfencftis: The virus is transmitted by the
fecal-oral route through ingeS' Lun. Inhalation or
entiy through conjunctiva of droplets ofrcspiratoiy
secretions may also be possible modes of cntiy in
close contacts of patients in the early stage of the
disease , The virus multiplies initially m the
epithelial cells of the alimentary' canal and the
lymphatic tissues, from the tonsils to the Feyeris
patches. It then spreads to the regional lymph nodes
and enters the blood stream ( minor or primary
vjVejKjd ) . After further multiplication i 11 the
reticuloendothelial system , the virus enters the
bloodstream agii i n ( nrajeror secojjdajr virem W
is carried to the spinal cord and brant. Direct neural
transmission to the central nerwus system may also
occur under special circumstances , as in
poliomyelitis following tonsiLiectomy,
In the central nervous system , the virus
multiplies selectively in the neurons and destroys
them. The earliest change is the degeneration of
Nissl bodies {chromatnlvsin ) - Nuclear changes
follow. When degeneration becomes irreversible ,
the necrotic cell lyses or is phigocjtnued by
leucocytes or macrophages. Lesions are mostly in
the anterior horns of the spinal cord, causing flaccid
paralysis, but the posterior horns and mtcrmedi .Ltu
columns mav also be involved to some extent.
Pathological changes are usually more extensive
than the distribution of paralysk In some cases,
encephalitis occurs primarily involving the
brainstem but extending upto the motor and
premotor areas of the cerebral cortex.
Clinical featuret: Following exposure to
. -
poliovirus 90 95 per cent of susceptible ndivi duals
develop only inapparejif iji /ecfion, which causes
. - -
seroconversion alone. It is only in 5-10 per cent
chat any sort of clinical illness results. The
incubation period is about 10 days on the average
but may range from four days to four weeks. The
earliest manifestations are associated with the phase
of primary viremia and consist of fever, headache,
—
sore throat and malaise lasting 1 5 days. Tkis is
called the minor iifness and in many cases may he
the only manifestation (abortive poliomyrlitis ). If
—
the infection progresses, the minor dlncss is followed
5 4 days later by the major illness. The fever comes
or again (hiphasic fever ), dong with headache, stilV
neck and other features of meting iris. This marks
the stage of vital invasion of the central nervous
system. Sometimes the disease does nut progress
beyond th i s stage of wcptic meningitis ( nonpwaJyr.'e
poliomyelitis ). In those proceeding to paralytic
poliomyeliiis, flaccid paralysis develops. Paralysis
is local in distribution ini dally hut spreads over the
next 3-4 days. Depending on the distribution of
paralysis, cases are classified as spinal, bulbar or
bulbospinal. Mortality ranges from 5-10 per cent
and is mainly due to respiratory failure. Recovery
of the paralysed muscles takes place in the next +
8 weeks and is usually complete after sir months,
leaving behind varyingdegrees of residual paralysis.
Laboratory diagnosis: Virus isolation in
tissue culture is the best method for specific
diagnosis. Many specimens can be used, Including
.
blood CSF, throat swab and feces. Virus can be
isolated from blood during the phase of primary
vircpiL, 3-5 days after i nfccricm, before neutral ini ng
antibodies appear. But this is of little practical
importance. Unlike other enteruviruses, poliovirus
can seldom be isolated from the CSF hue can bn
obtained from the spinal cord and brain ,
postmortem. The virus can be isolated from the
throat in the earlv stage of the disease. Virus
isol.ition from feces is usually possible from over
00 per cent of patients in the first week, 50 per
,
cent - t il ] 3 weeks and 25 per cent till six weeks. As
,
fecal excretion maybe intermittent, best results are
obtained by testing fecal samples collected on two
L l
jpyriaruea natena
 • Iricomavlmwe
separate days , as early in the illness as possible.
Fruluiigd fecal excretion may be seen in the
i mmurmdefic i en t, but permanent carriers do out
occur.
After appropriate processing to destroy bacteria
[centrifugation, treatment with ether, addition of
antibiotics), specimens are inoculated into tissue
culture. Primary monkey kidney cells are usually
employed , though any other human of simian cell
culture may be used. The virus growth is indicated
by typical cytoparhic effects in 2 3 days. - value .
Identification is made by neutralisation rests with
pooled and specific antisera. It must be remembered
-
that the mere T olation of poliovirus from feces docs
not constitute a diagnosis of poliomyelitis as
symptomless infections are so common Virus . monkeys, inactivated with formalin ( Brodie and
isolation must be interpreted dong with clinical
and serological evidence .
Scrodiagnosis is less often employed. Antibody
rise can be demonstrated in paired sera by
neutralisation nr complement fixation tests .
Antibodies appear soon after the onset of paralysis
so that even the first sample of serum may contain
appreciable amounts of antibody. Neucalising
antibodies appear early ;tnd prtsi-t for hie. In the
CF test, antibodies to the C antiuen appear first
and disappear in a few months, while anti- D
antibodies cake some weeks to appear after infection
but last for five ycars- The CF test is useful tc identify
-
exposure to poliovirus but not for type specific
diagnosis.
Immunity: Immunity in poliomyelitis is type
specific. Humoral immunity provided by circulating
and secretary antibody is responsible far protection
against poliomyelitis. IgM antibody appears within
a week of infection and lasts for about six months.
IgG antibody persists for life. Neutralising antibody
in blood generally protects agulnst disease by the
same serotype of the virus, but may not prevent
infection of intestinal epithelial cells and virus
shedding in feces . Secretory IgA in the
gastrointestinal tract provides mucosal immunity
preventing intestinal infection and virus shedding.
493
Breast milk containing IgA antibody protects infants
from infection.Poliomyelitis tends to be more severe
and virus shedding mure prolonged i :i those with
impaired humoral immune response. The virus also
induces cdl mediated immunity but its importance
appears to be uncertain as persons with defective
cellular immunity arc seen to respond normally to
puli,*V:rtiS infection.
ProphylflJcir Passive immunisation by cite
administration of human gammaglobulin is of little
Attempts at active immunisation with vaccines
date from 1910, Soon after the discovery of
poliovirus . The early vaccines were crude
suspensions of the spinal card from infected
Park) or riL inoleate ( Kolmer). They were not only
'
ineffective but often even dangerous, leading to
vaccination poliomyelitis. Folio vaccines therefore
became unpopular . Brudie is believed to have taken
his own Life, distressed at the suffering caused by
his vaccine. It was only after 1949, when tissue
culture was used for growing the virus and the
existence of three antigenic types of polioviruses
was recognised, that fresh developments in vaccine
preparation became possible. By 1953, Salk had
developed a killed vaccine. Almost simultaneously,
Koprowsky, Cox and Sabin ii tdependendy developed
live attenuated vaccines.
Salk 's killed polio vaccine is a formalin
inactivated preparation of the three types of
poliovirus grown in monkey kidney tissue culture.
Standard virulent strains are used. The three types
of polioviruses are grown separately in monkey
kidney cells. Viral pools of adequate ri tie are filtered
to remove cell debris and clumps, and inactivated
with formalin (1:4000 ) at 37 °C For 12-15 days .
Stringent tests are carried out to ensure complete
inactivation and freedom from extraneous agents.
The three types are then pooled and after further
tests for safety and potency, issued for use .
A nationwide controlled field trial conducted
in 1954 in the USA confirmed rhe safety of the
opy righted material
Hidden page
Hidden page
496 * Tc- xtbcok tyl M crobiology »
-
disadvantage in the advanced countries. Ptr' hii| s , it
"
may even be beneficial and may help to extend the
\~ .iL L iiiLk coverage in coumiies where vrild vims is
' '
endemic. Ideally, however, i r i s desirable to
vaccinate the whole community , n one time so chat
igT
natural diRaeirunarion is prevented. The strategy of
administering the vaccine to ail children in a region
on the same day { pulse stnmwthition: ) has been
found to be useful in the developing countries.
Eradication of poliomyclilia: By global
immunisation with OPV it was considered possible
to eradicate the disease . The World Health
Organisation Assembly in 19fiS had proposed
global cradicat ion of poli omyclir is by the year 2000-
Poor progress in immunisation in many countries
has been a set back to this objective.
Epidemiology: Poliomyelitis is an exclusively
human disease and the only source of virus is
humans, the patient of much mote cominoiilv the
symptomless carrier. Patients shed rhe virus in feces
for varying periods, about SO per cent for three weeks
and a small prnporriun far 3-4 months . No
permanent earners occur, 1 lowcvcr, the virus may
persist in the environment (sewage) for upto six
months. Virus shed in throat secretions during the
early part of the disease UI .LV also be a source of
infection for the contacts of patients.
Infection is, in general, asymptomatic. The ratio
of subclmical to clinical infections has been stated
as 100 or 1000 to 1 . The outcome of infection is
influenced by the virulence of the infecting strain,
the dose of infection and the age of the individual,
adults being more susceptible than children. The
following factors may influence the incidence of
paralysis: 1) Pregnancy carries an increased risk of
paralysis, perhaps due to the associated hormonal
changes. 2 } Tonsillectomy during the incubation
period may predispose to bulbar poliomyelitis.
3) Injections such as triple vaccine, especially alum
containing preparations, may lead to paralysis,
involving the inoculated limb- The mechanism Is
uncertain. The trauma mav «. lead to virus entrv into
d
local nerve fibres, or the segment of spinal cord
corresponding to the sire may be more susceptible
to viral damage due to reactive hyperemia - 4) Severe
muscular exertion or rraumadui mg the preparaJytic
stage increases the risk of paralysis .
Poliovirus type 1 is responsible for most
epidemics of paralytic poliomyelitis. Type 3 also
causes epidemics to a lesser extent . Type 2 usually
causes inapparent infections in the western countries
but in 1 nd i a paraiys i K due to type 2 is quite common.
Immunity is type-specific but there is an significant
amount of cross protection between types 1 and 2,
between types 2 and 3 and little or none between
tvpes 1 and 3.
COXSACKIEVIRUS
The prototype strain was isolated by Dalldorf
and Sickles ( 1943 ] from the village of Coxsackie
in New York. Several related viruses have been
isolated si nee then from different parts of the world ,
The characteristic feature of this group is its ability
to infect suckling but not adult mice , Based on the
pathological changes produced in suckling mice,
coxsackieviruses are classified into two groups, A
and B.
Properties of the virus: Coxsackieviruses
are typical enteroviruses. Following inoculation in
suckling mice , group A viruses produce a
generalised myositis and flaccid paralysis leading
to death within a week. Group B viruses produce a
patchy focal myositis, spastic paralysis, necrosis of
the brown fat and, often , pancreatitis, hepatitis,
myocarditis and encephalitis- By neutralisation tests,
Group A viruses are classified into 24 types and
group B into six types. All types in group B share a
common complement-fixing antigen - Coxsackic A
23 is the same as echo 9 and Cox&ackie A24 the
same as ECHO 34. Some coxsackieviruses ( A 7,
20, 21, 24 and B 1, 3, 5 6) agglurinalc human or
T
- monl«yr erythrecytcs-
Ho*t range and growth: It is necessary to
employ suckling mice for the isolation of
coxsackieviruses. Inoculation is usually made by
intracerebral, subcutaneous and intraperitoneal
Copyrighted materia
 < Pjcomsviruses
mutes. Adult mice are nm susceptible. Suckling
hamsters can be iiif^ued experimentally.
A|J coxsackic E viruses grow well in monkey
kidney tissue cul hurt , while ingroup A, only types
7 and 9 straw well. Group A 21 virus grows in
HeLa cells.
Clinical features: Coxsackieviruses produce
a variety of clinical syndromes in humans ranging
from trivial to fatal ] niece mos. The (bllowing types
have been recognised:
1. Herpangi us (VB > icular pharyn i r is ) i 5 a common
^
clinical manifestation of coxsackie group A
infection in children . It is a severe febrile
pharyngitis, with he .1 Hue hr , vomiting and pa: n
ill the abdomen . The characteristic Lesions are
small vesicles , on the fauces and posterior
pharyngeal wall , that break down to form ulcere ,
2- Ascpt iv men ingitis may be caused by most group
A and all group B viruses. A maculopapular ash
may be present. The ih:- ease may sometimes
occur as epidemics . Type A 7 had caused
outbreaks of paralytic disease in Russia , Scotland
and elsewhere, the virus for a time having been
erroneously referred to as Poliovirus type 4,
3 . HandjFoot “ and - Mouth- Disease { HFMD) was
identified in I960 as an exanthematous fever
affecting miiinly young children , characterised
by clusters of papulnues i c LLL .LT le > ions on the ski n
and oral mucosa. It occurs as sporadic cases and
as outbreaks . Coxsackie A-16, 9; B 1- 3 were
Common causative agents inn Lilly. It was a
henii n illness resolving in 1 - 2 weeks . The
^
situation changed drastically in the 1970 rs with
-
enterovirus 71 becoming a causative agent ,
causing extensive epidemics with serious
compl icsti ons like ascptiic meningitis*
encephalitis, flaccid paralysis , pulmonary
hemorrage, with many fatalities, particulirilv
in East Asla frumTaiwan to Siilgaput6 - HFMD
in now an important emerging disease.
4. Minor respiratory infections resembling common
cold may be caused by A 10, 21, 24 and B3.
5. Epidemic pleurodynia or Bornholm disease [so
I opyri
497
called because it was first described on the
Danish island of Bornholm ) is a febrile disease
with stitch-like pain in the chest and abdomen ,
caused hy group B viruses. The disease may
occur sporadically or a; epidemics.
6. Myocarditis and pericarditis in the newborn ,
associated with high fatality may be caused hy
group B viruses . The disease may sometimes
occur in older children and adults also.
7. Juvenile diabetes has been claimed to be
associated with coxsackie B4 infection but a
causal role for this virus has not been
establishcd -
.E Orchitis due to coxsackievirus has also been
reported -
9 . Transplacental and neonatal transmission has
been demonstrated with coxsackie B viruses
resulting in a serious disseminated disease that
[ ] 1 , LV include hepat i; is, meningoencephalit is and
adrenocort i call involvement.
10. Type B viruses have been associated with the
condition called posfriraJ farigue syndrome, hut
neither the condition nor the association has
been clearly defined.
Laboratory tlin noMB : Virus eolation from
^
the Lesions or from feces may be made by
inoculation in to suckling mice. Idenl ification is by
studying the hiRtnpathnlogy in infected mice and
by neutralisation tests . Due to the existence of
several antigenic types , serodiagnosis is not
practicable.
Jt- pidtrtjicHrtgvt Tike other enternviruses,
coxsackieviruses inhabit the alimentary canal
primarily .ind are spread hy the fecal-roral route .
Coxsackie B virus cpiikmics tend to occur every
2-5 years. Young infants arc most commonly
affected . Vaccination is not practicable as there are
several serotypes and immunity is type specific.
ECHOVIRUSES
When tissue cultures became routine procedures
i 11 diagnostic virology, several cytopathogen ic viruses
came to be isolated from the feces of sick as well as
S
/ <i
hted material
498 < Texlbook of Microbiology
health)' individuals. ' ITICSC viruses were not pathogenic
for bboab anirnakTheywere neutralised spooled
^
human gamma globulin. AH they could rot he
associated with any particular clinical disease then,
they were ailed orphans. They have been given the
, identification bv neutralisation tests laborious. IVis
descriptive designation 'enteric cytopathogcniL human
orphan viruses' and arc generally known by the sigh
echovi roses . Similar 'orphan:' Anises have also been
1
isolated from manv animats.
a
Properties of the virus: Echoviruses
resemble other enteroviruses in their properties. By
neutralisation tests, they have been classified into
34 serotypes. Types 10 and 28 have been removed
from the group , the former becoming a rcovirus
and the latter a rh in <>virus.
.
Some echoviruses ( types 3 , 6, 7 , 11, 12 13, 19.
.
20 ! 21, 24 , 29 30 and 33) agglutinate human
erythrocytes . Hemagglutination is followed by
elution , rendering the cells inagglutitiablc by echo
or ooanckijeviruses but not by myxoviruses .
( ifXfrwth nnd 1IL > SI ranf [c All echoviruses
well in human and simian kidney cultures
grow
producing cvtopathiceftectH. Echoviruses infect only
human beings naturally. rrhey are not pathogenic
to laboratory animals though occasional strains may
produce paresis on inoculation into monkeys and
newborn mice .
( llinical features: Though echoviruses were
originally considered orphans thevhave since been
shown to produce a variety of disease patterns . Most
infections ate asymptomatic. In general, the clinical
-
features resemble those produced hy
coxsackieviruses . Fever with rash and aseptic
meningitis , sometimes as epidemics , can be
produced by several serotypes, predominantly by
types 4, 6, 9, 16.20, 28 and 30, Echoviruses perhaps
constitute the most common cause of aseptic
meningitis . Echoviruses have frequently been
isolated from respiratory disease in children [types
1 , 11, 19, 20 and 22) and gastroenteritis ( type 18 )
but their etiological role his not been proved .
Occasional cases of paralysis and hepatic necrosis
havie also been reported.
»
Laboratory diagnosis: Feces , throat swabs
or C5 E maybe inoculated into monkey kidney tissue
cultures and virus growth detected by Cytopftlhii:
changes- The large number of serotypes makes
r
maybe simplified by hemagglutination and the use
of serum pools for neutralisation. Serological
diagnosis is not practicable except in case of
epidemics where the causative serotype has been
identified.
Epidemiology; Like other enteroviruses,
echoviruses inhabit the -alimentary tract primarily
and are spread hv the fecah-oral route. Epidemics
may occur, especially in summer. Vaccination has
not been attempted .
MEW ENTEROVIRUS TYPES
.
Ot the new enterovirus types, 68-71 type 6fi was
isolated from pharyngeal secretions of children with
pneumonia and bronchi ns.Type 69 is not associated
with any human disease . Type 70 causes acute
.
hemorrhagic conjunctivitis . EV- 71 originally
isolated from cases of meningitis and encephalitis ,
causes many other syndromes, including HFMD-
ActJTh HEMOHKIIACK;
CONJI XtJTIVrTIS
A pandemic of acute hemorrhagic conjunctivitis,
.ippaie nitty arising in West Africa in 1969 spread
widely involving several parts of Africa , the Middle
East, India , South East Asia, Japan, England and
Europe. The incubation period for this virus is
about 24 hours and the symptoms are sudden
swelling, congestion , watering and pain in the eyss.
Subconjunctival hemorrhage is a characteristic
feature. There is transient corneal involvement but
recovery is usually complete in 3- 7 days.
Rad iculo envelop a thy has been reported as a
complication from India. Sometimes it leads to
. paralysis resembling poliomyelitis .
Tire causative agent was identified as emerovims
type 70. It grows only cm cultured human cells
( human embryonic kidney or HeLa ) on primary
Copyrighted material
Hidden page
Hidden page
 lO Orthomyxovirus
in

The name Myxovinu was used origin illy for a
group of enveloped RNA viruses chincttriscd by
their ability' to adsorb onto mucoprotcin receptors
or ] erythrocytes, causing hemagglutination. The
name referred To the affinity of the viruses Co mucins
r
( from mt ra , meaning mucus). Included in chis
^
group were influenza, mumps, Newcastle disease
and parainfluenza viruses. The subsequent
recognition of important differences between
influenza viruses and the other viruses in this group
led to their being reclassified , into two separate
-
families orthomyxoviridae eon* sting of the]
respiratory tract which occurs in sporadic, epidemic
and pandemic forms. The name ' influenza' is said
to have been given by Italians during the epidemic
of 1743 , which they ascribed to the malevolent
influence of the heavenly hotlies or of inclement
weather . The modern histeiry of the disease may be
considered rodate from the pandemic of 1 RH 9-1 H 90,
during which Pfeiffer isolated Hiemophilua
influenzae and claimed that it was the causative
agent.The most Revere pandemic occurred in 191 K -
1919 , when it was shown that PfeilVerfs bacillus

influenza viruses and pnrasnvxavirid'Ac confuting

of the Newcastle disease virus, mumptvirui,
para influenzaviruses , measles and respiratory
syncytial viruses . Table 5 £.l lists the important
difference * between orthomyxovirus and
paramyxovirus.
INFLUENZA
Influenza is an acute infectious disease of the
Table 55 , 1 Distinguishing feslures of orthomyxovirus and paramyxovirus
was nut the primaiy cause of the disease , though it
might act as a secondary invader. The isolation of
the influenza virus in 1933 by Smith , Andrewes
and Laidtaw was a milestone in the development
ot medical virology. Tliev reproduced the disease
in ferrets by intranasal inoculation with bacteria '
free filtrates of nasopharyngeal secretions from
patients. Burner (1935) developed cluck embryo
techniques for the itropagation of the virus.
A notable advance was the independent

Property Orthomyxovirus n
JT MI d
. .r
UiJTJUJm U B
-
SlTEi? Etf Yfrkfl 80— 120 nm 100- 300 nm
Shape Spherical; filaments in fresh isolates Pleomorphic
Genamc Segmented; « ight pittC* of RNA Single linear
molecule of RNA
Diameter of mideocapsid Lf mm 18 nm
Site of synthesis of ribonudeoprotein . Nucleus Cytoplasm
Genetic reassortment Common Abienr
DNA -depcndent RNA synthesis- Required for multiplitftiiW Nor required
Effect of Actanomjdn D Inhibits multiplication Docs not mhibi. t
Aniigtnic stubility Variable Stable
Hemolysin Absent Present

JU righted materia
502 * Textbook or Microbiology
discovery by Hint, and by McClelland and Hare
(1941) chit influenza viruses agglutinate few ]
erythrocytes.The property of hemagglut i nut in m was
found to be a common feature of many other ’ 'iruscs,
Francis and Magill (1940 ) independently
isolated a serotype of Influenza virus which was
antigenicalfy unrelated to the strains known till then.
Thin was designated influenza virus type B , to
distinguish it from the original serotype, which was
named type A . Taylor (1949) isolated the third
serotype of influenza vi ms, type C . The classi ficat: on
ot influenza viruses into [he three serotypes, A , El
and C , is based on the antigenic nature of the
'internal or nbonucieoprotein and the matrix ( M )
1
protrin antigens -
Influenza occurs also in animals and birds in
nature . Indeed, the avian influenza virus was
demonstrated as early as in 1901, when Centanni
and Avonuzzi showed that fowl plague was a viral
disease . However, as fowl plague ( avian influenza)
is a septicemia, so different clinically from human
influenza, the association between the two remained
unknown till 1955, when Schaefer demonstrated
-
that the fewl plague viius was an igenicaHy related
to type A influenza virus. Shope (1931) isolated
the swine influenza virus- Not only did the swine
disease re- Kemble human influenza clinically hut
there was also epidemiological associ , i:inn between
the two. It was widely held that the virus spread to
swine from man at the Ume of the 1918 pandemic.
Influenza viruses have also been isolated from
horses, whales and seals.
Birds, particularly aquatic birds, appear to be
the primary reservoir of influenza viruses and
natural infection has been identified in several avian
species . In birds it is usually an asymptomatic
intestinal infection. The cloaca of healthy wild birds
is the best source for isolation of av:.un influenza
viruses. Ah isolates from oonhuman hosts belong
to type A - Influenza vims types B and t - appear to
be exclusively human viruses and natural infection
with them has not been identified in animals or
birds. Ordinarily, nonhuman influenza viruses do
not causehuman infection. But they may play an
,
important role in the emergence of pandemic
influenza.
INFLUENZA VIRUSES
Morphology: The influenza virus is typically
-
spherical , with a diameter of 80 120 nm but
plcomorphism is common. Filamentous forms, upto
several micrometres in length and readily visible
under the dark ground microscope, are frequent in
freshly isolated strains.
The virus cote consists of ritxmucleoprotein in
helical symmetry. The negative sense single
stranded RNA genuTne is segmented and exists ax
LIJTIL; pieces. Also present is a viral RNA-dcpcndcnt
RNA polymerase which is essential for transcription
of the viral RNA in infected host cells. The
nudeocapsid is surrounded by an envelope, which
has an inner membrane protein layer and an outer
lipid layer. The membrane protein is also known
as the matrix or ' M protein ' composed of 2
components, Ml .end M2. The protein part of the
envelope is virus coded but the lipid layer is derived
from the modified host cell membrane, during the
process of replication by budding. Projecting from
the envelope are two types of spikes ( peplomers) :
heifl & jizglutiijin spikes which are triangular in cross
section and the mushroom shaped ficLrramwifda.se
peplomers which are less numerous ( Fig. 55.1).
Resistance: The virus is inactivated by heating
at 50 °C for 30 minutes. It remains viable at 0-4
for about a week. It can be preserved for years at
-70 °C or by freeze drying. The virus survives slow
drying and may remain viable on to mites such as
blankets for about two weeks. Ether, formaldehyde,
phenol, salts of heavy metals and many other
chemical disinfectants destroy injectivity, Iodine is
particularly effective,
HcmaggluTinafiug, enzymic and complement *
fixing activities of the virus are more stable than
inactivity,
Huinagjluti upturn: Hemagglutination is an
important chatactefisric of influenza viruses. When
I rianiec ceria
Hidden page
Hidden page
Hidden page
Hidden page
 OrthHnyxavkus t
especially m type B infection in children, which
miv cn h present as an acute abdominal emergency
'
. to types A,
The uncomplicated disease resolves withiri about
seven LI .LV - .
The most important complication i
which is mainly due to
- pneumonia,
bacterial super infection or ,
rarely , caused bv the virus itself. Cardiac
complications ,. such as congestive fa Hurt: or
myocarditis and neurological involvement , such as
encephalitis, may occur rarely
Influenza, particularly infection with type B, has
been associated with Rcyc's syndrome. It especially
affects young children and is characterised by acute
degenerate change ^- in the brain, liver and kidneys.
Type B infections may sometimes cause
gastrointestinal symptoms (gastric flu ).
Laboratory diagnosis: of
the nrw antigen : Rapid -
Lliagno is on influenza may
be made by demonstration of rhe virus antigen on
the surface of the nasopharyngeal cells by
immunofluorescence. Detection of the viral RNA
by reverse transcriptase polymerase chain reaction
is extremely sensitive , but is of limited availability.
-
1imAttior) of the nrus: Vims l otarion is obtained
readily fmm pa Herts during the fir^ t two or three
days of the illness but less often in later stages.
Throat garglmgs are collected using broth saline
or other suitable buffered salt solution . If the
specimen is not processed immediately! it should
be stored at 4 ^ Ch or if the delay is long, at -70 cC .
The specimen should be ( reared with antibiotics
to destroy bacteria. Isolation may be made in eggs
or in monkey kidney cell culture.
The material is inoculated into the ammatic
cav i ty of 11-13 day old eggs, ir i ng at least six eggs
per specimen. After incubation at 35 °C For three
days, the eggs are chilled atid the amniotic and
allantoic Quids harvested separately The Quids aie
tested lor hemagglutination using guiiteapig arid
-
fend ells in parallel, at room temperature and at
4 ^C . Some strains of the influenza vims tvpc A
agglutinate only guineapi cells O i l initial isolation.
^
The type B virus agglutinates both cells, while type
507
C strains agglutinate only fowl whs at with amirem
K indC . Subtype identification is made
by hemagglutination inhibition test. Some of the
recent type A strains can be isolated by direct
allantoic inoculation of the clinical specimen: into
9-11 day old eggs. However, type B and C viruses
will be missed if only allantoic inoculation is used.
Inoculation into monkey kidney or other suitable
continuous cell cultures, such an baboon kidney, is
the preferred method where the lability is available.
Inoculated cell cultures art - incubated without scrum ,
and i u the presence oF trypsin, which increases
sensitivitvar of isolation . Incubation at 33 "C in roller
drums is recommended. Virus growth tan be
identified by hemadsorption with human O gimp,
iowl or guineapig erythrocytes., Rapid rtsults can
be obtained by demonstraring virus antigen in
infected cell cultures bv immunofluorescence.
-
IT
3. Serology? Complement Fixation and hem
agglutination inhibition rests are employed for the
semlogical diagnosis of influenza - It is essential to
examine paired sera in parallel, to demonstrate rise
in Here of antibodies.
Complement fixation tests with the RNPantigen
of Influenza virus types A, B and C are very usefeJ
as the antibodies are formed during infection only,
and not following immunisation with inactivated
vaccines. Complement fixation can also be done
using V antigens for the demonstration of strain
specific antibodies. Because of its complexity, CF
-
teats are now used only rarely.
Hcmagglutinatlon inhibition is a convenient and
sensitive test for the serological diagnosis of
influenza. However, it has some disadvantages. As
the antihemaglutmin antibodies arc subtype -
specific, it is necessary' to use as antigen the strain
currently causing infection. The may nr drawback is
the frequent presence in the sera of nonspecific
inhibitors of hemagglutination. The sera , suitably
treated for the removal of nonspecific inhibitors-,
are diluted serially in hemagglutination plates and
the influenza -vims suspension containing 4 HA units
added to each cup. Fowl red cells are then added.
opy righted material
8 dr w
Hidden page
Hidden page
510 * Taxlboon of Mic & tHCiogy *

separate , and also to keep pip away from them.
The practice nt keeping several species of birds
along with chicken* ill live bird markets is
potentially dangerous .
A unique feature uf influenza epidemiology wai
that once art antigenic variant emerged, it displaced
completely the pre -existing strain , Thus when Al
( HlNl ) strains atwc in 19*16-47, they became the
only viruses causing human disease , and the
previous Af > (HONI) strains disappeared completely.
The Al strains were displaced by Asian N I 2N 2)
strains in 1957 and they, in turn , by the A2 Hong
Kong ( } ! 3 lM' 2 ) strains in 196 E . However, thin rule
has not been observed in recent -vears. Even after
X
the re'emergence and wide dissemination of the
HlNl strain in 1977 , the A2 Hong Kong H 3N2
Strains continue to he prevalent. The reason for this
coexistence is rot known ,
There is considerable evidence to suggest that
there occurs an orderly reeve ling of the virus
subtypes at Irate with regard to their hemagglutinin
Table 55.3 Calender of appearance ol influenza A virus subtypes ( from 1 SS9 )
; Data wlcre 1933 based on Serci neciogf i
Dare Antigenic subtype
( H ) antigen , beroepidemiological (seroarcheo-
logical ) studies indicate that the severe pandemic
of 1889 was caused by a virus with rhe antigenic
structure H 2NE ant! that this was followed in 1900
by the subtype I 13 IV 8 which led to a moderate
pandemic. In 1918 came the most severe of al!
pandemics, caused by the ' Swine type" H l N l
( formerly Hsw Nl ) virus. Mild epidemics occurred
around iy33 and 1946 associated with minor
variations in the H antigen ( from HEW to HO in
1933, HO lo Hi in 19+6 ). The next severe
pandemic came in 1957 with the H 2N2 (Asian )
subtvpc. This was followed in 196 E by the H 3N2
( Hong Kong) virus leading to a moderate pandemic .
The year 1977 saw the reappearance of the H l N l
virus. Thus the sequence of variation in the H
— - — —
antigen baa httn Hl * H 3 * Hi » H 2 > HJ
111 from 1689 to the present time (Table 55.3) ,
From 1977, both H 3N2 and H l N l viruses have
beer circulating together . Table 5 5 - 3 lists the
sequence of appearance of these various subtypes.
^
Remajr&f

1 SH- 9- 1900 H2 N S r Pandemic and epidemics

190O“ 141ft H 3 NS Extensive epidemics
-
191 S 1933 Hi Nl firmer Hm Nil ‘Spanish flu'. The must severe pandemic
recorded; Heavy mortality -
193.1 - 1946 HI Nl (femur H0N 1 ) Discovery of influenza virus
r

- ^
( WS strain lWS Epidemics of ’AO* strains

1946- 1957 1 [ l Nl Epidemics of “ Al ' strains

1957- mE H2 N 2 Extensive pandemics of 'Asian flu' formerly
caJkd A2 ( Asian ) strain ., low mortality.
196 B H3 N2 Muiierate pandemic of 'Hong Kong flu'
co the present Turn: formerly called A2 rime ( Hong Kong ) strains,
very low mortality.
1977 HI Nl Re-emergenee of former Al strains First
ro the present time appeS/ed in Russia and China (‘Red flu *);
Mild pandemic, very lesw mortality

Copyrighted material
Hidden page
 Paramyxoviruses
The family PiramjntovLridue comains important
pathojrc ns of infants ajid children, responsible tor
* major part of acute respiratory infections
( respiratory syncytial virus and parainfluenza
viruses) and also for twin of the most contagious
diseases of childhood ( measles and mumps )
Though much less cochniont infections may also
occur in adults .
Paramyxoviruses resemble orthomyxoviruses in
morphology hut are larger and more pleomorphic .
They are roughly spherical in shape and range in.
size from 100 to 300 urn, sometimes with long
filaments and giant forms of upTa H00 Aiti The . Mumps is an
helical nudcaclpjid is much wider than in
orthomyxovi ruses, with a dismetc rnflSnm (except
in fVitLrnwnj- LT!; where it is 13 mrtJ. The geuOnte is
a molecule of linear single stranded RNA. Unlike
the orthomyxoviruses, in which the segmented
nature cif the genome facilitates genomic
rcassortments and antigenic variation so typical of
Influenza viruses, the paramyxoviruses with their
unsegmented genome do not undergo genetic
revoruhniaCioTL 'i or antigenic variations . Hence all
paramyxoviruses are antigenitilJv stable.
The nncleoclpiid is surrounded by A lipid
envelope which has rhe matrix (Ml protein at its
base and two types of tmumembrane glycoprotein
spikes at the surface. The longer spike is the
hemagglutinin (H), which may also possess
neuraminidase (N) activity and is hence known as
H or jHN protein. It is responsible for adsorption
of the virus to the host cell surface . The second
spike is the I' ( fusion) protein, responsible for fusion
"
of the viral envelope with the plasma membrane of
the host cells, which is the essential early step for
infection. It also bring about cell- lo-cell fusion,
*
causing Large giant cells or syncytia, which are
characteristic of paramyxovirus infections. The F
protein also mediate the liemnlytic activity of
*
. paramyxoviruses.
The family Paramyxnviridae in divided into four
genet a— Kubulsvirua f 3 a rainfluenza rirus,
Norbiliivirtu and Ptieumannu (Table 56.1),
RUBULAV1RUS
M l MPS VIRUS (G K M S R l Bt I. A V I H I s >
acute inleCtious disease commonI v
iffecring children and characterised by
nonsuppurative enlargement of the parotid glands.
As epidemic parotitis, it had been described by
Hippocrates in the fetch century BC. The viral
etiology of mumps was demonstrated by Johnson
mid Goodpasture ( 19J4) bv its experimental
transmission ro monkeys. Habd in 1945 cultivated
the virus in embryonated eggs. In 1955 , Hcnle and
Deinhardt grew LI in [issue culture .
Properties: The mumps virus is a typical
paramyxovirus possessing hoth HN and F proteins.
It agglutinates the erythrocytes of foiwl, guiueapig,
hum ans and many other specin. Hemagglutination
is followed bv hemolysis and elution at 37 C. The
'
virus can be grown in chick emhrvps - in the
amniotic cavity for primary isolation and the
allantoic cavity after adaptation. Eggsare inoculated
at b-S days and incuhated at 35 °C for five days
before harvesting.
Cell cultures are better suited for isolation -
primary monkey kidney being the preferred cell.
Copyrighted materia
 < Pgramyxgvirysgs 513

Table 56pi Primes

*1 Genera In the Family ParamyKCYiritee
Property Genus
Ptjntirf /Juenzsi'fnii Mumps Morbsllivirus Pncumovirus

Human viruses FirunBuean 1-4 Mumps Measles Respiratory syiicyria]

virus
Diameter of nucteocapsidtnm) IS IB 13 13
Fusiinl I F ) pfCtffcLQ 4 4 + +
Hemolysin + 4 + -
Ilcmagg]uTLnLn/Hcmad5orption + 4 4 =

Neuraminidase + 4 - -
InrrjetlliLdar inclusions in c c NX C
cytoplasm {CV nucleus (N)

The cytopathic effect is slow and consists of
syncytium formation and the presence of acidophilic
cytoplasmic inclusions. Growth is hqst identified
by hem adsorption .
The mumps virus is labile , being rapidly
inactivated at room temperature Of by cynosure to
formaldehyde , ether or ultraviolet tight . It can be
—
preserved it 70 "C or by hjfphili nation..
Tire mumps virus is aniigeniically stable and only
one serotype exists.Two complement fixing antigens
can be recognised , is in influenza viruses -the
soluble (5) antigen and the ' viral' ( V ) antigen.
Climeal features: Infection is acquired by
inhalation , and probably also through the conjunctiva.
The virus replicates in the ujjper respiratory tract arid
cervical Lymph nodes and is disseminated through
the bloodstream to vinous organs. "I he incubation
period is long, about 12-25 days.
Parotid swelling is usually the first sign of
illness chough it may sometimes be preceded by
prodromal malaise , Parotid swelling is unilateral
to start with Luc may become bilateral . It is
accompanied by fever, local pain and tenderness bur
the skin over the glam! is not warm orerytliematiOUS.
Parotitis is nonsuppurative and usually resolves
within a week. However, involvement of the
extraparnrid sites may be mote serious. Suck
involvement may sometimes occur even in the
absence of parotitis.
EpididymO -orchitis is a complication seen in
about a thirel of postpubertd male patients. The
festis becomes swollen and acutely painful, with
accompanying fever and chills . Orchitis is usually
unilateral but when ir is bilateral and followed by
testicular atrophy, sterility or low sperm counts may
result .
The central nervous system is involved til about
60 per cent of cases, as indicated by pleocytosis in
, the CSFbut only about 10 percent show symptoms
of meningitis- Mump bus been reported to cause
about 10-15 per cent of cases of "aseptic mcningi ti s',
Mumps meningitis and meningoencephalitis usually
re salve without sequelae but deafness may
sometimes result . Mumps meningitis may
occasionally occur in the absence of paioiitis, when
, diagnosis rests solely on laboratory evidence . The
virus can be grown readily from the CSF in the
early phase of meningitis.
Other less common complications are nrthriris,
oophoritis, nephritis , pancreatitis, thyroiditis and
myocarditis.
Kpidemiology; Mumps LS endemic worldwide
but has become Less common in the advanced
nations due to im muni Ration. It often occurs as
epidemics in children 5-15 years of age, and also
in young people living in groups such as in army
camps. Household spread is common . Humans are
the only natural hosts. The source of infection is a

Copyrighted materia
Hidden page
Hidden page
516 * .
TGKttaok ol M crabiolcigy *

Table 56.2 Parainfluenza virus types

Nomenclature o f human types

Current
Parainfluenza type I
PaninflueiRi type 2
Parain flmenacH type 3
Pa.raj.nflLie nza type 4 [4A 4 li
1 Related animal viruses
* Formet
Hrmads^ rprion type 2( HA-2 ) Sendai ( mouse )
Croup Misdated (CA) Simian virasGS SK 41 ( monkey)
Hemad &orpticin type I ( HA-1) Shipping fever (cattle )

hemagglutination inhibition .
Serological diagnosis in hampered by wide
antigenic cross reactions. Haired sera can be tested
by neutralisation, ELISA, HI or CF for rise in litre
of antibodies,
-
150 300 rnn . The viral envelope has two
—
glycoproteins the C protein by which the unus
attaches to cell surfaces, and the fusion ( ]’) ptitein
which brings about fusion between viral and host
cell membranes. The F protein is also rcspoisiblc

NEWCASTLE USEA 5E VIRUS ( NDV )

The Newcastle disease virus (avian paramyxovirus
type 1} is a natural pathogen of poultry in which it
causes explosive outbreaks of pneumoencephsdids
or "influenza ' with high mortality. In India it is
known as the Ranikhet virus. Control measures
consist of vaccination , and slaughter of infected
birds.
Human infection with NDV is confined to a
self-limited conjunctivitis ill poultry workers and
others in contact with infected turds .
Other types of avian paramyxoviruses cause
inapporenr infection in many species of birds .
GENUS PNEUMOVIRUS
kKSKEKATOm SYNCYTIAL V l l t l K
( R S V)
RSV was first isolated in 1956 from chimpanzees
with coryza and was called the "chimpanzee cuiyta
agent ' (CCA ). A year later , the virus was ohtained
from children with lower respiratory' tract infection .
Because it caused cell fusion and the formation of
rtUilTirtudeated syncytia in cell cultures , it was named
respiratory syncytial virus ( RSV ) It 15 nowr
recognised as the most important cause of lower
respiratory tract infection in infants, particularly in
the first few months of life,
RSV is pleomorphic and ranges in size from
-
for cell to *cc!1 fusion , which leads tc the
characteristic syncytial cytopathic changes inHJSV
infection.
RSV differs from other paramyxoviruses JI not
^ .
possessing hema lutinin activity It also dees not
have neuraminidase or hemolytic properties.
Another difference is that its nuclcocapsid diimeter
(13 nm ) is less than that of other paramyxoviruses
(18 nm ) .
RSV does not grow in eggs but can be
propagated on heteToploid human cell culture, such
as HcLa and Hep-2. It is highly labile and i s
inactivated rapidly at room temperature. Itcan be
preserved by tyophilisationr It is antigcnicaljr stable
and only one antigenic type exists. Howevtr.studies
using monoclonal antibodies have identifed two
subtypes called A and B.
Clinical feature*: Most RSV infecti + ns are
symptomatic. The virus can hardly ever h: hiund
in healthy persons.
Infection causes a broad range of respiratory
illnesses. In infants , the disease may begin a febrile
rhinonheu, with cough and wheezing , progressing
in 25-40 per cent to lower respiratory mvolrement ,
including iracheobronchitis, bronchinlrt .s and
pneumonia. In about one per cent , the i '.hrss is
serious enough to require hospitalisation. tSV is
considered to be responsible for about half TKL cases
of bronchi oil tin, and a quarter nf all pneumonias

