NAGPUR VETERINARY COLLEGE,

MAHARASHRA ANIMAL & FISHERY SCIENCES

UNIVERSITY, NAGPUR.

Laboratory manual of

Systematic virology

Course no.-VMC-605 (semester-II)

SUBMITTED TO
Dr. V.C INGLE
Associate Professor,
Department of veterinary microbiology and animal
Biotechnology teaching & research cell,
Nagpur-06
 CERTIFICATE

Certified that this is bonafide record of practical work done in the laboratory for the

course___________________________________________________________

Course No.____________ Semester________ of MVSc during the year_____________

By Mr./ Miss.______________________________________ Enroll. No.______________

Date:-

Signature of the Course Teacher

 Index

S No Practical Page No Date Signature

1 Isolation of virus in chicken embryos: Newcastle disease

virus by allantoic route,study of the lesions in
embryos,harvesting of virus
2 Isolation of virus in chicken embryos:Fowl pox/other
poxviruses by chorioallantoic route,study of the lesions in
embryos,harvesting of virus
3 Isolation of infectious bronchitis in chicken embryos,study
of the lesions in embryos,harvesting of virus
4 Isolation of virus in chicken embryos contd.....
5 Isolation of virus in cell cultures:Newcastle disease virus
in chicken embryo fibroblasts,study the cytopathic
effects(CPE)
6 Isolation of virus in cell cultures:Newcastle disease virus
in chicken embryo fibroblasts,study the cytopathic
effects(CPE) contd.....
7 Isolation of virus in cell cultures:Bluetongue virus in
BHK-21/Vero cell line,study the CPE
8 Isolation of virus in cell cultures:Bluetongue virus in
BHK-21/Vero cell line,study the CPE
9 Isolation of IBR/swine fever virus in cell cultures,study the
CPE
10 Newcastle disease virus: Haemagglutination and
haemagglutination inhibition tests

11 Rabies: Detection of negri bodies by sellers

staining,fluorescent antibody technique,mouse inoculation
test
12 Infectious bursal disease:Detection of antigen /antibody by
agar gel immunodiffusion test(AGID),detection of
antibodies by neutralization test
13 Mareks disease;AGID,CIEP
14 Bluetongue:detection of antibodies by AGID
15 Rotaviruses:Latex agglutination test,Electropherotyping
16 Foot and mouth disease: Typing of virus by ELISA
 Practical no.1

Isolation of virus in chicken embryos :New castle disease virus by allantoic route,study the
lesions in embryos,harvesting of virus

Aim- To isolate new castle disease virus from the given clinical sample

Allantoic inoculation
Many viruses such as fowl plague and Newcastle disease virus, grow readily in the endoderm of
developing chicken eggs. Other viruses such as influenza, may require repeated amniotic
passages before becoming adapted to the egg and grown in the allantoic cavity. Allantoic
inoculation is a quick and easy method that yields large amounts [8 15 ml] of virus-infected
egg fluids.
Requirements -
9 11 day old embryonated hens eggs and egg trays
Egg candler
Egg incubator at correct temperature and humidity
Pencil for marking and labelling eggs
Tincture of iodine or 70% alcohol and cotton swab for sterilising egg shell
Metal egg prick
1 ml syringe with a 3 cm long 24 or 26g needle (sterile)
Sellotape or wax to seal egg after inoculation
Virus to be inoculated, diluted appropriately
Method -
Candleeggs to ensure viability of the embryo
Allow 6 eggs per specimen plus 2 4 eggs for controls
Label eggs with the appropriate specimen number
Candle the eggs and mark the position of the air sac, and a point on the side of the
egg, which is not adjacent to any major blood vessels
Sterilise the egg shell over the air space with tincture of iodine or 70% alcohol
Flame the metal egg prick
Jab a small hole in the shell over the air space, just above the pencilled mark
Resterilise the area with tincture of iodine or 70% alcohol
Draw up inoculum in a 1 ml syringe
Plunge the needle through the theallanotic cavity
Inoculate 0.1 ml virus dilution/egg
Inoculate controls with 0.1 ml diluent (PBS containing Mg++ hole in the shell
directly into and Ca++)
Seal hole with sellotape or hot wax
Incubate for 48-72 hours, in upright position, at a temperature appropriate for the
growth of the virus
Candle daily. Chill eggs before harvesting
Harvesting of Allantoic fluid
Requirements -
Egg cups small beakers or damp cotton wool
Sterile Petri-dishes
Sterile scissors small curved
Sterile fine pointed forceps
Wide necked bottles: x 2 containing 70% alcohol
Wide necked bottles: x1 containing absolute alcohol
Sterile Pasteur pipettes and teats
Rack for holding pipettes
Sterile McCartney bottles 1 per egg
Sterile bijou bottles 1 per egg
Discard bin/container for eggs
Discard tray/bin for glassware and instruments
Bottle slope
Gloves, gown and mask
Bunsen burner
Bench cote - protective paper covering for surface of hood
Safety hood or biohazard cabinet
Disinfectant to wipe up any spillage e.g. 3% tegador and 70% alcohol
Sterile swabs and paper towels for cleaning and wiping
Antibiotic cocktail