Copyrighted materia
 i Paramyxoviruses
—
occurring in the fir** few months of life. Most
patients recover it ] 1 2 weeks but those with
immunodeficiency or cardiac defects mav have
protracted iUncs* and high death rattS-
RSV is an important cause of otitis media in
young children . A relation between RSV and the
sudden death syndrome in infants has been
proposed bur not proven .
In adults RSV infection may present as a febrile
common cold. It can cause pneumonia in the elderly,
Epidemiology: RSV is global in distribution , Prophylaxis No effective vaccine is available.
lr causes annual epidemics in the temperate regions
during winter and in tile tropica during rainy
seasons. Outbreaks are common in children s wards,
nurseries and daycare centres. Infection is most
common in children six weeks to six months of
age, with the peak at 2-3 months. The newborn!;
are believed To be protected hv high levels of
maternal antibody.
The virus is transmitted by close contact and
through contaminated fingers and fomites . Coarse
droplets of respiratory secretions discharged during
coughing and speeding arc more efficient in
spreading the virus than fine aerosols. The
incubation period is 4-6 day*, Virus shedding may
persist for 1-3 weeks , though children with
defective cell mediated immunity may continue to
sited the virus for months.
Reinfection with the virus is not uncommon but
the disease so produced is milder than in primary
infection. The role of the antibody in protection
against the infection is not clear. Secretory IgA is
considered more important than circulating IgG
in protection . Cell mediated immunity appears more
important than humoral antibodies in recovery from
infection . RSV does not induce high levels uf
interferon production.
Laboratory dLadnoaiu: RSV can he isolated
from nasopharyngeal swabs or nasal washings,
Samples should be inoculated in cell cultures
( HeLa or HEP-2 ) immediately after collection,
freezing of clinical samples may destroy the virus.
In cultured cells, RSV causes characteristic giant
» 517
cell and syncytial formation but cytopathic effects
take about 10 days to appear. Earlier detection of
viral growth in cells is possible bv immuno -
fluorescence tests. Rapid diagnosis nf RSV infection
can be made by the immunofluorescence tost on
JP
smear* of nasopharyngeal swabs ,
Serological diagnosis is by demonstration of
rising antibody litres in paired scrum samples bv
ELJSA, CF, neutralisation or immunofluorescence
tests.
Attempts at immunisation by formalinised vaccines
had to be given up as the vaccinees developed more
serious illness than the controls on subsequent
exposure to the infection.
Treatment: Management of RSV infection is
primarily by supportive Care . Administration of
ribovirin Hv continuous aerosol has been found
beneficial in hospitalised patients , decreasing the
duration of illness and of virus shedding.
GENUS MORBILUVIflUS
Mli \ SI KS C Its -
III . CI . A )
Measles is an ancient disease but for a long time
no dear distinction was made between measles and
other exanthematous diseases, including smallpox.
It was only in 162V that measles time to be
considered a separate entity. Thomas Sydenham in
1690 gave the first dear and accurate description
of measles in the English language.
In lf )4 b an outbreak of measles occurred in the
remote Faroe Islands,, affecting 75 per cent of the
islanders. The classic study of this epidemic by Peter
Panum, a Danish muJicid student, laid tile basis of
our scientific knowledge about measles.
The viral etiology ( it measles was established
bv Goldberger and Anderson in IV 11 by
transmitting the disease to monkey* through the
inoculation of filtrates of blood and nasopharyngeal
secretion* from patients- The virus was isolated In
monkey and human kidney cells by lindcrs and
I cblcs in 1954,
^
Men ales virus: The virus has the general
Copyrighted material
518 * ‘raxlbOOh OF Microfcwtogy *
morphology ff pifUflJMtwiniStt ( Fug - 5ft.2 ) It it u
roughly spherics] but often pleomorphic particle,.
—
120 250 run in diameter. The rightly coiled helical
nuricocapsid is surrounded hy the lipoprotein
envelope carrying on its surface hemagglutinin ( I J )
spikes. The envelope also has the F protein which
mediates cell fusion and hemolytic activities, The
merles virus agglutinates monkey eiydirocytes hut
there is no elution as the virus docs not possess
neuraminidase activity.
The measles virus grows well on human or
monkey kidney and human amnion cultures which
are the preferred cells fur primary isolation. Isolates
can he adapted for growth on continuous cell linn
( HcLa, Vcro ) and in the amnintic sac of hen's eggs,
Cyiopatlnc effects consist of multLnudeatie syncytium
formation, with numerous acidophilic nuclear and
cytoplasmic inclusions. Mulrinucleate giant cells
-
( Warthin Finkeldey cells ) are also found in the
lymphoid tissues of patients.
The virus is labile and readily inactivated by
heat, ultraviolet light, ether and formaldehyde . Jr
can be stabilised by molar MgSG + f so that it resists
heating at 50 "'C for one hour.
The measles virus is antigcnically uniform - It
shares antigens with the viruses of can inc distemper
and bovine rinderpest.
Clinical features: It lakes about y-1 l days
from the time of exposure to infection for the first
signs of clinical disease to appear. These consist of
prodromal malaise , fever, conjunctival injection ,
cough and nasal discharge . After 3-4 days of
prodromal illness, the rash appears. A day or two
before the rash begins, Koplik 's spots develop or
the buccal mucosa and occasionally on the
conjunctiva and intestinal mucosa, The prodromal
illness subsides within a day or two of the
appearance of the rash. The red maculnpapular rash
of measles typically appears on the forehead first
and spreads downwards , to disappear in the same
sequence 3-6 days later, leaving behind A brownish
discolouration and finely granular desquamation .
Most patients recover uneventfully but quite a
few develop Complications whit- h may he due to
'
the virus ( croup, bronchitis ) or to secondary
-
bacteria] infection (pneumonia* otitr media ). Ranch ,
the virus may cause a fatal gi .Liu cel ] pneumonia ,
'
particularly in children with immunodeficiencies
or severe malnutrition. Complications art more
common and serious in the developing countries,
The mos- t serious complication it
meningoencephalitis. Many survivors have
neurological sequelae . A nit late Complication is
subacute sclerosing pancncephiUds ( SSPE ) .
Protracted diarrhea is often seen as a
complication in children in the poor nations. The
virus may be recovered from the stools of patients
with measles enteriris.
There occurs a suppression of delayed
hypersensitivity after measles infection , which may
last for week* or a few months. Mann MIX and Other
allergic skin tests may be negative d u rin L!I i s period .
Underlying tuberculosis may become worse ^
following an attack of mcasie&. Recovery From
measles mav also be associated with an
if
improvement o i allergic e c z e m a o r asthma ,
,
Hodgkins disease or lipoid nephrosis.
,
Measles induces labour in some pregnant
2
'
Fig. 56.1 Measles res 1. Membrane ( Mi protein 2 Nuc -
leotapsid 3 . Lipoprotein membrane. 4. Spikes-
hemaggluiinin .
Copyrighted material
Hidden page
Hidden page
 Arboviruses
-
ArixwimsM (ttthnpod botDC viruses] axe viruses
<rf vertebrates biologically transmitted by
hematopKagnn-us insect vectnrs. They multiply in
bloodsucking insects and are transmitted by bite to
v£ i"i:ebr& T £ hosts. Insect viruses and viruses cif
vertebrates that are sometimes mechanically
transmitted by insects do not come into this
IT
category. Inclusion In this group is based on
ecological and C|i i Jen .loLoi-iL - aJ considerations and
hence it contains members that are dissimilar in
other properties. With better understanding of the
physical and chemical properties of individual
viruses, they are reassigned to more defined
tixodomical groups . Though tax on Clinically
unacceptable, the name Arbovirus' is a useful
biological concept.
A similar ecological group is that of the
rodentborne ( robo ) viruses which are maintained
in nature by direct transmission between rodents,
and sometimes infecting other species, including
humane, by direct contact without the agency of
arthropod vectors- Robovi ruses, I ike arboviruses,
belong to different taxonomical families, some of
them in common with arboviruses.
Arboviruses art worldwide in distribution but
are far more numerous in the tropical than in the
temperate zones. Over 500 viruses have beer listed
in the International Catalogue of Arboviruses
published in 1965. MOST of them cause silent
infections in rodents and ether wild mammals but
about 100 of them can infect humans. In India,
over 40 arboviruses have been detected, of which
more i ban 10 are known to produce human disease.
Arboviruses have been named according to the
disease caused ( Yellow fever ), the place of isolation
of the virus ( Kyasanur Forest Disease ) or the local
name for the disease (Chikungunya ) . They arc
classified according to their physical and chemical
features into CMNNOMINAL families. Arhoviruses have
a
been placed in Toga- , Flivi-, Bunya-, Reo- and
Rhabdovirus families (Table 57-1), Within each
family they are classified into genera, and antigenic
groups, based cm serological relationships. Some
viruses arc ungrouped -
Arhoviruscs have a very wide host range
including many species of animals and birds. The
ability to multiply in arthropods is their special
characteristic. The most important arbovirus vectors
art mosquitoes, followed by ticks. Phclhofomus,
COR -older and G m/cidae are less common vcctors.
'
In the laboratory, mice are commonly used for
growing arboviruses, intracerebral inoculation in
stickling mice being the most sensitive method for
their isolation. They can be grown in the yolk sac
or chorioallantoic membrane of chick embryo, in
tissue cultures of primary cells like chick embryo
fibroblasts or continuous cell lines like vero or
1 IcLa, and in cultures of appropriate insect tissues.
Most arboviruses agglminate the red cells of
-
goose or day old chicks . Hemagglutination is
influenced by pH and temperature , the optimal
requirements varying with different viruses.
Spontaneous elution does not occur.
Hemagglutination is inhibited specifically by
antibody, and nonspecific ally by lipoprotein
inhibitors In serum, brain and other tissues.
In general, arboviruses are labile , being readily
inactivated at room temperature and by bile salts,
.
/"H
LJ opy righted
B w
jr
material
$ 22 * Textbook or Microbiology »

Table 57.1 Tasonomy of some Important arboviruses

FBJTI jjy Genus Important species

Togaviridae Alphavirus Chikungunya^ Q nyong- nyung, Mayaio, Semliki Forest
.

Sindbis, Ross R i \rerp Eastern, Western and Venezuelan

. .

equine encephalitis viruses

Ftaviviridac Flavivi ms Japanese encephalitis , Murray Valley encephalitis,, VVesrNilc ,

Ilheus, St_ Louis encephalitis* Ydknr Fever , Dengue types 1 ,
. .

2, 3 , 4, Russian Spring Summer encephalitis complex,

Louping i£L, Fowassan , Kyasanur Forest Disease. Omsk
.

hemorrhagic few
Bunyaviridae Bunyimrus California eruxphaliris Qiopoucht, Turlock
!

Phlebovirus Sandfly fever viruses, Rifr valley fever virus

NurwiniE Crimean Congo hemorrhagic fever viruses,, Nairobi sheep
disease virus, Ganjam virus

Hantavirus Hantan, Seoul, Puumala, Prospect Hill , Sin Noinbre viruses

Rpovitidae Orhjvims Colorado tick fever* African horse sickness* Rime Tongue
viruses

Rhabdoviridae Vesiculovirus Vesicular stomatitis virus, Chandipura virus

ether iind other lipid solvents , Infecrivity may be
-
retained nr 70 °C or hv lyophilisatLOii .
Antigenic Structurei Three antigens are
important in serological studies - hemagglutinin ,
complement fixing and neutralising antigens all
integral parts of the virus pm tide . Considerable
- Arboviruses also cause a number of veterinary
antigenic cross reactions occur among arboviruses.
The plaque reduction neutralisation test { PRNT}
shows the greatest specificity.
Pathogenesis: The virus enters the body
tbrough the bile of the insect vector . After
multiplication in the reticuloendothelial system ,
viremia of varying duration ensues and, in some
cases, the virus Is transported to the target organs ,
such aa tlie central nervous system in
encephalitides, the liver in yellow fever and the
capillary endothelium in hemorrhagic fevers.
Arboviruses cause the following elinieal
s y n d r o m e f e v e r with or without r a s h a n d
arthralgia; encephalitis; hemorrhagic fever; and the
characteristic systemic disease , yellow fever (Table
.57,7 ) . All infections occur with varying degrees of
severity', subclinical infections being very common.
diseases such as Eaton, Western and Venezuelan
equine encephalitis in horses in America, Rift
Valley lever in sheep and cattle in Africa, Blue
tongue in asses in India, Africa and America,
Ganj am d isease of sheep in India and African horse
sickness in horses and mules in Africa and Asia.
1 aboratory diagnosis: Diagnosis may be
established hy virus isolation or scmlngy. As all
arbovirus infections arc viremic, blood collected
during the acute phase of the disease may yield the
virus. Isolation may also be made from the CSF in
some encephalitic cases but the best specimen for
virus isolation is the brain . Specimens are inoculated
intracerebrally i n t o suckling mice. The animals

Copyrighted material
Hidden page
Hidden page
 4 Arbcv
lesser extent in complement fixation tests . The
neutralisation test is more specific. They produce
epidemics of encephalitis in America and dengue ’ fever is typically biphasic with a period of
like fever in the tropics.
Enoephaililis viruses; Three members of this
group, Eastern, Western and Venezuelan equine
encephalitis viruses, cause encephalitis in horses
and humans , Eastern equine encephalitis ( EEE )
occurs along eastern Canada , USA and the
Caribbean , causing sporadic cases and small
epidemics. Western equine encephalitis ( WEE ) is
more widely distributed in America and causes large
epidemics. Venezuelan equine encephalitis [VEE) ,
prevalent in Central and South America, usually
causes an influenza - like illness, with encephalitis
in a small proportion of cases. Several species of
Culcx and Anopheles mosquitoes are the vectors
and wdd bLrds the reservo i ns. Formal ] nised vacci n en
have been developed for EEE and WEE and a
Live attenuated vaccine for VEE,
Viruses Causing Fchi Je Illness
J. Chrkungunpa wirm.. This virus was first isolated
from human patients and Aedes aegypii
mosquitoes from Tanzania in 1952- The name
'
chikungunya' is derived from the native word
for the disease in which the patient lies H doubted
up* due tD severe joint pains. Epidemics of
dukungunya have occurred in many African
countries . In 195 B, the vims caused a large
epidemic of hemorrhagic fever in Thailand . The
virus first appeared in India in 1963, when along
with dengue, it caused very extensive epidemics
in Calcutta , Madras and other areas.
Chikungunya outbreaks occurred at irregular
intervals along the east coast of India and in
Maharashtra till 1973. Since then the virus has
been quiescent.
The disease presents as a sudden onset of fever,
crippling joint pains, lymphadenopathy and
conjunctivitis, A maculopapukr rash is common
and some show hemorrhagic roam lest mums .
Hemorrhagic lesions were common in Calcutta
iu
^CS * 525
when the disease Erst appeared there in 1963
but have been extremely rare afterwards. The
—
remission after 1 6 days of fever. The vector is
Aidei legypli . Nt> animal reservoir has been
identified . Antibody to the virus has been
demonstrated in horsey cattle andother domestic
am main but its significance is not known. No
vaccine is available.
2. 1 fYUTtg-nyong virus . Th is vims was first isolated
^
in Uganda , litis is confined to Africa, is closely
related to the ehikungunva virus antigenically
and causes a similar disease. This is transmitted
by the Anopheles species. The Mayaro virus
causes a similar disease in the AAfcsr Indies and
South America.
3. Semi1.1, : Fonstvirut : This vims was. first isolated
in 1942 in Uganda from Aedes mosquitoes has
not been associated with clinical illness in
humans though neutralising antibodies to the
virus have been demonstrated in Africans, The
Sindbis virus , originally isolated from Culex
mosquitoes in the Slndbi -i district of Egypt in
1952 , has subsequently been recovered from
other puts of Africa, India, Philippines and
Australia. In Africa , it is known to be associated
with febrile illness in human beings. In India,
antibodies have been detected in human sera
but no association has been established with
human disease . The closely related Ross River
virus has been associated with epidemic
polyarthritis in Australia.
FUVW1 RUSES
Ike family Flaviviridae eontamh only ore genus,
.
FiavivituS They are somewhat smaller than
alphaviru«5 , being 40 nm in diameter, The name
/IsV7 virus refers to the type species, the Yellow fever
virus ( FJavus, L = yellow ).
There are over 60 arthropod -borne flaviviruses,
Reprecentarbre members af th i g r o u p are
dhtriliutcd in all parts of the world* covering all
the zoogeograph ic regio ns. They may be considered
Lu u 1 1
Hidden page

In India, I .Lpan-e -sc encephalitis was first
recognised in 1955 when the virus was isolated
from mosquitoes of the tufev Hshnuj complex from
Vellore during an outbreak of encephalitis in Tamil
Nadu , The vims continued to be active in Tamil
Nadu and Andhra in subsequent years also, causing
illness mainly in children, indicating the endemic
nature of the virus. Most of the cases occurred
between October and Nowmbcn
Jipanestr encephahLi ^ remained confined to the
southeastern parts of Indu Till 1973, when it caused
A large outbreak of encephalitis in West Bengal ,
The epidemic affected adults also, with mortality
rates approaching 50 per cent , suggesting that the
virus was freshly introduced into the area. Cases
occurred mainly between |une and October. Fro FT
1976, there have been periiwiical outbreaks of the
d ease in various parts of India— Dihrugarh
^ vaccine has been reportedly effective in preventing
( Assam ) in the cast, Gorakhpur ( Uttar Pradesh)
. .
und E E LCV H 11 ,L in the north .mdGiia and Maharashtra
in the west. In the south , outbreaks have occurred
in Kolar in Karnataka1 various areas in Andhra
Pradesh, I’irunelveli and South Arcnt in Tamil
Nadu, in Pondicherry and lately in Kerala . In
addition, sporadic cases have been reported from
different parts nl the country, excepting the
northwestern states . Japanese encephalitis has
become a mu jot public health problem of national
importance in India ( Fig, 57.1) ,
The natural cycle of the virus has been worked
out in detail in Japan. Herons act as reservoir hosts
and pigs as amplifier hosts. Human infection is a
tangential 'dead- end’ event and occurs when the
I n 11! i*. J iI if. i L .LL J I LI igk den xi 1 y. Tl i L natural
11:J enl EL 1
L ( ' '
cycle in India also may be similar. Natural in lection
has been demonstrated in Ardcid birds (berons and
egrets), 0 ' wdl as hird- ro- bird transmission through
QiJct tnVaen/orbync/ius, Other birds such as ducks,
pigeon) and sparrows may also be involved .
Vertebrate hosts may Include cattle and buffaloes,
besides pigs - The rmiior vector Culcx
tritucif '. fyi hynchus has a predilection for cattle and
'
bites them in preference to humans or pigs, but as
« AftHwusH p 537
cattle do not develop circniia, they do not -contriluitc
to spread of the virus. The high cattle- pig ratio in
India hasbeen suggested as a factor limiting human
infection.
Prevent ive measures include mosquito control
and locsti i ig pi ISeries away from human dwellings.
A formalin inactivated mouse hr ,Liu vaccine using
the Nakayama strain has been employed successfully
for human immunisation in Japan and. lit a small
scale, in India also. Two doses at two weeks" interval
followed by a booster 6-12 months later constitute
a full course . Immunity produced by the vaccine is
shortlived. A live attenuated vaccine has been
developed in China from JE strain SA 14- 14- 2,
passed through weanling mice The vaccine is
produced in primary' baby hamster kidney cells.
Administered in two doses, one year apart, the
clinical disease.
Vaccination nf pigs has been proposed in view
of their importance as amplifier hosts. During
major epidemics , slaughter of pigs have been
employed as a measure of conti inmentr A million
pigs were reportedly slaughtered in Malaysia in
1999 to stop an epidemic of encephalitis.
Yellow fes er: Yellow fever was recognised as a
clinical entity as early as in the seventeenth century
and was familiar to pirates as the 'Yellow Jack'. Jr
is a native of Africa and was transported thence
along the trade routes to Europe and America . The
most serious epidemics occurred in the Western
I Hemisphere' Central .America and the Caribbean,
and even as far north as New York. Since the early
' ' largely
twenik rh century, the rii & euiE IL.M: Keen
confined to certain areas of Africa and South and
Central America.
Carlos Finlay in Cuba in 1331 suggested that
yellow fever was spread by Acdes aegypti
mosquitoes. In 1900, the US Army Yellow Fever
Commission, under Walter Reed, confirmed this
observation and demonstrated that Aedcs
mosquitoes were infected by feeding on human
pati ents during the early vi remi L phase of the disease
opy righted material
8 dr w
Hidden page
 4 Arboviruses
KI bed me infective after an extrinsic incubation
, L:
period of 12 days. Thisled to the promptcidicition
of the disease from Cuba and the Panama Canal
;na by controlling the Aedes aegypti mosquitoes.
There were even hopes of ultimate total eradication
nf the di-ew but these had to he abandoned when
in 1932 outbreaks of yellow fever occurred in Brazil
Ln areas devoid of Actfes aegypti , It was then
recognised that the virus survives in another cycle
- the forest or sylvahc cycle - irevolving forest
; LNMINIS and mosquitoes.
The yellow fever virus was first isolated in 1927
by inoculating rhesus monkeys with the blood of
an African patient named Asibi The virus was
shown hv Theiler ( 1930) to grow well following
intracerebral inoculation in mice. The infected
mouse brain was used as a vaccine in former French
West Abie a ( Dakar vaccine) though this was
EncEphalitogEmv. IT was later replaced by a non-
ncurotropic (17D) vaccine.
After an incubation period of 3-6 days, the
disease starts as a fever of acute onset with chills,
headache nausea and vomiting. The pulse i & usually
h carries a high risk of producing encephalitis in the
slow despire a high temperature . Jaundice,
albuminuria, und hemorrhagic manifestations
develop and the pa r ient may the of hepatic or renal
failure . Most cases ate less severe, especially in the
endemic areas, and may present as undifferentiated
fever without |aundice.
Histologically, the liver shows cloudy and fatty
degeneration and necrosis which is typically
midzonah The necrosed cells coalesce and become
hyalinised leading to the formation of characteristic
eosinophil masses known as Councilman bodies.
1
Acidophilic intranuclear inclusion bodies (Tomes
bodies ) may be seen in the infected liver cells in
the early stages.The h niological p i cturc of ihe liver
is specific enough to be diagnostic., and this was
the basis of early surveys undertaken to detect areas
of yellow fever activity, A special instrument
(visceroTome) was employed for the collection of
liver tissue from fatal cases for histological
diagnosis.
529
The epidemiology of yellow fever was clarified
only after the recognition that the disease occurs
MI rwo distinct patterns. In the urban cycle, humans
at both as the natural reservoi r and as the definitive
case, the virus being transmitted by the domestic
Aedes w gypO mosquito. In the forest of sylvatic
^
cycle, wild monkeys act as the reservoirs and forest
mosquitoes [ tJacmagQgvs spegazrinii in Smith
America and Aetfej ifriianus and A. ArttpsOJl.1 in
Africa ) as the vectors. Human cases occur only
when humans trespass into the forest or when the
monkeys raid villages near the forest.
The control of urban yellow fever can he
achieved by eradicating the vector mosquito, as was
shown in Cuba and Panama early this century by
Gotgas butthi* is obviously impractiL-ablc with the
sylvatic disease. Two very effective vaccines have
been developed for human use. The French
neurutrOpi c vscci ne ( Dakar ) produced from infected
mouse brain was thermostable and administered by
scarifies hi m and hence convenient for use under
tropical field conditions- However, the vaccine
vaccinees, especially in children . A safe and equally
effective vaccine, the 17D vaccine was developed
by Theiler in hy passaging the Asibi strain
serially in mouse embryo and whole chick embryo
tissues and then in chick embryo tissue from which
the central nervous tissue has been removed . The
17D vaccine is thermolabife and is administered
by subcutaneous inoculation. Vaccination which is
mandatory for traivel to or from endemic areas is
valid for 10 years beginning 10 days after
vaccination . In lndi :i , the 17 D vaccine i *
manufactured at the Central Research Institute,
Ka &auli.
Yellow fever is largely confined to Central and
South America and Africa, Yellow fever does not
exist in India and it is important to us for this
paradox leal reason India offers. a receptive area with
a large population of Acdcs aegypd and nonimmune
humans. Strict vigilance is enforced on vaccination
and quarantine for travel from endemic areas. This,
>pyrighted material
H dr w
530 4 Tpittbaok
no doubt, has checked the entry of the virus ln.to
India through legitimate passengers. It is likely that
stray virus introduced may have been kept uut, due
to the prevalence in the local Aedes aegypti of
] Jetigue virus, and of anybodies to a wide range of
arboviruses in the local population. Another reason
could have been that in Africa, yellow fever was
ifi.iinlv in the west , and in India , Aedes mosqiutoea
were along the east coast , so that even stray
importations of vims by sea may not have found
suitable vectors. TIIK is no longer valid as yellow
fever has in recent years have caused epidemics in
East Africa , and Aides mosquitoes have spread all
along the west coast of India. If ever yellow fever
gets established in India* the consequences could
be catastrophic.
Dengue: Dengue virus in vilely distributed
throughout the tropics and subtropics. (The name
\ lengue’ derived front the Swahili KJ deiygap£ pQt
meaning a sudden seizure by a demon. The term
' break - bone fever’ was coined during the
lJhiLadelphi.i epidemic in 1750). Dengue fever is
clinically similar to the illness caused by
chikungunya and O' nyong nyong viruses. Four
* provides early diagnosis, as it appears within two
types of dengue virus exist: DEN 1 first isolated
from Hawn in 1944, DEN 2 from Nrw Guinea i n
1944 and DEN 3 and 4 from the Philippines in
195 b. Immurin is type specific so that it is passible
' test for IgM is available for rapid diagnosis.
fora person to have four separate episodes of dengue
fever. Dengue has been increasing worldwide over
the last few decades and today ranks as the most
important wetorborne disease, with about 2.5
billion people in 200 countries at risk .
Dengue presents clinically after an incubation
period of 3-14 days, as lever of sudden onset with
headache, retrobulbar pain, conjunctival injection,
-
p.Lin in the back and limbs ( break bone fever ),
lymphadenopathy and maculopapular rash . The
fever is typically biphasic (saddle . back) and lasts
for 5-7 days. Dengue may also occur in more serious
forms, wirh hemorrhagic manifestations {dengue
Hemorrhagic fever) or with shock (dengue shock
syndrome] . These complications, first recognised
o1 Microbiology
in Thailand, have since occurred in many countrics
in Southeast Asia and the Western Pacific . They
arc mote common in previously healthy children
in the indigenous populations of endemic areas.
They may be a hypersensitivity or enchancement
response to sequential dengue virus infection in
persons sensitised by prior exposure to other
serotypes of the virus.
Dengue virus is transmitted from person to
person by Aedes ae ti mosquitoes. The extrinsic
^
incubation period is fi-10 days. No vertebrate hosts
other than humans have beer identified.
Dengue was initially confined to the east coast
of India and has Caused epidtitt ica, some t i me i along
with the chikungunya virus, as in 1963 when
extensive outbreaks affected Calcutta and Madras.
Subsequently it has spread westwards and in the
1990s Surat and Delhi had major epidemics with
deaths due to DHF and DSS. All four types of
dengue virus are present in this country.
Occasionally, more than one type of the virus has
been isolated from the same patient.
Demonstration of circulating lgM antibody
to five days of the onset of illness and persists for
one to three months. IgM ELISA test offers
reliable diagnosis. A scrip imiriunoehrornaiographie
Control of dengue is limited to vector control
as no vaccine is currently available.
TICIBORNB GROUP
These viruses produce two clinical syndromes,
encephalitis and hemorrhagic fevers.
Tiokbome encephalitis viruses: A number
nf viruses belongi ng to the Ru»i; m Spring Summer
Encephalitis ( RSSE ) complex cause encephalitis
along a wide area ot the northern lindm.viH from
Scotland to Siberia.The names given to the disease
vary from one area to another depending on the
variations In the prominent clinical features. Thus,
in Scotland, it is called 'louping ill' as the disease
occurs primarily in sheep in which it causes a
Copyrighted material
Hidden page
 532 * Textbook of Microbiology
V
taj E
- JT*
%4T
_ ijr __
4
Fkg. 5?. 2 DI $|ribu1iQn of Kyasanur Foresl Disease (Courtesy : Nafmnal Inslilule of Virology, Pune ;-
BUN YAVIRUSES
This familv containing over 300 species is the
Invest group of arboviruses. The virus is about 1.00
rtm in diameter and has A complex Structure, with
a triple segmented genome of single stranded RNA.
Mosr bunyaviruses ue masquitobomc. Some are
transmitted by sandflies ( forexample, Phlebommus
fever) or ticks (Crimean Congo hemorrhagic fever).
Some arc established pathogeny causing natural
diseases, and even epidemics and epizootics while
many hive been isolated only from insect vectors
and have not been associated with any human or
animal disease . Bunjnvimgcs are HO named from
the type species Bunvamwtra virus isolated from
mosquitoes in Uganda in 1946.
The family Bunyaviridae contains four genera
of medical importance— Bunyavirus, Phlebovjrus,
Nairovirus and Hantavirus. A number of viruses
are yet ungruuped.
Gtitlus Bunyavirus: The genus contains over
150 specie , of which only a few cause human
*
infect ions. The clinical disease caused is
encephalitis, aseptic meningitis and fever, The
California encephalitis gjooip of viruses are endemic
in the USA. Large epidemics of fever with aseptic
meningitis have been caused by Ovopouehr virus
( member of the Simbu group) in Broad- The midge
CuJrcojde paracusia is the major vector for the
*
Oropouchc vims .
*
—
--
v1- i
fienus I hUboviniB: Tlte major members of
’
this genus are the sandfly fever and Rift Valley fever
viruses.
f Webufomufi fever or sandfly fever, also known
^ -
as Pappataci fever and three day fever, is a self-
limited, nonfatal fever nansmined hv the bite of
the sandfly Pftiefcotetfnu paparasii, It occurs along
^
the MetijterrjiTUfan Coast and Central Asia,
extending as far east as Pakistan and North Wsm
India. Cases have also been reported from South
and Ccn tral America.Twenty ant i genic types of the
virus exist , of which only five cause human
- -
disease — Naples, Sicilian, Punta Turu f Chagics,
Candlru.HIT»c vjrus has been isolated from sandflies
and patients in India. No vertebrate host other than
humans has been identified. Vbcife is evidence for
vertical transmission of the virus in sandflies.
Rr /r Valley fever is a inosqpirobot tie virus
causing enzootic hepatitis in sheep Laid other.
domestic animals in Africa, ft is named after Rir t '
Valley, Kenya,where it was first recognised., Human
infection causes a disease resembling influenza. In
1977-80, Rift Valley fever caused extensive
epidemics, with many deaths in Egypt and an
outbreak ofliicniorThagie fever with many deaths
in 1997-9? in Kenya. In 2000, it spread outside
Africa for the first time, causing epidemics in Vemen
and Saudi Arabia with hundreds of dcsths-
frMus Naimvinu: The uenus is named after
Copyrighted material
 H Arboviruses *
the type species Nairobi Sheep Disease Vims,
Memhen; of tfic Crimean Congo hemorrhagic
group are the major human pathogens in this genus
'
- -
fbe Crimean hemurrhagii l ^' ^ -ur V LT '.J ;, tir ? isolated
. ii Crimea in 1945, was subsequently found to be
'
,
]dertLical with the t !t ) fever vims isolated in 1956
in Congo {Zaire ), hence the name Crimean Congo
Hemorrhagic Fever ( CCHF) , The disease Is
endemic in Eiitcm Europe, Central Asia and many
parts of Africa. Cattle , sheep , goats and other
domesticated animals act as natural reservoirs. It is
transmitted by Hyalornma ticks. During the acute
phase of the disease, the blood of the patients is
highly in feet i uus and direct Transm issitm may occur
.
through contact. A related virus Hazard has been
isolated in Pakistan. It is also wulespread in Iran,
Iraq and the UAF- Antibodies to the CCHF group
af viruses have been detected in human and animal
sera from India.
Nairobi sheep disease is an acute, hemorrhagic
gasiroenteriiis caused by a Nairwirus in sheep and
goats in East Africa . It is transmitted by
Rhi|iicephalus ti . lts. The virus produces a mild
-
febrile illness in hepherds tending infected flocks.
The Ganjam v irus. isolated from ticks collected
front sheep and goats in Orissa, I nil i a, is (dosely
related to the Nairobi sheep disease tints, The
Ganjam virus has also been isolated from human
sources. Accidental Infection in laboratoiy workers
has caused mild febrile ilInc? -
( icniijh
-
Hantavirus: This virus causes
hemorrhagic fever uith renal syndrome ( HFRS),
also known as endemic or epidemic
nephros-onephri c i i , Manchurian epidemic
-
hemorrhagi .: fever, ncphrnpatli i i. epidemic a,
rodent -home nephropathy and other run ' if . This
condition first attracted attention in the early 1950s
- patents develop pulmonary involvement with - ^ rlv
ff
when a large number of US soldiers serving in
Korea got the infection mu it has been prevalent in
Scandinavia , Russia and China for centuries.
The disease occurs in two forms the milder
epidemic nephritis ( EN ) common in Scandinavia
and the more serious epidemic hemorrhagic fever
— The disease is caused by a newly identified
533
.
( EHF ) in the far east The clinical picture resembles
typhoid, leptospirosis and scrub typhus.
fhe genus least tour species
Contains at
Hantaan vims causing the severe HFft$ in the Far
East, North Asia and Russia, SCIHJJ virus causing a
—
milder type of disease and probably present
worldwide , Puumala virus responsible for
ncphropatb ia epidemics in Northern and Eastern
'
Europe, and Prospect Hill virus isolated from voles
in the USA, which has not been associated with
human illness.
Hantavirus species arc natural pathogens of
—
rodents field mice [ Apodemus agntr.' < ) being the
js
major host for Hantaan, rats f /iarms rantis and R,
nt > rve
^ . for Seoul, and voles for Puumala and
jcu s)
Prospect Hill viruses Vircmia is present in infected
.
rodents and the virus is shed in urine, feces and
saliva in high ritres. Transmission from rodent ro
rodent and rodent to humans is primarily
respiratory, by inhalation of the virus contained in
dried excreta. Domestic rats appear to be the source
of infection in urban cases of HFRS- Though there
are reports of the role of mites in the transmission
of the infection , this r- not confirmed. In the
absence of proved arthropod transmission , HFRS
should be consiilered a robovinis and not strictly
an arbovirus infection.
Demonstration of IgM antibody by ELISA or
of rising litres of immune adherence
hemagglutinating antibodies in paired sera are used
for laboratory' diagnosis .
A new syndrome, the HjrtWL jrtiS pulmonary
syndrome was i den Titled in south western USA in
-
1993. Afrer a prodrome nf fewr, malai e, myalgia
.LHJ gastrointestinal symptoms, lasting for 3- 4 daji -,,
~ '
radiological picture of pulmonary edema, hut few
L
physical findings. In severe cases, tachypnea ,
tachycardia, hypotension and hypoxia lead to death.
hantavirus, the Sm Nombre { meaning nameless )
virus, which is associated with the deer mouse and
other rodents of the sigmodontine subfamily. No
Copyrighted material
Hidden page
Hidden page
Hidden page
 H RtabdOVifUHE >
Tissue cu/fun : The rabies virus can grow In several
primary and continuous cell cultures such as diick
embryo fibroblast, porcine or hamster kidney but
cytopafhic effects are not apparent and the yield of
vnus is low. The fined virus strains adapted for
growth in human diploid cell, chick embiyo and
veto cell cultures are used for the production of
vaccines,
RABIES
-
Rabir ' has been recognised from very ancient rimes
as a disease transmitted to humans and animals by
the bite of 'mad dogs' . The name 'rabies’ comes
from the I . itin word rahfdus, meaning mu. I , derived
from the Sanskrit root mbhsSf , for frenzy. Reference
to rabies occurs in the Mesopotamian laws of
Fshnunna ( Circa 2200 BC ) . The disease was
tradiiinnally associated with the appearance of the
Dog Star Sirius in the dog days af summer when
dogs were considered to be prone to spells of
'
-
madness . The di -sease in human being is called
hydrophobia because the patient exhibits fear of
water, being incapable of drinking though subject
-
to intolerable thirst . Rabies in animals i not called
hydrophobia because they do not have this peculiar
feature.
The causative agent of rabies had, for centuries,
been associated with the saliva of rabid dogs but it
was only in 1304 that Zinke adduced proof by
transmitting the disease to normal dogs by the
inoculation of saliva from rabid dogs. In 1821,
Magendic and Brcschet infected dogs with saliva
from a human patient, proving the identity of the
agcoT earning human and animal T.IML :- . In a series
of studies dating from 1RE1, Pasteur established
that the rabies vims was present in the brain of
infected animals. By serial intracerebral passage in
rabbits, he obtained [he fodd virus .Lud demonstrated
that dogs could be rendered immune by a series of
injections of fixed virus of graded infectwity. This
vaccine was prepared by drying for various periods
pieces of spinal cord from rabbit ; infected with the
fixed situs. In July 1&8£, Joseph Mcbler, a nine
517
-
year old boy, severely bitten by a rabid dog and in
grave risk of developing rabirs, was given a course
of 13 inoculations of the Infected cord vaccine bv m
Pasteur.'Vhc boy survived, Thfr dramatic event was
a milestone in the development of medicine.
Pathogenesis Human infection is usually
caused by the bite of rabid dogs or other animals.
The virus present in the saliva of the animal is
deposited in the wound . If untreated, about half of
such case; may develop rabies. Rarely, infection can
-
also occur following non bite exposures such as
licks or aerosols or transplantation of cornea or
other virus Infected tissues. Humans appear to
possess a high degree of natural resistance to rabies.
The extent of inapparent or abortive infect i ' in with
rabies virus in humans is not known but the finding,
in a survey, of rabies antibodies in * ix per cent of
veterinarians without any history of anrirabic
vaccination suggests that it does occur.
The virus appears to multiply in the muscles,
connective tissue or nerves at the s- iCc nt deposition
-
for 48 72 hours. It penetrates the nerve endings
and travels m the axoplasm towards the spinal cord
and brain. The movement of the virus in the Ubtls
ii p; LH!- ivc, at a speed of about 3 mm per hour . The
infectit in spreads centripetally from the axon to the
neuronal bodies, and progressively up the spinal
COld through the napses of the neurons.The vi rut
^
ascends rapidly to the brain where it multiplies and
spreads centrifugally along the nerve trunks to
various parrs of the body including the salivary
glands. It multiplies in the salivary glands and is
shed in the saliva. The presence of the virus in the
saliva and the irritability and aggression brought
on by the encephalit i s ensure the tmnsmi ^ ion and
survival of the virus in nature. The vims ill ri [irately
reaches virtually every tissue in the body, though
the centrifugal dissemination may be interrupted
at any stage by death.The virus is almost invariably
present in the cornea and the fccid skin of patients
because of their proximity to the brain . This
provides a method for the antemortem diagnosis
- uJ Kumiin rabies. The virus may also be shed in
/"H .
LJ opy righted
Bw jr
material
538 T xiboei of MitrcSio
i
^ *
milk and urine. Vircmia is not clinically significant
though ir has been donon ^tnled under experimental
conditions.
In humans chi - incubation period is usually from
1-1 months, though it may be as short as 7 days or
as long us dime years. The incubation period is
usually short in persons bitten on the face or head,
and long in those briten on the legs, This may be
milted to the distance the virus has to travel to
reach the br ;Lm . The incubation period is generally
shorter in children than in adults.
The course of the disease in humans car be
—
classified into four stages prodrome , acute
encephsill IC phase, coma and deftch. The omei is
marked by prodromal symptoms such as fever,
headache , malaise, fatigue and anorexia. An eftrly
symptom is often a neurluc type of pain or
paresthesia and fascicularion at the site of virus
entry. Apprehension, anxiety, agitation , irritability,
nervousness, insomnia or depression character:?- r
Excessive libido , p n .L p i i m and spontaneuus
-
the prodromal phase, which usually lasts 2 4 days.
ejaculation may occur rarely
The acute neurological phase usually begins
with hyperactivity, which is characteristically
intermittent, with bouts of bizarre behaviour,
agitation or seizures appearing between apparently
normal periods . Such hyperactivity may be
spontaneous or pteripasted by external stimuli . The
pathognomonic feature is difficulty in dririkiug,
together with intense thirst. Patients may be able
to swallow dry solids but not liquids. Attempts to
drink bring urt such painful spasms of the pharynx
and larynx producing choking or gagging that
patients- develop a dread of even the slight or sound
of water ( hydrophobia ). Generalised convulsions
to respiratory arrest during ccmvubions.
-
follow. Death usually occurs within 1 6 days due
Some patients progress to paralysis. In rare cases,
hyperactivity may not be prominent and paralytic
features dominate from the beg inning- Such
paralytic disease is mare common in La:in America
and Trinidad after exposure to vampire bat rabies . rabies Is an important source of human infection in
and in those who have received postexposure
vaccination. Patients who survive the stage of acute
neurological involvement lapse into coma , which
m a y last for hours o r days. Death i s due t o
respiratory arrest or other complications.
Some persons exposed to real of imaginary risk
of rabies develop a psychological disorder which
-
ha ? been called tyssaphobia or h ydrti photlop hobia.
Patients present with anxiety, irritability and
exaggerated hydrophobia , They are afebrile.
Serial ion and reassurance are generalk all that are
called for.
In dogs, the incubation period is usually 3 6
weeks but it may range from 10 days to a year. The
-
initial signs arc an alert, troubled air and a change
in disposition w i t h restlessness , snapping at
imaginary objects, licking of gnawing at the siie of
the bite. After 2-3 days of tliri prodromal stage,
the disease develops into cither the furious or dumb
type of tallies. In filrioux rabies, which is much
more common, the dog runs amok, biting without
pfuvucaiioh and indiscriiinnately. The lower MW
droops and saliva drools from the mouth. Ptnlysii .,1
convulsions and death follow, The second type,
dumb rabies, is the paralytic form In which the
animal lies huddled, unable to feed. The dog may
not hilx but attempts to feed it are dangerous. The
dumb form Is as infectious as the furious type. About
60 per cent of rabid dogs shed the virus in saliva.
Rabid dogs usually die in 3-5 days.
There have been reports of persons developing
rabies after being bitten by apparendy healthy dogs.
However, in countries like India where stray dogs
are so common, it is not always easy to exclude the
possibility of such patients having been bitten earlier
by other animals. The presence and prevalence of
rabies vims carrier state in dogs has been a matter
of controversy and concern . Ifar all, this is a very
rare event. The possibility of carrier dogs has nor
so fir altered the recommendations tor postexposure
treatment.
Rabks in cats is similar to canine rabies. Reline
Copyrighted materia
Hidden page
Hidden page
 4 RhandouiruFieF:
. .
I Ltnir LIIJJI -LIII India manufacture BPL vaccine .
3. inf int brain vaccines; Tire cncephalitogenic
factor in brain tissue is a basic prorein associated
with myc - in , It is scanty nr absent in the
'
nonmyelinated neural tissue of newborn an i m a Is
So vaccines were developed using infant mouse,
rat or rabbit brain . Occasional eases of
neurological reactions Lave occulted following
infant brain vaccines also- Infant brain vaccine
is impractical in India due to the very large
quantities required.
Neural vaccines are Lins at is factory for many
reasons. "J’hcy are poor immunogens as they contain
mostly nuclcocapsid antigen, with only small
quantifies- of glycoproLi; i 11 tj,. which is the sole
protective antigen. They may contain infectious
agents which may not he inactivated during vaccine
preparation and storage. They are encephalitogenic.
Neural vaccines have been abandoned in the
developed countries. The onlv reason for their
continued production and use in a few developing
countries is that they are cheap.
-
NON NEURAL VACCINES
1. Egg racemes: (a ) Duct egg vaccine prepared
from a feted virus adapted for growth m duck
eggs and inact i rated wi [ h beta propiolactone was
used, hut was discontinued because I its poor
immunogen kiry, A purified, more potent duck
egg vaccine was developed, but was supplanted
bv.
JT
tissue culture vaccines which became
available then. ( b) Live attenuated chick embryo
vaccines; TWO types of vaccines were developed
-
with [ lie Flury strain rhe Low Egg Passage
—
( LEP) vaccine at 40 SO egg passage level for
immunisation oi dogp and the High Egg Passage
{ HEP) vaccine at ISO passage level for cattle
and cats these are nor in use now.
, does nor take into account the rare possibility of
2 - Tissue culture ncc. us: The first cell culture
vaccine was the human diploid «11 ( HDC )
vaccine developed by Koprowslcy, Wlktor and
Plotkirt . It IH a purified and concentrated
preparation affixed rabies virus {Pitman-M' lore
BAl
strain) grown on human diploid «Els ( Wl dfl
or MR.C 5 ) and inactivated with beta
proplolactone or tri- n -butyl phosphater It is
highly antigenic and free front s-erious Side
effects. Tts only disadvantage is its high cost .
Other equally effective and more economical
vaccines have been developed. Ihcsc include;
primary cell culture vaccines grown on chick
embryo, hamster ki dney and dog kidney cells and
conr .' jjunus cell culture vaccines grown on Verio
cell line derived from the kidneys of vervet
monkey or African green monkey ( CcjTopjYjftecws
artfrinpr).
In India the following cell culture vaccines are
available: Human diploid cell { HDC } vaccine
chick embryo cell ( PCEC } vaccine and
Purified verv cell ( PVC ) vaccine. All three of
them are equally safe and effective.
3. Subuoii raceme:The glycoprotein subunit on
the v ] rus surface, whith i the protect i ve ann:gcn,
has been cloned and recombinant vaccines
produced. They arc still in the experimental
stage.
VACCINATION SCHEDUI L -. S
Antlrabic vaccine should be administered when
person has been bitten, scratched or licked by an
animal which is rabid or cannot be apprehended.
When the biting animal can be observed, it should
not he destroyed but should be kept for ten days.
The observation period often days is recommended
hecausc the vims may be present in tile Sldivfl 3 4
_
days before onset of symptoms and the animal
—
usually dies within 5 fi days of developing the
disease. If the animal remains healthy after this
]K rind , there is no li * k of rabies and vaccination, if
already starred , may be discontinued - This, of course,
the carrier state in dogs.
In cases where the vaccine is started with the
hiring an i mil kept under observation , art alcernat i ve
recommendation is to stop treatment after five days.
The animal is observed for a further five davs*
opy righted material
8 dr w
542
* Texiboc ; of Micro Dio ogy
vaccine being starred again if die animal becomes
ill ar dies during [be period.
NtLIlMl VfljMiilttK The dosage [ ]f the vlccirte
depends on the degree of rink to which the patient
has been exposed. Accordingly; pat ients are classified
as follows:
Class I: Patients in whom the risk i -. estimated to
be slight. These include:
a. Licks, including direct contact with saliva on
definitely remembered fresh cuts or abrasions
on all parts of the body except the head, face,
neck or fingers.
b. Licks on intact mucous membrane or
conjunctiva.
c. hides or scratches which havciai -ed the epidermis
but have not drawn blood , on all parts of the body
except the he .L:L 1.LCC . mck or fillers.
d. Consumption of unboiled milk or handling raw
flesh of rabid animals.
Class II : Persons who arc e& Timated to he at
moderate risk. These include:
a. Licks on definitely remembered fresh cuts or
abrasions on the fingers-.
b. All bites or scratches on the fingers which are
not lacerated, not more chan half a centimetre
long and have nut penetrated the true skin.
c . Hites or scratches on all parts of the body except
the head, face, neck or fingers which havedrawn
blood but excluding bites which have five teeth
marks or more, or in which extensive laceration
has occurred .
Class III : Persons in whom the risk is estimated
to be great . These include:
a. Licks on definitely remembered fresh cuts or
abrasions on head, face or neck.
b. All hues or scratches on the head, face ur neck.
c. All bites or scratches on the fingers which are
lacerated, mure than half centimetre long ur ,
hive penetrated the true skin.
d. All bites penetrating the true skin and drawing
blood , when there are five teeth marks or more.
C, All li i tes on any part of the body L .LUH i ng extensive
'
laceration.
-
'
f All - nicka] and wolf hires
,
g. Any Class 11 patient who has not received
treatment within 14 days of exposure.
The ren >mmcndcd schedule of vaccination for
the different classes is as follows:
Semple vaccine BPL vaccine
Class I 2 ml x 7 days 2 mi x 7 days
L iass II
'
5 ml x 14 days 3 mi x 10 days
Class III ID ml x 14 days 5 mi x 10 days
The above schedule for the BPL vaccine is
recommended by the Pasteur Institute, Coonoor.
The Central Research Institute , Kasauli ,
recommends a slightly different dosage for its
vaccine, ( The manufacturer instructionsshould be
followed in even' case ). ^
The immunity following vaccination with
neural viaincs is expected to last far six months
only and any exposure later should receive fresh
treatment . The vaccine is ad mi nistcEcd
subcutaneously on the anterior abdominal wall.
Anti Tab i c Vaccine may cause certain adverse
reactions. These range from minor local reactions
to serious ncuroparalitic cam plications. The latter
may he of the neurihe type, the doreilumbar type,
the Landry type of ascending paralysis or
encephalomyelitis. The etiology of neurological
complication is believed to be immune response to
the injected brain Tissue resulting in organ specific
iinmunologicaJ damage .LH in experimental allergic
encephalomyelitis. These complications usually
occur within 1-4 weeks of commencement of
vaccination. The incidence of complications varies
with different vaccines, one in 2000 to one in 12,000.
When such complications are noticed during the
course of vaccination , further vaccination should
he withheld and the patient started on
corticosteroids. If further vaccina tion is considered
imperative, non -neural vaccine should be used.
Severe exertion and the use of alcohol during
vaccination have been said to increase the risk of
neuralogical neactiinns.
( lull culture vaccina: All three cell culture
vaccines available in India ( HI JC , PCEC and PVC )
opyrighted materia
 < Hhabttoviruws *
'
+
I
t $ Ik
F|gP 53 - 2 Negri btwltes : Oiiuio inclusiors in Hie Cytoplasm ! neurons In Tahiti dog train
have the same dosage schedule, which is the same
for both adults and childrer .
Pre -exposure prophylaxis requires rhrw doses
of the vaccine injected on day 0, 7 , 21 or Q, 28 and
5fi. A booster dose is recommended after One year
and then one every five years,
Fostexposure prophylaxis requires five or six
doses, on days 0, 3, 7, 14, 30 and optionally 90.
Tins course is expected to give protect ion for at
least five years-, during which ]TL'rioJ any further
exposure may need only one or two brmsrrr doses
(on days 0, 3) depending on the degree of risk .
After fi %' e years, it is advisable to give a fitll five
injection course if exposed to infection ,
The vaccine is to be given IM or SC in the
deltoid region , or in children on the anterolateral
aspect of the thigh. Gluteal injections arc to be
avoided as they are luund to be EeaS jrtiiminugCniC.
It has been shown that a dose af 0 , 1 ml
administered intradermailv is as effective as a 0, 5-
1.0 m! dose SC ot IM and that i uwnurii &adon may
thus he made more economical . However, this is
not rccommrnded a routine practice , as
imradermul tnjtxtion is technically difficult , and it
will be ineffective if this dnse is given
subcutaneously by mistake.
543
*
Pittivr immunisation : Antirablc scrum is
manufactured by hyper immunisation of horses .
Cnidc equine antirabies scrum is not to he used as
it is liable to induce anaphylactic reactions, Purified
equine rabies immune globulin ( LRlG ) is much
safer, though not completely free from risk. Human
rabies immune globulin (11RIG ) is fret from the
danger of ttneidtatkm but should be ensured free
Inom ElIV and hepatitis viruses. HRIG is much
costlier than tRlG,
Passive immunisation is an important adjunct
fn vaccination and should lie invariably employed
whenever the exposure is considered of high risk .
The recommended dose of H RIG is 20 lU/kg body
weight, half the volume infiltrated at the site of
the wound and the other half injected in the gluteal
region. Passive iinmuni -sarton should be given before
or simultaneously with the first injection qf the
vaccine, but not after it. In persons receiving the
serum and vaccine , a booster dose of cell culture
vaccine on day 90 may he given.
Recommendations for postexposure prophylaxis,
as endorsed by the WHO in 1988,, are shown In
LVaccj' ne faifores' ( persons developing rabies
even after a full course of immunisation ) are not
uncommon with neural vaccines , while they are
pyriq
544 H Textbook of Microbiology
--
Tab lc 50-1 Recommendalion for postexposure prophylaxis ( WHO]
Category o f if si Typi of expo&utt
i Touching or feeding of animals;
Licks on intact skin
u Nibbling of uncovered ckin »
minor rente he* or tbrenoai without
bleeding; Ikk* on broken skin
m Transdcrmal biles Of BCraJCdlti
contamination of mucous membrdne
with saliva
— Ruhies. Immune globulin vaccine to he
extremely rare when Immediate Inral treatment has
betel followed by rabies immunoglobulin and a full
course of i cell culture vaccine. In view of the safetv
of the cell culture vaccine, U would be advisable to
recommend the vaccine even when there is the
slightest risk of exposure to rabies.
Vaccine lor animals: Anlirabic* Lmmunisarkm
in animus is to be done as pre-exposure prophylaxis.
PostcxpoHure treat me ot is nut generally of much
use, Neural vaccines arc not satisfactory as they are
not adequately immunogenic, need nuiiciplf doses
and have to he repealed every six tn ( jn. Clie .
Concentrated cell culture vaccines containing
inactivated virus are nuw available , which give
gnod protection after a single IM injection .
Injections are given at 12 weeks of age and repeated
—
at 1 3 yeux intervals. Rabies vaccines mav he given
separately or as combined vaccine for immunisation
against other common veterinary infections also.
'DwalitienC Until recently, rabies was considered
to be invariably fatal and no serious attempt ar
treatment was made , apart from sedation. It has
now been demonstrated that Complete recovery Carl
occur from established rabies , with iotensive
supportive care and management of complications.
No specific an 11 rabies ugeiU is available.
Epidemiology : Human rallies is A [lead end.
Direct person - to- person transmission of rabies has
Recommended prophyl* xit
None iif history is reliable)
Stan vaccine. May be discontinued if
animat is well AFIRET 10 days
siHJt
^dr May be di*eontinu(d if animal
is well alter 10 davi
not beeo recorded, though the virus is present in
the saliva of patients. Therefore, there is nn danger
in examining or nursing hydrophobia patients
provided suitable precautions arc taken. An unusual
mode of tranum iss- ion of rabies has occurred in some
recipients of corneal grafts- The donors had died
of unsuspected rabies and the infection was
transmitted through the cornea.
Rabies virus is present in terrestrial animals in
all parts [if the world except Australasia and
Antarrica, and some islands like Britain. Two
-
epidemiological types of rabies exist , [/rharg
transmitted hv domestic animats like dogs and cats;
and syfvaitru , involving animals in the wild, such as
jackals, wolves, foxes, mongooses, skunks and bats.
-
Most cases of human rabies follow dog bitc fi but in
endemic areas almost any animal can transmit
rabies . In India , anti Tabic treatment in to he
considered following rhe bite of any animal except
.-
rats. Where urban i r i mestic rabies has been
controlled , as in the USA, the majority of infections
are due TO bices bv wild itiimali .
The primary source of lire rabies virus in nature
seems to he in the mustel 'Js and vtverrids, rhe
ermine in rhe northern coniferous forests, the skunk,
mink and weasel in North America, the mottled
pole car in the USSR, rhe elver and pole cat in
Africa and the mongoose in Asia. Rabies vims has
Copyrighted materia
 i Rhabaaviryses *
been isolated repeatedly from the bruin jind salivary
glands of apparently healthy wild rodents.The virus
survives in this reservoir population by achieving
i start: of latency with occasional activation nucb
that only a small proportion of them will be
shedding the virus at anv one time. I 'rom tbe
reservoir species, wild vectors such as tbxes, wolves
ind jackals acquire the infection and occasionally
epizootics occur in these species . Carnivorous
Quintals may acquire the infection by eating carcasses
containing the virus. From these species the disease
spreads to dogs and other domestic animals.
A smouldering epizootic of rabies in. the red
fox in Europe had spread westwards steadily from
Pbland to France during the list few decades.
Vaccine baits ( chicken head or other meat
containing live attenuated rabies vims) have been
used to immunise the red feu in an attempt to check
the epizootic in the forests ot Europe .
Another natural cycle of rabies concerns bats .
A fatal paralytic disease of cattle and humans was
noticed in Central and South America and the West
Indies early in the twentieth Century, This was
identified is rabies only years later. rEhc disease was
shown to be transmitted by vampire bats riiat sweep
dtiwn on their prey at night. Vampire bat rahics
had taken a heavy toll of cattle. Vampire bats may
Table S&.2 Lyssavlcus serc -'genolypes
Genotype/
Serotype Virus Isolated
i Rabies Warm blooded animals
2 Lagos bat/ Natal bat Bat/ cat
3 JVlokoIa Sh rew/cat/dog /humari
4 DuvcnhaRe Humart / bat
= European bat lyssa.
Type I
: Bat - ’ human
6 European bat l sEavinisi Bat /human
Type 11 ^
7 Australian bat Lyssavims: Bat / human
545
shed the rabies vims as sympenniless carriers over
a period of several months.
Rabies alio occurs in insectivorous and
-
frugivuxous bat . Infection in insectivorous bats is
symptomatic , while frugivorous bats become
asymptomatic carrier!. While in canines rabies is
ncurntfopic, in bats the vims is primarily adapted
to the respiratory tract . Humans may be infected
by aerosols iif they enter caves where infected hats
colonise . Pncutootropic rabies virus strains have
been obtained from hats. Bat rahiics is largely
confined to the Americas, A few strains of the rabies
virus huve been isolated from bats in Europe but
their epidemiological significance is not known.
Rabies i '- endemic in 1 ndia . 11 has been estimated
that mote than 30,000 people die of rabies in India
every year and more than 700,000 receive anti rabies
vaccine. Human rabies can he checked Hv control
F
of rabies in domestic animals , by registration ,
licensing and vaccination of pets and destruction of
stray animals. With the dog population in India
estimated as over 1.6 million , the problem is
immense. I iowever, rabies can be eliminated only
if the wild vectors Such as jackals and foxes, and
tlte reservoir mustelida and viverrids are controlled .
Rabies has been eliminated from islands like Hritain
and Japan bv rigid quarantine. Australia which has
from Distribution
Worldwide with few exceptions
Nigeria/Central and SouihAfnea
.
Nigeria/other African countries
South Africa
Eumpe
Europe
Australia
Copyrighted material
 546 i Ttfidbaoh Oil M . ::rnbiotogy

no native mustelhd nr vivemd population has no
rabies . Eradication of rabies from countries Like
India wirh abundant wildlife may not be practicable.
RABIES RELATED VIRUSES
The genus Lyssavints consist* of the rabies virus
and other serologically related viniHS. Lyssavirusts
have been classified into seven serotypes.
clinical rabies after being bitten by a bar. It is
classified as Lyssavirus serotype 4.
5 . 6. Rabies- bke viruses isolated from European
bats have been classified into two groups:
European bat lyss &vi rus types 1 and 2 . They can
infect humans, as was found in the UK in 2002,
when a wildlife worker fell ill with 'rabies' and
diLid. Tins was the first "rabies death in the UK
1

1. Rabies virus is classified as Lyssiums serotype I in a century.

"WT

2. The Lagos bat virus, classified a* Lyrsivirus
serotype 2 . was isolated in 1956 from the pooled
brEiins of frugivnnous bats from Lagos Island,
Nigeria, It causes a rabies-like illness following
intracerebral inoculation. Negri bodies are found
in infected monkey brain but not in mice or dogs.
3-. The Mokula vims , first isolated in 1968 from
shrews captured near Ibadan , Nigeria, has later
been found in many wild and domestic animals
m Africa, It was also recovered from two
children with central nervous system disease,
one of whom died. A case of laboratory infection
with the v: nis occurred in a person possessing
high tittes of antibody to the rabies virus. 1c is
classified as Lyssavlms serotype 3.
A . The Duvenhage virus was reported in J 971 from
the brain of a man who died in South Africa of
7. Australia was considered free of rabies and
related viruses till 1996, when a lyssavirus was
isolated from a frtigivorous bat . Since then a
number of similar isolates halve been obtained
from ; Litferent types of bats in Australia. Fatal
infection* have occurred in persons having
contact with bats. The virus antibody is widely
prevalent among Austral i:tn bats which appear
to be carriers. The virus, named A u f ndfim bat
^
lyssavieus is closely related to, but distinct from
the rabies vims. Antirahic vaccine and scrum
appear to protect against experimental infection.
The relevance of rabies related viruses in human
disease is not dear, though some of them have
caused illness and death in humans. They are
considered to represent a biological bridge between
the rabies virus and other rhabdoviniscs.

Further Reading
GM . 1991 . The Nimfil I h .-.icn of
]i , ii.Lr
FiEhijcsii . CRC Pre -^.
BD wd LE RubLnaun I 99.V Rubies, Vpw fi- nglj Afwf 329:1632.
Rupprecht CE ei al. 1994. Lyaavinuea . CufWrlf Tfiprcs in Micrvbioi Immunol 167 .
Smith JS ct al . 1990. Unexplained rabies in three irtimLgrjnTt in The United States. New EngJ Med 324 :1990.
Smith JS. 1996. iVew aspects of rabies. Clin Miembio! Re* 9:166 .
WHO. 1992 . Expen CflunturnM u»t Rxhiex , 3rb Kep«) rT . Technical Serin No . 924 , Geneva.

Copyrighted material
 Hepatitis Viruses
The Derm viol licpafi IH -H' re fere Do a primary infection
'
of the ljvicr by any' one of a heterogeneous group of
litpibtu viruses’. , which currently consists of types
A , B, C , D, E and G. (The designation ’type P had
Iwen proposed for a putative vima believed lo cause
transfusion- associated hepatitis , distinct from type
A to E. Hut it proved to be a mutant { HBx ) of type
R virus and not a separate entity, Type F was
therefore deleted from the list of hepatitis viruses. }
Hepatitis viruses arc [ .cto nominally unrelated.
Except for type R which is a DNA vims all the
others are RMA viruses . The features common to
them are their hepiTotropism and ability to cause a
similar icteric illness, ranging in severity from the
Tinapparcnt to the fulminant fatal forms.
As alt types of hepatitis viruses cause a clinically
indistinguishable acute illness, their differentiation
is based on their serological and molecular markers,
E Ecpacitis may occur incidentally during many other
viral infections, such as with yellow fever , Lassa
fever, Marburg, EB, cytomegalo, herpes simplex ,
varicella zoster, measles , rubella or coxsackic
viruses. These arc not included in the category of
viral hepatitis.
By epidemiological mu I clinical criteria, two
types of viral hepatitis had been recognised for long.
One type occurred sporadically or as epidemics,
affecting mainly children and young adults, and
-
transmitted by the fetal oral route . This was called
infective or infectious hepatitis , later termed type
.
A hepatitis A second type of viral hepatitis ,
transmitted mainly hy inoculation was originally
olnserved in persons receiving serum inoculation
or blood transfusion. This had been given various
names such an hornAognus srium jaundice , -serum
toepafiffs ( because of its association with human or
homologous antisera so commonly used for
prophylaxis or therapy early in the twentieth
century) and tfitvtsfilHUfl hepatitis.Tt wan later called
type ft hepatitis ,
Kx a rime it was believed that ah viral hepatitis
was caused by either of the two hepatitis viruses,
type A accounting for all infectious hepatitis and
Type B fur all |JOS[ - transfusion or serum hepatitis .
However, with the development of techniques fot
identifying type A andi type B viruses, if became
apparent that in many cases of infectious and post -
tTransfusion hepatitis no evidence could he found of
infection with either type A 01 B viruses. It therefore
Eitcame evident that the clinical syndrome of type
A or R hepatitis could also be caused by one or
more other unchaiactcrised viruses. The term fion-
A non -U hepatnii was applied to this group . Soon
a type C virus was identified as causing many
transfusion - associated hepatitis cases. A defective
virus which depends m the helper functions of type
B vims was called delta or type D hepatitis viruses.
Yet another type of hepatitis transmitted by the fccal-
Oral route, provident mostly in the developing
nations was found to be caused by hepatitis E vims.
The sixth member of the group, hepatitis G virus
can also cause hepatitis, hut its role lias not yet
been adequately understood.
TYPE A HEPATITIS
Type A hepatitis ( infections hepatitis} is a subacute
disease of global distribution, affecting mainly
children and young adults.
Copyrighted materia
Hidden page
Hidden page
550 i Textbook of MicrDtupIpgy
third ( ]t the wi?rld|' population is csti mated to he
have been infected by hepatitis R virus ( HBV ).
About a quarter of them hecome HRV carrieni. A
quarter of these develop serious liver disease ,
including chronic hepatitis, cirrhosis and primary
hepatic career. As there in an effective vaccine
against HBV, hepatocellular carcinoma becomes
the only human cancer which in vac cine -
preventable . The WHO estimates that HBV
infection causes more than a million dearths a year
worldwide .
Clinical features: The incubation period is
-
long, about 1 6 months. The clinical picture of
hepatitis R if - similar Co that of type Ar hut it tends
to be more severe and protracted. The onset i ?
insidious and fever is not prominent. Ejttrahepatie
complications I Lice arthralgia, urticaria and rarely
polyarteritis or glomerulonephritis may occur .
These are ascribed to circulating Immune complexes
containing the viral surface antigen.
About 90-95 per cent of adults with acute
—
hepatitis R infection recover within 1 2 months of
onset and eliminate the virus from the body within
about six months , remaining immune thereafter .
Mortality Is about 0.5 to 2 per cent , but may be
-
more in post transfusion eases. About l pet cent of
patterns particularly those having simultaneous
della virus infection develop fatal Ruminant
hepatitis.
A proportion of cases fl- UJ per cent ) remain
chronically infected - I'hcy may be asymptomatic
carriers or may progress to recurrent or chronic
liver disease or cirrhosis. A few of them may develop
hepatocellular carcinoma after many decades.
The pathogenesis of hepatitis appears to be
immune mediated. Hepattxylcs cany viral antigen!;
and are subject to antibody -dependent NK. cell and
cytotoxic T cell attack. In the absence of adequate
immune response, HBV infection may not cause
hepatitis, but may lead rn carrier state. Therefore
infant ? and irmnunodeficicnt persons arc mote
likely to become asymptomatic carriers following
infection.
i r i
»
Hepatitis It virus ( HBV ): HRV is a 42 nm
DMA virus with an outer envelope and an inner
conL , 27 n:n ul di&neterj enclosing the viral, gtibtmW
and a DNA polymerise ( Mg 59, 2), Because of its
unique features. HRV is assigned to a separate
family .' iepadnsviridae (hepatotropic 1 > N A viruses),
which consists of two genera, O r r h o£ VJJTAS
CI in tain mg HRV as welt as the wnodfhuck and
ground squirrel h e p a t i t i s viruses , and
AvjhepadridWmS cunt .Lining the Pekin duck and
grey heron hepatitis viruses. HRV is HepaflntviniS
type 1 ,
The discovety of HBV was serendipitous. In
-
1965, Rlumhcig, tudvi n g Inj ma n se rum Lipoprotein
allotypes, observed in the serum of an Australian
aboiLgmeh a new antigen which gave a dearly
defined Line ol precipitation with sera from two
hemophiliacs who had received mulii [ile blood
transfusion*. . his was- flamed the Australia jtn ycn.
By 1969 the 'Australia antigen was found to be'
^
associated with serum hepatitis. IL was mbsequendy
shnwn to be the surface component of 1 J B V .
Therefore the name Australia antigeo was changed
To hepatitis B surface airmen [HBsAg ).
EftwSrtQpn i HHsAq ;
Nuclwcap
iHBfcAgl
^d C- .r ^
i
y
DNs* P&yfW
Plus SlrfiifcJ
-1 DMA
Mims SlmndJ
Fie. 59.2 Hepatitis S virus struclurs
 * Hepatitis VirUi&S
* 551

Under the elect rnri microscope, ncra from type

H hepililk patients show three types of particles
( Figs, 59.5 , 59.4). The most abundant form is a
Spbtiidil partible, 22 nm in diantrter. The accord o L
type of puticle is filamentous or ruhular with a o
diameter of 22 nm and of varying length These . c c o
rwo particles art untigenivalty identical and art
surface components of HliV (HBaAg) which are © o oo
produced in great excess.The third type of particle, Q o o
tar fewer in number, is n double walled spherical
o
structure, 42 nm in diameter. This pardde is rhe
Complete hepatitis R virus. It was first described hv
Dane in 1970 and so is known) an the &tnrpartide
Tiie envelope proteins expressed or the surface Fig 59.3 Dilferont types ml particles seen Ln serum
of the viriiirt. and the surplus 22 nm diameter of pattern whh type 8 hepatitis : A - Spherical 22
sphericJ and filamentous particles constitute the mu particle. 8. Douhle shelted AS mn particle i & ane
P5 rficl^i- C , Tubular 22 nm panicle.
hepatitis R surface antigen. HRsAg consists of two
major polypeptides, ore of which is glycosylated,
HHsAg exhibits antigenic diversity, It contains
mn different amigcnic components the common -
group reactive antigen a, and two pairs of Type
specific antigen '; rf-yand ir r, only one member of
Ku-b pair being present nr a time, tlBsAgcan thus

he divided into four major antigenic subtypes.: jirfw,

adr, AJTV and JJT. The subtypes do rot seem to be
important in immunity because of the dominant
antigen a shared by all. The subtypes breed true , Fig . 59.4 Eteeiron micrograph ot H0 Y -Pooied plasma
and the index cu^ e and contacts in an Outbreaks MU . -flOOl [ Courtesy : Natmnal Institute of Virology ,
have the same suhfypc. They show a distinct Pune|
geographical distribution . Subtype jnrisi-umirain IllicAg, though immunologically distinct, are coded
from West Asia through the Middle bast, to lor by the same gene .
Western and Northern India; udw is common In The nucteocapsid encloses the viral genome
Europe, Australia and the Americas; adr it consisting pf two linear strands of DNA held in a
prevalent III S- Ihi. ilIn jml I-.. -. - r InJlli HnJ I he I .nr I - . .ml ; .. c:,. 111 LLC 1.1 . i l l i u c u l
1 ' leij . One LPI [ hr htr ,L .J.|H ( t i n- piiijs
[]

Ayr is very r:irc . A no mbrr of other su rfacc antigenic

^
strand) is incomplete, so that rhe DNA appears
reactivities (a, x. ft trj, n. g) have been reported, but partially double stranded and partially single
not adequately studied. stranded. Associated with the pha strand is a viral
Mild detergent treatment disrupts the viral DNA potymerue, which hits both DNA- dependent
envelope and exposes the corr or nurleucips id.The DNA polymerase and RNA -dependent reverse
antigen expressed on the core is culled the hepatitis Transcriptase functions. This polymerase cun repair
B am antigen (HRcAg). A fhind antigen called the gap in the plus strand and render the genome
the hepatitis W e antigen ( URcAg ) is a soluble frilly double stranded (Fig 59.5) . r

nonparticulate nuclenvapsid protein. HBcAg and The genome has a compact structure with four
552 * Texibook o Microbiology *
'

overlapping genes, The b gene codes for the surface -

Pra il -
Pn «
antigen , It consists of the S region and two Pre-S =
-
regions, FtC S2 and Pre- S 1. The protein coded for GENC S
by the S region is called the S or major protein .
When translation begins from the Pre- S2 region , t -- - -\\
\ V Slrfll’ J
i

the M or middle protein is formed . When the entire I -:

gene from Pre-Si is translated, rhe L or large // / *P

Li£ l

protein results- The L protein is present only in

f Nt G
the virion * while the M and S proreins ate found c MNC K

in the circulating HBsAg particles also .

The C gene has two regions , C And Prc C.
When the C region alone is translated * [ he cure
- Fig . S 9 .S HBV genes and gene products
antigen ( HBcAg) is formed . HBcAg is assembled
as the nudeocapsid core particles. It is not secreted
and does not circulate in hlood, hut cm be
demonstrated in hepatocytcs by immuno
fluorescence . Antibodies to HRo, both LgM and
- S+ Pre- 32
TgG appear in blood. LgG antibody to HlieAg
persists in blood long after all other serological
markers have disappeared and so provides a usehd
marker of prior infection with HBV. If translation
begins fiom the Pre-C region, the requiring protein
is HBcAg , a nun particulate soluble an M geo
possessing a signal protein winch enables U [ u be
secreted. Ir Is therefore present in circulation. The
presence of HBeAg in blood provides a convenient
and readily detectable marker of HRV replication
and high infect!vity,
The P gene is the largest and Codes for the DNA
polymerase enzyme- The X gene codes for a small
rtonparticulate protein fHBxAg ) , which has
Trans activating effects on both viral and some
cellular genes. This leads to enhanced replication
of HBV, as well as of some other viruses , such as
the human immunodeficiency virus. HBxAg and
its antibody are present in patients with severe
chronic hepatitis and hepatocellular nrcinomn-
A few cases of infection by mutant viruses have
been identified . Two types of mutations have been
studied . One type * initially identified in
Mediterranean countries, presents as severe chronic
-
hepatitis, caused by pre core mutants unable to
synthesise HBeAg. Those infected with precort
Genes Regions Gene products
s Major protein ( S ) ~
Aliddl.epfoteL:n.( i ) 1 HB*Ag
S+ Pne’SlBtS2 Large protein ( L) J ^
C t: HbcAg
P
X
C+Pne-C HbcAg
DNA poh mcrase
HBxAK
cnay he positive for anti- H Be and anti-
mutants
-
HBc. The second group of so called 'escape
mutants' huve been seen in some infants bom 10
-
HBeAg positive mothens* and in liver transplant
recipients who had received combined
immunisation with anti-J JBV immunoglobulin and
vaccine . They show mutarlon in the common ; t
determinant of HBsAg, preventing them from
being neutralised by and-HBsAg antibody. If such
mutants become more common , they may pose
problems in hepatitis B prophylaxis.
HBV replicates within hejiatocytts . Viral DNA
exists in the heparocytc nucleus in the free
evtracbromosnmal state or integrated with the cell
chromosome. Replication resembles that seen in
retroviruses, in that DNA is synthesised from an
RNA template by reverse transcription .
f LBV DNA and protein havealso been identified
in cxtraheparic sites such as hoot marrow, spleen *
lymph nodes and circulating lymphocytes * but
apparently no damage is produced in these locations.
The significance of this extrahcpatic presence is
HOC understood.

Copyrighted material
Hidden page
Hidden page
 * HG|M1I1IS Viruses f 555

L»f the high oust of imported vaccine. Now Thai the
meant is manufactured in India , and is available
at lower cost, it should be possible tn include this
in the national immunisation schedule.
1 , Elbetrulor \ din nosi ^: Specific diagnosis of
^
]ic[iatiri. s J J rests on the serological demonstration
of the viral markers- It is therefore necessary to
its antibody. sutti - HBc appears in serum a week Or
two after the appearance of J JBsAg. It is therefore
the earliest antibody marker to be seen in blood ,
long before anti - fEBe or anti - HBs , As anti-HBc
remains: lifelong, it serves as a useful indicator of
prior infection with HBV, even after all the other
viral markers become undetectable. Initially, anti'
,

understand the sequence of their appearance in HBc is predominantly IgM, but after about 6
blood ( Fig. 59.6), months, it is mainly IgG . Selective tests for IgM
HEsAg is the hret marker to appear in hlood oi IgG anti - H Re therefore enable distinction

after i nlwtinn, being detectable even before delation
of transaminases and onset of clinical illness. It
remains in circulation throughout the icteric or
symptomatic course of the disease. In the typical
case, it disappears within about 2 months of the
start of clinical disease, but may sometimes last for
6 months and even beyond . When it is no longer
detectable , its antibody. anti-HBs appears and
remain* for very long period*. Anti- HBs is the
protective antibody
HR cAg isnor demonstrable in circulation
because it is enclosed within the HRsAg coat , but
between recent nr remote infection respectively,
HBeAg appears in blood concurrently with
HBsAg, or soon afterwards. Circulating HBeAg
is an indicator of active inirahepatic viral replication,
and the presence in blood of UNA polymerase ,
HBV DNA and virions, reflecting high infectivity.
The disappearance of EIBcAg coincides with the
fall of transminase levels in blood . It is followed by
the appearance of anti HBc. -
For the diagnosis of flBV infection detection
of HRsAg in blood is all that ordinarily necessary.
The simultaneous presence of IgM anti - HBt

JAUNDICF

LIVER ENJYMEE 1

- lt- HBc IqG

A'

Antl +tac loM

Anrti - HRt
nBeAq
AnliHBA
.
*
f

\ \\ /
.* * n wm M . .
wA pc **.
^ -
a %
^ it
1 '
0 j
12 6 2Q 2* 26 32 36 50 ICG
* I I h
Week! iflei ftntechnr

Fig . 55.6 Typical course oi aculc hspalilis type 0

Copyrighted material
 556 4 Texib&ofc of Microbiology
indicates recent infection and the presence of IgG
anti-HBc remote infection, Occasionally when the
level of H RsAg LH too lew to be detectable, diagnosis
has to be made by testing for IgM anti-HBc.
HBeAg provides information about relative
in fee rivitv. Its presence denotes high infertility and
its absence, along with the presence of anti-HBe,
indicates low inftetivity. As it i* invariably present
during acute hepatitis, its testing is indicated only
in chronic infection and carriers.
-
The presence of anti HBs without any other
serological virus marker indicate? immunity
following vaccination. Table 59.1 shows the
interpretation of various serological patterns in
hepatitis B.
Like HBeAg, HBV DNA is also an indicator
of vital replication and inactivity. Molecular
methods such is ON A : DNA hybridisation and
PCR, at present used for HBV DNA testing ate
highly sensitive and quantitative. HBV DNA level
in serum reflects the degree of viral replication in
the liver and so helps to assess the progress of
patients with chronic hepatitis under antiviral
chemotherapy.
Prophylaxis! General prophylaxis consists in
avoiding risky practices like promiscuous see
injectable drug abuse and direct or indirect contact
with blood, semen or Other body fluids of patients
and carders. Health education, use of the disposable
syringes and needles, screening of blood, semen and
organ donors, have all helped to an extent, bur these
alone cannot eliminate the risk altogether,
particularly in the devdopuyg .
countries The only
certain method appears to be universal
immunisation.
Both passive and active methods of
immunisation are available. Hyperimmune hepatitis
B immune globulin (HBlG) prepared Bom human
-
volunteers with high title anti HBs, administered
IM in a dose of 300-500 i.u. soon after exposure to
infection constitutes passive immunisation. It may
not prevent infection, but protects against illness
and the carrier state.
Active immunisation h more effective. The first
vaccine introduced in 19fl2, was prepared from
pooled plasma of healthy human carriers with high
level antigenemiia. The 22 nm HBsAg particles
separated by ultracentrifugation were treated with
proteinase, urea and formaldehyde and used as the
vaccine. This was immunogenic , but became
unacceptable because ire source was human
plasma, limned in availability and not totally free
from possible risk of unknown pathogens. It
continues to be used in tome countries because it is
cheaper.
The currently preferred vaccine is genetically
engineered by cloning the S gene of HBV in baker 's
yeast. It consists oi nonglycosyiated I IBs Ag particles
alone. It is given with alum adjuvant, IM into the
deltoid or, in infants into the anterolateral aspect of
the thigh. Gluteal injection is not recommended as
it may result Ln poor immune response. Three doses
giver at 0T 1 and 6 months constitute the full course.
Seroconversion occur* in about 90 pc cent of the
vaccinees. A special vaccine containing all antigenic
components of HBsAg (Pre-Si , Prt - S2 and S) has
been developed, which gives greater seroconversion.
Screen version can be checked by testing for anti'
. HRs which is usually detectable for about 5 years.
Clinical protection is believed to last much longer.
Booster doses are needed only for those at high
risk
For nonimmune persons exposed to HBV,
combined immunisation is recommended. For
babies bom to carrier mother?, a single injection of
0.5 ml of HBlG given IM immediately after birth,
is followed by the full course ufvacdncat a. different
anatomical site, the first dose being given within
12 hours of birth . When HBlG is not available,
the vaccine given alone has been reported to provide
protection.
Treatment; No specific antiviral treatment is
available for acute HBV infection. Interferon alpha,
alone or in combination with other antiviral agents
such as lamivudine and famcyclovir, has been
beneficial in some cases of chronic hepatitis.Them
i
[J LJ
J riantea feria
 * Hepatilis Viruses 557

is no effective treatment tat the carrier state, though
^ pnntuneauK mnlution takes place in some of
thCtflr
TYPE C HEPATTHS
Attempts to identify the group of 'non -A nou -
s' viruses by experimental infection in chimpanzees
led to the discovery of hepatitis C vims ( HCVJ. It
is now the commonest cause of past - transfusion
high risk. Sexual transmission is probably less
important. Vertical transmission from mother to
baby may take plact-
Thc infection occurs throughout the world, with
carrier rates varying from 1 2Q per cent. HCV
infection is prevalent in India too, with an estimated
12.5 million cases- A quarter of all chronic hepatitis
eases in India is believed to be due to HCV
infection*

hepatitis in the developed countries.
Clinical features: The incubation period is
long* 15-160 days , with a mean of 50 days. The
acute illness is usually mild or anicteric. Overt
jaundice is seen in about 5 per cent of patients only
The important part in type C hepatitis is the chronic
Illness. About 50 to SO per cent of patients progress
to chronic hepatitis, with some developing cirrhosis
and hepatocellular carcinoma ,
lipidtinioluiiy : HCV infection is seen only in
humans. The source of infection is the Idige number
of carriers, estimated to be about 200 million
worldwide- In general the epidemiology resembles
Hcpnlilis C virus { HCV ): The virus has
not been grown in culture , bur has been cloned in
—
.EscfiericJuz coii. HCV is a 50 60 run virus with a
linear single stranded K.NA genome , enclosed
* within a core and surrounded by an envelope,
carrying glycoprotein spikes . HCV resembles
fhvivinucs in structure and organisation, and has
been classified as a new genus W( jM(iviras in the
family Flaviviriiae.
Thc virus shows considerable genetic and
antigenic diversity. At least six different genotypes
and many subtypes have been identified, indicating
high mutability Some genotypes are seen
*

chat of hepatitis 11 . worldwide, while others are localised. Because of

Infection is mainly by blood transfusion and this diversity there is litde heterologous or even
other modes of contact with infected blood or blood homologous postmfection immunity in hepatitis C.
products. Injectable drug abusers, transplant Laboratory diagnosis; The standard method
recipients and immunocompromised persons are at of diagnosis is antibody detection by ELISA. The

-
Table 59.1 Inlerpn&talfon of common, serological patterns in HBV mfeelion
Virus/ Antib&dy Mlfkttt
Interprets lion
HBsAg HBcAg snd - HBc unti - HHs
+ IgM m Amhc HBV infection; highly infectious
+ l G Lare/chionk HBV Infection or carrier state;
*
* highly infectious
p- IgG * w- Latc/chronic HBV infection or cumer state;
Low infcctivity
+7 - IgM + /- Seen rarely in early acute 1 IBV Infection ;
infectious
IgG */ - Remote HBV aafedkicis infetrivity nil or
very Low
= *
+ “ J

Immunity following HBV vaccine

Copyrighted material
558 4 Textbook of Microbiology *
antigens used art variant structural and non-
structural proteins cloned in E. CGJJ . There have
boon three successive ger it ration > of such antigens,
introduced Do improve iicrts ] ti^ iity and specific!ry of
Krotagb-a] diagnosis , Even the third generation
ELISA currently i :i use, employing NS- 5 region
protein and sy ntheri c pept i dea becomes pc * 11 ive only
months after the infection and shows mmspecific
reactions. Confirmation. % iinmunoblot assay is
therefore recommended . In HCV infection
antibodies appeal irregularly and late, limidng r I itir
diagnostic utility,
Ideoiiticatniii of HCV RNA in blood provides
more sen* i ri ve and speci tic results wi TO i n a few days
of -exposure to HCV. Molecular methods like PCR
and branched DNA assay arc employed for the
purpose,
Prophylaxir Only general prophylaxis , such
as blood screening , is possible , No specific active
or passive immunising agent is available .
Trefllraenti Ptolongcd treatment tvirh interferon
idphu, eilher alone or ML combination with antiviral
agents like ribavirin has been reported to be useful
in some eases,
HPED ( DELTA) HEP ATTTB
In 1977, Rfewtto and colleagues in Italy identified
a new viral antigen in the liver cell nuclei of patients
infected with hepatitis B virus. This lus been shown
co be due ro the hepaiotropic virus Delta or
Hepatitis D Virus ( HDV ). Delta is a defective
RNA virus dependent on the helper function of
HBV for its replication and expression. Therefore,
it has ild independent existence and can survive and
the host,
-
replicate only a.; ( png as HBV infection pCTMSft itl
HDV r i Spherical , .lb 11 ITL particle with an
outer coat composed of the hepatitis B surface
antigen surrounding the circular single stranded
RNA genome . Though it resembles some plant
viruses, such as viroids or satellite viruses, it has
been proposed to be classified in a new genus
Deltaiirus, because of its special features.
Its mode of transm i ss ion K the same as for HBV.
Two types of infection arc recognised, coinfection
and .‘ irf 'tTjrift' L rHur . In coinfection, delta and HBV
'
,
are transmitted together at the same time . In
supennfcctian, delta infection occurs in a person
already harbouring HBV, Coinfection clinically
presents as acute heparm;- IS . ranging horn mild ro
fulminant disease . Super imfecLKsn usually leads to
more serious and chronic illness, with deterioration
of the underlying HBV infection. No association
has been noted between HDV and hepatocellular
carcinoma .
Delta amiigen is primarily expressed in liver odl
nuclei , where it can be demonstrated by
immunofluorescence . It is only occasionally present
in scrum. Anrfeddta antibodies appear in senim
and can be idem ified by ELISA . The IgM antibody
appears 2 3 weelu after infection and is soon
replaced by the IgG antibody Ln acute delta infection.
However, in chronic infection , the IgM antibody
persists for years . Delta RNA sequences have been
cloned and DNA probes have been developed for
the ra|ud identification of deha particles in
circulation. The woodchuck has been found to be
a suitable experi mental model for the study of HDV
infection.
HDV is distributed worldwide but is more
common m certain endemic areas . In the
Mediterranean countries, where it is endemic,
LiifecLion is spread commonly by nonpcicutancous
mutes, cspccialy by close personal contact . In the
noncndemic areas, such as Northern Europe and
North A me [ feu , infection is more often through
blood and blood products and is commonly seen in
drug addicts and hemophiliacs. In [ reduction of
HDV into nonendemic areas where HBV infection
,
i 5 common may lead to outbreaks of severe hepatitis
with high mortality.
No specific prophylaxis exists, but immunisation
with the HBV vaccine is effective as HDV cannot
infect persons immune to HBV. Screening of blood
donors for HBsAg automatically limits blood home
HDV infection.
pyri nted materi
J
^
J
Hidden page
 TaWe 53.2 Viral hspatilis ifpm : comparalive features
s
A B C D E
Virus- HAV, 2 7 n m RNA,
Pleornavirus
HBV, 4 7t a t i DNA
{Hepadnavirus)
-
HCV, 30 Mfim
RLNA, FkvdviriiJis
.
HUV i $ mm
Defective RNA.
HEVn 32- 34nm RNA
Herpesvirus
{ Hepatcmruii) -
( hepamini-s ) Delta.vii. ru5

Modbs of lofiufiQp lpKa 2 oral Pfemitstneoiis Percutaneous Percutaneous Fetal -Oral

Verdcii, Sexual

Age Affected Children Any age Adults Any age Young adult*
*
15-160 30 - 180

Textbok
IntiabiCitin 15 -45 3EMS0
Period(dap )
1S
*0
Ontti Acute Insidious Inekti&u* Insidious Acute of
Illness

Carrier scare
Mild

Nil
Occasionally severe

Common
Moderate

Present
OccaskariHlIy severe

NiKunljr with HBV )

Mj](tr
lurvcy

Nil
except in preg

Microblgy
*

Oncogenicity Nil Present speddly after Present Nil. Nil

neonatal i n fection

Prevalence Worldwide Worldwide Probably woddwidb Endemic areas Only developing

O ( Mediwrraneini . N.Europflt ounfrics India , Asia
^ ^
mouAdo
1

Centra] and W. America) Afrjsia , Central,

America}

& Specific praphylj-xis Ig md Vaccine ; Ig and vacc i ne Nit HBV vsrixine Nil
p

leut i
 * Bopflftta Viruses 561

FUfttl -flT Kstri!J Ilpj

j

Alter HJr 1997- Anrte nnn A- E hfipflirttiai in the USA and Elbe rale ofkepaddr; G ifeiis iofeelkn, NfiW EngfJMed 336:741 -
British Midkal Bulletin , 1990, Hepatitis 2:46
Brown JL. 1995, Hepabria C. J fnfict 3KW5,
-
Collier L (ed ). 1996. Heparirie A and E Tbpfch &pls'VEJfsoiriiAfianbwJqgv and Mm a] /n/pctj»ons 9*«hi , London;Arnold
'

^ .
Dhenstag JL and Kj IsseUbachEr 1998. In Acute Viol Hepatitk. Hinisons jFWncfpJks aflbwal AfoJrdne 14* cdn. New
Yoflc Mc^Gcnf HiU.
Hbllinger FB andTj Liny 2002, Hepatitk B vims, In Field’s Virology, Vbl 2. 4* ed . Philiiddlphia: Williama and WffldnL
1989, Type D ( Delta ) Hepatitis, J Amer Med A*wc ML1321.
Krawoyraiski 1993. Hepatitis E. Hepa udogfr 17:932
EC
--
[ .cn :: r $ M and DL Thomas 1997. VfiGdm to prewnc viral hngbm NewEnglJ Mtd 336cl 96.
MtiPtrd ME and JR Gels 2003. Suppressing Hepatitis R . NgwBngJ Med 348; ML
.