Method
Candle the eggs and mark those containing dead embryos
Chill the eggs at 4C for 2 hours to constrict the blood vessels and kill the embryos
Prepare harvesting area and label one McCartney bottle and one Bijou bottle per egg
Set out several eggcups or small beakers or damp cotton wool on sterile Petri-dishes
and prepare egg pipettes
Harvest eggs individually in the following order:
1st :alive control eggs
2nd:alive infected eggs
3rd:dead control eggs
4th: dead infected eggs
Remove the first egg to be harvested from the fridge and place in an eggcup. Keep
the other eggs at 4oC until harvested. If the eggs warm up, the blood will start to flow
and contaminate the harvested egg fluids
Flame the curved scissors (warm, not hot) and cut eggshell below the pencilled line
marking the air sac
Pipette the allantoic fluid (5-10 ml) into the appropriately labeled McCartney bottle
Flame the forceps (warm, not hot) and pierce the amniotic membrane
Pipette, using a clean pipette, the amniotic fluid (1-2 ml) into the appropriately
labeled Bijou bottle
 Tip the embryo out onto a sterile Petri-dish
Using clean scissors and forceps, decapitate the embryo and open its chest cavity
Remove the lungs and bronchi and pool with the amniotic fluid. Titurate the lungs
using a Pasteur pipette to release the virus
Discard the rest of the egg material and the dirty glassware
Use a clean Petri-dish for each embryo and change egg cups frequently
Rinse forceps and scissors in 70% and absolute alcohol, flame and allow cooling
before harvesting the next egg
Add antibiotics to the egg fluids:
approximately 100 units/ml of penicillin and streptomycin, and 50 g/ml of
gentamycin.
Test egg fluids individually for the presence of virus by haemagglutination using the
appropriate red blood cells, such as guinea pig or fowl red blood cells.
Store egg fluids at 4C do not freeze during passages.
Passage egg fluids at a 10-1 or 10-3 dilution. Do not inoculate undiluted egg fluids as
inhibitors, such as interferon, will inhibit viral growth.
 Practical no.2
Isolation of virus in chicken embryos :fowl pox/other poxviruses by chorioallantoic
route,study of the lesions in embryos,harvesting of virus

Aim- To isolate fowlpox/other poxviruses from the given clinical sample by using CAM route

Chorio-allantoic membrane inoculation

This method has been widely used in veterinary virology because many viruses grow readily or
can be adapted to grow on the CAM and produce visible foci or pocks, inclusion bodies,
oedema or other abnormalities. Viruses include the herpesviruses (infectious laryngotracheitis
and Aujeszksys disease virus) and poxviruses (fowlpox, ectromelia and vaccinia).

Requirements
10-13 day old embryonated hens eggs on egg rack
Egg candler
Pencil for marking and labelling eggs
Egg incubator at correct temperature and humidity
Ether for sterilising egg shell at site of inoculation
Dental drill with rounded burr head
Sterile injection needle
2 ml rubber teat
Sterile Pasteur pipettes
PBS warmed at 56C
1 ml syringe with fine short needle
Sellotape to seal holes
Bunsen burner

Method
Candle large white eggs and choose those with well-developed CAM membranes
Mark the air sac and a point on the side of the egg not adjacent to any major blood
vessels, which will be the site of inoculation
Sterilise both areas with ether. Iodine is not used as it may cause non-specific pocks
Drill a hole through the shell and shell membrane, above the middle of the air sac
Drill a lengthways line through the shell at the site of inoculation leaving the
underlying shell membrane intact. Drill slowly to avoid producing heat that can
cause non-specific pocks
Blow off shell dust with a Pasteur pipette and flame pipette lightly
Pipette a drop of pre-warmed PBS onto the hole at the site of inoculation
Stroke the fibres of the exposed shell membrane with the sterile needle
The fibres of the shell membrane run transversely across the egg, while those of the
CAM run lengthways along the egg. Therefore, by stroking the shell membrane
along the drilled line, the fibres can be parted. This allows the PBS to run in-between
the two membranes and aids in their separation
Apply gentle suction, using the rubber teat, to the hole overlying the air sac. The
suction will pull the air out of the air sac, and, as air is pulled into the hole at the site
 of inoculation, the CAM will drop. Too vigorous a suction may pull the membrane
down too fast, causing haemorrhage and non-specific pocks