Sarin 5K and AK Singil (« d*). 1996. Hepatitisi & in /ndis. New Delhi - CBS Publishers ,
Van DuiHM P m al 1.997. ImgnfiQn of heparin* B vaednarion into narimaJ imniunLy arinn nmruillIKt. iJAI / 314il 033-
. .

opy righted material

8 dr w
 Miscellaneous Viruses
PAPOV A VIRUSES
The term LFapova' is a sigla indieating the names
f viruses included ] ii tKii- group: pipilloms virus
of human beings and rabbits, polyvtna vi & of ni : c
and vacuolating i rr -uj of monkeys. The famiiy
Papovaviridac has two genera - Potyvmzv / rux
which contains the simian vacuolating virus ( SV
4 (1 ! and polyo mai l ruses , and Pdpiifojn^rrnii
containing human and animal papilloma viruses.
They are small, nonenveioped, icosahtdral DNA
tumour viruses. Most of the naturally occurring
papovavi rus tumours JTLL benign, but some such as
rabbit papilloma are potentiallymalignant , Polyoma
and the vacuolating virus SV 4f) produce malignant
tumours when inoculated into newborn mice or
hamsters, These viruses havebeen widely employed
in the study of viral oncogenesis.
Myoma virus often causes latent infection in
laboratory mice. However, when inoculated into
newborn mice , it produces a wide variety of
malignant rumours. Hence the name polyoma.
The simian vacuolating vims CSV 40) was isolated
from uriinoculated rhesus and cynomolgus monkey
kii.lncy tissue cultures. The virus did not produce any
cytnpaihic effects in the original cultures but when
fluid from such cultures was inoculated into kidney
cell cultures from other simian species (African green
or griver monkey ), cyiopathic effects occurred,
consisting of prominent cytoplasmic vacuolatiun. SV
40 isoncogenu in newborn hamsters. Its only medical
importance is that because of its oncogenic potential,
live viral vaccine ?; should be mamifectuied only in
monkey kidney 1issue cultures tested and found free
from SV 40 infection.
Papillomaviruses are spedcs specific and infect
squamous epithelial and mucous membranes,
inducing different types of warts or papillomata in
their hosts. Human papillomavirus (HPV ) infects
only humans and grows only in organ cultures of
human skin. Over 70 types of HPV have been
recognised based on gCnetiv homology. There i-S
correlation between the vims type and the type of
lesion produced. Common warts (verruca vulgaris)
usually feund on the hands and feet of children
and adolescents are mostly caused by types 1, 2, d
and 4 Condyloma acuminatum or genital wart
r
which is a more moist, soft, pedunculated wart
found on the external genitalia is usually due to
types (J and 11. This may be transmitted venereally
and may occasionally turn malignant. There is a
close association between specific HPV types and
genital malignancies in both sexes. HPV types 6
and 11 are associated with intraepithelial neoplasia ,
while HPV 16 and 18 sue ctiologically related to
more severe invasive malignancies such as uterine
Cervical cancer. CnfaCtnr? appear to be important
in the induction of HPV associated malignancies.
Human papovaviruses have been isolated from
a number of patients with impaired immunity. The
| C vims was isolated in 1971 from the brain of a
,
patient with Hodgkin disease and progressive
^
multifocal leukocmccphalopathy (PML). Isolation
of simitar strains from PML cases has been reported
feom the USA+ L h and France. The JT virus grows
only in human fetal glial cell cultures. It is
oncogenic, producing [naiignant gliomas following
intracerebral inoculation in newborn hamsters.
Another human papovavirus is the BK virus,
Copyrighted materia
Hidden page
Hidden page
 * Miscellaneous Viruses 565

- I- T
i' V.
-A r*
>
- ft »

,TH .

Sr V '
.
- .-
r Jr 6
k .J
i ' 1

^ -

-
loo nm
.-

* * * 00 om
*t
J
/- - i SiKrw * » JQp
«8-
r
-s

* .- ar
- «
i -

.
:^ -
>? UP
l^r _ 35! '

j
1

^J\ . ®
.
..
-
p
»^ jS
* JHI
,j
.

* SI

" ¥aH; Iq
’W
'
. JJ
.-

r: #J§
J

-#.
1 r - wa v:

w -r
* mV *
)

It 4
jr '4

Wh
ur ?

4
^
/if

& i

.
F q &D 1 Electron Tiicrograph & pi viruses associated with diarrhea ft 20O QOQI 1. Rgtavirga. . 2- Adengvirua ;
.
3 SmalE round structured virus [Mcrvialk -like calcivirusesi : 4. Corgnevirus

Copyrighted material
Hidden page
Hidden page
Hidden page
Hidden page
Hidden page
 * Miscellanea us Viruses * m
coronavinis' in memory of Dr Carlo Urban! who a common complement-fixing antipen but can be
identified rhe new epidumu , initiated cAfiy ^trps dilTefmtiated by neutralisation ( ES( t . Reoviruses
_
'

for its Control and died of i [ within a month!
REOVmiDAE
The [anuiy Reorirldac derives its name from the
jirmoiypi; virus wltieh was known as the Respirator
1
aggiuti natc hum an tryth rnryte v Hcmaggluti nation
is type specific and is neutralised by the specific
antibody. Rrovimses- have not been proved to muse
any human disease.

Kntcric Orphan virus, because it could be isolated 0HBMRU&

frequently from the respiratory and enteric tracts, but 'ITiesc have a double shell in which the outer layer
was not a^soeiarcd with any disease. Members of diis is fuizy and indistinct. Tin? inner layer has 12 ting
tun Lily are double shelled iuosahedral Anises, 55 57
naTi in diameter, The genome amsists of double
— shaped rapsomers. The nmc nrhivirus is derived
ft[]ni Oifn’ in Latin , meaning ting . Orbiviruses
i

stranded RNA in 10-12 pieces , a feature unique multiply in insects and ndrcbmte* thns Qualifying
among animal viruses , They are nonenveloptd a ]td as arboviruses. They are responsible for veterinary
restslant to lipid solve nis . Tlit fan i ily contains it ] ret diseases such African horse tick] Less and blue
as
—
genera Rcovim^ , OrMvmn and Retinnu. tongue. The only known orbivitus infection of
human beings is Colorado tick fever.
flEO VIRUS ROTAVIRUS
The genus Rcovirus contains three mammalian These double willed viruses prune nt a eharaeteriStic
serotypes ( Reovirus types;\ y 2 and 1 } which possets appearance under the electron microscope ,

t O
* f i

®S&
|

o si'Sk o
Hi
6 5
2
3 4

Fig . 40.3 : SAftS - a $ H c i M Ipti cororisvitus. [ diagrammatic reptescniatiofl) 1 Envelope : 2. RbA -. Kud?oeap?ld
nueleoproleifi . A , spike glyenpruleln ; $. HoftiagglutinIn- acetyi ealafafit glycoptoEstn; &. aroaN envelope
glycopr & toin ; 7. membrane glycoprotein.

Copyrighted material
Hidden page
 < M '- scsIlaneaus Viruses * 573

Rotavirus vaccina have been developed, bat are adenoviruses can he only grown with LliftiL-ultyin tissue
not in UK row. culture ' Iliey have been designated types 40 ami 41 .
,

OTHER VIRUSES CAUSING DIARRHEA
Reside rotaviruses, the following viruses arc known
or ^iLspccled to cause diarrHcal disease :
Norwalk vime A 27 rm virus was shown to
be responsible for an epidemic of gastroenteritis
affecting school children and teachers in Norwalk,
Ohio* in 1972. The virus induced diarrhea in
human volunteers. Serological surveys have
shown that infection with Norwalk virus is
widespread in many countries . Extensive epidemics
of Norwalk virus diarrhea associated with
consumption ofrawoyseen have been reported from
Australia and America. It appears to be an important
cause- dt LJI .LRRLIL~ JI iit adults aod children.
The virus can he demonstrated in feces by cJeCtTO©
rtiiooseopy, Antibody to the virus can be detected by
immune electron micmsjctipy and radioimmunoassay
It Jus not yet boat propagated in cell cultures. Little
i s known about the properties of tlie virus. 11. lilts been
included in the family Cdicftinditc; consisting of
small round RNA viruses, 22-30 nitl in size, many
ofwhtL^ h have been reported from diauheal feces.
The name caUcivirus LF derived from the presence
of 32 cup shaped depressions on the vims surface
(fnom CJJIT, meaning cup).
Adenovirus Several outbreaks of L arrhea in
children haw both associated with the presence
^ Large scale slaughter of pigs had been undertaker
of latgc number* of adenoviruses in feces. These
Adenovirus associated diairltwiL lias been seen more
often in summer months.
Astro virus: These star shaped, 2 % nm isometric
particles have been associated with sortie epidemics
of diarrhea in children . Similar viruses have also
been identified in lamb and ealf diarrhea .
Corondviruc These are well established causes
of acute diarrhea in calves , piglets and dogs. They
have been observed in human feces also but their
relation to diarrhea is unceitiLtl.
HenJrji a. nd Xipnh viruses: Two new
zoonotic viruses, closely related hut distinct are
Hendm U994) and Nipah (1999) viruses named
after the place they were first isolated in , in
Australia and Malaysia respectively. They have
been classified under the family Fammyxovi ridae.
They arc prevalent in Australia and in Malaysia,
Indonesia. Philippines arid the Pacific Islands .
Their natural hosts arc fruit bats in which they
cause silent infection. Symptomatic infection occurs
in horses , pigs and many other domesticated wild
animals. Human infection is usually saibctinieal , but
may he a fiulikc fever which may lead to fatal
encephalitis, sometimes ILS outbreaks . Hendra virus
outbreaks have been minor, but nipah virus bw
caused large outbreaks with case fltalitY rates of
up to ?0 per cent in persons in contact WLth pigs .
in order to stop outbreaks.

Further Heading
LSerlin 11 iftd U Dciselbufgef 1996 . Rotavirus paih^flenHis . Lancer 21 R : £99.
Ciitcf MJ and \VD Cubitt 1995 . Norwalk and related Cun- In&er iiii 8:401
.
Fields BN er al . 1996. Virology edn , PhUaddphia;Lippinccirt - Rivcn.
Johnson KMet al. 1939. Clinical virology of LlW fmr.J Infect Dis 155 :45*.
Kapikian AZ - 1994 , Vlr^l infa-titm* ipfthe gatizoijirHnii tract New York; Mircel - Drkkrr
Lee HW and Li Van der < Irom 19 H 9. Htiruvrhagi .: fever with nenal syndrome. /Ye r Aled Vjni/ 16:62.
Holmes KV. 20U 3. SAftS . New En$ J Med 348: 1948. ^ ,
Pnisancr SR . 199R Priona of humans and animals. In Tofitcy tnd W41h? fl * MuQWfrbygyurf MicraMaf Jnfec CIOFFT, 9 r edn .
London: .Arnold .
Richmond JK and DJ Baglole 2003 , Lu;i Fm(. Hr MW / ttttUTl.
Wenzel RA and MB Edmond 2003. Mansions SAKS. New ] MeJ .14S: 1 SM 7 ..

Copyrighted materia
Hidden page
I
* Oncogenic Viruses
Table 61.2 Lia1 cl onCDgenic viruses
RNA VIRUSES
I . Rc trovi ruses 1. Avian leukosis viruses
1. Murine leukosis viruses
3 . Murine mammary
tumour virus
4. Leukosis -sarcoma vims
of various animals
5 . Human T cell leukemia
viruses
DNA VIRUSES
I . PipOvavirus 1 . Papillomaviruses ol
human beings , rabbits
and other animals
2. PoSomavirus
A Simian vims AO
4. BK and JV viruses
IT . P& Kvirui 1 . Molluscum coniaginisurti
2. Yaha virus
3. Shope fibroma
Ill . Adenovirus Many human and
nonlwman types
TV. Herpesvirus 1. Mareki disease virus
2. Lwcke’s frog tumourvirus
3. Herpes virus pau, papio,
attic* and saitnui
Epstein-Barr virus
4.
5 Herpes simplex virus
. carcinoma and used widely in various laboratories,
typci 1 und 2
6. Cytomegalovirus
V. Heparitii B and C
limn
(Table 61.1) . Transformation from normal to
malignant cell is a multisKp process anil may he
partial or complete. For example, some viral agents
C ; m ' immortalise ' infected cells, SO that they become
capable of continuous multiplication in culture,
without possessing other features of malignancy.
Transformation is recognised primarily by a change
in morphology of cultured cells. Transformed cells
are altered in shape and lose the property of 'contact
1
inhibition HO that , instead of growing as monolayer,
they grow piled up, one over another, forming
'miciotumours'. Foci of transformation can easily
be made out: and are used in the assay of oncogenic
viruses, such as cite Rous sarcoma vims.
About a quarter of the 600 or so animal viruses
575
possess oncogenic potential {Table 01.2). The
viruses associated with cancers in human beings
are shown in Table 61.3. Both KNA and DNA
viruses art oncogenic. While all oncogenic RNA
viruses (formerly called oncornaviruses) belong to
a single family ( retrovirus), oncogenic viruses occur
among ail major groups of UNA viruses, except
parvovirus. Retroviruses arc responsible for naturally
occurring leukemia and sarcoma in several species
of animals . Among DNA viruses , some
herpesviruses and hepadnaviruses cause malignant
tumours in their natural hosts.
ONCOGENIC DNA VIRUSES
V A P u V A V i m. S E S
Papilloma viruses cause benign tumours in their
natural hosts but some of them (e.g., condyloma
acuminatum in humans, rabbit papilloma ) may turn
malignant. The association between human
papilloma vims ( HPV ) infection and cancer of
cervix uteri, particularly HI > V types 1.6 and IS ha*
been established. The continuous cell line He La ,
derived many decades ago from a cervical
has been found to contain HPV- lfi DNA. In
general , infectious vims particles cannot be
demonstrated in tumours induced by DNA viruses
but papilloma in the wild cotton tail rabbit is an
exception . Rabbit papilloma virus , or DNA
extracted from it , can produce papilloma in rabbit*
following subcutaneous injection.
Tbe polyoma virus causes natural latent
infection in laboratory and domestic mice .
However, when injected into infant mice or other
rodents, it induces a wide variety of histologically
diverse tumours . The virus can be cultivated in
mouse embryo fibroblasts or baby hamster kidney
cells, in which it induce* transformation . The
polyoma virus produces a hemagglutinin .
The papovavirufes BK and JC, which cause
widespread asymptomatic human infection, can
induce tumours in imjmunodelkient subjects.
Simian virus 40 ( SV 40) was discovered in
Copyrighted material
576 ^ Textbook oi WicrDbiology
Table 61 .$ Viruses associaled wilti human cancer
Virus Family Vf /ut
Papovaviridae Human papilkuna virus
HcTpesviridif F,- B Virus
HSV type 2
Hcpidrtavirtdu Hepatitis B virus
Flaviviridae Hepatitis C virus
Rctmviridaee HTL virus
apparently normal monkey kidney cultures u ^cd for
rhe production of tire polio vaccine, It causes an
itl apparent infcctno in rhcHUH anti cynomoljus
monkevr; anti tIoeH out t^use uvtopatbic effects in
cdl cultures from such monkeys. Hirwever , when
fluid from such cultures is inoculated into renal
poll cultures derived from African green monkeys,
cytopadlic change with prominent cytoplasmic
vacuolatiou results, 1 njection into newborn hamsters
produces tumours. Transformation is induced in
cultured cells from several species , including human
cells. Millions of doses of polio VKOSt prepared
in monkey kidney cultures that may have haibourrtl
SV 40 virus had been used before the virus was
discovered. These individuals have been followed
up for over 25 years and no SV 4Q - related tumours
have been reported . There was considerable
apprehension when die oncogenic effect of SV 40
was discovered. However there is no evidence that
the injection of vaccine containing SV 40 has
induced cancer in humans.
POJCVIM 5
Three 1 embers of tile pOJtvitUS group induce
[]
benign t u m o u r s , rabbit fibroma , moll u s c u m
contagiosum and Yabs virus . The last causes
naturally occurring benign histiocytomas in
monkevti . It is apparent I v transmitted by insects.
Similar t u m o u r s can be induced ejeperimentally in
many species of primates , including human beings .
The numoun; regress spontaneously in a few weeks .
Nonpritmres are unsusceptible .
I}pes o f cancer
Cfirvicai , vulvar, penile cuhtert
Squamous. rail carcinoma
Nasopharyngeal carcinoma
African Buriatia lymphoru
B eel] Lymphoma, Cervica] carcinoma
Hepatocellular card noma
Adult T cel] leukemia
AlJl ' N O V l H I S
Though i t u n e types, ( l 5 r l n 21 ) of human
adenovirus may produce sarvomtiH in newborn
^
rodents after experimental inoculation, they do not
appear to have any association with human cancer.
HKRFH » VJRL IS
Many herpesviruses have been associated with
natural cancers in animals and humans.
LI . MLiruk s discus This is a find contagious
'
ncurdymphomatosis of chickens . Mo infectious
virus particle can be isolated from the lesions or
seen under the election microscope . However, sick
hirds shed large quantities of virus from their
feather follicles. The virus is a typical herpesvirus.
MarekV disease can be induced in young chicken
hy the injection of the virus. The virus grows well
in chick embryo fthroblasts producing cytopathic
change* but no evidence transformation . Manet's
^
disease car lnL prevented by a live a virulent vaccine .
Tins ii tile fiftst inn, tirtce of LI L 3 iJ.hgo .mt disease
being controlled by a vim] vaccine.
h . I . ucke' s tumour of fm su A herpesvirus
^
is considered to be the etiological agent of a renal
adenocarcinoma in frogs.
e - Herpeflvirui snimiiri : This virus was
isolated from a culture of Squirrel monkey kidney
cells . It causes fetal lymphoma or reticulum cell
sarcoma when injected into owl , monkeys or
rubbles. Herpesvirus saimiri infection has been
,
suggested LLH n primate model for the study of
Copyrighted materia
 i QntpgeriLC Viruses
interactions between the EB virus and human
beings .
J. Epstein-Barr virus: A herpesvirus , called
the EpSteiil-Barr virus, is found regularly in
cultured lymphocytes from Burldtt 'f- Ivmptioma
patients. In the body, the tumour cells contain no
virus but cell lines established fmm them contain
5-20 per cent of cells that produce the virus. The
virus multiplies only in human lymphoid cell lines.
Serological surveys show chat Infection with the
virus is worldwide. Infection is usually
asymptomatic . In young adults without pne- existi ng
antibodies, EB virus infection induces infectious
mononucleosis. Lymphoma is believed to occur
when the infection takes place in children whose
immune systems are compromised, as tor instance,
by chronic malaria, EB virus associated lymphomas
have been reported in transplant recipients. EEJV
has also been linked to nasopharyngeal carcinoma
in the Chinese male population in southeast Asia
and East Africa,
e. Herpes simplex nnd canter cervix:
An association has been proposed between herpes
simplex type 2 infection and cancer of the uterine
cervi though not proved. It has also been suggested
^
that herpes simple--* type I infection may be
associated with cancer of the lip . Herpesvirus
type S has been linked to Kaposi’s sarcoma .
f. Cytomegalovirus infection has been
associated with carcinoma of the prostate and
Kaposi Sarcoma.
HEPATITES B VIRUS
HBV has hcen chimed to be directly or indirectly
involved in the etiology of hepatocellular
carcinoma . Studies in many countries have
demonstrated an excess prevalence ot markers of
HBV infection in patients with primary
hepatocellular carcinoma as compared with
matched controls or with the general population.
Hepatitis C virus infection lias also been reported
to lead to hepatocellular careinoma.
57 7
ONCOGENIC RNA VIRUSES
RETRU \ lit t s
Retroviruses are enveloped , splierical virus® that
are released by budding through the host cell
membrane. They are approximately 100 run in size.
The genome consists of two identical , linear, -single
,
stranded RNA molecules . The icosahedml
nucl cocaps id core encloses the helical
rlhomiLleuprotein and is MiHDundcd by an envelope
composed of glycoprotein and lipid.
The characteristic feature of retroviruses is the
presence within the virion of the unusual enzyme
RNA dependent DNA polymerase or reverse
transcriptase (hence the name retro , meaning
reverse). Unlike the classical transcription of generic
information from DNA to RNA, the reverse
transcriptase cn-cyme prepares a DNA copy of the
retroviral RNA genome— initially an RNAtDNA
hybrid and then its double stranded DNA form.
The double stranded DNA form of retroviral
genome, called the provt /us, is integrated into the
DNA oftbf nltected host Odll, It is front the prnvims
that all retrovirus proteins are translated . Infection
with oncogenic retroviruses does not lead io
cyrolysLS or death of infected cells but die peprinu
remains integrated with the host cell DNA for the
rest of the life of die cell .
While all oncogenic RNA viruses belong to the
family Retntviridae , all retrovirus- es are not
oncogenic, The family /teflrQvjriJaf is classified into
three subfamilies.
1. OnCovirinac comprising all oncogenic RNA
viruses ( formerly called oncornavirus).
2 . Spuimnirinxe containing the nononc-ngenic
' foamy viruses' (
spuma = foam ) causing
asymptomatic infection in several animal species,
and presenting as contaminants of primary cell
cultures in which ihevinduce Ibaittv degeneration.
3 . I enmirinne including the viruses causing ‘slow
^
i rtfoctions' ( fantut = slow) i n animals ( see chapter
60) , as well as rht human and related animal
immunodeficiency viruses ( see chapter 62) .
Copyrighted materia
 57S * TOKIDOOK of Microbiology *

Retroviruses arc widely distributed * being found quantifies of interleukin - 2 < I'hc closely
in nearly silt vertebrates, including am mils , birds related HTLV-1I is also u with T cell
and reptiles. R .Lsed on tbc host range and types i >f mjljgruiLCy. HTLVr infection it tu be spread
disease caused , oncogenic retroviruses can be through blood transfusion and methods of
considered under the following groups : transfer of leucocytes .
1 . I 'ltL \ \ i in i euko^ ia ( iotTiplux: A group
"
HitSl Specificity: RetToviruM usually infect
of indgenivaUy related viruses which induce avian only one ho t species, the specified ^ being
^
leukosis ( lymphomatosis, myeloblastosis and co]iditioned mainly by the i u st r )c e of viral receptors
erythroblastosis viruses ) or sarcoma in fowls ( Rom ( i n the host celt surtacc. ! icndi on their ability
sarcoma virus* R 5V ), to grow in cells from different . . . retroviruses
2 . M u r i r i L I euka[HG Viruses: This group have been classified into (1) ecotiopi ( multi fliving
consists of several strains of murine leukemia and in cells of native host species nl ' in pic

sarcoma viruses* named after the investigators who
first described them (e .g. Gross* friend * Moloney*
Rauschef ) .
O M b i m r i v j m I i n i r i n v : r i ^ ' i! I I I I L T ; This
r Virus ' i wmission; Two : pi of retrovirus
virus occurs in certain strains of mice having a high
natural incidence of breast cancer. It used to be
known as the Hmilk t actor' or LRinner virus'. It
multiplies in the mammary gland and is transmitted
from mother to offspring through breast milt Mice
can be infected by oral * subcutaneous or
iritrjjieritonf .Ll mutes . MamtUiy cancer occurs otilv
in susceptible strains of mice, after ; L Latent ]ieri( jd
of 6-12 months.
-
4 . Lcukugi & Kstnit)ina viruses ol other
mirunlsl A number ot viruses have been isolated
from leukosis and sarcomas in various specie of
( multiplying in cells of native i Iron species);
and (3) xenotropic ( m ldptymg mi? to ceils of
foreign species but not of native host species).
transmission occur. Exogenous ernoviruM are
spread horizontally. Most cutcugeni retroviruses
.ire exogenous. Endogenous rerrnvi are
transmitted vertically from parent to offspring* by
the pro vims integrated with the germ Line cell
genome . The endogenous reiiovirus provirus
behaves tike a cellular gene ind is subject to
'

5.
-
animals cat , hamster , rat, guinea pig and monkey.
Human T cell leukemia
( lymphotropiii ) vinisrt : ( HTL\ ) :
Retroviruses named humanT cell leukemia viruses
were isolated in 1980 from cell cultures from adult
patients with cutaneous T cell lymphoma ( mycosis
fungmdcO and leukemia (Senary syndrome ) in the
USA. Similar viruses have hcen isolated f r o m
patients with a d u l t T cell leukemia in japan and
the Caribbean . HTLV lype ] it pnesent worldwide
hut the disease is limited to endernit areas. Besides
Fig. 6i .t Mouse rnarmriity cancer , stained wih
adult T cell leukemia , HTLV- 1 Ins also been uranyi acetate and lead nitrate . melure
associated with tropical spastic paraparesis * a bud dine E type virus ( arrow ] intracellular
demyelinating disease- The vims preferentially
infectsT4 (CD 4 ) cells . Infected T cells express large
Immalura A type virus (1 rlpl
iCcurk sj -: Carter RssE- .srtli
* 1 .
(* ISO OQO )
Mumbai,]
' !
 * Oncogenic Viruses 579

A LTR LTR

itWV

B LTR LTR

mg peif env fiat

C LTR LTR
snR
line

. .
Fig 61 Z Provirus genomic structure ol different s of reTrcvirusfS
-
A. BasiK irtmvinis Jjemimc e -J'. Avilli li .ilstuLia viruses, sLiiw transforming viruses
'

B . TrmsjcpiJicing retnovirasei-e.g . HTLV. EIIV

C. Amt* traiwfonningTttrovinis-i oncogene tepLacjng part of basic genome. Rcplirabon deFecrivie
regulatory control by the host cell - Endogenous
retroviruses. are usually silent nd Ho not transform
^
cells or cause any disease- 'I’hey can be detected
1
cither by 'activation after ntposuie to radiation or
chemicals, or by nucleic acid hybridisation
techniques-
Resistance: Retroviruses are labile , being
inactivated at >6 "C in 30 minutes, by mild acids,
-
ether and formalin. They are stable at 30 ’5C .
Antifjcnb: Two types of antigens ate present
the type specific glycoprotein antigens on the
— after thr env gene. This in a transsctiViiEiug gCnC
envelope , and the group specific nuclcoprotein
antigens in the virion core . Cfoss-reacrion > do not
occur between surface antigens of retroviruses from
different host specie.
Genomic structure; Retroviruses have a
relatively simple genomic structure ( Fig- bl .3).
The provirus- of a standard. retrovirus (such as a
nondefeefive avian or murine leukemia VUUt)
consists of three genes required for vital replication
- gag, pol, and env in .that order from the 5' to 3'
end . The gag gene codes for the nuclcocapsid core
proteins which are group specific antigens ( hence
the name gag ) ; the pol gene encodes rhe RNA
dependent DMA polymerase; and the env gene
encodes the envelope glycoproteins. Long terminal
repeat ( LTR) sequences are present at either end
of the provirus and linked direedy to ll it host DNA.
The LTRs exert regulatory control on the piovirus
gene functions .
Some retroviruses (iransrcgulating viruses) such
as- HTLV or HIV carrv a fourth gene , lex or rat,
that regulates the function ofviraE genes.
The standard oncogenic retroviruses, such us
chronic Leukemia viruses;, are slow transforming
viruses , i .e., they have a low oncogenic potential
and induce malignant change , generally only of
blood cells , after a long latent period. They do not
transform cultured cells . They are capable of
replicating norm ally. In contrast, ffte Scute
EnuJonnillgrinilH arc highly oncogenic a nd cause
malignancy after a short latent period of weeks or
months . They can cause different types of
-
malignancies sarcoma , carcinoma, leukemia -and
also transform CC-UE in culture. However, most acute

Copyrighted materia
580 * TembooH oF Microbiology
transforming viruses are unable to replicate
normally because they carry on their pnaitir an
-
additional gene* the viral oncogene { V onc gene)
which replaces some of the genes essential for viral
-
replication. Such Y onc viruses can replicate only
if co- infected with a standard helper retrovirus.
The Rous sarcoma virus which carries the
oncogen ic sre ( pronounced 'Sark1) is the best known
among acute nans tor riling viruses bur it is different
in being replication competent, that is it can . to be identical with cellular oncogene*.
replicate normally because it posseses the full
complement of gag, poL, and me genomes. Most
acute transforming viruses axe replication defective,
ONCOGENES
-
Viral oncogenes ( V onc) , commonly known as
‘cancer gen^ arc genes which encode proteins
1
triggering transformation of normal cells into cancer
cells. Oncogenes are not csscn ri al for the repli cation
of the vims and mutants lacking them occur, which
replicate normally without being oncogen ic.
Genes closely resembling viral oncogenes are
found in normal as well as cancer cells. Oncogenes
isolated from cancer cells arc called cellular
oncogenes (C-onc). Similar genes found in normal
cells are called prom-oncogenes. They are not of
, the exact mechanisms of viral oncogenesis are not
vital ORIGIIL. On the contrary vi rai oncogenes appear
to be of host cell origin. Cellular oncogenes contain
intnons characteristic of eukaryotic genes, whereas
viral oncogenes do not. Apparently viral oncogenes
originated at some distant past from proto-
oncogcnes by recombination between retroviral and
cellular genes.
Proto oncogencs are widespread in vertebrates
—
and metazoa from human beings to fruitflies.
They arc well conserved in their genomes ,
suggesting that they serve some resentin ] functions
in normal cells. They have been found to code for
proteins involved in regulating cell growth and
differentiation. The presumed functions of many
oncogenes have been identified. For example, the
oncogene sne related to tryioeine-specific protein
kinases, sis to a platelet derived growth factor, and
myc to DNA-binding proteins, all concerned with
regulation of normal cell growth .
A useful method for the study of oncogenes is
transfection. Certain mouse fibroblast ccD lines, such
as NIK 3T3 , can take up foreign DN A, incorporate
them into their genome and express transfection.
By this technique, DNA extracted from human
rumour cells has been shown to transform 3T3
cells, and such transforming genes have been shown
ANTE- ONCOGENES
A class of genes has been identified in normal
retinoblastoma ( Rb) gene, the loss of which is
associated with the development of retinoblastoma
in children. The p53 gene appears to be a tumour
suppressor gene with a wide range of effects.
Specific chromosomal deletions, recognised in
association with certain types of human cancers may
reflect che loss of tumour suppressor genes.
MECHANISMS OF VIRAL ONCOGENESIS
While it is known that oncogenic viruses are able
to transform etUs in Culture and Induce tumours in
animals, under natural or experimental conditions,
well understood. Malignancy is a stable heritahfe
change and , as such, should be the result of a
modification of the host cell genome .
In the case of oncogenic DNA viruses, the viral
DNA ( or a portion of it ) is integrated with the
host cell genome.The viral DNA being incomplete
Or 'defective , no infectious virus is produced.
1
However, under its influence, the host cell undergoes
malignant transformation . A virus transformed
cancer cell is in many ways analogous to a
bacterium lysogeniEed by a defective phage. In both
cases , the cell is not destroyed and no vims is
produced. Acquisition af new characteristics by the
transformed cell resembles lysogenic conversion in
bacteria.
—
In general, retroviruses induce tumours by two
mechanisms cither by introducing into the
/” *L
u. name* ri -
J V I
 i Oncogenic Viruses S 81
Table 6 l.i Sflme oncogenes' and ( halt chrwioMrtL* l location in humans
V,rirHJOinCOg-CDC Origin Nmturml Human gene Chfomnsomal location
tumour in humtn beings
V- re chielspn C arc 20
V-^ra*
^

rat Sarcoma C-tit 11

V-myc chicked Leukemia C -myc &
v-r« cat Sarcoma C- fe* 15
V-( is monkey Sarcoma C-sis 22
V-cKVOS mouse Sarcoma C-fflus »
1
Oncogenes ut fpvrn three letter cwltf from the animal or tumour from which they are derived , preceded by
tidier V- or C-, for viral or cellular penes reepectrvely;
DC sarcoma of chicken, ras -. r it sarcoma , sis - simian sarcoma ,

cellular genome a new transforming gene gene product may lead abnormal growth .
to
( oncogene ) or by inducing or altering the expression Recombination between retroviral and cellular
-
of a pre existing cellular gene , Several molecular genes , promoter insertion, chromosomal
mechanisms have beer suggested for the conversion
of benign proto-oncogenes to cancer genes. The
-
t ran location , gene amplification and mutation are
some of the genetic processes relevant in this
genes may get overevpressed and the overproduced connection.

Further Rtiidiiijj
Bnhdjj JM. 10 R 9 . The mulet-idlr gerittu:£ ufOUBK Siicnif 235: 305.
Friend , SH etal. 1900. Oncogenes and tumour suppressor genes. New Engl J Med 318:618 .
Nejl MS and JA Wyke 1990 . Viral onooRenicity In JbpJ r and Wiltons Microbiology and Microbial Infections , 9 , k edn .
Vc' l . l . LondoruAmcdrL ^
Varmus Hr 1980 - ftctrovinises . Sdener 7-
WeinbeiR RA 1998, tlndinf ; the antioUCpRtiK , Siieur 4 I!?9: i-i.
njrr
-
Vfanbrip RA . 1989 . Oncogenes , intioncogenes and the molecular basis of carcinogenesis , CIIKIT fie-v 493713,

Copyrighted material
Hidden page
 4 Human ImmunotteficienCy Virus: AIDS
o
i
-
o
>
FlJ, 62.1 Slrutture Ol HIV (dbflrammiUc repre $flrvtatlcn|
E- Tfansmemljrance pedicle glycoprotein (gp4i|, $ . Outer Icosahedral shell of nuclecicapsid (p16), 4, Cora
shaped cone ol nucleocapsld \$ 24\- , 5. Inner core. 6. Viral proteins associated with RNA . 7. Viral iRHA,
.
0. Reversa transcriptase, 9 Envelope lipid bilayar .
in 2003 , Three mill inn patients died of AIDS in
2003.
HUMAN IMMUNODEFICIENCY VIRUS (HIV)
HTV, the etiological agent of AIDS, belongs to the
Icntivinjfl subgroup of the family R-ntruvifidae. The
lentivirus subgroup includes the causative agents
of the slow virus diseases visna/ maedi in sheep and
infectious anaemia in goats and horses. Besides
HIV, the related animal immunodeficiency viruses
also are assigned Co this group (Table 62.1 ).
SJTftUtTUKB
HIV is a spherical enveloped virus, about 90-120
not in ( f ig - -62- 1 }- The nucleocapsid has an
si 7 c
outer ictisahrdrill shell and an inner coneshapcd
core, enclosing the ribonucleoproteins.. The genome
is diploid, composed of two identical single
stranded, positive sense RNAcopies. In association
with viral RNA is the reverse Transcriptase enzyme,
which is a charaeterisric feature of retroviruses.
When the virus infects a cell, the viral RNA Is
transcribed by the enzvme, first into -single stranded
DNA and rhen to double stranded DNA ( provirus)
583
l. Etivdopfl glycoprtfeln spike ( gp 120),
which is integrated into the host cell thro mosunue .
The provirus can remain latent for long periods ,
though it influences host cell functions. At times,
in response to viral promoters, the provims initiates
viral replication by directingsynthesis of viral RNA
and other components
During viral replication, when the naked virus
buds out through the host cell surface membrane ,
it acquires a lipoprotein envelope, which consists
of lipid derived from the host celt membrane and
glycoproteins which are vims coded. The major
virus coded envelope proteins ire the projecting
knohkke spikes on The surface and the anchoring
[ ransmcmbranc pedicles, llic spikes , constitute the
major surface component of the virus which binds
to the CD4 receptors on Husceptihle host celts.
Tmnsmembrano pedicles cause cell fusion .
VIRAL CJKNEB AND ANTHIRNS
The genome of HIV contains rhe three structural
genes [ gag, pot and env ) characteristic of all
retroviruses , as well as other nonstructural and
regulatory genes specific for the virus { Pig. 62.2).
The products of these genes, both structural and
Copyrighted materia
Hidden page
 c Hyman Immunodeficiency
prevalent ail over the world belong to HIV type 1.
HIV strains, first isolated from West Africa in 19B6,
which react with HIV type1 antis cnun very weakly
or not at ill have bun termed HIV type 2. The
envelope antigens of the two types one different,
though then COrt polypeptides show some emss
-
reactivity. HIV 2 has only 40 per cent genetic
identity with HIV-1. It is more closely related to
simian immunodeficiency virus than to HIV-l. It
is much less virulent than HIV- I . It is largely
confined to West Africa, though isolations have
been reported from some other areas, including
western and southern India.
-
HIV l strains have been classified into at least
ten subtypes based on sequence analysis of their
gag and rnv genes. TTiese subtypes are designated
as A to J and constitute Group M ( for ‘nuiicr’),
-
which cause the large majority of HIV 1 infections
-
worldwide. A few HIV 1 strains isolated from West
Africa (Cameroon, Gabon ) do not fall within the
Group M and have been designated Group G (for
-
'outlier'). Some later isolates of HIV 1 from
Cameroon, distinct from M and O groups have been
called Group N (fill new),
HIV-1 subtypes show a geographical
distribution, though this is often blurred by viral
trafficking. All known HIV virus groups and
subtypes are present in Cameroon , West Africa,
which may perhaps be the site of origin of the
virus. Subtype A is the most prevalent, being found
worldwide, while B is the most common in the
Americas and Europe. The common subtypes in
Afnca are A, C and Dh while in Asia the common
subtypes are E , C and B . Subtype E is ihe
commonest in Thailand. In India and China,
subtype C is the must prevalent.
Antigenic difference? between HIV strains may
be important in serudiagnosis. Infection by HIV 1 -
or 2 may not be identified unless the corresponding
type is represented in the test antigen. Even with
HIV-1 strains , the differences between the
subgroups are significant enough so that Group O
strains may be missed if the test antigen contains
Virus: A.IDS £85
Group M only. Even subtype differences may cause
discrepancies so it may be
advantageous to use
antigens containing the prevalent subtypes in
different countries.
The subtypes seem to vary in frequency of
cransmissibility by different mutes. The suhtypes
common in Asia and Africa (C and E ) are more
readily transmitted by heterosexual contact than the
American strains (subtype B ) which are
preferentially spread through blood - by injection
and homosexual contact ( homosexual transmission
is considered to be bloodbome as the virus is likely
tn enter directly into the blood through minor
mucusal lean ).
Differences in growth characteristics are
sometimes observed between HIV isolates from
asymptomatic carriers and from AIDS patients. The
former grow slowly and infect only the peripheral
blood lymphocytes, while the latter grow faster and
yield high litres in established cell lines of
lymphoid and monocytoid. origin. Strain varLutidns
may account for differences in clinical course of
HIV infected persons.
Resistance: HIV is thermolabile , being
inactivated In 10 minutes at 60 :: C and in seconds
-
at 100 °C, At room temperature (20 25 aC ) , in
dried blood it may survive lor upto seven days. A,
At autopsy, HIV has been isolated from various
tissues upto 16 days after death.lt withstands
lyophilisation. The virus in lyophilised blood
products can be inactivated by heating at 68 °C for
72 hours and in liquid plasma at 60 aC for 10 hours,
Table 6S,a Major atrtlfltfla al HIV
A Envelope antigens;
-
1. Spike antigen gpl 20
( Principal envelope antigen )
2. Tran ? membrane pedicle proieiri ” gp 41
B. Shell antigen
C. Core antigens:
-
1. Nudrocapsid protein plB
1. Principal care antigen - p 24
2. Other core antigens - pl 5, p55
D. Polymerase antigens - p31 , p51 , p66
opy righted material
8 dr w
Hidden page
 • Human Itvimunodflliciencf Virus . AIDS *
HIV can be isolated from die blood, lymphocytes,
-
cell free plasma, Kfflen, cervical secretions, saliva ,
tears, urine and breast milk .
The primary pathogenic mechanism in HIV
infection is the damage caused to the CD4 +- T
lymphocyte. The T4 celli decrease in numbers and
rhcT4:T8 ( helper: suppressor) cell ratio is reversed.
Viral infection can suppress the function of infected
cells without causing structural damage. Infected
T4 cells do not appear to release normal amounts
of inierleukto - 2 , gamma interferon and other
lymphoid nes. This his a marked Jumping effect
nn cell mediated immune response .
Though the major damage is to cellular
immunitv, humoral mechanisms also are affected .
, 62.4 ) .
Helper T cell activity is essential for optimal B cell
function , particularly in responding To thvmus
dependent antigens . AIDS patier- ts arc unable to
respond to new antigens- An important feature in
HIV infection is the polyclonal activation of 13
lymphocytes leading ro hypergammaglobulinemia.
All classes of immunoglobulins arc involved hut
levels of IgG and IgA are particularly raised , lit
infants ami children, IgM levels also are elevated .
The hypergammaglobulinemia is more a hindrance
than a help because It is composed mainly of useless
immunoglobulin’ to irrelevant antigens and also
autoantibodics. This may also be responsible for
allergic reactions due to immune complexes ( type
.3 hyperseosirivirv ).
"
Mnnocyte ^macrophage function ' s also affected
apparently due to lack of secretion of activating
factors by [ lie T4 lymphocytes . As i result *
cbemotaxis , antigen presentation and intracellular
killing hy monocytes/maurophsgpi are diminished.
The activity of NK ceils and cytotoxic T
Lymphocytes is also affected . The principal
immunological abnormalities seen in HIV infection
arc listed in Table 62.3,
Clinical manifestations in HIV infections are
due not primarily to viral cytopathology but are
secondary to the failure of immune responses- This
renders the patient susceptible to opportunistic
SB 7
infections and malignancies. An exception to this
IIL .LV he the dementia and other degenerative
neurological lesions seen in AIDS, 'Hiese may be
due to the direct effect of HIV on [ lie central
nervous system .
ACQUIRED IMMUNE DEFICIENCY SYNDROME
( AIDS)
CLINICAL FEATURES ut HIV
INFECTION
AIDS il only the laxt -stage in the wide -spectrum of
clinical features in HTV infection. The Centers for
Disease Control ( USA ) have classified the clinical
course oFI ilV infection under various groups ( Tabic
The natural evolution of HIV infection can be
considered in the foLtowi og stages:
1 , Acute HIV infection: Within 3 6 week* —
of infection with! IIV, about 50 per cent of persons
experience low grade fever, malaise , headache ,
lymphadenopathy, sometimes with rash and
arthropathy resembling glandular fever . Rarely,
there may be acute encephalopathy, Spontaneous
resolution occurs within weeks . Tests lor HIV
antihodics arc usually negative at the onset of the
illness but become positive during LLS course . Hence
this syndrome has beer called ' seroconversion
illness* , though in many of those infected , ‘acute
retroviral syndrome ' or seroconversion occurs
without any apparent illness. HIV antigenemia ( p 24
antigen ) can be demonstrated at the beginning of
this phase. The pathogenesis of seroconversion
illness is believed Cu be due t» immune complexes
as well as to the direct effects of viral multiplication ,
2. Asymptomatic or Intern infection; All
persons infected with HIV, whether or not they
experience seroconversion illness , pass through a
phase of symptomless infection {clinical latency)
which may last up to several years. They show
positive H|V antibody tests during; this phase aod
arc infectious. The infection progresses in course
of time through various stages, CD4
Lymphocytopenia, minor opportunistic infections,
Copyrighted material
m * T£ xJbD3k Gf Micmtunlcgy
pcrsisfcrit generalised Lymphadenopathy, A[D $ -
relatcd complex ( ARC), ultimately terminating in
fall blown AIDS , with it? characteristic infection;;
and malignancies. The median rime between
primary HIV infection and development of AIDS
lias been stated as approximately 10 years . About
5-10 per cent of the infected appear to escape
cli nical AIDS for 15 yearn or more . Diey have heen
termed ' lung term survivors' or ' lung term
nonprogrepsors '. The mechanisms for such
prolonged survival are not dear, though many viral
and host determ inant* may he responsible.
This period of clinical latency however dues
out mean microbiological latency an virus
multiplication goes on throughout . ') IK host mounts
an immune response against the virus , both
hu moral and cellular, wh ich can only limit the virus
Load, but not clear it completely. A chronic persistent
infection with varying degree* of viral multiplication
is the result. The CD4 + T cell count decreases
steadily; from over 10OQ per microlitre to about
500 or less by the stage of acute infection. When
the count falls to 200 or less, clinical AIDS usually
sets in . For this reason the case definition bv GDC
includes all HIV infected cases with CD4* T cell
counts nf 500 or less, irrespective of their clinical
condition .
3 , Persist tut generalised lymphadeno-
piLtlly (. Pfil . ): This has been defined as the
presence nf enlarged lymph nodes , at least lem , in
diameter , in twn nr more noncontiguous
extrainguinaJ sites, that persist for at lease three
months, in the absence of any current illness or
medication that may cause lymphadenopathy. This
by itself ifi benign but the cases may progress to
ARC or AIDS
4 , AIDS related complex ( ARC ): This
group includes patients with considerable
immunodeficiency, suffering from various
constitutional symptom? or minor opportunistic
infections.The typical constitutional symptoms are
fatigue, unexplained fever , persistent diarrhea and
marked weight Loss of mure than 10 per cent of
hndy weight. The common opportunistic infections
arc oral candidiasis , herpes zoster, hairy cell
Eeucoplakia , salmonellosis nr tuhercnlo-sis .
Generalised lymphadenopathy and splenomegaly
are usually present . ARC patients arc usually
severely ill and many of them progress to AIDS in
a few months.
5, AIDS: This is the end stage disease
representing the irreversible breakdown of immune
-
defence mechanisms , leaving the patient prey to
progressive opportunistic infections and
malignancies.
The clinical severity of AIDS varies with the
type of infection or malignancy present. In early
AIDS, many patients are ill only during episodes
of infection , which may respond to treatment.
Between episodes they may be relatively well and
able [ L. I resume normal life. Patients with Kaposi 's
Table $2.$ Immunological abnormalities in HIV
in lection
[ . Featuresthat characterise AIDS
1 Lymphopenia.
.
2. Selective T cell deficiency’
.
Reduction in number nf T4 [CD4 ) "
cells Inversion ofT 4:T9 rario.
3. Decreased delayed hypersensitjvjiy
on skin testing.
4. Hypergammaglobulinemia -
predominantly IgC and IgA;
and IgM also in children.
5. Polyclonal icrivjricn of H rtllx ami
increased spontUUOUl KCretitffl
oHg
II- Other consistently gbwnd feature ;
-
1. Beer eased in vitro lyTnphcKjte
,
proUierntive response to mitogens
and antigens.
2. Decreased cytotoxic responses bv
T cells *ndNK cells.
3. Decreased annbody response to
new antigens.
4- Altered monccytc/matmphage
function.
5 , Elevated levels of immune
,
complexes in serum ,. .
Copyrighted material
Hidden page
 590 * Tsutbook ol Microbiology
pathy leading to loss of higher functions,
progressing to dementia.
7 . I Vdkltrie AIDS: About a third to half the
number of babies born ro infected mothers arc
infected with HIV. Virus transmission may occur
to the fetus in pregnancv as early as the first
trimester, but infection is more common perinatal ly-
Many of the infected children may not survive for
a vtar . Children may also acquire the infection from
blood transfusion or blood products.
There are manv. differences between adult and
X
pediatric AIDS. Children develop humoral
immunodeficiency early , leading to recurrent
bacteria ] infection!. Failure to thrive , chronic
diarrhea , lymphadenopathy, tuhrrculosis and
opportunistic bacterial infections are common
manifestations in pediatric AIDS. Lymphocytic
in [end la] pneumonia in seen mostly in children,
while Kaposi sarcoma , toxoplasmosis and
Cryptococcosis are less common than in adults.
LAHOKATOKY D I A C^O S I ^
Laboratory procedures for the diagnosis of HIV
infection include tests lor unTTlUOOdcfiCrenty Et5 well
as specific tests for HIV.
A . Immunnli itnl nests: The following
^
parameters help to establish the immunodeficiency
in HIV infection.
1 . Total leucocyte and l ymphocyte con nr io
demonstrate leucupenia and a lymphocyte count
usually below 2COQ/ mm \
2 . T cell subse [ assays . Absolute CD4* T cell count
will he usually less than SCKVmm '- T 4:TS cell
ratio is reversed .
5. Flatekr count will show thrombocytopenia,
4 . Raised lgC and IgA levels.
5. Diminished CMJ as indicated by skin rests .
6 . Lymph node biopsy showing profound
ahnormalities-
B, Specific tests for HIV infection : 7
These include demonstration of HIV, its antigens
or other components and antibodies and isolation
of the vims,
1. Antigen detection: Following a single massive
infection , as by blood transfusion , the vims
antigens may he detectable in hlood after about
two weeks, The: major core antigen p24 is the
earliest virus marker to appear in blood and is
the one tested for, IgM antibodies appear in
—
about 4 6 weeks , to he followed by IgO
antibodies [ Fig, 62.3; Tabic 62.6).
If the infecting Jose is small, as following a
needle - stick injury, the process may he
considerably delayed. The appearance of p24
antigenemil and viremia, followed by IgM
antibody response coincides with the acute or
seroconversion illness . Afterwards, free p 24
antigen disappears from circulation and remains
absent during the long asymptomatic phase, to
reappear only when severe clinical disease sets
-
in . However intihody bound p24 antigen
continues to be demonstrable, after dissociation..
The p24 antigen capture assay ( F.L1SA ) which
uses anri - p24 antibody as the solid phase can be
used for this. The test is positive in about 30 per
cent of HIV infected persons. With prior
-
dissipation of the antigen antibody complex,
the positive rate increases to about 50 per cent .
In che first few weeks after infection and In the
terminal phase, the test is un i fermly positive. The
test is most useful in persons recently exposed
to risk of infection, in whom antibody test ia
negative. It LS oirrentlv used for screening blood
donors in the USA, along with HIV ELISA.
2 . Virus hoL non:Once infected with HIV, a person
remains infected for life . The virus is present in
circulation and body fluids, within lymphocytes
ur cell-free. Virus litres parallel p24 titm, being
high soon after infection, low and antibndy-
bound during the asymptomatic period, and
again high towards the end . An infected person
may therefore be infectious throughout , the
infectivity being highest in the early phase and
again when the perwn becomes terminally ill.
The virus is present in many parts of the body
and Can be isolated from the p e r i p h e r a l
Copyrighted material
Hidden page
Hidden page
Hidden page
Hidden page
 * Human immunodefiofmcy Virus AIDS m 595

have spread to Europe from America, us well is of mitochondrial DMA from human and
directly from the (briner African colonics of the chimpanzee viruses . It shows rhar the progenitor
European nations. of HJV-1 entered the human fropulatiou from
Conclusive evidence hus now been obtained chimpanzees of the subspecies Pan tragladfTti
from molecular studies, including genetic typing troglodyte living in equatorial west Africa
(Cameroon*, Gabon, equatorial Guinea) , where the
vimh it considered, on other evidence , to have
M
emerged in humans . HIV's are believed to have been
3S
present in monkeys for over lQ0 h 00Q years ,
NS
Transmission to chimpanzees is a far more recent
.J

^ {*r« »r- fl-- rh- Th- tP

“i*
fa
- - E' *'
-
P ’' *
1 P" IT ® ® ®
* Wfl q? 4 «* ffl K phenomenon and may have hapjjened through [hnr
^ ^
>

Of3 M
* s,
^ killing an.d eating monkeys. Human infection could
have conic from chimpanzees who were hunted by
ftp 12 11 them and killed for meat. The cpz 51V may have
taken root in humans by becoming HIV through
IPS
ffil
“ = mutation or recombination.
W *1 r* * - i
PH

Pi
— flp 1 w
90120 =
pfiS
piv- 4^ - *

-
P 5A P Sf
TlLLCHt] It U VT - -
gp-41

Fig. 62 , J Western blot lest tor HIV antibody . N | (ro-

^ -
c JJuloae strips conlainlng separated HIV IP 3- 1
proleina are reeded wi|h lesl toes.. 4 to 27 :
arto eonlfgj strong positive, weak positive and
negative | nos. 1 to 3 i aer(. flniitiotfy bound
.
speed ic Hly in HIV proteins Is visualised by
usinn goal anhhumnn IrnmunoglpbuHn-biolin
conjugate and 4- cMoro 1- naphtha I substrate.
A set uni is considered positive if it shows
antibody binding lo el least 2 gene preducts
of 1 he 3 maigr HIV genes , namely gag ip 17, p Qam&
24 , p 55), env ( gp 41 . gp 1ZD . gp i c- O i and poi '

HIV- 2
ip J i . p Jj l and p 6 f . Examples «
typical results
are sera ngs . 4 , 5 . £ for positive .. 7 & for . «C I " *P % I IH
negative and ft , 21 tor week positive rations, '
iWi wniTTwr

( Courtesy: Prol T, Jaccb John . Depahmenl of

Virology and Immunology , CMC Hospital .
Vellore.]
fig, «z. 5 HW
negative i
— Wesfarn bioi test strips poswv* ( pud

pyriqrEGt IQ
596 -
* Tc^ltioc k of Microbiology *

The three groups tit HIV-1 {M , O, N } miy
represent independent transmissions from
chimpanzees to tinmans. The source of HIV 2 Has
already been established as SIV from the sooty
- affected - In some places, more women arc femnd
mungaher rmsnkev Ctrcocebus a tvs.
The virus has spread virtually all over the world,
though the prevalence rates in different countries
vary widely. Initially North America , Brazil ,
Western Kurnpe, Australia, Centra] and West Africa
had high prevalence, while Eastern Europe and
Asia, were only sparsely affected . However this
soon changed . By vigorous measures and active
public participation, the developed countries have
succeeded in reducing spread of the infect ion. Hut
in many countries of Africa and Asia, the infection
women. However, the situation is very different in
Africa and Asia where men and women are equally
infected due to the high rate of infection in
pnxSticuMs. Transmission m the developing countries
is almost always heterosexual and can take place in
both directions.
The besr method of checking sexual
transmission ( if infection is health education
regarding the danger of promiscuity and other high
risk activities. Some changes in life style and sexual
attitudes have already taken place in the USA and
the incidence in homosexuals has come down.
Persons indulging in high risk sexual practices and
spouses of infected persons should be counselled
,

has spread unhindered reaching epidemic
proportions. Differences also exist, both in the
modes of infection and in clinical manifestations,
between the affluent and developing countries.The
epidemiology of AIDS has been studied mostlv in
regarding safer methods. The use of condoms
sex '
offers considerable , though not complete ,
protection. The risk of HlVtransriuSsion increases
with multiple planners.

—
the developed nations and only sketchily in the third
world - HIV is spread only hy three modes* sexual
contact with infected persons ( heterosexual or
homosexual ) ; by blood and blood products ; and
Table G 2.7 Common modes of transmission of

Type*
HIV and Iheir TEiahve risk

of expoturt
Approxiuiitc cfuitK of
infeawa per txpvtuiv
from infected mother to babies ( intrapartum ,
.
perinatal, postnatal} There is no evidence of HIV I ScaTyaJ inf -ercminse: -
O. l UW
anal^vagma] ora1
transmission hv other means including casual
contact or through insect* , The mode* ol
I] Blood and blood ^ * 90%
products. Factor VII
transmission ol HIV and their relative risks are CTC Blood transfixion .

shown in Table 62.7. III [ issue and. organ

’ '

5 Q-9Q%
HIV is primarily a sexually transmitted infection. don-jition: sem^- ri , comw.
Inline USA it was transmitted predominantly among bone marrow; kidney etc
male homosexual*. The danger of infection is more
IV Injections and o.s-i m
injuries; shared needles
for the passive partner because mucosal tears are by drug addict
very frequent during anal intercourse and virus laden Injections with unsrerile
lymphocytes in the semen can directly enter through syringes and needles
these . One reason for che high incidence of HIV .
Needle- s rjck and other
infection in male homosexuals may be the large iniunes in health staff r
Surgical wounds
numbers of sexual partners they are reported to V Mother to baby 3096
have. In the affluent Countries, homosexual and Trutplacenol
bisexual men ire infected far more often than the At birth
heterosexuals. For this reason, infection was found After birth
predominantly in men and only occasionally in Bteasr milk

>pyrighted material
Hidden page
 * Tesrtiook nl Microbiology
-
5 million HIV infected people, the second largest
such population alter South Africa,
PrapklriMs; The prevention of AIDS rests at
present on general measures such as health
education, identification of sources and tliminairon
of high risk activities . No specific vaccine is
available . The bi b mutability, dr.nefte antigenic
^
types and subtypes, long latency and persistence in
infected cells as provinis pose serious problems in
the development of vaccines. An ideal vaccine
should not only prevent infection hut also have
therapeutic application in asymptomsti c
^ ropositivesr
The lack of a £ uLiable experimental aminal is a
severe constraint. Chimpanzees, monkevs and
rabbits car be infected but they do not develop the
disease. Infection with the simian immunodeficiency
virus is a convenient model . Several possible
strategies have been explored for vaccine production.
These include immunisation with fa} modified
whole virus; ( b) subunits, based on envelope
glycoproteins expressed in animal cells, bacteria,
viruses - or as synthetic epitopes on adjuvant
carriers; and (c) target «11 protection by anti-CD4
antibody or genet ically engi neered CD4. A number
of candkiate vaccines arc being tested in clinical
tri .Js in humans.
Treatment : Approaches to the treatment of
AIDS include: (1) the treatment and prophylaxis
f l i r t h e r It t a J i n n
ftmder SC arnlTC Mtiilpii led}. 1993, JblfuK of AIDS Mrdkine . London: Williams and Wilkins.
^
Fauci AS . :: . l HC 1-ane 199 R , HIV Ubease , En Harrison* Principles of Internal Medicine, l 4, h edtL VoL 2 . New Yorlc
i
McGraw - Hill .
CenneH for DL
. -
iase CoriTrtJ nu: Pyrvcnri . ni , 1993.
Widely KL-| M: I . Ll
of infeci ions and tumours; ( 2 j general management;
(3 ) immunorcstomivc measures; and (4) specific
anti -HTY agents.
IWnpt diagnosis and appropriate treatment of
opportune he infections and tumours In the early
stage or AIDS can be very useful and the patient
'
mav be able resume normal life in between
to
if
episodes of illness. General management of the
patient retires the understamiuig and cooperation
of the health stafil in fhe hospital and of relatives at
home. Groundless fears about imaginary risks have
to be allayed and reassurance giver that the patent
car be kept at home or treated in the hospital
without danger co contacts, if proper precautions
are taken.
Steps at immunoreslorativc therapy such as
administration of interleukin-2 , thymic factors ,
leucocyte transfusion and bone marrow
transplantation have not been very helpful .
Specific treatment with antiretroviral drugs Is
the mainstay in the management of HIV infection.
A number of effective dmgs have become available
in recent years. These include nucleoside analogues
like Zidovudine ( Azidothmidine , AZT ) ,
Didano^ ine, Zalcitahine, Lamivudinc and protease
inhibitors like Saquinavir, Ritonavir , Indinavir,
which have been used as monotherapy or in various
COMBIIIBTIONS. Adverse reactions and high cost
restrict their wide use in poor countries.
Revised clawifiniticHL system for HIV infection Morbidity Mortality
Copyrighted materia
 CO Normal Microbial Flora of the Human Body
CO
lInman beuigs [ike other animals, harbour a wide
JTT .LV o t microorganisms both on and in their bodies.
The normal m i cnobial flora are more or less consta n r
for each species and arc broadly divided into
residents and transients. The former constitute a
constant population which cannot be completely
removed permanently while the larrer vary from
time to time and are temporary. The residents
prevent permanent colonisation of the body br -
other organisms. A knowledge of the normal flora
of rhe body is essential ro an understanding of the
interaction of human beings and their pathogen
laden environment."The normal microbial flora play
an important role in body economy. They can
(1 ) become pathogenic when host defences falter
( 2 ) prevent or interfere with colonisation/!nvasion
of the body by pathogens > (3} raise the overall
immune status of the host against pa I hope ns having
related or shared antigen ? , and {4 ) cause confusion
, meningitidis and Group A Streptococcus and
in diagnosis due ro their ubiquitous presence in
the body and tlieir resemblance to some of the
pathogens. Members nf the nnrmal flora form part
and parcel of the host and include saprophytes „
commensals , fa cultstive pathogens, and true
pathogens.
The microflora of rhe intestinal tract synthesise
wf
vitamin K and several B vitamins which supply on
OCCSHftQ the body's needs . 1 he antibiotic substances
'
produced by some , fur example,. coQicins , haw a
harmful effect or pathogens . The endotoxins
liberated by them may help the defence mechanism
of the body by triggering the alternative
complement pathway , as long as they arc not
produced in excessive amounts.
On the contrary, the opportunistic pathogens
among tliem cause disease when the body's defence
mech an isms faiL Thei r abnormal multip]ication c an
cause diseases such as enteritis and endotoxin shock,
PenidJlinaJc producing organisms can aggravate
infection by interfering with therapy. Certain
streptococci of the mouth cause dental caries.
In environments Laden with pathogens for
example, hospitals, a shift in the normal flora of
the individuals there can cause an increase in
carriage of antibiotic resistant staphylococci. It has
also been shown that such people can he
rccolonised with penicillin sensitive staphylococci
of strain 502 A which arc harmless and thus
overcome the damage done , When large numbers
of people congregate from different parts of the
country as in army camps , the new recruits
experience increased colonisation rates of Nafferi*
viruses ^ uch as rhinoviruses and adenoviruses
sometimes resulting m epidemics.
NORMAL FLORA OF THE SM %
The human skin is constantly and Lontinuouafy
bombarded by organisms present in the
environment. It is also contaminated by one 's own
secretions and excretions, the extent depending on
the individual's personal hygiene . The Dora depend
on the area of the body, the clothing one wears,
one 's occupation and environment. Transient
mktoflora tend to occur more frequently on the
skirt.
Cultures front the skin have frequently
demonstrated diphtheroids ( including
Copyrighted material
600 < T^ rfrnok ol Microbiology
pno piOnibaCterii ) , itaphjlococci (aerobic and
anaerobic); Gram positive aerobic spore bearing
knit-ill i; StT. viridsn?. Sir. /aevrafhr, Gram negative
bacilli smJi as E. coht Proteus, and other intestinal
o rga n i H m > ; m i mieae \ roycobac teria ( nciin -
pathagcnic) ; Candida aihicanir, cryptococci and
PiryncMponiJi] omit.
Often the skin uJ cKe face, neck, hands and
"
buttocks carries pathogenic hemolytic
streptococci .md staphylococci . Penicillin resistant
staphylococci are seen in individuals working in
hospitals-
] lair frequently harbours St .iph . aureus and
forms a reservoir Ihr cross infection.
NORMAL FLORA t > r THE
CoKJirNCTiYA
The conjunctiva is relatively free from organisms
due to the flushing action of tears 'fhc predominant
,
organisms of the eye arc diphtheroids
(Cafynehucterium xerox's) , MonunUa species ,
staphylococci and nonhemolytic streptococci .
NORMAL FLORA OF THE NOSE . Within 12 hour: after birth alpha hemolytic
NASOPHARYNX AMJ ACCESSORY
SirvtiHus
The floor of the nose harbours corynebacceria.
Staphylococci anti streptococci. Hacmttphi} uf
species and Momotlli IKIUUII may also be seen .
The nasopharynx < ht the infant is sterile at birth
but, within 2-3 days after hirth , acquires the
common commensal flora and the pathogenic flora
carried hv the mother and die attend ants.. The
nasopharynx can he considered the natural habitat
of the common pathogen it bacteria which cause
infect ions of the nose, throat, bronchi and lungs-
Certain Gram negative organisms from the
intestinal tract such as Pseudomonas aeruginosa,
E .ioli, paracolons and Pmteus are also occasionally
Found in normal persons. After penicillin therapy,
they may be the predominant flora.
K
NORMAL FLORA OF m i : MOLTH AM )
UPPRR RFKPJRATORY TRACT
The mouth contains a plethora of organisms -
pigmented and nnnpigmented microCOcci , some of
which are aerobic, Gram positive aerobic s|n > re
bearing baulk, culi forms, Piute us and lactobacilli ,
The gum pockets hetween the teeth , and the crypts
of the tonsils have a wide spectrum of anaerobic
flora - -anaerobic micrococci, mlctoaerophilic and
anaerobic Streptococci , vibrios, fusifurm bacilli,
COryncbactKnum species , autinomyces, leptothiijc,
mycoplasma, ueusem, and bacieroides arc ail found
in varying extents . Among fungi, Candida and
geoCrichum have been reported.
The mouth of the infant is not sterile at birth.
It generally contains the same types of organisms
in about rhe same relative numbers as those present
in tlie mother’ s vagina , that is a mixture of
micrococci , streptococci , coliform bacilli and
Doderlien’s bacilli . These organisms diminish in
—
number during the first 2 5 days after birth and
arc replaced by the types of bacteria present in the
mouth of the mother and nurse.
streptococci arc found in the upper respiratory tract
and become the dominant organisms of the
oropharynx and remain so for life . In the pharynx
and Trachea, flora similar to that of the mouth
establish themselves . Few bacteria are found in
normal bronchi. Smaller bronchi and alveoli are
normally sterile,
NORMAL FLORA OF THK INTESTINAL
T k vt :T
-
In 80 90 percent newborn infants , the meconium
is sterile but in 10-20 per cent a tew organisms,
probably acquired during labour, may be present.
In all cases, within 4-24 hours of birth an intestinal
flora is established partly from below and partly by
invasion from above. In breast fed children the
intestine contains lactobarilli { E bdidiis constituting
99 per cent of total organisms in the feces ) ,
Copyrighted material
 'i MnmaJ Microbial Flora d the Human Body
enterococci , colon bacilli and staphylococci. In
artificially fed {bottle fed ) children L, acidophilus
and colon bacjlli and in pan by enterococci, Gram
positive aernhic and anaerobic bacilli . With the
change of food to the adult pattern, the flora change.
Diet has a marked influence on the relative
composition of the intestinal and fecal flora.
In the normal adulth the microorganisms on the
surface of the esophageal wall are those swallowed
with S .LIL 'LM and food. Because of the low pH of
the stomach , it is virtually sterile except soon after
eating . In patients with carcinoma of the
Stomach OC achlorbvdri .i nr pylon -c obstruction ,
there is proliferation of Gram positive cocci and
bacilli.
The number of bacteria increases progressively
beyond the duodenum to the colon , being
comparatively low in the small !uterine. In the adult
duodenum there are HF-106 bacteria per gram, in
—
the jejunum and proximal ileum 1(F 10* batten A
per gmm , and in the lower Ileum and cecum
bacteria per gram of contents . In the
duodenum and upper ileum , lactobacilli and
enterococci predominate but in the lower ileum
and cecum the flora resemble the lucal flora. There
are about 10" bacteria per gnam of contents in the
colon and rectum, constituting 10-20 per cent of
— —-
the fecal mass In the adult normal colon, the
resident bacterial flora are mostly {‘96 99 per cent )
anaerohes anerobic streptococci anaerobic ,
lactobax il!!, dostiidbi., and bacteni cs and about
-small —
1 4 per cent aerobes enterococci, conforms, and
numbers of Proteus , Pseudomonas ,
lactobacilli , mycoplasma , Candida and others .
N RM
' . F LORA OF I HR
GENITOURINARY TRACT
Mycobacterium an i egrt M ff >. a harmless commensal,
in found in the smegma of the genitalia of both
men and women. This may, by LTH presence in the
voided specimens of urine , cause confusion. From
apparently normal men, aerobic and anaerobic
bacteria can be cultured from a high proportion,
» 601
including lactobacilli. G .irJ. vaginalis , alpha
hemolytic streptococci and Bacfero.'Jes species.
Chkrn. nachumatis and UftaplaantA NREALYTJ cum
may also be present. The female urethra is either
sterile or contains a few Gram positive cocci.
The vulva of the newborn child is sterile but
after 24 hours it acquires a varied flora of
nonpathogenic organisms from the skin, vagina and
intestines. The nature of the flora in the vagina
depends on the pH of its secretions and its enzyme
content. In the first 24 hours it is invaded by
micrococci, enterococci and diphtheroids. In 2 3 -
days , the maternal culrin induces glycogen
dept iM Cion i n t i LC vaginil ep i theliurn . This facilitates
the growth of a lactobacillus {Doderlien 's bacillus)
which produces acid from glycogen, and the flora
for a few weeks is similar to that of the adult. After
the passively transferred estrin has been eliminated
in the urine, the glycogen disappears, along with
Doderlien's bacillus and the pH of the vagina
becomes alkaline. This brings about a change in
the flora to micrococci, alpha and nonhemolytic
streptococci, coUfbrms and diphtheroids. At puberry
the glycogen reappears and the pH changes to acid
due to the metabolic activity of Doderlien's bacilli,
.
E eoli and yeasts. This change probably helps in
-
the prevention of colonisation by pos .iblc harmful
micro organisms. During pregnancy there in an
increase in Staphylococcus epidermidis , Doderlien's
bacilli and yeasts. Occasionally other members of
the intestinal flora maybe present. After menopause,
the flora resembles that found before puberty. The
normal vaginal flora often includes anaerobic cocci
and hacilli, lixteria, anaerobic streptococci, tnimeae,
mycoplasma, GardnereUa vaginalis , neisseriae and
spirochetes.
BACTERIA IN THEBLIKID ANU
TISSUES
The commensals from the normal flora of the
mouth , nasopharynx and intestinal tract may get
into the blood and tissues. They are usually quickly
eliminated by the normal defence mechanisms of
opy righted material
Hidden page
Hidden page
Hidden page
 * BartetiQlngy ol W-Hlor Milk and Air
Durham* rubes contain E. CKJ /J - From the number
of positive tuba obtained, results are read nfF the
probability ts ’cs . Further confirmation of the
presence of E , coli can be obtained by testing for
indole production and citrate uriliutum.
L
) A /:‘mh . anr filtration method A measured
' 0.5 mg per liire, for a. minimum contact period of
volume ol water is (titered through a millipore filter .
All the hacteria present anq retained on its surface,
It is placed on suitable media face upwards and
incubated at the appropriate temperature, and the
cola diet that develop on the surface of the membrane
arc counted. After 1 ft hours of incubation the
presumptive coliform counts and E . coli counts can
be directly made.
3 . Detection of fecal sirepiocnoci -
- con Cam i rati on of water Coliform courts arc rot
Subcultures are made from all the positive bottles
ir the presumptive coliform test into tubes
containing 5 ml of glucose azide broth - The
presence of Srr. focc-Alit is indicated by the production
of acid in the medium within IS hours at 45 C . "
Hie positive tubes should be plated onto
MaeConkey 's agar for ennfirmarinr -
Millipore membrane technique can also he
adopted for this purpose.
4 . Examination for Cl - perfmgent; This
is tested hy incubating varying quantities of the
water in litmus milk medium { anaerobically ] a[
57 " C for five days- and looking for storm v
fermentation .
5. Tests for pathogenic bacteria: Linder
special circumstances, specific pathogens such as
typhoid bacilli or cholera vibrios may have to he
looked for in water, This used to be done by adding
the water samples to tenfold concentrated liquid
media , incubating and sub Culturing onto
appropriate solid media . A simpler and more
sensitive method is ro filter the water sample
through manbnne filters and incubarc the filters
cut appropriate solid media.
VIRI SES WATER
IN
Methods are available for the isolation of
605
enteroviruses and other cyiropathogenic viruses front
warer bur rhev do nor form part of routine tesrtrg.
As a general rule, it is assumed that the viruses in
water are destroyed by eh lor i ration , when the
concentration of free residual chlorine is at least
50 minutes at pH below 4 and a turbidity ol
1 nephalomctrle turbidity unit nr less-
PROTOVOA IX WATKK
Entamoeba histolytica , Ciiardia species and
ftoiantrebum coir can contaminate drinking water
However, there is m > good indicator for pruioioal
reliable as indicators of protozoal contamination
of chlorinated water as they are more resisrant to
mf
chlorine than are coliforms-
BACTERI0L0GY OF MILK
T V I ' I' S u f. |1 \ C T E H I\ I X Mu K
These ear be diLss- ified as- below:
1 . Acid forming bnclcria: The commonest
are lactic streptococci including Srr. hetia and 5rr
faccaJji. Ijcrobjcilli are also found. Tb»e ferment
'
laetoHe in the milk , producing aeids- , mainly lactic
acids, which lead to the formation of a smooth
gelatinous curd.
2 . -Atknli forming bacteria: These consist
of alkaligercs sjip, -some aerobic spore bearera and
Achromobacter. These render the milk alkaline.
3, Gai*forming bacteria: Coliform bacilli
are the com monesi . Others are Cl. perfringens and
Q fctifyricum* Acid and gas are produced , A smooth
gelatinous curd riddled with gas bubbles. is formed.
Cullfonn bacilli am responsible for the ropiness in
mult -
4 , Proteolytic bacteria: Sporc - bcaring
aerobes such as B. subtilis and B . cereos, Proteus
vufgam, staphylococci and micrococci come under
this category.
n . Inert baclerin: bacteria which produce no
visible change in milk arc called inert. These
Copyrighted materia
Hidden page
Hidden page
Hidden page
 iJ .
Bftc e 'iQlQ? / d ter . MilH fend Air 609

further Rcadiaf
Linton *\I I and HM Dick (cds ). 1990. Bacteria in the Environment. In Toplcy &. Wc/soni Pdodfim of Bacteriology
J

\riroiogy and fmmunrrv, Veil I General Bacteriology and Immunity. Section B. 211-291. London: Edward Arnold .
^
World Health Organization, Geneva: GupdWjnes fer Drrnijng Vtirer Quality.
'

VdL 1, Rtrarnmendatnems; (1983)

VolIL Health Criteria (1984)
Vol , 111, Drinking wafer quality control in small community supplies ( 1995).
*

Copyrighted material
Hidden page
Hidden page
Hidden page
Hidden page
Hidden page
 * Mecical Mycology r 615

Liib4 ii,al4 i,) di

) i
Flrf rOiitin*. method
of diagnosis is by the examiner n ; t
'

Scrap!np are taken from tl:. .. f ringworm

lesions . T h e specimen TS mix I with a lrop of Ltflfc
KOH on a slide, and after p acini; .:. cen rslip, fh
]ireparation is gently heated tn M . !
<
'
1

MktOscopy reveals branched pc .Ln l -_

65.5 ). Selection of infected 1 .

lntccted liair will be tluoresci i . :

'
« =
facilitated b y exposure to U\ I : it Wood '? limp}*
- i '
B
infection may be di 9 tingubbed tn wet mounts ,
'
'Wpthrt * in which arthr< p< res are seen as ,1
sheath surrounding the hai and 'cndotl
which the spores are insidn r : . "i
hair h , 'i : FJ 1.;.
.
65.6) Demonstration of the Ltngju* tails may
difficult and may he po & s ib 1 ;i 1
'
le 1
with KOK for a day or two.
Species identification is orJy by ^U LUJC . Fig H 5 Derm.Uopbylc hypba -o in akin scraping , KOH
examination . Specimens are inoculated on
mount , Partly digested epithelial cells form The
Sabouraud ’s medium { with antibiotic ^ and background.
cycEoheximitfe) andincubated at room temperature .

I -

- *

Fig. 65 - 5 Etlothrk and endglhrli lypes of Heir Infection .

Fig. 65.4 Trlchaphylnn utbrum culture (noun) showing 1 , Bclolhrhx type with Fungal arthrospores
n fcroc on Idid along sid ^ a DI r - vp^ ro and long surrounding hair 2. endothrlit type showing
cylindrical macroconma . arihrasporea inside hair shaFl,

Copyrighted material
 616
^ or Microbiology
* ToxlbOd

Table 55.2 Clinical types of dernfialophytoses and Trentnu ' iil : Topical antifungal agents arc usually
their common causative agents effective. 7? rubrum infections may be resistant to
jDlSCilSC Common causative agents treatment . Oral grbeofulvin is the drug of choice.

Tinea capitis Mkntpomm any species, i: wmutisis

Trichophyton most species Gaudidosis ( candidiasis, moniliasis ) is an infection
Fav US T. schoenkinii T
M . gy' pscum of the skin, mucosa , and rarely of the internal
Tinea barbie T nibmmr T.mcntJgFophytcs, organs , caused by a yeast like— fungus CWxb
Tr \Trrucosum idfciican.s, and occasion . LLLV by other Guufidl species .
TJHSM ippiivjtTTfii Tr Luncentticum Candid* alb/csAf is an ovoid nr spherical
TJJICJ oxpoA T. nthrum jnd any « IIHT huddmg cell, whidl produces pseudnmycclia both
dermatophyte in culture and in tissues { Fig. 63,7) . Candida species
T!cruris E.Ax,LErxwjr » T! ruftfiun
I

T. i*di$ X! mbnutu E, fimwm
Ectotlmx ha.ir Mktosp&tmn speriei,
infecliurt T rufcnjjT] TlffKiuiyqpJtyw
F the rnm monest pred i sposi ng factor being diabetes .
Endorhrix halt T ^choenJemi , Tr hymirojii,
infection T, viuheemn
Growth is slow and colonics may .LpjiCiLr onlv in
1-3 weeks.
Epidemiology: Dermatophytes!B occurs
throughout the world but certain types of disease
"
and some specie* d fungi show geographically
restricted distribution. Social and mltuml patterns
also influence deroiatoph ^ toses. Tories petlix , so
common in the temperate climates where all went
shoes ]* fare m die tropes where most walk barefoot.
• frequently in pregnancy , and oraf fjfirusft found
Many factors, such as age, hormones and intcrcuncnt
diseases, affect rhe susceptibility to dermatopliytosi;; .
Depending on theii natural habitat ,
dermatophyte* may he dlassfied as anthropophilic,
zoophilk JIUI gcophiliL species. Human beings arc
the main only hirsts for n nth r lipophilic
dermatophytes. T. rutrain, E. fbcouian and M.
auJoufrtJj are examples . They Ciutc mild hut
chronic lesions. Zaophilic species are natural
parasites of animals. Examples arc 7 ' rerrumsum
in cattle and A/. CAtiii in dogs a[]d cats. Human
infections with zoophilic dermatophytes cau ^ c
severe inflammation but are more readily curable.
Geophilk species, which occur naturally in soil ,
are relatively Less pathogenic lor human brings*
Examples are A/, gypseum and T. jjethii.
arc normal inhabitants of the skin and mucosa.
Candidosis isanopportunisticendogenous infection ,
Cutaneous candidosis may be intertriginous or
parooych i al.The form cr i s a n c rythc rnatous, sealing
or moist Lesion with sharply demarcated borders ,
where papular Lesions arc most prominent.The sites
ilflpcred are those where the skirt is macerated by
—
perspiration groin , perineum , axillae and
infra mammary folds. Paronychia and onyehia are
seen in occupation* th.ir lead to frequent immersion
of the hands in water.
Common mucosal lesions arc va firjjris
characterised by tun acidic discharge and found ^
commonly in bottle fed infants and the aged and
debliiated. Creamv white patches appear nn the
tongue or buccal mucosa, that leave a red oozing
surface ou removal.
Intestinal candidosis. is a frequent sequel to oral
antibiotic the rap v and may present as diarrhea not
responding to treatment , bronchopulmonary
eandidosis is seen a & a rare complication ot pre
existing pulmonary or lytttmic disease. Systemic
-
infections, such as septicemia, endocarditis and
meningitis may occur as terminal complications in
severe generalised diseases such as leukemia and
in persons on prolonged immunosuppression
Candida granuloma and chronic mucocutaneous
candidiasis arc serious manifestations seen in
ImriuDodeJicicTideE.

Copyrighted material
 * Medici Mycology * 617

therefore, of greater significance. Cultures can be

J?
'

obtained readily on Sabourand 's and on ordinary m

J bacteriologies] culture media. Colonies arc creamy

white, smooth and with a yeasty odour. Candida
j/bieans ran be identified frum other Candida

0 . species LC. .tfoliamidxe , C - tropicslis , C .

pscudoiropicAli C . krusci, C, guiHicrmondu, C .
I L ^
parap&ilosis , C . vj'swanar/ jj j ) by growth
'

diaUCtensrin and sugar Hsmmildtion and I or men Lai in ML

tests.C. idbj( T[in.H alone forms chlinnyduspcires on
Z J/ com meal agar admits at 20 °C . A rapid method
of identifying C. iWuesns is based on its ability to
FI& $5.7a Yeasts and chiemympores or Candida form germ tubes within two hours when incubated
albicans
in human serum at 7>7 f'
C ( Reynolds - Braude

>
phenomenon ) .