Figure 2: Schematic diagram of the inoculation of the chorio-allantoic membrane

Candle the eggs to ensure that the CAM has dropped. The original air sac should
have disappeared and been replaced by one at the site of inoculation
Incubate the eggs for an hour to allow the membranes to settle
Do not leave the eggs any longer as the membranes will become refractive and
develop non-specific pocks. This will lower the membranes sensitivity to the
inoculated virus
Inoculate the virus directly onto the CAM. Do not attempt to touch the membrane, as
it is fragile and easily ruptured
Roll the egg gently to distribute the inoculum evenly
Seal the two holes with sellotape
Incubate the eggs, inoculation side uppermost, at the appropriate temperature

Harvesting chorio-allantoic membranes

Requirements
Gloves and gown
Cotton wool eggcups on Petri-dishes
Curved scissors
Sterile forceps, several pairs
Two Petri-dishes with sterile PBS per egg

Method
Candle eggs and record any deaths
Mark the position of the artificial air sac with a pencil
Chill eggs in the fridge for at least 2 hours to constrict blood vessels. Do not freeze
them
Work under a hood, wear gloves and a gown
 Place egg in eggcup and cut eggshell with a sterile pair of curved scissors. Start
incision at the hole above the true air sac and cut egg in half below line of artificial
air sac
Trim the shell around the artificial air sac while holding the eggshell with a sterile
pair of forceps
Using a 2nd pair of forceps, peel the CAM from the shell
Wash the membrane in a Petri-dish containing PBS and then place in the 2nd dish
with clean PBS
Examine for pocks over a dark background
Count number of pocks

Lesions seen after incubation-pock lesions on the body surface of embryo

 Practical no.3-4

Isolation of infectious bronchitis in chicken embryos,study of the lesions in

embryos,harvesting of

Virus

Aim- To isolate infectious bronchitis virus and study of the lesions using embryo inoculation

Chorio-allantoic membrane inoculation

This method has been widely used in veterinary virology because many viruses grow readily or
can be adapted to grow on the CAM and produce visible foci or pocks, inclusion bodies,
oedema or other abnormalities. Viruses include the herpesviruses (infectious laryngotracheitis
and Aujeszksys disease virus) and poxviruses (fowlpox, ectromelia and vaccinia).
Requirements
10-13 day old embryonated hens eggs on egg rack
Egg candler
Pencil for marking and labelling eggs
Egg incubator at correct temperature and humidity
Ether for sterilising egg shell at site of inoculation
Dental drill with rounded burr head
Sterile injection needle
2 ml rubber teat
Sterile Pasteur pipettes
PBS warmed at 56C
1 ml syringe with fine short needle
Sellotape to seal holes
Bunsen burner

Method
Candle large white eggs and choose those with well-developed CAM membranes
Mark the air sac and a point on the side of the egg not adjacent to any major blood
vessels, which will be the site of inoculation
Sterilise both areas with ether. Iodine is not used as it may cause non-specific pocks
Drill a hole through the shell and shell membrane, above the middle of the air sac
Drill a lengthways line through the shell at the site of inoculation leaving the
underlying shell membrane intact. Drill slowly to avoid producing heat that can
cause non-specific pocks
Blow off shell dust with a Pasteur pipette and flame pipette lightly
Pipette a drop of pre-warmed PBS onto the hole at the site of inoculation
Stroke the fibres of the exposed shell membrane with the sterile needle
The fibres of the shell membrane run transversely across the egg, while those of the
CAM run lengthways along the egg. Therefore, by stroking the shell membrane
 along the drilled line, the fibres can be parted. This allows the PBS to run in-between
the two membranes and aids in their separation
Apply gentle suction, using the rubber teat, to the hole overlying the air sac. The
suction will pull the air out of the air sac, and, as air is pulled into the hole at the site
of inoculation, the CAM will drop. Too vigorous a suction may pull the membrane
down too fast, causing haemorrhage and non-specific pocks

Figure 2: Schematic diagram of the inoculation of the chorio-allantoic membrane

Candle the eggs to ensure that the CAM has dropped. The original air sac should
have disappeared and been replaced by one at the site of inoculation
Incubate the eggs for an hour to allow the membranes to settle
Do not leave the eggs any longer as the membranes will become refractive and
develop non-specific pocks. This will lower the membranes sensitivity to the
inoculated virus
Inoculate the virus directly onto the CAM. Do not attempt to touch the membrane, as
it is fragile and easily ruptured
Roll the egg gently to distribute the inoculum evenly
Seal the two holes with sellotape
Incubate the eggs, inoculation side uppermost, at the appropriate temperature