)$s
Agglutinins appear in the sera of patients but as
thev are frequent in normal persona also, they are
not helpful in diagnosis. Delayed hypersensitivity
to Candida is so universal that skin testing with
m Candida extracts is used as an indicator ni the
functional integrity of cell mediated immunity.
Management of cindliiotif it mainly by
removing the predisposing causes . All Candida
v strains art sensitive to Nystatin bait as it is poorly

* '
%
absorbed from the gut, it is not useful in systemic
diseases. Amphotericin R, S - flunrcKTtnsine and
clotrimazole may Le used for disseminated
candidonih .
4

>
\ i \ *

Fig . £$.71) Cand/ tfa atbicans in a balnea speeimeh o!

spwium The presence of hyph « l olomenu in
DEEP MYCOSES
Doqp mycotic infections may be classified as those
that afreet mainly or ndlisivtly tlie suheuMucous
r r

tissues ( subaitaneous or intermediate mycosis ) and

those that involve tlie internal organ.* ( Jeep scaled
addillon to yeait lorms indicates colonisation ol
lissut fay the organism.
or systemic mycoses).
Sfi &i iifjot^itrs rn i roses:
•

Diagnosis can be established by microscopy and 1. Mycotic mycetoma

nillpt . Wet films; or Grain stained ymearx Imiti 2 . Chromoblastomycusis
lesions or exudates show budding Grant positive 3. Sporotrichosis
edit - AH Caodilbl can be neen on normal nkin or 4. RhiuosjHiridiusis
mucosa as wclL only its abundant presence is of 5 - Suhcuraocous phycomyensis
significance. Demonstration of mycelial forms Systemic iqmwrs;
indicates colonisation and tissue invasion and is, 1. Cryptococcosis

pyngh
Hidden page
Hidden page
620 ^ Teubcok o \ M ' crobiology
df
0
m SQ
o
d^t
£7, 0
^0
Fig. 65 - 5 Sporothrix {Sporolrichum/ schenftii: culture mount showing line branching hyphne and pear shaped
com dia borne in rosette I ire clusters at tips of lateral branches and singly along sides of hyphae
U n i N o s l ^k l D H i s i S
Rhino spot idiusis is a chronic granulomatous
disease characterised hr the development of
friable polype usually confined ro rhe no&e, mouth
or eye but rarely seen on the genitalia or ocher
mucous membranes. Though the disease wan first
identified in Argentina, rhe large majnricynf cases
come from India and Sri Lanka. While the disease
is generally confined to MUIIOUH membranes ,
hematogenous. dissemination has been recorded
very rarely.
E listologically the Lesion is composed of targe
mm hers of fungal spherules embedded in a stroma
of connective tissue and capillaries. The spherules
are 10-200 prn in diameter and contain thousands
of endotpon (Fig 65.10) .
*
The causative fungus Rh rnosporidw 171 xeberi
'
has nnt been cultivated in media . Tire mode of
infection is not known though infection is
believed TO Originate from stagnant water or aquatic
life.
Si. HCl T tM '. m sPtl VtitiM ^ ciirtlg
In this condition, originally reported from Indonesia
and subsequently identified in many Asian and
olo
rQ
0
o%>
olG
African COUOtriesh a painless subcutaneous nodule
develop which enlarges to involve a whole limb
*
or large areas of the body The causative agent IS
dtsidioboltti haptosporus , a saprophytic
phycomycctc found in decaying vegetarian and in
the intestines ol many reptiles .ind amphibians . If
has been suggested that the infection may be
acquired by insect bites.
< ] H Y I P T( X : < H I T I O S I S
.
Cryptococcosis (torulosis) is a subacute or chronic
infection caused by the ve.isr Crypfococcus
ncoformans. It is a round or avoid budding cell, 4 -
20 pm in diameter,with a prominent polysaccharide
capsule ( Fig. 65.11). It is .L soil saprophyte and is
particularly abundant in the fc«S of pigeons And
other birds .
InfeCQOfi is usually acquired by inhalation but
may sometimes be through the skin Of mucosa. Most
infections arc asymptomatic Pulmonary .
cryptococcosis may Lead to a mild pneumonitis. As
OO calcification occurs , healed piiltuonarv lesions
are not evident radiologically. Dissemination of
infection leads to visceral, cutaneous and meningeal
disease . Visceral forms simulate tuberculosis and
Copyrighted material
 * Medical Mycology * 621

hy culture, The capsules stand out in India ink

r r ' lAfcTSft
preparations - The fungus grows readily on
Sabouraud 's agar forming smooth, mucoid, cream
coloured colonies-. The ability to grow at 37 ®C
* *
r* C and hydrolyse urea differentiates C- mwfojmm.sfrom
* : jinnpathogenic cryptwocci . Pathogenicity can be
V
demonstrated by intracerebral or intraperitoneil
H
y
»
*
#
A inoculation inco mice , which develop a fatal
infection . Capsule ted budding yeast cells can be

h& n
/
* demonstrated in the brain of infected mice .
i
Two perfect stages of the fungus have been
discovered.They belong to the class Basidiomyeetes
wt TL «- '
and have been termed FHobstldidh DCofomans
and F. bnsilispora.
? - - 7 ^S
*
Four Knlogkil types of ttyptocoecal capsular
-
polysaccharide A, BH C and D - have been
Fig .65.1 D ncsponclium Mature Sporangium ideiititled. Demonstration of the capsular antigen
(com tally loeatcdi in the stroma of a lesion.
by precipitation can sometimes be valuable in
cancer clinically. BorKH ^ nd joint? may be involved. diagnosing some cases of vryptncnccaI meningitis,
Cutaneous cryptococcosis vines from small ulcers when the CSF is negative by smear and culture.
ro large granulomas , Ciyptocoeczl meningitis is the Amphotericin 13 , o -lluofOCytosine. clotrimazole and
most serious type of infection and can resemble miconazole are useful in the treatment of the disease.
tuberculous or other chronic types of meningitis. Cryptococcosis is worldwide in distribution . As
Its onset it iAtidimi and. the cOUfJc slow .ind it Was originally reported from Europe , it used In
progressive, [ r is ofren seen in AIDS . he known HR 'European blastomycosis'. Several
Diagnosis is established by demonstration of cases of cryptococcos i s have been idenrifled in 1Lidia ,
Cipsukttd, budding yeast cells in the lesions and this being the onlv deep mycosi -s common in this

r . **

rs

. .
Fig S 5 fi Cryptococci s nfiafai st CPUs surrounded by
a Ijtg o capsule-
iopyrightet
Hidden page
Hidden page
624 4 Textboah DI MicrotJiO ogy
Amphotericin B has been found useful in
therapy.
OPPORTUNISTIC SYSTEMIC MYCOSES
Some saprophytic filngi which arc ubiquitous in
the environment are important in medical mycology
for two nelsons , Firstly, they are common laboratory
contain LTia. nts tin culture media— AHf >Crgilhli ,
PcniKiUivm , Mucor and Rhizopus species grow on
virtually anything , Secondly, they can produce
serious and even fatal infection in persons who are
otherwise debilitated . Aspergillosis and
mucormycosis are Lcnportanr opportunistic systemic
rnycuses -
A S P K t t t j t L I . UK IK
Aspergill! and fleniclltia constitute the commonest
MOULDJ teen on dump bread nr almost -Uiv other
organic matter. Qf the 300 odd species of aspergilla,
A . fomigafus is highly pathogenic for birds, and
occasionally Causes invasive disease in human
heings - A few other species may also cause
opportunistic human disease . The commonest
human disease caused by aspergillj is otomycosis .
vm
jf
(
Flgr 65.13 HlsJgplasma COpsulalUVf )
Systemic aspergillosis occurs as die following
clinical types:
1. Pulmonary aspergillosis
.
a. Aspergillus asthma
b. Bronchopulmonary aspergillosis
c . Colonising aspergillosis ( AspeiyiLlnma )
2. Disseminated -aspergillosis
Aspergillus asllima occurs in atopic individuals
following sensitisation to inhaled aspergdlus spores.
Er bronchopulmonary aspergillosis , the fungus
grows within the lumen oi the bronchioles, which
may he occluded by fungus plugs. The fungus can
be demonstrated in sputum . The condition Is made
worse by the development of hypersensitivity to the
fungus . Colonising aspergillo- is usually develops
in pre - existing pulmonary' cavities , such as in
tuberculosis or cystic disease . The fongus grows
into large "bails' (aspergilluma}. Surgical removal
becomes necessary as the disease commonly causes
massive hemoptysis. In invasive aspergillosis, the
fungus actively invade* the lung tissue. Disseminated
aspcigillosis involving the brain, kidney and other
organs is a fatal complication sometimes seen in
i
opy righted r rprla
debilitated patients on prolonged treatment with
- Madlcat Mycology *
antibiotics, steruids and cytotoxic drugs.
Diagnosis may be made by microscopic
examination and by culture. The fungus s tOwS rafiully
^
on culture media . Identification of Aspergillus is easy,
based on growth tbafi£te nit MH and morphology.
AspctgjUi have septate hyphae. Asexual conidia are
arranged in chains, carried on elongated cells called
‘
stengmata , borne on the expanded ends (ve -nudes) of
donidiuphurts (Fig . 65.14).
As asper dh are such common contaminants,
^
their demonstration in exudates and isolation in
cultures have to be interpreted with care .
P E M C M -M O a l M
Pcnicillium species have been very rarely
incriminated in opportunistic human infections E
mjfincfJeJ has been reported to be an Important
opportunist pathogen in the HIV infected .
Members of this genus can be identified In the
brashlikc arrangement of ennidia ( Fig. 65.14).
Mi <:n I* MYCOSIS
Mucormycosis in an invasive disease Caused by
phvtOTTVVCetes-, m .iinly by species of Rhr/ opus,
Mucor and Absidiia. It used to be a rare terminal
complication of uncontrolled diabetes and other
chronic debilitating diseases. TTie incidence of the
-
disease has increased con .i.. Lerah ]v as a result of
the widespread use of antibiotics, steroids and
ajitimctaboliicx. The fungi am normally avifulenr
and are able to invade tissues only when general
resistance is extremely low-
Thc primary infection ii usually in the upper
rcs]i i ratory tract or nose, where the spores gcnt -inaie
and the myceiia invade the adjacent tissues the
orbit , sinuses and the brain. Primary in tectum may
— MYCOTIC POISONING
also occur in the lung, the fungL mvadingthe arteries
to cause thrombosis and infartLion. The disease is
fatal.
Diagnosis is usually made during histological
examination of autopsy material, by the presence
of hrnad, nuresepTate myceiia ifi tissues. The fungi
"
can be grown easily on Sabuuraud medium -
625
without cvclohcximide . Mucor shows branched
ST
sporangiophores arising randomly along aerial
mycelium . Rhi /oids are absent. Rhizopus has
rhizciids, and sporangiophorcs arise in groups
directly above the rhizoids ( Fig. 65.14) .
( ) fOM YCOSIS
Otomycosis is a fungal infection of the external ean
It is a very common disease and is usually caused
by species of aspeigtili ( A. iwgfr, A. fumjganis) and
penkiilia. The symptoms ore itching, pain and
deafness. Secondary bacterid infecrion , commonly
due to Pseudomonas and Proteus , causes
suppuration . Diagnosis can be made by
demonstration of the fungi in scrapings and by
culture.
OCULOMYCOSIS
Mycotic keratitis usually follows corneal trauma.
Fungal spores colonise the injured tissue and initiate
an inflammatory reaction, leading to hypopyon ulcer
and endophthalmitis. If not recognised and treated
earlv, enucleation may become necessary. The
widespread use of corticostcnods in ophthalmology
has resulted in an increased incidence of
keratomveotis .
Many saprophytic fungi can cause ocular
infection , Aspergillus species , Fusanum and
Candida diiiomt being most often responsible.
Diagnosis may be made by examination of
deep scrapings. Superficial swabs may not show
the fungus. Local application of amphotericin B,
Nystatin and Pimaricin ( Natamydn ) may he
useful.
Many fungi form poisonous substances. Mycotic
poisoning is of two types - myccrism in which a
fungus which is eaten for itself causes toxic effects
and mycotoxicosis in which fungal toxins
contaminate some article of toed .
Copyrighted material
626 4 Tesftbook ot Mierobiotagy *

ft 0 v:

* J

r
I f

PENJClLLlUMSp ASPERGILLUS Sp.

.
r

MUCOR $P RHIZOPUS SP.

fig. eS-14 Common contaminant fungi. culture mount) . PetfcMium showing the 'peniciitus or brajahh cgnsisung
'

-
of dui ns nt spares ertehding from I he ends nr shori br inchgs oi conicfinphares . Aspetgfflus showing
unbranched non ^Bplite conidicphores. terminal mg in a gtobose vesicle bearing phtallds from which arise
chains gl conldis . JWucor showing nonseplate mycelium without rhliolda irootlikc- structures) .
SflOrdtitplophorea, which miy be branched terminate m forge globose sporangia containing numerous spores.
,

RhUopus showing nennseptate mycelium with rhlzolds . UnbrAnthad sporangiophcirea arise opposite rhizoids.

Copyrighted material
 4 Medical Mycology r 627

Mycctism has been known from ancient times, com and peas. IT is highly toxic to animals and
several varieties of poisonous- mushrooms having birds, and probably to human beings as well It can
been identified as inedible, Mycctism may cause cause hepatomas in ducklings and mss* and its
gastrointestinal disease1 dermatitis or death. The possible carcinogenic effect in human beings has
-
hallucinogenic agents (d lywrgic acid, pwilocyhdn )
produced by the Psihcybe species and other fungi
caused great concern. There have been several
reports of aflatoxicosis from India, involving human
have Attracted much attention in recent years.
-
The best known my corns in is aflatoxin '
produced by Aspcrgiltus ffrvm It is frequently
beings and animals.
Ergotoxicosis (ergotism ) is due to the toxic
alLdoids produced by the fungus C/awceps ppuin,
present in mouldy foods,. particularly in groundnuts, while growing on the fruiting heads of rye .

F u r t h e r keailulft
Evans EG V et al (eda). 3989. Mndol Mfrologft Oxford: Oxford University PVess.
Kibbler CC ec ah 1996. fVincipIcj and Aactrcc osf Climod Chkhem Wiley.
-
Kwun Chung KJ and JE Bcnnen 199i . MadSssalMycohgyr, Philadelphiar Lea and- FGfei cr-
^
Riduidson MD and DW Warnock 1993, Fungi/ /nfretfon. Oxford- Blackwell .
Rippon JW . 1981?. Medical Mycology. Philadelphia-; WB Saunders.

opy righted material

8 dr w
 CO Laboratory Control of Antimicrobial Therapy
CO
ANTIBIOTIC SENSITIVITY TESTS
from rare exceptions like Strep. pyogenes,
Afiart
pathogenic bacteria exhibit very great strain
viriftidpi in susceptibility to antibiotics anti
CbClDOtllcriptUtk agents. Thin is particularly
marked in the case of Staph. aitmrs and Gram
negative bacilli. Therefore , it is essential TO
determine die Susceptibility Ot isolates of pathogenic
"
bacteria to antibiotics that arc likely to be used in
treatment. Antibiotic sensitivity tests are of two types,
diffusion rests and dilution tests.
Diffusion test: Here the drug is allowed to
diffuse through a solid medium so that a gradient
is established, the concentration being highest near
the site of application of the drug and decreasing
with distance. The test bacterium is Seeded Ort [he
medium and its sensitivity to the drug determined
from the inhibition of its growth. Several methods
have been used fur rhe application of the drug. It
may he added to ditches or holes cut in the medium
or to hollow cylindeni ( Heady cups) placed on it.
The method most commonly employed is to use
filter paper discs, impregnated with antibiotics.
The dj.se diffusion method uses filter paper discs,
b . O rnm in diameter , charged with appropriate
concentrations of the drugs. The discs are stored
dry in the cold , They may be prepared in the
laboratory or purchased commercially. A suitable
dilution of a broth culture or a broth suspension of
the test bacterium is flooded DO the surface of a
solid medium (MutOfir- Hinton ugar or nutrient
agar). The plate is tilted to ensure uniform spreading
and the excess broth pipetted off. Inoculation may
also be performed by spreading with swabs . After
drying the plate 0 ? "C for 30 mins), antibiotic
discs ( four or five per 10 cm plate) arc applied with
Sterile forceps. After overnight incubation , the
degree of sensitivity is determined by measuring
the zones of inhibition of growth around the discs.
Growth will be inhibited around discs containing
antibiotics to which the bacterium is susceptible
but not around [hose TO winch it is migrant.
The diameter of the zone of inhibition is
influenced by a variety of (actors, such as diftusibility
of the drug, the disc concentration, the nature and
composition of rhe medium, its thickness, presence
of inhibitory or sti m uUtory substances, pH and rime
of incubation . It is , therefore , necessary TO
standardise all the variables. It is also necessary to
check rhe potency of the discs periodically using as
control a standard bacterium of known sensitivity,
such as Staph, aureus Oxford strain (NCTC 6571).
There are several recommendations regarding
the antibiotic concentrations to be used in discs.
The Kirby-Bauer and the ICS methods art in
common use , Table 66.1 shows the disc
concentrations and the critical zone sizes for
antibiotics in common use.
A suitable method for routine use in diagnostic
laboratories is the technique originally described
by Stokes . This incorporates built - in controls
against many variables and therefore provides
dependable results, A standard sensitive strain of
bacterium is inoculated in the middle tliird of the
culture plate. The standard strains used are Staph ,
aureus ATCC 25973 . K . cnii ATCC 25922 or
Ps.acruginoaa ATCC 27853 , depending on the
bacterium to be tested . The test bacterium is
Copyrighted material
Hidden page
630 * Medical Mycology

inoculated, After overnight incubatmn, the
'minimum inhibitory concentration’ ( MIC) is read
by noting the Inwest concentration of the drug that
inhibits growth . The "minimum bactericidal
concentxatinn (MBC) is the W»t concentration
of the drug that lulls the bacterium. It can be
estimated by subculturing bom the broth tubes that
show no growth on to suitable solid media.
The ‘igar dilution’ method is more convenient
when several strains are to be tested at the same
time. Hem * serial dilutions of the drag ux prepared
in agar and pouted into plates. The advantage is
that many strains can lx inoculated on each plate
containing an antibiotic dilution. Automated
versions of sensitivity tests are available and are in
use in large laboratories.
Antibiotic aaauya in body fluid*: The & c
arc required to verily whether adequate drug
concentiarinns arc achieved in blood and other body
fluids, and to guard against excessive blood levels
of potentially toxic drugs. The assays are generally
done by making semi dilutions of the specimen
and inoculating standard suspensions of bacteria of
known MIC . Assays can also be done by the agar
diffusion method - This depends on the direct
relationship between antibiotic concentration and
the diameter of the zone of inhibition with a
Standard sensitive Strain of bacterium.

Purther itfe . id i
L«uan V. 19 Bb Antibiotics in laboratory Medicine. Baltimore: Williams and Wilkins.
Murray P et al («h). 1995. Mwual of Clinical Microbiology. Wuhlngran: ASM EYC&L.
Willi um RJ and D1 Heymann 1998. Contajnmcnt of antibiotic resistance. Science 2?9il 153

Copyrighted material
 Is
co - Immunoprophylaxis

An important contribution of microbiology to Table £7.1 Nalmnal Immunisation Schedule

medicine has been immunisation, which is one of the (INDIA )
mo& t cfFecrive methods of controlling infectious Age Vtecine
diseases. By systematic active immunisation , many
BCG, GPV-O
developed countries have virtually eliminated Lvaccine At birth 1 --- -- r

preventable diseases ' [ VT'D ) such as diphtheria, fa weeks

pertussis, tetanus, measles, mumps, rubella and PT- l ,OPV-t
|}

poliomyditiSr The global eradication of smallpox, of Ifl weeks DPT- 2 P -

QPV 2
course* has been the clowning glory of irmrami«ri«i
Immunoprophylaxis may be m the form of
14 weeks .5 i

-- J .. DPT- 3 GPV 3P -
( I ) routine immunisation, which forms part ( it’ basic 9 monitu Mettlei
health carei or ( 2 ) immunisation of individuals or lib-24 inentht .r , P ,P I 3 IJT, OPV
selected groups exposed to risk of specific infections . -
l fii years
( wJiKKd entry)
P B P H I P m
ROUTINE IMML NISATION
10 ynn . TT

--
< k <> fi i

Routine immunisation schedules have been

developed for different countries* and modified from
lb years
Fur prrjfiunl
— s . TT
TT 1 titf blotter
time to time , based on the prevalence of infectious !
HBiKia
diseases* their public health importance* availability
of suitable vaccines, their cost benefit factor?, and On* month aftei TT 1 - TT- 2
logistics. In India, the Expanded Programme or
Immunisation ( EPI ) ,ind the Universal Nulr: 1. Fuf institutional bifthi wily. OPV-fl as
Immunisarion Programme ( U1P) have been able AddiEimalt and noi to be couDicd fur
the primary cown* of l dowa srarcing air
to afford protection for much of the target
IS w« ki .
population against VPD$ r 2. Only for infanta mil: glim. BCG iE
The National Immunisation Schedule in force birch .
3* . A accural (LCHC uf DT to be jpvcn ID
in India is shown in Table 67.1 . children with ns dwumcniary evidence-
In India, EFI and UTP have led to a significant Dr hraCury af primary DPT imununiutian.

decline in the recorded incidence of VPDs* as well 4 A second dcra of TT to be given after
tstc inuiich tu thuac willi rccutfd dr
as of infant and child mortality, for example * it has hutory of prior DPT DT w TT
been reported that in 1992 alone* 1.7 million lives immunisation.
5. Fw pnzvenhun of tetanus in the necmaEe
of children under five year? were saved , sis compared primariEv, but dro in iJic mother
to the mortality figures tr 1984, the year before
UIP was started.

Copyrighted materia
i>32 ^ Texluook of Microbiology
Immunisation with three dows ofOPV has not
been consistently effective in India and other
developing countries, with hi li rates of
- ^
seroconversion faiLures. Thi is sought tn be met
through the strategy of lmop uph rounds by giving
OPV to all the children in an area on the same day,
expecting natural spread of the vaccine virus among
the children to reinforce immunisation . These
rounds arc preferably held during October to April,
as the polio season in Indio :.s from May to October,
-
with a peak in July August,
-
E Jdh’rvrit cocritrie eriijiluv different immunisation
schedules depending on thrir priori tics.
1 NDI \ mi \i . IMMUNISATION
Vaccines offered under national programmes art
limited bv. economic considerations and so some
important vaccines may be omitted because they
are costly These maybe supplemented by individual
initiative, whenever possible.
Hepfttitia h vaccine: Many developing
countries, including India , have hii t cmk minty for
^
this virus. Perinatal cfansmissidn and acquisition of
the virus infection in the first five tars of life are
r
^
common in such areas, in contrast tY> low endemic
areas where infection is usually acquired in
adolescence or adulthood from satual or household
contactst contaminated needles, blood or blood
products or occupational exposure. Besides the
morbidity and mortality due to acute and chron ic vi rus
infect ion „ chronic carriage which may be very
prolonged i -. itself a serious public health problem - It
has also become an economic problem as carriers are
domed entry oremplcymcnt in many foreign countries.
Inclusion of the hepatitis B vaccine in routine
childhood immunisation will therefore be benehdaL
The feet that a quarter to half the adult dose of the
vaccine is adequate for children bongs down the
cost . Till it becomes part of the national
immunisation schedule, it would be desirable to
have the vaccine admi matured tn as rn .mv children
im
and adults as possible by individual immunisation
or through voluntary agenesis. The recent reduction
in cost of the vaccine as a result of indigenous
manufacture, has made mass vacanafi on mote feasible.
MM It V -.i L L i i i f ; The composite tneaslct-
mumps- rubella vaccine is employed in the affluent
countries but in the developing countries only the
measles vaccine is given at nine months, the earliest
age when it is likely to he immunogenic in the
presence of maternal antibodies in the baby.
Whenever possible, a dose of MM R vaccine may
be benefic ial at 16-24 months or later, not only to
reinforce immunity against measles but also to
protect against mumps and rubella.
Varicella vatu: inert Chiclrenpcot is very roiild
disease in children, but in adult it can be serious
and even fataL In most parts of the world, chickenpoK
is very rare in adults, but in some areas m the tropics
it i ^ not uncommon . The age of incidence of
varicella is reported to be rising. Varicella vaccine
had been used for many years in
immunocompromised children. Recently, with the
development ofa more stable and effective vaccine,
its scope has been extended for general use for
prevention of varicella and herpes zoster The live
attenuated vaccine is recommeded as a single
subcutaneous dose in children 9 month? to 12 years
of age, and as 2 doses at an interval of at least 6 weeks,
in those older, Pregnancy is a contraindication.
Typhoid vaccine, Typhoid fever continues to
be a maj^ KT public health problem in the developing
countries. Immunisation against typhoid is areal
need, particularly in view of die spread of drug
mi slant typhoid strains . The original typhoid
vaccine is not width used because of its uncertain
jf
benefit and frequent adverse reactions. Two recent
typhoid vaccines, the live oral Gal-E mutant
vacc ine and die i mectabfe pui lied Vi polysaccharide
]
vaccine may be acceptable because they offer
prolonged protection and are free from reactions .
Thev are recommended for Immunisation of those
a
five years old or above and so may be employed at
school entry
Immunoprophylaxis of individual diseases has
been described in the respective chapters.
Copyrighted material
Hidden page
Hidden page
Hidden page
 « Textbook of Microbiology
Tetanus an a result of ]i i > > ii i C al iflfei^lionf iS iWW
fire hur should he kepr in mind and toxoid
administered to nonimmune patients before elective
surgery. Many oases of neonatal tetanus have
occurred due to the use of contaminated umbilical
cord ties.
2 , I rinjiry trsiel inftctionif Even with
adequate precautions, catheterisation in hospitals
leads to LirinuTT infections in about Two per cent;
with Indwelling catheters, the tilt gtn'S Up to 50
per cent or more. E. coli , Profm-s, Ps . teusvginott
and other Gram negative bacilli are the causative
agents . Mixed infection is common. Infection can
he prevented by strict asepsis during catheterisation.
Indwelling catheters arc to be used only when
unavoidable , and then only with proper closed
drainage.
3, Respirator} infections'. Aspiration in
unconscious patients and pulmonary' ventilation nr
instrumentation may lead to ousccoinial pneumonia,
particularly in those with pre - existing
cardiopulmonary disease. Multidrug resistant iVapfi.
auJTWS and Gram negative bacilli arc the common
pathogens. Antibiotic treatment is unsatisfactory.
Postural drainage is useful in the prevention and
management of mch oses.
4 , Buotcncmia and MpTioentia: These may
be consequences of infections at any sire but are
camirKmlv caused hv infected intravenous carmulae .
The longer the cannulae life kept in situ, the greater
the risk of infection . 'Gut -downs’ on the leg veins
in infants or children with diarrhea generally get
left in place for long periods, the site being bathed
in diarrheal SUHJIH . Phlebitis sets in with Consequent
bacteremia . Many a child admitted with diarrhea
thus dies of septicemia . Gram negative bacilli arc
the common pathogens. 'Cut-downs’ arc safer on
the arms than on tegs. Intravenous rehydntiem in
diarrhea should be restricted TO emergencies and
should be replaced bv oral fluids as early as possible .
Infection can ho prevented by proper skin toiler
before 'cut -down ' and rhe i ] se of stainless *ted
needles instead of plastic canmilic ,
»
Staph. bacteremia is seen commonly
in patients with artificial heart valves. Bacteremia in
those with valvular defects may lead to endocarditis.
DIAGNOSIS AND CONTROL OF
H O S M T A I . I \ F K t :T I O N
Hospital infection may occur sporadically or as
outbreaks . Etiological diagnosis is by the routine
bar tend logical methods of smear , culture,
identification and sensitivity testing . When an
outbreak occurs, the source should be idem tried
and eliminated . This requires the sampling of
fioysible sources of infection SUCIL as hospital
personnel , inanimate objects, water, air nr food .
Typipg isolate - phage , hacteriovin, antibingram
-
or biotyping from cases and sites may indicate a
causal connection . Obvious examples of sources of
hospital outbreaks are nfrsll carriage of
staphylococci by surgeons or pseudomonas growing
in hand lotions. Cirrieiy should be suitably mealed.
Sterilisation techniques have to be tested. The
cause of infection may be a defective autoclave or
improper techniques such os boiling infusion sots
inward sterilisers. A cardial analysis of the pattern
of infection may often reveal the source bur
sometimes it eludes the most diligent search .
IT must be emphasised that control of hospital
infection should be not merely i spasmodic exercise
to be employed when an outbreak occurs but rather
a permanent ongoing activity :n any large hospital,
Evciy maior hospital should have 'infection control
1
teams consisting of microbiologists, medical and
nursing scoff and hospital administrators. Besides
investigating and controlling outbreaks, their
functions include formulating appropriate
guidelines tor admission , nursing and treatment of
infectious patients, aurveillance on sterilisariun and
disinfectant practices , determining antibiotic
policies and immunisation schedules, and educating
patients and hospital personnel on infection control.
Such measures help in reducing the Incidence of
hospital infections, even if they do not eliminate
them altogether.
Copyrighted material
 H Huso . tn infection
Unfortunately, In many hospitals, infection
control is attempted by resorting to more and, more
of antibiotics. This is not only futile but may even
r m
be positively harmful by encouraging selective
cdoniiiJticm by imJtincsLHiant pathogens. In the timd
analysis, prevention of hospital infection rests on a
proper undemanding of aseptic practices and
meticulous attention to hygienic principles. Sir
William Osier's aphorism that 'soap, water and
cymmonsense are the best disinfectants applies even
"
today in the contest of hospital infection .
HmMt ujc M WASTH M V N V ( I I : M I - \ i
'
Biomedical or hospital waste means any waste
generated during health care , research , resting or
related procedures on human beings or animals
conducted in hospitals , clinics, laboratories or
similar establishments. This is far more dangerous
and offensive than domestic waste. It contains
infectious or other hazardous materials that may
injure , infect or otherwise harm patients , their
visitors, hospital personnel and the public at large
in several ways , Biomedical waste if kept untreated
would ferment, attract flies and other insects , birds
and animals, malting the place filthy and unhygienic .
It contains ’sharps such as needles or broken glass
"
that can cause injury and infection. Discarded wane
attracts ragpickers who may repack disposables or
drugs and sell them. The waste mar contain harmful
chemicals and radioactive materials. Liquid waste?
can spread , seep into soil and contaminate wells
and tanks polluting them . Unless carefully nunsged,
biomtdicul wjsrc can be serious pollutants of soil ,
water and air.
With public opinion rising against
environmental pollution, governments across the
world are forced to bring legal restraints in this
area . The Uovcrmcrt of India has promulgated the
Medical Waste { Management and Handling )
Rules, 1998 tinder which the persons who are in
charge of medical and other institutions where such
wastes are generated {called 'occupiers') are held
legally responsible lot maintaining the conditions
¥ 637
prescribed LEI the rules , which have come into- effect
from 1 January 200.T . The occupiers halt to obtain
due authorisation from the prescribed authority
after selling up the retjuireil waste management
facilities.
Qi i v r m ANJI TVPHS OK
UloMBDlCAL WASH:
"
Hie amount of waste generated in hospitals under
Indian conditions has been estimated a-s ] to 2 kg
per bed per day. This is composed of different rypes
f > ( infectious waste , not all of which is infections.
On an acenige about ptr cent i ;* harmless and
IS percent hazardous.
Harmless WBffte is pajler , cardboard , cartons,
flowers and ordinary office , or kitchen waste akin
to domestic waste .
, /nfecridJlS waste is anv waste likely to cam - anti
transmit any type of pathogenic microbes. This
includes human or animal tissues or organs
removed at biopsy, surgery or autopsy, placenta anti
Other jMroductsof conception, anv pathological fluid
or diachurges, dressing , swabs and Other soiled
items, laboratory samples sent for microbiology,
pathology and biochemical tests, at ) microbial
Cultures, used syringe needles , used SC& Eped blades
and other shaipS-
Ncwintectidc# hazardous waste maybe chemical
{ toxic ,corrosive, inflammable , reactive and
otherwise injurious ), rjJjipjctjve {handling and
management of which arc under the direction of
the Bhabha Atomic Research Centre ) , and
phanrtacrWHjyff.CiJ ( surplus or time expired drugs) .
Waste management A primary prerequisite
for effective waste management is a clean and tidy
environment . Waste tends to accumulate in dirty
surrounding. The hospital and its premises should
be kepf ina clean and hygienic condition . This
requires frequent soap and water washing., mopping
And good housekeeping.
The objectives of hiowaste management are to
prevent harm resulting from waste minimise its
K
volume, rttrive reusable material and ensure safe
^
Copyrighted material
Hidden page
 INI >KX
A aerobes 21 anabooeptor 102 124 k

a type particle 178 aeromonas 317 ambulant typhoid 29ft

abiogenesiB 1
abortive: infection 438
jhsidEa 621
aceJJukr pertussis vgcdncv 141
Adholepksma taidfawii 595
adugmobibcier 606P 607
acid fast bacjlb 151
fcinrtobactcr 22^ , 229 32 L 405., 603
acquired immijiKiddntiencY syndrome
(AIDS) isa* M. Ml Ml Ml 5 K2-
5M
AIDS relblcd yimi ' lps 5 RR
actidjo
- ^
( cydc LiimidL ) 611
actin binding protein deficiency 1 53
ai- cin sbHcillus 122
^
aetinamadhira 402
actinomvces 266, 400. 64 IK )
auELiiL Hiiyviqps ( lumpy jaw ) 400
'
clinical forms 400
epidemiology 401
laboratory diagnosis
tie atm cm 401
artimmycotic mycetoma 402, 6 IB
acute hemorrhagic cystitis 4BS
acute infantile gaaFfEKrnterritiis 371
acute I ' hii.sL- IUISI achT 213
JL-UEE respiratory diw-aEcs (ARD) 487
acute t "a - L
- i «:> r 3 n INJU;
viruses USA
acyclovir 459^60 478, 430, 432 . 122
afferent inhibition 1. 111.
afipk 421
afiamxin 627
african hoarse uebw 523, 534, 521
agar (sgar-agw) U
agamma kitiidLi irink 1 5 j
^
Ewiss- type 153
, X - linkisd 152
aggiutinaFiofl 92, IRS
agglutination reamoas/rests 4, .114,
334 142, 383, 392
applications «f 2B
- hetefophik 44
late* dates fixation ) EDO
passive 100
reversed! passive Ull
slide agglutination 98-49
tube 33!
agglutinin 9J
agglutinogen 92
4411 air, bacteriology of 607
alastrim 468
Alberts stain 2 3 2 1 5
aJcaligenes sp, 603
akaligenjes face-alls 41)4
aJcxine (complement) 4, 1H1
alkaline bik ult agar medium
(BSA) IQfi
alkaline peptone water medium! 30
amidase test HJ
ammonia production test 46
amniocentesis 1211
W marker 91
rinaerobesB nonsporirng 258
anaerobes, aporing 264
anaerobic bacteria 411
waerobk cellulitis (wound
contamination ) 2 ^ 2 , 255
anaerobic cocci 266-67
anaerobic (clostridial ) myositis
anaerobiusis 40
anaeropksma 345
anaeroplasmaraceae 395
analjffle ( ligand ) JUS
anamnestic reaction 140
anaphylactic reaction 160, 161
anajdiytKiic shock 1M
anaphyberoid! reaction 161
anaphylaroxins 163
anaphylaxis 160
atopy 164
Cutaneous 166
\ncii ( hx-al whcal-aTxl-flare
response) 162
mechanism of 162
pXSaiVe riiitM. iiL'ou ^. 161
in vitrt> L 62
ijnemuy

AxldLjiims disease dkliimcnpjiiv genes 185 fk1-utc hErVicdvtk 169

admitLi allergic rests 360 aplastic 15ii
mesenretic 330, 488 allergy 4, 159, 360,. 364 utoimmune 121
AJladherifl boydii 618 harulwe 152 5.171. 3%. 397.
syppunifive 20K
adeno a.swx Laced virtLVB ( AW ) 447
' AJJodermafifysflus sanguineus 4 Hh- 481 ^
adenosine dkanunw ( ADA ) allograft 129.126 hypoplsfes-tic LS5
deficiency 1.S3
r
reaction 166,. 122 angiomatosiE 421
adenusatellovinjiE 447 alknypk rperifirities 31 angstmim unit S
adenoviridac 446, 486-489,. 522 alopeda 614 artimaJia minuta 1
adenovirusts 4 B6 4B9, 512
adbesins, piracy of 340
- alph&fetopmrcLn L 79 181
aiphagJohuJin LSI
m anthracoid (or pseudoanthrax )
—
bacilli ? 46 47
adjuvants 76, 141) alpha lysine ( alphatojcin.) 195. anthrax L, 2x li 244 606 L

adoptive imuuiinigaHOfl 177 alpha methyl cbpa 122 antibiotic 39, - jjj.

immunity Z1 alpha neuranunoglycopixuein 112 antibiotic associated colitis 261

immunotherapy Lfi2 alpha roxin 250, 253 antibiotic resistance 18
adult respiratory distress seadrome alpha virus 447 antibiotic sens-mvity teat 628
( ARDS) l££ alternative pathway of complement antibio'i ic rheflipy 5£
Aeries (C) 112 antibody 84
aeppti 527 529 _ 330 . alum absorbed vaccine 143 albumin Hgglutmanng 185
afrkanus 123: aluminium paint appearance 119 antiheimjggfuriination 504
simpwni 322 alum precipitated toxoid 745 antinuclear ( AMA ) IZi
AjtPrthae®er acK geneK 2 R1
^ ambJycrmma ticks 437 atopic 164

J monie eria

autoimmune 371
blocking: ( n agg]urinatingj
9g. 99. 149
sLytcjEfcjjik: 160
dependent cell mediated
cytotoxicity ( ADCC) cells 126
humoral 452, .453
ineompkfe fmoiwalenc} 98,. 158s
143
measurcnwint of 9i lill
IGL liidteti! LirilBLUrdlV
^ (CFT) im
(AMI) 117, m
in viral infections 4 2
monockmd 135, 122 ^ absorption 302
’ nauirsf Lfii
pf -oduaiovi of 133 , Hi
reaga me 1M
secretory 9
iJiiCiJms VTitK ^cni.ijc. lcu.si; B
'
(DNAase B) 2lfl
anti- gas gangrene 256.
antigens fill ft4 —
autologous (self antigens ) S2
biological classes of 83
T cell dependent £TD) B3
P Otil mdepciiLlcn I ( 11 j 83
'C somatic 2 !8
euniplcre BQ
cross- Tractisig 1ZQ
face 4? f, in tissues 133
feral Ul
foreign 17W IS2
tllTWIUflil 82
If ilagcllar 221
beat labile 469
beat stable 469
heteiaphiJe ( hctcrogcnctic ) £2
histrxompatibiliity 82. 129
HLA ( human leucocyte anrij ti )
82, U£3
1region associated 1 M )
major HJSTOCAMPATIBLLIIEV antigen
130
measurement n( IQS.
neoantigens JL2Q
"O' 2ES
nboriudcopiutnii ( RNF)
antigen 504, 5 Q7 508 U
sequ£SE md. ( hidden ) 121
>
soluble (S) 504
surface S04
tumour 1S1
associated transplantation
antigens (TATA) LEI
specific transplantation
antigens (TSTA) LEI
4 TEXTBOOK OF MICROBIOLOGY >
viral ( v) 504, an antineuramintdase 453, 504 , 508
Vi 291-91 - antisera. 32
WiistriTkiiin 302 antisepsis 21
antigen-antibody neaetioni 97- ltW anriaraphylolysin (anriaJpbalysin} 199
agglutination reaction antistreptolysin O titration test 103,
antiglobiilin ( Coombs ) test 33 206
passive agglutination test 1QQ an rite ran i,is immunoglobulin , human
!
^
slide ig utiMiion 38
tube agjrlurinaGiMin ^23
complement fixation tern
(Tig) 2hl
an rite ran us serum ( ATS) 263
antiTosin may car 311 , 314
aphtbewiruses 430
congl urinating complement apcd.cm.ui agiarius {field mace ) Hi
apoptosis 12b
indiraT ci jmpkirierif Hxaticai 1£[2 antbinewe 405
other ttsrnplcmraE dependent awhnis pfOptMtica 100
serological testa LQ2 arboviruses 484-96
enzymc immunoassay ( EIA ) 10 h antigenic structure 522
ELIbA life control! 523
genital. Icaturcs epid m »nllng}- 523
=
diaracrerisacs ) of 9 >-9 t alpha virus 525
immimoekctroblor techniques lllfl (Hasdvirus 525
1
immunodectronmicracoplc
tesfe l£l3
laboratory diagnosis 522
pathogenesis 522
immunofluorescence 104 syndromes, associated with 524
direct test 104 taxonomy of 522
indited EL NE jJH!
artiriavrfLi&Ess 447, 568, 6-35
iwaufttftent of antigen , Argentinian hemorrhagic fever 569
antibody 105 inuinsi -
ha-iilh 1 'V li
neutralisation tests lfLl arfhrospciire 612
upsimisaEimi 104 Aeth-ite reaction 114, 165, 2Z3
pf expiration reactions 34 amifickl virus 469
applications of 35 aryl sulphatase test HI
eledtrnimmunedifiusi 32 ascending tetanus 259 , 265
i mmu nnd5fFusi.43n Aftberff iteduki 203
( precipitation in gd ) AscoLi s cbermoprecipLEin rear 95^ 245
tests 36 ascomycetes 611
radioimmunoassay ( R1A) 106 ascospores 611
antigenic asepsis 21
compecLEbon MU ASO im 206, 210.
^ determinant (epitope) SO aspergillosis 624
'drift 505, 509
1
aspergilhis asthma 625
505, 509
‘shift 1 assays
antigenic specificity Si ajitibicnJc till I-
antigenicity determinant! SI binder -ligand 105
antigLohulin ELHC (see H!H > Coomb* hemaxgtutinadk^ n 434
EMl ) 33 nf infestivity Ml
C.
antihemagglutmin 44 j. 504 plaque M2
anrtilympkircyte scrum (ALS) 141, pock M2
HE quanta) 442
antimeiabolire Ml quaniEinitive 44 ?
antimicrobial therapy, laboratory radioknmuno 1115
contro] of frJfl-lfl transformation M2
assays iai body fluid* 630 viral IM
seriKitiviEy feists 623 asteroid bodies 619
difiiisaon rest 62fi asEftwicus 523
dilufi-on. rest 629 ataxia ndangLeera^ia 153
j
/ name eria
 4 INDEX ft 641
iihkie'i foot 614 reaction) H gene 53, 5ft, 60
Miipy 164, 3S1 . eett envelope 12 generic mechanisms of drug
itHMUxn 2, 62 «11 wall & 1L 12 niPtintt in bacteria SS
SLlypiraJ SBjjnfaaCIHtt ( ariuilyiTKMift/ defideni ftwms J99 gene iransfer S3
- genome 53, 54

—
unclassified/ non tuberculous cell wall deficient bacteria 13
mjrabaercTia) 351, 361 cytoplasm 15, H genotype 53, 54
-
,
Australia antigen 5 5*0 551
Australian ' X disease 526
1
cytoplasmic inclusions 12 14
cflapmaaL lAasma membrane 12, 13
- iusertion KqueflOH 6Q
innons 51
All E Dill] 169 episome* H mucagctu 54
aucoanribodics, cold HI Fimhri 15 mutation 51
autoclave 25 27 *26
^ ^
(uirncrtne effect 144
fbgdla 14 -1 ft mutational v? transferable type of
drug resiseanire 52.
autoimmune diseases
rnH
^ocapsufes ll
nucleus 14 molecular genetics 60-61
class ini cation of 171 spore liL blotting techniques 62
entnia for 169 types of bacterb DNA probes 62
mcduuiem of aurtoimmuiitsHbon 120 carbon dioxide, requirement genetic engineering 53* 5iL 6D
aurojmmunity 1 ft 9-7 S of 22 molecular epidemiology 63
autojnterfcrence 445 colonies 12 niicfeotides S X 6fi
autolysis 2 t 7 continuous culture of 1£
female 16
- PCR 62
uutertrophs 2D phenotype 53
aviuleiKTvirusc 44fa freeze drying ( IjxsphilUadofO 22 polypeptide SI , 53
avian leukemia virus 579 Gram positive n Gram protein synthesis 52
avian leukosis 52ft Kgldw 2 replica plating technique 55
growth and mi drip!ration of l£ replicons* independent 53
B growth curve 14 resistance fnuwfcr factor 52
idealised bacterial cell Ijf 12 transduction 55
B-cclI activating factor 1.44 UVtVBcytOfPlNlIkic indusions 11
R-etll grnwih brim 115, HI transmission of gen«k
invohinon forms 2£l material 55
R-eells 115, 1S2
.
R virus 47B 494, 4%
Babw- Ernst granules { m also volutin
male 16
microscopy £
moLunint 22
tnmpoHUe genetic
dements
granules ] 231 transposition 60
nucleus 14 tnuispovons 6Q
ha he no. 41fi nutrition 2Q
bacillary angiomatosis 421 momma S3
bacillary dysentery 3ft5' BB =
osmotic effect cm 23
cuygen requirement and
bacterial idenrifieaEion 44 4ft
bacterial lysis 461
-
bacillary pdiosi-s 421 metabolism of 21
bsdle CalnHEie ct Guerin (BCG ) 354 bacterid tMonomy 48-51
pleomorphtsm 12 classification M.
bacillus 14, 15 1454, pH KnalinCj 23
^
jn[lmcL5 241-46 ncifflcncluvre 5D
shapes 10 ape»cLe£ EtcHieejits 4fl
differentiating features MW of Z
between an thru and type OIITULECS >Q
temperature requirement of 21 bacterial vaginosis 410
Hnthracoid bacilli 246 bacterial genetics 51-64
medusa head appearance of 242 bacteriodn 57> 301. 466
chemical structure of double
eereus 246
unadfid BNA 52 -
typing 47 467
bacteriological index 370 » 325
lichmlformis 2ft codons SI
tapsubtUH 2M IwiLtcrialyHB 110, 113
phlcgmonis emphysematosae .249
FtcarorhcrmophilujiE 22* 2£
coldnogenwz (col) factor 52
conjugation 56
bacterinphnge 4 , 55a 450 67
assay 466
-
subdLis 64 24ft. 6fl5
^
deoCTribofinick-k: Mid ( DNA.)
double helix 5L 52
.
iiTi i' vf •: LUJVE 464
badnacin 300, 204, 205, 2lfl, 261, 26i recombinant dna til lifeeyek 442
bacteremia 70, 197 , 200„ 216.219, enzymes- 5A morpholiDgy 462
im, 237, 296., 297, 3.11 , Hi puma 53.56 typing 466
hacieria exons 55 bimteriuria 27 ~~ 77
anatomy of 22 txErachjomoMiiud genetic baricroides 267-69, 4008 ft^ll
morphology and pbvrio bogy 7-21 efemenu S3 bactcroidks liaplis group 26 ft . 269
bueroides. niel Miogefikus -ordis
captide 1L L2 , 14
capsule dwelling (qpjellutig
F facEur 56, 5Z
fllKtHtlHI HSLT 55 gfotip 26
^ ^
: i
yrrante aieria
«
642 * TEXTBOOK OF MICH06I0L0GY *
balantidium vnli t£lS HnunMbXt pmmgiop of 1SZ cams 145
Baliensp-Bcthesdh group 28ft. 291 inheritance LH.7 mclitcnsi* 348
Barnxndb badUifomis 42ft blood transtiisiofi 181 BBtnBB 346
banunella iM blue pus 12Q twis 346
bortondlosis ( Carrions disease) 120. blue tongue drug 522, 521 sun M&
baEsichobolu-s haptospoms 62Q body EDUK brucellins 150
basidiomYtctcs 611 ,. 621 boivin antigen 12^ 291 brucellosis 25Q
bandiojpuncs 6-11 Bollinger hodia 449 bufTalo pux 468
haney bacillus 366 Bolivian hemorrhagic few 569 buLbolderia
B cells ( lymphocytes) 90, 121, 122. bone marrow culture 2918 ccpacca 321
characteristics of 125 ^- -
booster dtwe 7 jJ L 2£d
IkideNjfifigffFil modium 339. 341, 342
mallei 321
pseudomaUei 121
growth factor (bgf ) 111 WIeKlIa 119 44 BtiflyuMHi vim* S32
marunrion 124 bronchkepcjea 344 bunyavMdLae 447r 522-24 512-14 p

BCG vHdffl 148, 154, 362, US epidemiology 342 buny^Ttims 447

beds^h 422 laboratory diagnosis 342 Bufkirt ’
s tvmphoma ISO 483, 574,
,

bejel 385 parapertu** i* 344 ilti

Renee Jew* mAi 2Q pathriHgenieity burst 4JB
beneekj vdnifica 117. pernissii (earlier Hjemophilus hutyrivibrios 266
bomlmuwn cUoridv 2&1 pernusEis) 341 Bran) selective media. 447
iMMja glutaialdehyde 320 feossn ( FT) 1M bystander cells 112
r

betsdysin 13h prophylaxis 343

b propLobpcfcm: ( BPL ) 32 434
^
, treatment 144 c
536, 540, 564 variation 141! calcLviridat 565
B IT reaction ( biological false
3
Bornholm disease (epidemic camp reaction 211
positive] 381, 406 pleurodynia) 497 California encephalitis virus 532
hharn.L virus 534 borrelia 266. 377, 386-OT
Bhatnagsu strain 290
biiMobacrerium 267, 269, 400
burgdorferi 389
duvtorui 386
epidtmirtlngy
Cfl
^ffUBfaflCEdro
eamelpox 46SP 471
groradomatas 4M

campy BAP Kkctiw media 407

dentiurft 400 Campylobacter 406-407
bile Hluhiliiy itsi 217 hermsii JM ciruedi 402
biomedical waste disposal tilZ
bind flu M12
laboratory diagnosis 388
morphology 390
.
coli 406 402
condiseus 4416
btwnuA wbwlkykit iMIE parkeri 3B6 fennetliae 407
Bimr rins 57S paibogenkity 391 bw 402
BK vims 563 prophylaxis 393 jejuni 406
black death 326 rrajurentis 386 pylori ( H pylori ) 407
bbrstogenic ( mitogenic ) factor (.BF7 treatment 1B9 yilOMID illlt
CBurypai 469
MR 141 virtemtii 388
Candida 61 Hi
blutornyves dermatitidis 622 batryomycostt 402, 618 albicans 155, ItK 601, 610, «12,
blasTomycosu 621 botulism 263 "
613, 616- 17, 627
bbstoepores 612 bovine spongiform encephalopathy
blood groups 567 guilliermondi 617
A and its subgroups 184 85
Abo hemolytic disease 189
- bndytinki 163
bnHihftfnetft 229
krusei till
pOrapsilcwL* 617
Abo system 1M braaihamdJa catarrhalk 2 X 410 paeudowopic-ali* 617
steJiartHcLac 617
anql disease* 15LI henry 254 mupkidis till
trots matching 188 kwaeilian purpuric fever ( RPF) 137 viffwimdiy 617
Lewis sysrem 187 breast cancer 578 Candida granuloma 616
medical applications of 187 BriU-Zinsser disease 415 candid 5 B9 616-17
P
mn system 182 bromovinyl dcoxyuridiix ( BVTOJ) 459 capnoejTiDpJiaga 41ft
O’ group 187 brownian movement 15 capniMsx virus 469
‘GIFT ( Bombay ) M2 he Licet la 345-50 capsid 444, 445
Rh sywem 186 abortus 345, 346 capsomm 446
antibodies 186 mtigaaC stricture 346
antigens 1M bacteriophages 346 221 . 334. Hi ^
capsular pp fiscdliride 217, 2 IS , 219

n r
yrigm i
ri
cuanoeiDlHjmHC antigen 191
cardiobacterimn homini 410
-
dijwf hone typhus 416
INDEX y

thikunguma virus 522, 524 , 525, 530,

closTriditim
Jrttobucylicum 24S
643

cnj-djn lijifcn antigen 179 3R1 534 aerofoe tidum 255

' p

ta. rd.iEm.njBEl 490 chimpanzee coryza agem 516 bifitmenians 255

carriers 2%. 303
chronic 6L 22Z
rhlamydiiic 422-29
classification 424
buiuJinjuTn
buryricum
249, 250 258, 263 64,
248
. -
rantaet 65, 29? human diseases caused by 424 chauvoci 250
convalescent tL. -39" M3 » laboratory diagnosis of codile-arum 250
healthy 6£ 2fi2 chlamydial diseases 424 difficile 251, 265
paradoxical 65 morphology and growth cycle 422 faliuE 251, 255
temporary 65 297
^ pneumoniae 422, 429
hirtolyticum 251 254.
ncpvyi ( Cl oeefemabens) 254
-
Cary-Blair medium 303, 3liS, 4fl" psittiuci 424 -
|it: rP:i ri|rL"!: 254 , 255
rattmecU metlwd 29B, 34BP 3-1' 1. 134, »
mdlOfHdl 424 septicum 251, 254
425
ase- pe[oxidase tot ( tubercle
. li— ri ivi H.i.- : , genital 427 Kmldli 255, 265
^
cal chlamydospores 6l2„ 613 612 P sphenoides 250, 26Q
bacillus] Hi Lhlorhciidine ( hibitanc ) 32, 19H .. 275, ipocogcnts 251, 255
L Lcalaftc produdHU teiir 4 277, 407,
^
4m ^ 2M
chkortwylenol 320
Etrrium 24 K , 255
reiani 249, 25 L, 251
cat scratch disease 421
cd marker 133
chocolate igu 224 225 , 22S . tetarwmorphum 250., 256, 260
cholera cluster ot difFerentiaricm ( CO)
cJ t cell marker 121 epidemiology 111 mirktrs 122
cell cultures 439 immunity 315 CD4 T cells 122
cell death (cytocidal eflbd) MS EnKuhit period 306, W , 312, 315 CDS T cells 122
cell medialed immunity (CM] ) 117. "

.uaguiue 69, 192, 193, 194

laboratory dijignurik 313
133.142, ] 52, 151156, 302, 370, coagghiiinaiicin IM
371 , 372, 371, 184, 453,. 458,. 475, pathogenesis 309
coccidoidil granuloma 623
m 514, 517, Hi prophylaxis MS
-
cuctidoidw immitis 622
detection. -of U6. tmutmaK 316
CIKCIDHJIDLRTIMUSU 143, 623
Junctions seopc ) 143 vaccines US
^
induction ®f 143 vibrio 2a H O
codon 52
coinfection ISA
Transfer factor of 146 cholera red reaction MS colkinogctuc: faijior 5Z, 467
in viral infections
irhurdopoxvirijae 44$ Lolicina 599
ihorioGtEanioic membrane ( CAM )
ffftihf
change* (cytopathic effects) 449 .
337 m, 3M» 439
EDlifboBI 271- 4 ^
colifbrm bacilb contaminedorj tests
ChristenscnE unease medium M 64)4
injury 449 cristispiia 377
proliferation 449, 455 coliFtin sulphpte 205
chromobacterium 603 collagen disease l 7 jt 174
cerarophytlus fasdatus 139 L- hromobacEcrium violaccum 404
|

calonisaiion factor antigens 273, 279

cercopifhecus swthwps 41
^
ceoimide (ctraulnn) 13, 283, 323, 321
chromoblastomycosis (verrucous colony ^cimuklii : Ektuv; 127, 143, MS
CF antibody rest 42S
dermatitis} 617. 618
uhruntoiriveuais 61S
^
Colorado rick fever 522, 524 534 571 h fc

chancre 379
!!
colostrum
chronic fatigue syndrome: 464 commcn^J" 64
chanuTHd 33? = chrunic granulofrHta (gumnrme?.) 3W
chandipura vim* 533 534
Chang CVHL EDC Ml - chrome granulomatous ctiMuue 15?
chrnc i m IIL u nosuppm si ve therapy
common cold ( sec also rfmoviiuses) 42Z
compknaencaDon 445
complementuirn*’ determining region*
Chediak-Higiwhi Fyudrume 153, 157 LSI 137
chemiluiTunncence immuiH ay IM
chcmo- organotrophs 397 ^
chronic obstructive pulmonary disease complement (C) aysrem h 110-16
(COPD) 402
chcmotacrtic factor 1.43 acrivarion 111
dtfVtt utiluuitiun EfcSB 46, 2 .1, 2*M)
chemotrophs 2i(
chicken chuJetfa 2
ritrobacter 274 28Q.
citron bodies 256
- alternative C pathway 112
biological effect? erf 113.
bacillus 2 cladosporivm 61B 619 buqtihem of Hi
cascade 112 ,. 113
?

vaccine i clavLceps purpurea 627

chick embryo ttchnJcjucib 506 clone 49 classical C pathway 114
dhickcnpuK i 44 47B 81, 635
^
ihii- k matEiEL EcaE 33
- clostridia 243-65, 262
clostridial myonecrosis 215
components of (C1-C9) 115.
conglutiruring component lli

.1 ,
J rionte ate nai
644 4 TE5CTBOOK OF MCTOBJiOLOGY t
deficiencies 11 5
general properties 110
M 3, 444
•

croup 335 , 515

- depcmWmis 4SS
Dermacentor andersom 416, 534
irthihiftiE , dfcfidtrtijY 113
1
Cgwrzfdklr-JflJub diseut 566, 567 derhutontyeo^if 613
regulation nf C Htm H Hi
^ cfjfigfelwiwflnii S l , SQ - dermitomyo*tis 173
oompfement fixation lest 110 Cryptococcus neotbimans. 6AJ dmrriarGphyres 611-16,
ccrncaruvilin A 121 Ctenophalides fclis 421 dcnnatoptulus 4QQ
concentration methods 35" tulicoides 532, 534 •dEfmUopfaytidi ("id " racoon ) 614
condyloma acuminaiurn 562 cultt fiflMfJfiMl 526 dermatoph >ttrtis 616
conglutination 115 culture .
dtsenssrisirioii apecLtlc
congjurinin (K) III cdl 439 ( hyposeiisdtiaaifon ) 165
iLdiji type* 611 indications for 32 desert rhcumatkrn (valley fever) 623
?

conjugation 57
conjunctivitis
methods 39 43
anaerobic dU
- detmit-6 cell line 441
deutoromycetos ( hsphomyceiM/fongi
acute follicular 485 continuous 4fl imperfccd ) 611
acute hemorrhagic 498 pour plate 39* Mi demin 232
angular 410 stab 32 dapsone 325
fnllirttlar 477 streak smote 32 dktwcbm 385
cenwirotive eruyenes M. mw plate dHJ Dkk tec 201
contact dermatitis (hypersensitivity ) culture media, types 33^ :15 didcmyadcnDSLne (DDA) 459
i59, i6.i , m cutareous hmopfau hyptam ii-irtY 166
contact inhibition 575 cyrocidal (cytolytic ) rears 103 ^ didemeytyridine ( DDC) 459
dkleiwinQsne ( DDI ) 459 -
contagious disease 66 cytokines 143* 455 dkne% m.ettwd 396
cnfitpgintki neumlymphomatcwi* 576 cytolytis 112. 449. 4,52, 511 Djp| « B syndnrMine 154
oragiouf p&i&niar dnulitB 4faSp 472 cytolytic metim 1M. dilute carbnl fkurhsin. stain 388
comagiLLm vivum ] cyun dawia JJH, 4S1-S2, 577, 635 dimne^phsc fungi 619, 621, 622
Cwmb? teFt 99, 172,
191, 349, 3«
mmm ^
clinical features 431
laboratory diagnosis 4B2
dkiiirochloTCiJbeinjzeQe 186
diphosphopfiidincneucIntidAse
coproantibodies 31 5 cvtupclTiic dfeis (CPE) 440. 487.. 506 (DPNiie) 2HZ
cold factor 171, 352 qrtopltttn 12, 13, 121 435 p diphtheria 606, 60S, 633
commeal agar 6-11 cytoplasmic knchitians 12 a 234
eulnoieoy

IOK 240
JjpMwk 240
—
coio navi ruses 448, 569 r 573
onrwehfl£teriu:m 231 4Q
-
cjToaine arabiimide 141 , 459
cytotocric reactions 16fl 165,
CMpdc-DteK medium 611
>
B
mm& 04, 140a J33, 237 39
J61.332
diphtherions 466
diphfemids. 240.334
-.
cqui 240 diplncwnn ( see pnesumooeccus ) 216-
monutissEimuin 240 D 21
parvum 2Mi dakar vaccine 529 1 dipbid cell strain 440
p-ssudodiphtheriticu.iia 2441 O antigen 136 dip dide culture mediEwk 277 .

pseudoiubercuJMis 240 dane pcrtkfe S51 ddslrifeclajits 249, 250

Male 240 dangerous Q group 137 chemical 502
ulccrans 240 Danysr phenomenon 212 ideal 30
Kerosis 2 =40 dhiYpUi lUVimdnetUJ 371 testing of 28, 33
cough pl3.1t method 342 deae ctectran 425, 426 disLnfeciacHn. M
cnundlmin bodies 522 defective virus 438 l>lyseigic acid 627
counTtrimmunwlectrophorcsis 92
CPWJIY type A inclusions $5il -
dtbytd |- iVf!c-rM -:: itiMly skin test 3L50 D marker 494
DNA 5 P. Il 141 ISDg 314, 391,
oMpos L 4, 468« 472
.
iMt-alysin 196
delta virus 558
,
^
430 , 435, 445, 446,^ 449 , 462, 466
Ccracllfl burned 26.412, 41f , 420
ccmckic viruses 49ft, 491.495, 496
497, 4gjL 5 j2
. detFisifoecw (fomented? mi fungi 619
dendritic cells 122
dengue (break- bone fern ) 53fl
synthesis 442
Bwkskkti baciUua 267, 600,. 601
D^ncrwiia granubimaEia
Craigie's lube 43 292., 293.
^
C-ffactive pnrteirt 218
hemmrhagfe fever 530
shock syndrome 530
( Calyrruram
- ^ ^ glllltasiMBs) MA
doncft aiiiMssia 404
Crecfc"s method 221
Crimean Congo hemorrhagic fair
.
viruses types 1 to 4 524
licujcychubhfc citrate Tnedium 272.
DPT wdoe 611
IDooeA egg medium 293. 352
fimet 532, 533 285, 29ft. 319 Drej^er s tubes 299
critical r.mM 3.80 dsir rdxTrrtJsckaffi (aveputtevkuc ) 207 drLnldiiig water, cbusificati 604
cross- reactivation (or marker rescue ) ^
deoxyribonucleic add (1>NA ) drciplEi nuclei 607

Dvrianiec ri
 * INDEX *
drug resistance ( R ) factor 57 cm rn mopoxvinruK 468 erysipelas 202, 208. 404
mutational £8 enteric fever ( typhoid fever ) 295- 03 ^ eiysipJothrii rhuskpithiae 404
trjTLNluLihli.: (epnsi i mal/i ntuitionsJ 5ii bacteffcophage typing 300 erytluuiu
dubos medium 152 8 di&gnotis of carriers 30Q anhritkiim epi <kmicuni
dysgammaglobubneinia 154 drug RUftinoe 1L12 ( Haverhill fever) 406
dysgonk 2S2 epidemiology 296 chronicum migrahs (annular skin
labonnEEKry diagnosis 297 lesion ) 389
prophylaxis liVl mu hi. forme 4ZZ
E
early antigtiis 484
^ ^
treatment302
nt robacto 281-84, 603, 635 eryrhri. ru l 345 , .347
-
iiEKhssum 33®, 3311, 3 62

carty proteins 452. aefogenes 231 erythroblastosis fetalis 165, 188, 190
Eaton agent 398 cloacae 281 ttyffort&L«fflQ ris virus 578
tbenhdk typhi J9fl
Eh nuclear antigen 434
_
entoobacreriatceae 771-SU
] USKI li . aCirnj 271
tfydiTDCfft ( E) antibody ( A ) complex
ill
ebok fever 569 enterococcu* 203. 234 aychrqgenic fdiclk, scaikiaml) mxbs 2Qi
echo viruses 491L 49h enterotcodni 1%. 273. 2K 6 E$therichid ccJi 3L 54-57, 59 67,h

eclipse phase 43BB 465 enteiwimses 434. 436, 442. 445, 447, flg, 224, 252, 271 , 272-BO , 286, 287,
Ectliyiiniij gnngtfenresum 320 490-500. 635 326, 346, 462, 466, bOiL 602, 635,
ecnjchm hair infection 615. clinical s)rndiamc 497 6.36.
Eczema herpcticum 476 envirnurinc 460 antigenic stETacnure 273
Edmcmstoiir-Zagrdb strain 520 enzootics 526, 531 bncKrhemicaJ reactions. 2Z1
ifidwititibdli 2!? 274* 2SQ enzyme immunoassay (eia ) 106 . clinical in&eiicMii 275
efferent Inhibition 128 enzyme Linked immurujtocbem assay cultural characteristics- 212
egg yaUk medium react ion 46 ,
(ELISA ) 106, 457 582, 590 .
enteroa j reganve ( EAEC ) 279
Egyptian ulcer 231 enzyme multiplied immunoassay ^
eniemhaiwiTha c ( EH EC ) 222
Ehrhch phenomenon 2J2J
Ehrlichia 412, 1LZ
technique ( EMIT) lfifi
enzyme neucialisation ccs;s 508
^
enterobvuivE ( ElEC) 278
HiKcTopathogHiijc (EPEC) 228
aenrteBu 417 .
et ^ inyphilia tropical 382 enterEHEraiggriir: ( E^ PEC) 278
EjchwaJd-5 ilmsct cffccr 178 L- qrfarii.Vj.ThiE L lici iiul jdiC tiij:;:tn|
'
r?i of enterotoxinmethodfs for
Eijikman toe 604 anaphylaxis , ( ECF - A ) 363 detection 223
EiknelLa ennodpens JLH) eosinophilk: hyaline indusksei hi id i LN 473 virulence factors 223
- —
cledrobn mu clodiifluson ( see
antigen anybody reactions )
epidemic
diarrhea of infant mice ( ED1M)
Escherichia 272
^
esophagitis, HSV 477
election rnkporseopv 431 551 568*
, , 572 KSS (emhtcscyte sensitising
57ft diseases Zli mbstance ) 418
tlek pel pm E pi Cation 2M hcmoflrhagic fever 525 esthiMKiK 42$1
ekrncnmy bodies 422, 4J1, 4612 kierrtiileiinju.nctivitis ( EKC ) 4B8 ether 432, 434, 502, 513, $18, 563
elution 434, 435, 503 nephritis- ( EN ) 533 ctiivknc diiminc tetracetic acid
EMJH medium 390 nephmsnnephritLi iil ( EDTA ) 212
EM USA 12±
mctphalid* 397, 475, 477, 478, 479,
parotitis ( fcsse JIMS mumps ) 512-13
pfeurndyinia 497
eubacterium 266 267
eugonk 352
.
.
492, 4%, 522 523, 524, 525, 536, polyarthritis 525 eukaryote L d3ll
539 typhus 413 exaltation fix
central eurepean 525 epidermuphytun 614 exanthem subitnm ( roseola infantum
fail 523 episodic angioneurotic edema 114 or aixih dbease ) 4R 5
hsv 477 episodic lymphopenia 1 exfoliative toxin 197
vaedme 527 cpisemes 34 exophiaJa wcmcckii 612
ciKephaJornycUtis 140, 143 epitope £li exmxin a Hi
encephalopilhy’ 343. 344. LLEi epizootics 328, 329„ 53lp 534, 545 exotuKins 67, 68
ndmk disease? ZQ
cr-idcri me ( or nib-uiuj] ) typhoid 297 574, 522
-
Epstein Bair vims 12 3, 474, 482-85, , expanded programiiK on
immunisation 631
endemic typhus 413 equine encephalitis virus 52.3, 525 , expanded rubeUa syndrome 564
cndofhrix hair infection 615 equine rabies immune globulin experimental aJkrgic
eikdcrinufri 67, 63 (imp si] encepba]omyelirii& 173
Entamoeba! histolytica 6Q5 6.15
Entamoeba maritirurtnlhim 481
P

erwinia 233
-
ergotism (efg oroxkosia ) 627 expkht cuilures 439
extrapuLmunary' infeerculoaia 1. 60

j name nai
646 4 TEXTBOOK OF MICROBIOLOGY *
F rularenrsis 12L 331, 332 Mh
P versus host .re-action 180
fab fragments 85 freeze diving 434 Gmm stained smear technique % 111
fattia sp 402 freeze etching & Graves disease (see rhyxotooskosis}
favut (Trichophyton fchonleinu) 6l0^ Fra * test 42 b, £2& gritSth typing 201
614, Mi] Freunds adjuvant 140.169, 173 gri&eoiuJ. 61b
k (rapiiflnt 85 Fuller’s method 2li5 Grim nJuite test 212
_
fclix lubes 229. fungi 143, 229, bl0-27 -
group specific nudeopiutcin antigens
k rii ) L'ijCri >.H 11 21 fiuigi impeffeeri (dcuCCTLiniyCetesi/ 577, 522
Fernandez 's reaction 121 hyphomjttMes) 611 growth inhibition ter 397, 398
fanmaternnl A HO incoiinpnbi% 182 fusiform badlh 235, fcQD Guamlen bodies. 449, 470
FfrrtW 56 fasubacterium 267, 268, ZZD GuillakHtfane syndrome ( idkspmfhiic
fibrinolysins 207 fliJH-wpiriKhcCnsis 388 polyneuottii) 173.477
fibroma 468 gummata (chronic granulomam) 370,
fibronectin (binding protein ! 127 G 321
FICVTC bouionneuse 414, 41 b gi£f\cf 11
Fikiba-iidiclla banllispora 621 grille rrtuCanl wiodrte 612 Id
Filobas.jdjelIa nco-formam fall gammaglobulin 153, 154 haernogogus apegizzimi 529
fitdes 334 gAA&dbfrii1

fiLoviridjic 448, 569
filtration 2H
fimbriae 272, 223
Fira-Hugh Curtis syndrome 427
Dahlia (see bacteria cell will )
flavivirus 447,. 525-26
flai'obatleriuTH 603
lEbCELiii iM.'
^
fleabotne typhus 4111
flea index 328
fb? h eating butfwia 208
Fletchers medium 390
flocculation M
fluctuation test 55
flyaKtoem antibody test
Ciepmeaual antibody test 104.
fluorescent dye*. 351, 357
-
5 fluoxoujraciJ 390
5-fluorocytosiine 617 , 619, 621
fluey stmias 439,
IbILioiJjria 589
t i.4iscLLii.Li ( harmod mdru m. ) 618
Fontana’s method 378 »
food poiaotiing 252, 27 H£
hacmaphysalis
gSnjam diseww £rflimp) 522 Icachi. 416
ganjam virus 522, 511 spmigera 531
gatdnetelk vaginalis 229, 410-11, fefil
gas gangrene 248. 249. 251. 252 =
ticks 530 31
hacmciphilu* 224, 11,3-38, 60fl
anaaobie myoma 252
endogenous 254
prophylaxis and therapy 25b
-
acgypticus 337
aphruphiluK 118
ducreyi HZ
piicum 4414 gauspak tytteEti 41, 269
GB vims 559
haemoljtLcuE 338
influenzae 311
generic engi neering (see blCttria] p M aherEiol yr ic us 117
generic*) panunfluenzae
genital chkmydiasb 427 paraphrophilus 116» 339
genotypic muring 445 HafFkine 's ,acdnc 130
gentamicin 4Q3 , 405 ^
haiViia 281
UK dffij gcotrkhum tiflQ HiJtKrscacdicr Pkcrtrt ^.Lk (HP) bodm
german merles, ( we also rubella ) 563 425, 426, 126
germicide 2i halophibc vdbmos 316
ghan focus 355
giardia sp. 6il5
hand , foot and mouth disease 497
hantavirus 447a SH
5.16, 541 Cirms stain method 412. JlZ hanraan virus 533N
girmaa stain 124, 178, 186, 3&EP 1W , haploijpe 112
540 ^ . .
396, 424, 426, 449, 47 480 519

Girard M E V strain! 3.30

hapeens 80* Si* 83, 379
hard chutiLJE 179
foot and mouth cLsc-ise 606
forbidden clones 149
^ . I Lkshlmofut discjic ( IVEI iphaihiLoid
glandular fever 81, 170, 171, 189, 182 gcume) 122
4U 3, m HmiU's corpuscle 118
fimddetiyde 30, 3k 243, 249. 258, globulin {see i nn TU u noglohu Li n ) HAV vamne 548
291 300, 330, 414 aiz, 513, 518, . glucm 182 Iw-axa IT.rus 513
548, 5M glularaUJehytk 31, 249 281
s
Heat's multiple punmut cechuikfiie 161
formed Kutoid ( fluid BOBaid) 218 glycuptforein 185, 435, 504, 511 heat labije rosin (HLT) 340
forscarnet ( trisodium GM (gamma marker) sysE’cnn 91 hear arable toxin 2/ 4, 279» 281 » 111
phosphonofimmle) 460 graUpooc 468 hrjvy diiin 86
foranun antigen 82, 484 gulgi apparatus 125 dhca^t 90
fowl plague SXJl2 gonncociJUi ( see also neisseria) 2.17 hcla cell line 412. 425, 426, 444 444,
tbwipox 468
F ( fusion ) protein 5 86
22, 225
gui KuriiLa 225 29
.
475» 479» 5H» 516
. ^
I Id J cefe 287, 425, 436 475, 479, m
framboCMLa (see yawg) G protein SM helper vinu 418
francisdla 331 graft 176 - HO hcmadsorptu m 44U, 449, 504, 506, 507
 * INDEX 647
hemagglutination 16, 432, 434 » 4.3fr , hih PRP vaccine iJ5 Hunterian chancre 379
519, 521^ 536
.
493, m-5 fK 507 512, 1R 516 . hide porters disease 244
Hikqjcma strain 307, 314
hytkxnnu. ticks 416, .533, 534
hvtuidomas LSI
.
inhibitions ierf 397* 39B, 457, 504, hinge region 86, 37 hvdrogcn sulphide production test ±6
507. 523* Hippolates paliippes 385 .
hydrophobia 2 537, 538.5M
indirect 321. 398 HE* serum water mdrum 38. 217.222 hy fh rp|jirhini T ^ hi i (ly^ saphish:^ ) ^
1 ' ' "

passive 172, 239, 329, 397, m timMUOt sentitHpR fklftr (HSF) 34T! h }JK* IgK sypchtime 158
*i£il 4 U
hemagglutinin 504
hi&riucytE (m mat rt phage)
(

hiffmcompatihLlity antigen* 1 7 H
hypenetisiiivLry 92, 10 L 159 6B, 261
atopic 164
-
filarnenttMi’. MB hirtupluma 623 classification of 159

^
hemolysins 6 195-96,, 251
hemolytis 110. 3 88, 250. 258
alpha, hera 258
tapsititum 623
duboisli 623
hisroplasmotis 623
delayed (or cell mediated ) 159
immerduaCE 1.59
immediate vs delayed 160
hemolytic disease of newborn 16-5 , afiican 623 types of mctirnnF and their
mm hisfnry nf hact nil ]logy 1-6
"
feature!!: 1.60
hrcnmnirhapir femrs, viral 563 HIV siiFertixm 89, IKE, 545 hvpnthyruidiKm ( myxedema) 172
hcinorrhicic Fever with renal HLA molecules 12L 13Q hypa*anthine ph »hinribnsvl (

syrdrnme ( HFRS) 563 alpha L 2* 3 chains OH rransfenue (HPRTj 13Z

kvendra virus 5 / 3 1 ILA typing 1.31 hypha 610
-
hep 2 cell line 2S7 412 440, 447
M

hepadTWiTijK 442, 447

, Hodgkins disease 158 . 518, 562.589
hepatitis viruses 27, 188, 434, 547-61
lira! hepatitis 5JZ
hepatitis lype A ( infectious hepatitis)
ill
hepatitis A tint* (HAV )
hepatitis type R (serum hepatitis) 549
antigen* and antibodies 55-3
hepatitis B virus 43BP 550. 577
and hepatocellular carcinoma 552 ,
553, 554
hepatitis type C 552
hepatitis type D (delta) 43-8, 553
-
hepatitis type E (E NANB) 559
hepatitis type G virus 559
hepatitis, NAME 559
hepatitis vaccines 549
hep Lifted I ubr cantinoma 552, 553,
( human immuTHidcfidencY virus 432-48
554, 572, 5ZZ
hmdilary anporancitk edema 114- Hit
herpanginn ( vesicular pharyngitis ) i9Z
bihmncytfttrtjpimri 3Q 1
' hnitw BIIKfflkrf B1 la antigen 134.1
hospital infection 634-38
common types of 635 iatrogenic infections 634
diagnosis and control of 636 1CRC bacillus 3Z£L 325
idSQtiS
factor? contributing to 634
micrnbmlngy nf 634 idnxyutidine ( 1 DE. J ) 478
W ttniann (s*e dermatophyTids )
srETilianf; eqotpffKftt iuvd tlhcuS viEta 526
techniques, testing of 634
hospital wrote disposal 63b immune adherence IQL Ifl3
imnni, Liie C' jimpls ^c (nr hsxjir uimplcK
Hugh- Liefain's teal Zfll, 308
human body louse {pediculus diseases) 155, lM. liil
immune cytolyFis 111
humaram corporis ) 387,, 413
imctiLine: elimination 75, 135, 261
human diploid cell iTriift (HDCS)
nmmuM response 111-51 -
vaccine 541
cellular (delaved hypenensitinnly}
human embryonic kidney cells M3-
human gammagJnbulin ZZ
: 4. 149
eflSsi^ of antibody 11
AIDS related vims 587, 5S&
'
effect of malnutrition 139
variation and diversity genes 124 122
antigenic humoral 1441
t

herpes febrilis 476 irnmunolnricil abnormalities of in malignancy IBP

herpes letiHi 589
m iMpLcive phase 134

--
Instiviruatt 594 primary 133
herpes simplex 476* 478.481 pathogenesis 586 secondify 133
herpcFvind ^e 474 85 resistance 5S5 immune RNA 132
herpesiimses. 474-35 tiles, of isolation 590
immune system 117-3T
herpesvirus simile ( B virus ) structure of 5H 3
immune tolerance 122, 123
(eirenpithicine herpes virus, 1) 478
herpes zoartr (shingles, MM ) 4flO
h*rpt* irater, nph[ hafinicu* 481
human herpesvirus type 6,
human leukocyte antigen ( HLA ) 130
complex Uffl
^8 485
immunisation
oumbanj&d 78

human papilloma virus ( HFV) 521 routine 611 '

herpetic whkknr 476

hcreraphMe antigen ( see Forasman human rabies immune globulin individual immunisation 632
antigen ) ( HRIJS) Ml Echeduks for 611
heterotriBpha 212 human T cdl Ihuhcmk vims (HTLV) immurury 4, 71-79
heuons 486 m acquired- (adaptive) ZS
HFR cel] 52 human T cdl hnnholnpic ^tnii- lll S2B herd 79
Lfiiruce Z1

nie< ceria
MS
Iral 7g
cntiiui iTitnr
^ of 78
BTTTifBiriifiMfrTwiglufttiin (IK ) 11 5
immunodeficiency diseases 152-58
primary LtnmunodifiCLeftCLes 152
dutlfic&tkn of 111
disorders of uompkmem 153r ISfi
cbsoniers of phagocyrais 3LS2
secondary immunockfianiaH JLifi
Lmmujsodiffusicm fprecipitation in
»6036
radial m i l j
immuiindectTnn mpcfoBUQpy
immunodectrophorcsis SI
immunocnTyme tnt 1139
inU}iui>0tKn.nic £*n« toft 104, 22®
* bacterial appendage? 63
233* 245* 277* 382„ 39Db 392h 197, *
398, 40 L 405 , 409, 417 , 416, 420,
4B0, 484, 507* 5K 515, 5 T7* 519,
523
immunogldbLns (IQ) S4-91
]ga 78, S3, Sfl-B9, 113, 152, 154,
.
164 315. 343,
ami atopy
i ihlmiiy paste 89
"
^ biological actinide? of
Bum \gk ($IgA) W
IjjrD W, 115,
%E 90* 115, 153, 158, 160, 162,
i64. m cwotuxins vs endotoxins 62
IgG 87.112, 113.114, 152, 154,
Mk 162, 183, 189 m 315 , 5b4,
511
.
r
IgM 89 112, 115, 134, 152, 154,
160.185, 190, 315, 381, 53a 533,
564, 521
immurniginhulm gene superfimily 121
imflnriiiirthcrnicialugy 185 91
immunological paralysis 340, 147
immunological MrvciUlsincc 5* 181
kmmunc4ogka] tolerance 147
iimmunolpgkallv LYKmpcctric
(ICC) 121
bTiuuuralyELn test 223
LmmunpmeCric te ts
* IflE
LmmLiJiEipnnphyUxb 6.32 1. 3
immunoauppresawc: agtnca 141
immHttdlcrapy in malignancy 182
impetigo 197, 589
IMVK tests 46
inri tit ran on 25
Lndu.5i.0 n. bkfMvtaM 427
inclusion bodies 423, 424, 425
iddrtphilic 449
basophilic 449
Ccgwdry type A / B 450
eosinophilic 449
4 TEXTBOOK OF MICROBIOLOGY »
gusumieri bodies 449 morphology ID2
HtncjtQplwHC 449 onhomjfluvmu and
intranuclear 450 paramyxovirus 501
inclusion conjunctivitis, monara! form pandemic 5012, 505
of 427 psAfOcernt 50S
indole production test 45 , 273, 278, prophykm 511
2811, 319 sporadic 508
infection 64-70 treatment 511
clarification of 64 types A B, C 502, 305, 507* fII
stead).' state infection ( eeDuibu mterference 441, 443
injury) 449 anesrfimrs (IFN ) 143. I45r, 445, 454, 460
immunity 384 interleukins (IL ) 124* 127, 144 45 -
Mft mode* of transmission 66.69 intrauterine infections 89, 481
microbial parhogemcit factors 66 intravascular coagulation,
IABHOD 61 ^ disseminated 114, 127
iodine 31, 502
baduml products 68-69 staining 424
bacteriophages 68 iddophofca Jl
communicability 6fi idoxuiidinc ( 5 - iodfl - 2 deoxvundIne )
*
infecting dote 63 425, 418
inva&ivcncss 67 isuinChgtin* 82. 190
plasmids 6E HJAT ( international union against
route of infection 69 tuberculosis) 352
toxjgenicity 68 Iuat-LJ medium 352, 358
Ixodes dammiru 389
ewtoitfan* 62 UKKIUI ticks 419 a 531
ttdkmunf 6Za 62
euoroxLns 67^ &fi. J
Japanese encephalitis 522, 526
sources of 65
types 69
infections, vinu
cbemutherapy cif 459
host response 451
jafisth
—
Japanese UB' encephalitis 526
Jc virus 56,7-63
-
HeftdicimjT rcptficifif 384
Jen tiers' cowpox vaccine 457
immunity in 451 network hypothesis 149
ertncr s
-
tryl Lyiin strain 314
immnuoprophylaxts of 4 7 ^
- laboratory diagnosis 455
nonimmunologkal ^spumes 45.1
h virus 499
Zitas syndrome 157
[itini* v bacillus ! M-
pathogenesis of 450
screening of virus 453 parafubeinJcHsis)
cell Spedipms lo be sent to diagnosis 456
kifeettout ituniam.ideiflis (glandular
fever) S3, I 7P, 171, 189, 582, -«13, 48:3 K
infective hepatitis (see hepatitis ) K antigens 273 , 2Z5
- inflammation 2A
LnfWnu 3, 434
antigenic classification SOS
factor )
<
^
KAF (set oofl ntudHl aerivaiing
killer K) cells (see ADCC cdk)
antigenic smjctme 304 Kiatpnn phenomeirison 316. 317
antigenic variation 504 Kip "- sarcoma 4778 485, 577. 582,
1
dhokfil features 506 589, 590
epidemic 508 Kaposi s viri.eetliForm eruptions 477
epidemiology 508 kiiba vipw 534
hemagglutination 502
host raxige 506
KmiffhfffmrWhkt scheme 293 .
KCN medium 273, 280, 2M
immunity 508 Kdev stsfains 536
laboratory dLagnosas 507 Kelley 's medium 389
r
pyrigniet i
ri
 « INDEX t fi49
kerion 614 icremhemnrfhagrie 389 lymphocyte tnmsformafion tets 372
kingelk 410 intrarogans 389 fymphoqrosk producing factor 340
-
Kirby Bauer method 628 laboratory diagnosis 391 lymphocytic dhoriomeningirii ( LCM )
kflMinp d isease 4ft 3 serological diagnosis 392 virus 447, S/J1
kkbsKlla 15, 2BO-81, morphology 390 lymphogranuloma
—
KJcbs Leefl1irr bacillus 231
Koch's phenomenon 4
pathngEmdty 391
prophylaxis 333
inguinale 428
venereum ( LGV ) 424 228 k

Kochs postulate iL resistance 190 lymphoid organs, central 117

Kj&ehrweds bacillus 321 therapy 393 bursa of fabridus 119
Kofihk* 'spue* 51S. 564 leptospirosis 391-93 bursal lymphocyte
KorriioF s medium 390 leprorhrbc 600 (B cells) m
Koser's citrate medium 4j& lepECHirichia 267. 268 bursa dcpcndcnC -
area i 119
KttvRc'p reagent/method. 45* 46, 223 leucocidin 6i 196 thymus 117
kuru 450, 567 lLru.o:>::>te ajirtivatirng feet nr ! I jVKl 141 mesenchymal stem cells LIZ
kupffer cells 1 leucocyte diffiaenriarion antigens 122 runt disease HR
Kyasanur forest disease (KFD) 521. leucocyte GfiPD deficiency 157 thy antigens 118, IRQ
522, 524 -
lirukosi: ^ afCMVVa virus 573 lymphoid organs, peripheral 119
linskntTa UTIL'S 163 bronchus associated lymphoid
levamisole 182 tissues ( BALT) 12Q
L
.
l- jh-E mco (commercial meat
^
extract ) 32
—
l^nnthaHZok lilfe (LOJ bodies 42-7
LevinthaFs agar medium 334, 336, 4Lti
-^
jj0iE a &udiftjfcsd
(GALT) L2Q
Evmphoid tkxuca-

LiLEY s DFP medium 342 40S. 41 ]

UdribaalhM 2L 266.267. «M, till
.
Lancefield groups 205 204, 7 H1
add extraction method 2lii
Langerhans cells 122.
-
Lsuge Sadhs group 286
bssa fever 569
lassa virus 447, 547. 569
Late fixation rest 100. 224. 402
lattice hypothesis. Si
Lazy Icucocwe syndrome 153, 158
LD 5Q £L9
L£ body LZ=L
LE cell phenomenon173, 174
legfonella dLS
kgioniwiLTes duem iQ2
Iccirivirtijcs 566
Leporipox virus 466'
lepra (foamy) cell 170
Lepra rtacwnn 373
Lepromin KH 373 74
Leprosy LLL 367 , 370-?h
.
.
L forms of bacteria liLlS 193. 399. lymph nodes 11 *9
mucosa -associated lymphoid
and inyLY >]ila!-iia;i 399 tissues (MALT) l 2l~li
limes nul [B
|dose 219 mucosal (or itereriary) immune
limn told Q+ ) 232 system 12Q
limulus polyphcmus 69 apken 120
lipopolvsaccharide (LPS) toxin 226. germinal centers 120
ir iighian OCrpUtdfi or folfckf Lift
211
Lipschuitz inclusion bodies 474
hqwti& m
],, ^
cells 12& Hi JUS2
*
infrfi,::, ki IIL ucthnited killer (1..AK )
limed 6fll lympWmas 574
IktcrLa monocytogenes 224, 403 EB afeiixiaicd 4 H2, 5H
lirterwfls I 43r 4J11 _ lymphomatosis 2ZE
litmus miJJk test 45 > 249. 2511 lymphopoiesis 122
lirver infimion media 345 lymphorecLcutr syscem,, ceLk of 12&3
ll ^ xiiffleris methylene blue 231 357 Jymplwcyte 120.125 , 127.129 .
Loeffler * serum slnpe 26, 231, 23 F , 137. 146, 159, 164
252 lympholeLMi 122, 123, 127, 160.
long acting thyroid! stimulator L62
( LATS) IfiO, LZ2 ph3 c.x,"ytic cells 117.120
louping ill 524, 52fi ^
lymptvitrixin ( TNK-beta} L45
louse of human body (pedSculus- tyophiliaadon 293. 424
humaruu earporii) 413 ly H niL- cycle 56, 462, 465
^
'

classification 371
culcivjciun 3711 ^ iwusis- lem PunsEii ( LJ ) medium 26 ,
[. lysogenic haurteria 56, 462
epidemiology 372 11352, 367 lymgenic conamion 2hx 232 , 465
Lwkes tumour frogs 226 lysogeny 5 465
immunity 372
laborafcsTY diagwris 374
LucHami medium 193. L2R Imigemsadon 223 ^
Ludwig's angina 2QR lyuoi 32 329
morphology HU ^
lumpy skin disease 46 B Ivsosomes 21
prophylaxis 1Z5. lupus vulgaris 362 Jpostaphin 194
redsranee 371
Ijgrwmm 22B lysftcyme 12
beph 3xpi. nL 3R9 Lyme disease 389 lyssaphotwa ( hjdrophobkiphdbia) 528
antigenic properties 390 lynnphadeiicipathy associated virus lyraavirus 447, 545
odfirfal characteristic? 3*90 {LAV ) 582 lytic (ar virulent ) cycle 56^ 462
epidemiology 392 lymphoblasts L2i 'Ij'sis from Tvithout ' 564

Copyrighted materia
650 4 TEXTBOOK OF MlCHOBlOLfKiV
M 446, 447, 449, 451 458, 517-520, lethal dose (MID) 69, 238.239
,

MicCoofae/i medium 3Z 561632 reacting dose (MRD) 239

rtuthiwellft swain* 432 , 424, 426
machupo varus: 569
nttcrngfobulirtcntii 84, hiO
mamoconidb 612-14
macrophages 119 , 124, 135, 140 , 143
1M, 167 , 295 . m
Htmted 124
activation/ aggregation factor
( MAF) 141
in blood ( manocym) 74, 126
charaizccruto of ill
dbemxiak factor ( MCF) Ml
migwion inhibiting ficm (M1F)
143
clinical features 518 mink encephalopathy 567
laboratory diagnosis 5l9 mitogenic factor (MF) Ml
VHQEK 520 Mirsuda's crude antigen 374
virus 517 Mirandas late Teai-tinn 374
mediterranean fever 1. 45 mixed leucocyte reaction {MLR ) 1 .TO,
medusa head appearance (sec bacillus ) 12a
melioidosis 322 mixed lymphocyte edfim: (MLC ) 179
rrxmury oelLi 143, 146 mnhiUincw 266, 267, 4] 0
meningococcemia 223 , 22A molecular buk>gyr basic principle of\
meningococcus ( pec N meningitides) 51
222 molecular epidemiology 61
metaholie inhibition. 398, 440 AMJILCUEB 395
metachrcwiiaEij 11
meracliiPOTiiHnc granules (sec Babes-
EMSI Rfjinules )
nfif &nm 48
momtiiai 589 616-17
^-
MnUpannn contag Dsum 449, 4> D, 441

BMKMJOCletff 326 h

in tbeusc ( histiocytes) 74 L 12fk methanamine silver stain 611 monkey fever 531
mad cow disease 567 nKthartobaetmA 266 monkeypox 468, 471
maduranijcosis (nuduia foot ) 402,. 618 mdtjfctt blue rtduLiicHni EE?W 46, 449,
606
monoclonal antibodies 122 135 .
madurdla 61S manocytt (see DHCRHIUHBJ)
maisdi ( progressive pneumonia ) 567 methyl red ( MR ) re*t 45.273. 3%, monokines 143
MAI complex 366 MTadyean's Traction 242 monoma-fphism 51
major harimompaiibLliry complex
{MHO
antigen
m
122.
^-
mcrrMfc hj]!! . : bacteria 23 6flfi
microbial flnr ? of human bodv,
nnrmd 599 602
^ Momrk CTTA medium 306
ManrAmridd hsdlius 410
moraxelb 230.600
genes 129 micrococci 194, 200* 600, 601, 605, iruwbiDirkrujt 447, 512, 517
molecule 129 micmconLdia 612 63 3, 61 4
V mctfgrmelia 272.274 , 262. 2S3
restriction 1 V, I „ 02 niernn Z morgani i ( formerly Prorms
msuor outer membrane protein miaophage 74* 122 maiganu )
(MOMP) J24 microscope
dark tldd (dirk ground ) 8, 314,
rnorpohnfogksd irrfew 370 32S .
fWHudi ( aiptcgillut, nuoar) 607, 610,
nsai-hi-wraj furfyr 612
mrfigfwwy 386 , 391 , 406, 301 624,
immwB response in. 178
knimianolGgy of 180
malignant edema, 254
direct examination 311
electron 8.
interference S
-
mousepox 451, 466
.
M proteins 9Q 205 206.209 402.
504, 512.513
.
optical (or light) 371, MS murker 494
malignant pustule 244
Malkin BEST 322 phase contrast 9^ 407 ^ mucormjTOSdB 624, 625
maTta fever 2» 345 polarisation 8 nSHiW spedcs 612, 624 625
^
mammary tumour virus (of mice) 578 micrtwpwuiiT 614 Mueller-Hinton medium 22.1, 225 ,
numiomc rear 361, 518 microfilmours SIS 223
Marburg disease 569 Mddkhuk'i medium 352 multiple myeliHiui 158
Mnki disease 576 migration mJiifcision factor ( mif ) 143 mumps 143, ]2L 436.448, 453, 458,
MnHFiw monkey mammary
Linde r 576
mitk 605 60?-
h ft jitter if ik ^gii^aiJ ^.'om inhaininrs 606
501 512
»

clinical features 511

mast cells 127, 02
iflnCidcHmM 446
Muted $ method 205, 2lfl
imypfP virus 522F 524, 525
iruaucchi vaccine 245
'
Mobride s intratypac antigenic
fHlktf 494
McCoy veil W 425, 426, HI
Mdiriitiosh-Filkks' jar 41
diseases, miMKMTie 606 epidtmifiLigy 513
milker 's nndcf ( paravaccinia ) 468, innmumry 514
472, 60A laboratory diagnosis 514
fflUfamriff mokude 89 properties of the virus 512
mima polymorpha 404 prophjhrii 514
msmeae 405, 601 mump orchitis 172
rnkumum bactericidal Liirttenti'ation
{mom
murine leukosis virus 575 578 .
murine typhus (*ee endemic typhus)
minimum hcraalytk dose (MHD) 102 faMiffty viileir etictplulLib virus 526

Mdnod1. l medium 37* 232 minimum infecting dose {MID ) 68 muv muscuJus 416
measles 3.143, 152, 158 , 436 , 440, inhibirorv ctHKeniruifHi ( MIC) mutation 443
630 mitogens 142

r
vrianiet i
ri
 4 INDEX i 651
Mnumj grajiLilocoq udjts £M
^ N nrtrtrtJk virus 571
nmoorvnial infection 634-39
tny
^dbcJib gravis 172
mycelium *10, 614, 625.
Npgkr* * medium 3£
nuelcocapfiipd 431, 435, 437, 446. 512
Magler reaction 251, 252 , 256
mycctism 627 nairoviru* 447 nuU cdls 12i
myccToma 402 618 r Nfljrobt sheep disease virus 513, numenr agar 1Z
mycobacterium naKphaiyTipsal carcinoma 4S1. 574,
africanum {'alrkan type1) 351, 526 O
354, 36B native ( N ) antigen 491
asiaticum 365 , 368 Oaklcy-Fulcbjopc procedure 96
natural killer ( NK) cells 126 , ociiLnmycarsis 627
atypical 365, 366, 36ft, nccr-nrising LTIJLEU
avium 366 Ogiwa and wrocypw 3QZ
uteriju 212 old tuben.TJin ( OT) 361
hdnei 36ft hsmm im Omsk hemorrhagic fever 522, 531 ,
btjrvift 351, 1
. 66
iejuniris ( aiceriris flcoodmw, oncogenes
hufgli
piRhel ) 252 cellular nnenpene* (c-^ine!i 580
huiyfLcum rerinitii 477, 4ftl
chdonds 351. 367 proto oncogenes 5 Ril
negri bodies 449, S 36 , 539, 540, 543 sre cmL ogeji S 5 SO
clAssification of differentiatum 146
* '
^
viral oncogene ( v -onc} SBD
between M marinuiYi jnd M NeLH-McKnrr fhinica} redi-lion 415 an.ti -nncngei¥rs SSQ
uker-ans 166 Jirisser 's Ftrun l 3 j 211 chfamr^onrul lncUion in human
ffllbur 368
formicum 367
3i 222- SH
eatarrhalis {Branhamelta
m m m 601 beings SSI
mechanism of oncogenesis 581
gnrdonae 365
hjcm&philum 36S
eatarrhalis) 43.129. 2M
dAnatill dunuxerisek* ot 229
oncogenic viruses 574-S1 -
intraccllulare 366 ashicLfeEAd viith human cancer 5 / 6
N flm 221 dna vinisa 525
kansasli 165, 366, 369 N flavcKmE 221 ma viruses {formerly
Iqnc 370-76 N gonorrhoeae 225-29 oncomavimses) 577
,

Icptnc murium 175 N imupdidis 43* 222- 25 , 599

mabnocfisc 366 ,
imwiflM 577- Rll
N sieca 229 '
one-?4ep growth cum 465
4

marinum 366, 367 Messier * reagent 46

1

o nj'ong - nyong 'inis 522, 525, 53fl

'
imnori 3M » 153, 354, 366 neuraminidase 170 431, 434 443 , , ,
oo^pcHcs fell
paratubcrajkiFis | lohne's bw/ iJlus)
166
447, m . 503 504. 505, 506, 508, ‘opacity' associated' ( OPA) protein 226
Slip. 511, 516, 518 ophthalmia neonatoruin 22Z
phlei 165, .366, 167 neumtoKin 258 261, 286
schimoidei 16ft
,
opsonins 127
tueuEraJisancHi. ECSIS 4, 103. 239. 250. opsoniutvon 104
scrofulaceum 365 , 366, 368 «7, 48 488, il£. HL 522.523,
SLITIHUE 365
stnegtnacii 367, 151
525 ^
al redEM 3S2
optoclun test 216, 7. H 7
oral polio vwrisw ( opv ) 494, 495, 531
EH-hivirus 448 , 5.14, SZL
neug ir
stercoris Newcastle disease nua IM off ( contagious pustukr dennatids )
STulgai 366, 368 NezeLuf synchDrive 155
ulcerans .366-68 468, 472
niacin USE 351 nmiTbodonn: 3M
xenopi 366 ,. 367, 3 9 * nipah virus 571 onuthoias 428
I Z -. X i ih. i . -fr - rii in i tubefoulm 351 iv . 589
* niwl bodies (ehmanjitilysisJ 492 offophar>T! jiitis (vincrot's jingina) 1. 88
mwolotf 610-27 nifr* feduotHH tear 46.2\\ 404. 07 mpoude virufl 522, 524.522
mycoplasma 1 L, 171 . 395-99 ,. 430, nocardia 4W , 401 oroya Fescr 42 f >
431, '600, Ml tookfe 402
nqQpktmfll pneumonia 397 brasilknsk 4152
* "wphdn’ viTusts
iSfi
myrows 612, 624
mriioftijwoi'irus 433, 43fe, f f l l -1 1
caviae 402 orthopoxvirus 46 R , 471
filrtgoidw iZE nocardiasis, svsnemk 402 otoiAyv u 625
mycotic keratitis *25
. r 4B~

riairneEicbnijre, bacterial ( see bacterial ^

Otwn's Tjiwidej s strain 330
mycotic poisoning 627 taxonomy) mchrtsribny pErocodurrc Ma 91
tyHMBmu 627 nnffgnAAcncat urethritis 427 Oudin promfore Sfi
myeloblasiosas 78
myeloma (M ) pranrins 90
^ nCHfnshetodinillQffm 365 , 366 oxidase iacdOD 46i, 404.405, 402
aonrapoifider* middiion- P0di*.Tl.Ms (isdos) putenrial 22
mj.'dopcp^cddnsc! deficiency 157 mxnEe-creton: IM
myJucmtiUtwis 445 nonspecific inhibitors 503 504 S'
?
myxDiiiJvirufi 445 nonsporing anaerobes 266-70 pandemic TOj 501 , SOI
,

mymni 435, 503

11ELU r i
ria
§52 H TEXTBOOK OF MICROBIOLOGY
papilloma virus 446.563. 571.574
pfipDVtVtfUM 437, 444 P 445 , 446,
~
pett pnini medium ! ~T !
^ * i»«W 357
Htnoffh
-
plasmids 14 53,56^ 52
plasmdj'Sis 23
.
5 f 2 _ 575 ftp's punches ] IT 12& 2 % Vfl
• .
plssmoprysis 23
and cmc-er of the uterine PfsiffcnUa ill platelet activating factor (FAF) 161
cervix 562 PftiffeA barillas 333, 501 ptomorphism 17, 51
paracoccidioidcs brasilic-nEis 622 FfcLfFeA phenomenon lilt plesbmneias 3fllr 317
paracoccidioidomycosis 622 phuoMiMphjdaxiE 171 shigeUkiLdes 312
pUHokof 2 7 [ „ fiin phttobyphomycosit 615 Pkt medium 242
pninfluenu virm 515-16 pfcuKe pleuropneumonia- like organisms
m
^
pn rtk poliomyelitis 490, 422
pnpqffmridte 427. 447
ptqgmffrinv 468
466
ttOTveiuijQrt
DNA 462. ifil
463. 465
(PPLO) .
psin^osii- lyiTiphogriiEi.gbma
trachoma ( PUT) 422
piOtyphoid 290. 606 susceptibility test .107 pfieumoeoecus (air. pMumgniaae ) X
parmcriiua (m milkerPE nodes) typing 466 14, 216-21.224
paroxysmal cold hcvnogtobinruria 169, .
phagocytosis 126.205 225, 226, 223 pnciunocyFtii car ini: 582.389, 592
LZ1 dividers of 153 pocumolysln O 219
pariusymtfd WKtnn) hemogfebinurti phagplywsome IX 121 pneumovirus 447 , 512, 516
114 phagosome ( vacuole) 74^ 126 pock assay 448
pgHOTiliklfiC 447 phenetk system 42 poliomyelitis (uafifctirik paralysis) X
72* 76. 23B , 44Sr 458, m 491
"

parwriiniLs 431* 433> 437 , 447h 563 phenols 2A. 31

psscbsm bodies 422, 431, MS phenotype mixing, viral 445 abortive 452
passenger virus 482.574 phMopboxa 619 cradi carion of 4-96
jpssEeurdJa 124-30 phJdbonMr&ui 532 nonpanlytic 492
avrscptica 124, 111 phlebotomo* paptt&ii 532 paralytic stage 452
bwiseptLca 331 phJidbovirus 447 strains t y p& L 2 and 1491
lepis- cpbca 331 phosphatase lest 607
multorida U? 4. 331 photochromogens 365 virus 491
pCSEi* (wM yenimii) pturtutrnphs 20 poliovaccints 491
pmdotubemildsi ( s« yersinia phycomyceiies 6ll polioviruses 491-94
pseudotubemitosis) phiiCcunjTrwk, stibmtaneous 620 poij^orvianiide gd dcc impi^ reHiK 571
PW Bvwd test 83, 99, 484 pk'ummridae 447 poljdcarytMitosls {syncytium.
pedionlus human us corporis {body pfcnmavinucs 432. 433, 490-5Ofl formation ) 449
louse ) 4JL 336 . 413
. piedra 613 pdhpTOirs^ chain metfea 591
pmkillmasse {beta kccamase-) 194.222i puritan horfcae 613 pollyoowrinc 446
pemrilfiows 625
pemriflium 612, 624, 621 626
marndfri 62i
pigeonpos 468
Pike's medium 210
pili ( see fimbriae)
^
po riboqrl rihikot phosphate (PRP)
antigen 333
pdfysacchsjndle vaedne 2l£
pentons 436 nun 377, 3S5, Ponders stain. 231
,

pMEaae fevpr 4ffl

perpbmtrs 431 Pitman-Moore sixain 341
peptidoglycaji 16? 195 , 205.221
h pfijdhai vesakcfcr ( tinea wssmtir) 612 pooled human gammaglobulin ZI
peprtKoed 266, 262 pitymaFporum poscnasal mb ( West * pTHttWid
pqari^oqstococ-eus 267. 270 ofbkukic (Milwrfi* furfur) 612 swab ) 342
anacrdbius (formerly P puiridiis) Wile 600 posE -trsmsfusLoin. mononucleosis 4&2
262 plague 326 30— pownssan virus 523, 531
pconiridac 446 468
^
wcchin tiac 262 ,
bubonic 322
PPA reaction 282
magnus 767
prcvoti 267
tetradius 2fiZ
domntk 328
mild ( pestis minor ) j ?7
,

pneumonic 327
^ 1M
-
VtmmutB tbulua (PfQ Beac»D ffl,

predpftBcloa ncdona h, 9 623

periodic arid schiff stain ( PAS stain)
611
permetbllkj factor (PF) 111
prophylaxis
septicemic 322
plague assay 44 448, 466 pnsuriurtifiofsi 76 3B4
^
PmssE-Nocsird bacillus 240. 322

pernasal swab 342

perosidase test 107, lOft
persistent tolerant infection 148
pertacbn 341
. plaques 466
pestig mirtur 327
^
plaqu£ mhibition tot T 03
plaque reduction neutralisation
, tesE
^
pngsumpti« cctfbm count 19, 6QQ
primary atypical prunjun^La { PAP)
(PRNT) 522 S3r 397
pfinuy rampks 355
plasma cells 117.119 122 . prion 448 , 567

,
pyrign
i J J
i
ri
 4 INDEX * 653
progressive multifocal pyoern typing 320 rhipkephaJus ^snguk^ua Ticks 4!l 6, 53.1
-
Leukoeivcephal* AEHY ( PML) 562,
566, 56E
j i%
- pyoderma 2u 4, .208
pyrimidine* 397
rhizopus. 624 625
ribosOnnix Li
-
prokaryotes 7^ 430. pyr wsc 204. 2115 ribtnnrus 446
properdin
pathway HI
rickemiu m, 412 21
dari 415
-
M?
Q
system
. Qfeirr J12. J 1 S. J 19-30.606 bufflitii 419
prophage 5 465 conori 415
^
prcrpiuriibaeterium 240.267. 269. fifiO
qurilung reaction 218. 335
quinsy 2lM parkeri
pruSodEmu: diieue 70 prowazelui 413
Prospect Hill viruses 522 ill
proFtaglandinrs 161
. ft
nckensii 415
Siberia 415
proteinase 207 rabbit fibrnnu 574 576p tsutsugamushi ( R oricntalis) 416
protens- 272, 2?i 275, 282, 231, 413 . rabbotpsK. 46E typfcu ( ft moused ) 417
418, 467, 60ft 601, 605, 625, 635 . rabies IQi 430, 432,, 439, 440.447,
535-46
weiWeLx reaction in rickettsial.
6JII diseases 41.8
'hauch.' farm 2fl2 rabies relaxed viruses %46 rickettsial pox 416
ohne haudf form 2fi3 lyasavirus 546 RLd»L Walker test 13
mirabilis 283, ilfl nluet virus 535-37 Rifr Valley fev« 522, 524, 521
mtirgiinii 233 radial immunodiffusion 96 . 508
radkiautive antigen - hiiiJjr&g test
. dngwwm ( tinea ) 612, 614
stnins Ritter's disease 131
ox H 40, 418 radicallergosorbent test {RAST ) 164 RNA viruEcs 447, 569
o^ 2 413, 431 radioimmunoassay ( RL.\] 105.1 Robertsons cooked meat medium > S,
WE k 413, 413 221.457 ., 572, 198.205. 24S, 350.256. 2.57,
Mul ix 418, tfiS Ramsay Muni syniirume 481 ruboviruscs 521, 533
protista 7± 4£ ^ Rants and Randall i method 205 ruchalimaea quintBita 412 420 421 - -
protoplasts 10* 12* 199 ratb-,re fever {RBF) 405 RsxJcy MounEadn sponed fever 414,, 416
pro trachoma 426 mgin antibody 164 , 379, 33L 382 Roa-Waaler test 100, 174
provklenck (formerly PfOBcus receptor djestroyiug cmtvme (RDE) Russ River virus 524, 525
inconstans) 274 , 283. 434.503 MSWUi 10 !107, 448, 5ZL
-
aJk alifaciens 283 rouomhdnckiit DESSA technology 61 *
nxis sarcoma virus ( RSV) 438, 578
rengerai 2B3 feenmbarmtnnn viral M3 routine test dose ( RTD} 466
stumtii 283 Reiters syndrome 229 398, 427 . card test (rapid jih&m t in ) Ml
cpf
^
provirus 577, 578, 531
pfHjutne phenomenon 94, 382
pscujJciinrhraji Ktd ]]i 246
relapsing fever 382, 3R6-88
.
mn'iros 436 443, 571
resaiuriri test 606
ruhelb 89, 148, 451, 455 54 563
-
66 632
rubeola {see measles )
^ ^ -
pwtjjdolywgeny 354 reservoir hoars 65 rubivnrus {rubella vinii) 447, 523, 563
pseudomonas 2X 275.319-23, 6CX). resistance transfer factor (RTF} 57 Runt diseau ( M lyrnphuUl otgsnt,
603 625.63S. 636
- respiratory syncytial virus 5 l 2, 516 central )
icrugirwH (ps. pyoevanea b. .- RVtridiiHi endonucltaw Russian spring summer encephalitis
fpxymm) 22, ST, 191* 319 21.
466, 600, 635
-
polyincirplruam CR>1 P) 179
reticula re body i :mrial. body) 422
(ESSE) WLplm 524, 53 . 531
RSSE vaccine 531
a
ntkukr ' eneas of de Vkal 153, 156
ceparia 321
mallei 321
makophila 321
^^
rctiwirus 433h 436, 575 577-Rfl
Bcnomk structure 579
. S
pscudomallfi 322 base speaficirv 578 Sabin 's vaccine 76, 458, 494
morphology 474 S HDuraud's glucose agar medium 611
peeudomycekum 610
-
pscudoviripon 445
virus transmission 578 ^
Sadis buffeted glycerol saline medium
psilncybc sp- 627
reverse transcriptoe 437 r 577
reversed passive kfet agglutination
> m
pattuau 422„ 42S Salk polio vaccine 491
( RPLA ) teats 196 salmonella 27k HI* 790-304
psyrhrophilic bacteria 22
purified protein derivative ( PPD) 361 Ktycii xyndnTme 507 antigenic structure 291
purpura 330
Rrjnokk-Br-aude phenomenon 617 andgenic variations 292
R (actor 304 bacteriiDphage typing 300
fbnmboLytopenic 165
puumala virus 522., 5.13 rtuhdorifusa 535 46 -
diinofiporidioaia 617. 63-0
biochemical reactions 291]
Ktuftm^WkiS schesiw 291
pyocm 466
.
duxwviruEes 4-34 447 499 . .
paxaryphii Ag B C 293-301

L ' ii
i J riahte feria
654
[MEhogHlLCLty 225
ptuRC cnnverakn 3£H
typhi 37, 69, 167, 2?i IffiL 290
93. 300, 303. 466
lyphimurium Ml, 3Q3. 655
salmonellosis 606
Smardlj phenomenon 162
sandfly fewer ( pappataci fever) 522,
524, 112
virus 522
saprophytci 64
sardna 607
satoUitisni Jill
Saulon's medium 352 . Suft SfTTC 337., 379
Schick am 7ft, 103, 232
SditilEz ehaekon reaction 207
Skhulvz-Dale phertotrtenon 162
Schwartzmin iE5KSrav, phenr.mn¥?ri 115
,
tcfemic cells 619
KrnrthrhrosnrvisjncnK .365
scrsrpk 450, 566, 567
scrub typhut 414.416, 418.
FcutuJa 6 H
secretory immune system 78
Kactocy piece 8£
Sellers technique 540
Semliki forest vini* 522, 525
Semple vardne 540
*endai infua' 454, 455, 515
H
reneny lest 379
soubQiun -
(5 bydhncy nypramine )
scrmtJj 282,. 603fc 635
marcescens [bidlliH prodigioRis )
282
serum sickness 114* 1M
severe acute respiratory syndrome
(SAKS) 570
SARS WWH iirw 569
aKwr mb techniq/w 3QQ
1
taduction 58
Sezary syndrome 578
Shanghai fever 320
sheep-pox 468
shiga-lik* main 273, 2Z£
shigella 271. 275, 273, 285 2S9
bnydii 2M
epidemiology
flexneri 285
schmifzi 2S6
shigae 336
sonnei 286
shigelo*U 286, 2 R 7, 2M
shingles (see herpes zoster)
shipping fever 515
Shwachmans disease 158
simian ^enabling vims {SV 40 ) 525
1 TEXTBOOK OF MICROBIOLOGY
sindbis virus 524, 525
Sjogren's syndrome 173, 174 chemical agents 24.3Q
-
sterilisation 17* 22* 27 36
- skiii blueing icsr 111
slide ctKlguLix KSt 192 , 193
pastcurisanon of milk 26
flash process 26^ 419
slime layer 12 holder method 26
alow reacting substance of anaphylaxis physical 24.
(SRS-A ) 163 cold sterilization ill
slow virus diseases 566 controls 25
slow mm&rmtng viruses 579 iniermirrenr ( tyndallisat ) 27
-
srndlpsn 1 4. 446. 46BF 611
vaedne 468
physical agents
drv hear 25
r
Smith-Noguchi medium 12 hot aix erven 25
sodoku 406 incineration 25
moisr heat 25
Sokhcy 's vaccine 330 radiation 29
apedfie soluble substance (SSS) 217, ionising 29
21R amma rays 29-30
spermine ZI ^high energy electrons 30
spheftjpbsts 12, 13* 322 infrared ftyi 29
fpidJhjm minut 405, 406 nonionifing 29
iporuuioipore* 611
-
iptrochtfa £ li 235, 377 394, 601 ultraviolet rays 29, 31
sunlight 24
Sporovhrix (Hporatridhum ) schenckb ul masonic and sonic vibration 343
619, 620 srtme vaccine 744
spotted fever? 412. 413. 415 sEofnoxys caldtrans 7.44
staining techniques 2* 9-lfl stormy fermentation 45* 25fl
differential 9 strain 49
impregmafirm method 9 straus reaction 322. 348
street virus 536n
negative 2
simple 9 strepCitbaalluF- raooofifecmit 17, 399,
162 staphylococcal .
405 606
Food poisoning 1%, 138 streptococci MW-%14
lesions 133. dmifiettrion of 202
pneumonia 197 alpha hemolytic 2fl2
scalded skin syndrome 197 beta hemolytic 202
septicemia 132 enterococcus group 203, 214
staphylococcus 2* 192-24)1 , 224 , 333 , gamma nonhemolytic
334, 461 , 466, 601 (indififererif} 203
iibw m bimfidid group! 204, 205
aureus ( tormcrLy. fttaph pyogenes) virkkns group 214
.
192-94 195,
, medically important 204
bacteriophage typing 197 streptococcus
cultural characteristics 193 pyogenes 204. 205
epidemiology 197 umuintf 2W* 2Ii
taboomy diagnosis 198 sanguis 214
morphology 192 viridam 214
pathogenicity 194 stftpeoeoccus mg 214
toxins and virulence factors 195 streptodo-mase 207
treatment 1M streptokinase 202
intermedint 2£MJ streptolysin o 206
pyogenes 132 strepm ] n a 206
upmphjtitij* 200 streptomyces 4i K). 402
'
auphylakinaae 195 strepwxjme iest 210
steam , sterilisation 2i .25 , 22 string test m
. IE. 316
steam sterilisers 2, 22 Stuart's transport medium 38
SCcnoITupbumemw maliophik 321 subflcute Klercwin pBnencepihalitis
sterigmata 625 . ^
( SSPE) 518 519, 568

subacute spongiform viral
encephalopathy 566* 567
tub-unit vaccines 458
bugar fcrmcntatkin ( wt J5
suipuxvirus 468
Sub's medium 152
sulphur granule^ 40(J . 401
SuLzberger-Chase phenomenon 119
supermtigens 142, 197 , 202
superinfcction SSS
nperinfccboQ inumiittcy 465
auppnsaof (cjroimk ) cells 122
surface phagfttyiosis 219
sushmtH samhita 17 h
SV All virus 494
Svedberg unit 84
sweep plate technique 60S
swimming pool conjunctivitis 427
swimming pool granuloma 365, .169
swinepox 4fii
iwiss type jgajiamagli 4ra Line Alia 15 -1
jyrn pathetic EPphthalmia 17.1
syphilis 95, 102. m HP?. 377-85
congenial 3R 5
endemic 1. 85
latent 380
nonvenercaJ 380,. 383
neuro 383, 3M
primary 379
scLoisdirv 37V
“
d
'
serological tests 381
aulnnuled njud plabiEul
reagin ( RPR ) tear Ml
Kahn test 381
reagin antibody test 181
Reiter protein complement
fixation (RPCF) lest 382
Rl*R t«c 3B1
VBRL teal IS3
siantWi iifKts fur (STS) Ml
tertiary {quaternary) 3W
treatment 3W0, 3B 21. 363,. 3M
‘
ire puitcrEiai lots 379
venereal 379, 380 , 3M
syphiloma 379
fjtfUC ulcer ( Egyptian ) 231
systemic lupin erythematosus (SLE)
20, 114p ] ALl?h m 382,
T
tab vaccine 299, 301
tacaribe virus complex 4*l7 569
unpat 468 472 ?
tarshas nbedium 352
T cells (T lymphocytes ) 118 120
2 L 122± 152, 1 S 5 , 4£i 4E5
iINDEX * 655
duarsurteriatk 121 ceU culture 439
cytotoxic 135 otpUnt culture 439
effector 12CK 121 oigan futKR 439
OVwdk fktW (TOR 143, 145 tobacco mosaic virus 411, 432
helper US Todd— Hewitt bmth 205.
killer ( KJ *d 3sli5 rogaviridae 447, 523, 563
nuturaiion Mi Torres bodies 523
memory 143. 14fi ( OKLC thnelc syndrome ( TSS ) 168 ,
natural killer (NK) Mi
nuH «lb Mi
196 97 -
rain (TSST) 196
retailors (TCR ) 145. MM Tca&opbsma gondii 72, 635
regulatory 143 , 141 T protein 205
surface antigens 135.137 , tracheal cytolcnin (TCT ) 340
T «11 leukemia 2 tr4ChE>rtiir 422, 424, 42&-27
tecanolysan 256, 26Q epidcmioLog}- 426
tetainospflETnm 259 -, 253 UnmCDiy mitosis 426
tetanus 259 transoiipflpdaEkin 445
ivmnujiLutuin 260 iTsuiscTibsdjLi'nilt II deficiency 154
incubation period 111 transduction 5i 55, 465
laboratory diagrams 256. transfection 464, 5S3
OtQBflJC 259 transfer factor (TF) 77^ 79 147 , 166
p
prophylaxis 260 transferrin ( binding prord &n) 122
toxoid 261 ErarufcMlruiEiuci 5^, 441
EincatnIiniE 262 Eransfarnruiut er-cMth factor- bera
tetanus nconanmirn 259 (TGR 3ii
tetrathionate broth 32 CransknC hypt mmj ahLdLiicrrtkj
T even phases 462
Thajra^Martin medium 223, 2?- 5. 228 .
infancy 194 ^ ^
fraisaJaticHS inhibition prorein {TIFjl
Theobald-Smich phenomenon 1M 454
thermal death point 22 (ransplantadon , Lmmunolog) 176^-78 1
thermolabdle exotoxins 122 (raiKplant ( or graft ) 126.
thermophidjes 22 dassitkation of 126
tfrermopredpkin test 245 iran rt piece 8S
Thumsen-Frddenreich phenomenon ^
eranspcison 60
121 travellers disurhea 2ZB
(finish 616 trrnch fever 412. 418 420 421
, ,
thymk hypoplajja (DigHKgePi; tBepnBmft 2ML 377
syndrome ) 15.1, 1M carjuenjim 377, 3B6
thymocytes 117 , 124 emlcmicum 377s 385
thymoma 1 ' H paJilidum 377
thymosin HZ 186
]Mrti uiseurticuli
pertenue 377 , 385
thymus ( see under i vI : I •i I • : i i organs,
'
,
phiigedenis ( ROCCE erciponeme 'i
1
ccmral ) m3B6
dtyroidiri* 1.41, 1Z1 nefifingene 186
di ToliiJULTHiUi ( Cji'a-Vtis dLleatej 172 strains
^
1
tieftome viruses SM nicfv.il 's 378, 3M
tinea reiter IZi
>
baibye (buWf itch ) 614, filfi trejiuntmaioAes 38.1! 385
capitis 614 616?
TWC agenifi 424, 428,
corporis (gjibrusa) 614 trichophytin ( firngus antigen ) 614
cruris (>14,, 61 6
inhbrLcact 614, 616
- -
• ri . 1 1' n In !. 614
'
trkhosporon beigcllii 60
h
nigra 6 )2 triphenyJ tetrazoljum chloride I I' lt- ).
pedis ( athlete's toot) 614, 616 test 277
vtnitEtkir ( »e pityriasis triple ujiEifljeia (vsKone ) 218 , 26 L 143
.. .
vehicular) 612 fl
Eri|k rUgvc iron 'SR LT medium 46
tissue cultures 438 4Z ^ ^
Copyrighted materia
656 i TEXTBOOK OF MICROBIOLOGY >
trombiui ];i ilh vector hi embryonated eggs 439
tsutsugamushi disease 416 biological 65 tissue culture 439
tuberculin allergy 111 mechanical hi defective 4-39
{infection) type hypersensitivity 366 vcilbnc iliae 266 fixed 536
tubewulopMftifl 356, 1. 59, 360 venkatFanian-ramakrishnan ( VR) general properties 43ft 43
tuftsin deficiency 158 medium 306 . geneties uf 443
tularemia 3.24,. 331 VETO ceQ line 441, 444 growth Ln cell culture 439
I L.ICRI : ^ r necrc isihi JaCEcrf
yi . WOCytOWA ( car vErnlminJ 275 , 2 79 hemaBRturination of 434. Ml
< L- iK - lM!rtin ) 12fc. 327, BO , Hi verruca vulgaris ( human WJJESI 162 incomplete 439
futltcjpoat 468 verruga peruana 420 morphology nf 431 33 -
—
,

turlock virus 522 vesicular sranantis vmis 454. 522 oncogenic 574 Si
TWAH. ( Taiwan acute respiratofy) rcskukrnruj 522, 534 535 h spread in the body 45D 51 -
strain 423 v (actor 133 vaccines 453-. 457
TWE-IKN- Rft Jii viable count 19* 606 vista 566
lYndallLuEttm {see ^ i ^ rillscjciiiit ) Vi tut Eigen 69, 346. 464. 466 Vcsges-Proskausi ( VP) test 45273 .
typhoid fever (see enteric fever) vibrio 50S - -UH 3Q7,
typhoid vaccine alginolvticus 317 volutin granules. (Babca-Emst
injectable ( lyphim-Vi) 222 chnlene 3lii granules) H
live ( typhoid) 3G2 classification 36Z Von Magnus phenomenon 438, 506
typhus fever S3, 99, 295, 416 20 el tor 308-15 ,

endemic (murine or fleabome

trohwH17, llfi
( nonaggRititiablc )
vibrios 301
w
epidemic ( classical, gaol fever) NCY (nondnolen) vibrios Wa patsuma agar llh
1US 3flZ Waldenstroms macmplobulinacinia 90
recrudcsceEt ( Brill-Zmsser differentiation from allied wartuwri vino? 534
disease ) 4 t .3 415
p gencTa. 303
Wtftkif Fifiktldey cells 5IS
waits, human (verruca vulgark) 56-2
Tzanck cells 477, 479 morpholegy 305
comma 303. water, bacteriology of 603 605 -
presumptive colifoim count 604
u manuals 316
parahacmoIvocus 316 - warercan. perineum 227
ultrasonic vibration 30 vulnificus 31 7 Waterhouse-Friderichsen syndrome
ukraviokt rays 24 „ 25, 22 vibffan huryfLque 266 162* 222
uttdulam fever 345, 346 wei ls vaccine dlfi
universal donor 387
universal Lmmumsacjon programme
vihrion sepcique 254
vidarabine 459< 478 ^
rf* ditetfe 389, 391
Wfeil-F 2KT 419.421
( UIP) 631
Vincent s angina 235, 263, 388
Vi polysaccharide vaccine, injectable
.
^ ^
western blot test 592, 595
universal rtdrpkirE 132 west rule vims 522, 524, 526,
purified 632
urmphsma HI viral diarrhea 635 west 's posmasal swab 342
urrapiasma urealvtieum 229. 3»98 Virchow's Lepra (foamy) cells 370
‘wet kissing ' 597
unease rest 4! whey afgglutination test 350 , 607
^
useless immunoglobulin 5~6 v
virion 431 , 435, 437 , 433
viroids 448 whiEe graft TCSpmiB 1 79
virulence 61 whitlow, herpetic 476
V enhancement of ( aranudHuii ) 67 whooping cough 340 , 342 , 610
marker antigens ( VMA ) 279. 232 Wi - 3S cell strain 441
vaccine Widal test 94 , 292.29«, 299-300
hourter dose 76. 134 541 , TeduL iicHk of ( cKaltflCkm) 62
'

test 236 Wilson and Blair medium 37 , 290,

egg S41 2W , m
subunit 541 virus (es )
abnormal replicative cycles 43 B Widoorr-AHfi.ch syndrome 146, 1S6
tissue culture 5.41 woods tamp 615
hepatitis b 632 artifidal JfrS
assay 441 wiooSsartcrs disease 244
wane preventable tfeeases ( VFD) 631
Wright 's stain dll
.
vaccinia 434, 439, 468 463 fusing diarrhea 572
varicella (diickpar) 438, 47 K HO— chemical properties of 4.12

- -
varicella zoster ( V Z ) 473-ftO classification and nomenclature of X
w»lt 469 71, 4Z5 846 xenografts {formerly heEcaeigrafti) 177
validation 4 Cultivation of 438 xenopsylJa
VTlRL antigen 379 animal Lnocubriuri 439 asoa 323

i
j monte eria
 * INDEX 657
dicupiE 32Z
X factor 333h 33B
X- linked aguuiiigkibuKjHfflntt 152
,

X ]inked l}TnphopraliferatiYe
“

syndrome 4S 5
xeroderma 589
xytose 116. 334, 40S

¥
yaba vim & 468., 575.. 576
yaws (framboena ) 377* 385
yarn jjOlj, 610
yeisc - ILkc fyi gi 610
^
ydlaw Fewr (yellow jack) 43BP 439 p

446 , 521 , 522, 523, 522

vaccine (17d strain ) 439, 450
virus 438, 521
yersinia 272, 324,
cnterocnJirLea 330
pestis (prasteumdJa pcaris) 324
pieudoftibcrculoEis 330
yersinias 330

z
Zichl-Ncd ^ ni sraiming technique 10,
17, 44, 351, 357,
Zinssers unioariui hypothesis 92
zona lomerukiKL, cells of 172
^
tortt uf inhibition 628, 629
%&m phannaHfl 24
J

HNU« -345 „ 392, 419

zoonotic ECEML 416
zoster immLmogkibuJFn| ZIR} 4SO
zygosity 1M
ZygsstpsFS 611
zymosan 512

Copyrighted material
Copyrighted material
Hidden page
 Gf
^ nonthonaroyari and xanikei s
Textbook of Microbiology
^ i
i

Microbiology has become un increasingly important discipline, sot to face new

challenges, ftaf jid developments in (hesjbject bavr made a now edit inn necessary.
.
The seventh edition ni this Lisste textbook has relevant new information and .ill I
i hiipiors have been thoroughly revised and updated . I; contains r urrent
material < m
H1Vr hepatitis virus . SAKS, loronaviru ^ , bird flu virus and many of the newer agents
responsible for human in I eelions. I he epidemiological patterns have been
rorksL ritied and updated.

Other books from Orient Longmjn

Textbook nf Milica I Parasitology
R Panjaraihinsm

Clinical Gynecology ( Fourth Edition)

J
K faster flop N \ Roy Choudbury I
TexthL >rill of Medicine
R S Vasanr Sudbd Seshadri

Textbook of 1’alhnlogy
V Krishna
Mudatiar and Mtrnuns Clinical Obstetrics i Tenth Edition )
Ediied by Sarj/a CopjLn A V'an/ta Uin

I
I

e
C3

i
Rt 395.00 _ D
"

L
B
>
3
www ordefiilenpinan .com

0
Orient Longman
iSBN U 1 250 ?soe 0

Ananthanarsytti $ Panther 's Textbook of toternbiafayy

7 H K I : 5 H\ ;H; 0 S 6

Copyrighted material