Harvesting chorio-allantoic membranes

Requirements
Gloves and gown
Cotton wool eggcups on Petri-dishes
Curved scissors
Sterile forceps, several pairs
Two Petri-dishes with sterile PBS per egg

Method
Candle eggs and record any deaths
Mark the position of the artificial air sac with a pencil
 Chill eggs in the fridge for at least 2 hours to constrict blood vessels. Do not freeze
them
Work under a hood, wear gloves and a gown
Place egg in eggcup and cut eggshell with a sterile pair of curved scissors. Start
incision at the hole above the true air sac and cut egg in half below line of artificial
air sac
Trim the shell around the artificial air sac while holding the eggshell with a sterile
pair of forceps
Using a 2nd pair of forceps, peel the CAM from the shell
Wash the membrane in a Petri-dish containing PBS and then place in the 2nd dish
with clean PBS
Examine for pocks over a dark background
Count number of pocks

Candle 9 to 11 old embryo.wash the egg mark two spots on egg,one spot on side of egg and other
over the center of air space .Apply spirit and drill thehole using 16 guage needle at both spots
scrape off shell membrane gently without puncturing it at spot on the side.

Keep the egg horizontal and with the help ofrubber bulb placed over hole on air sac suck out the
air.this

Harvesting of chick embryo-

After inoculation by any route,the eggs are incubated at 37c and candled daily to check whether
embryos are dead or alive.Once the embryo dies the daed eggs are transferred to freeze between
4-10c for chilling of the contents.After 3-4 hrs the eggs are removed and placed on an egg
rack,with air sac facing upward.the chilling prosess is done to contract blood vessel,so that the
RBCs do not leak out into surrounding fluid during harvesting procedure.the shell over air sac is
broken gently with forceps to expose inner shell membrane.
Shell membrane is pealed off gently and CAM is picked up with forceps and Pasteur pipette is
introduced and 5-10 ml of allantoic fluid is collected from 10 day old and placed in sterile
glassware which is then placed in deep refrigerator till further use in serological test or
haemagglutination test.

The chorioallantoic is then cut open and other contents of egg i.e yolk sac and embryo are
eemptied into sterile petridish.the embryo and yolk sac are observed for any lesions of virus
which was inoculated.the chorioallantoic membrane is removed off from shell and observed for
lesions(pock lesions)after placing it in petridish .the embryo,yolk sac ,embryo and CAM are
preserved in deep freezer for further use.

inhibitors, such as interferon, will inhibit viral growth.

Lesions seen after inoculation In I.B Virus,dwarfing and curling of embryo

Yolk sac inoculation

The yolk sac route is particularly suitable for the cultivation of Chlamydia, Rickettsia and certain
viruses, such as louping ill and other togaviruses.
Requirements :
Embryonated eggs, 6 8 days old, on tray
Punch or drill
Tincture of iodine and swab on stick
Egg candler in darkened room
Pencil for marking eggs
Sellotape for sealing hole
Syringe with long (2) fine-bore needle
Petri-dishes
Scissors and forceps
Saline
Glass slides
Giemsa stain solutions
Microscope with 100 x objective, microscope oil

Method
Candle the eggs and mark the air sac
Label the eggs (virus inoculum and controls)
Disinfect the shell above the air sac with tincture of iodine or 70% alcohol
Punch or drill a hole through the shell over the air sac, in the centre
Using a syringe with a long needle, introduce the needle vertically through the hole
for 1
Inoculate 0.1 0.5 ml virus material directly into the yolk sac
At this age the yolk sac is large while the embryo is small and unlikely to
accidentally damaged
Withdraw the needle and seal the hole with sellotape, wax or nailpolish
Incubate at the temperature suitable for the virus
 Candle daily and discard embryos that die within 24 hours

Harvesting
Chill the eggs to kill the embryo
Cut the egg-shell open and empty egg contents into a sterile Petri-dish
Gently empty the yolk sac of yolk using forceps and scissors
Remove the yolk sac to a clean Petri-dish and rinse in saline

Harvesting the Yolk Sac

 Practical no.5-6

Isolation of virus in cell cultures:Newcastle disease virus in chicken embryo

fibroblasts,study of the cytopathic effects

Aim- To isolate new castle disease virus in chicken embryo fibroblasts by using cell culture

INTRODUCTION:-
Viruses are obligate intracellular parasites that require living cells in order to replicate. Cultured
cells, eggs and laboratory animals may be used for virus isolation. Although embroyonated eggs
and laboratory animals are very useful for the isolation of certain viruses,
cell cultures are the sole system for virus isolation in most laboratories. The development of
methods for cultivating animal cells has been essential to the progress of animal virology.

MATERIAL REQUIRED :-
virus suspension , tissue(chicken embryo fibroblast), media, trypsin, petri dish, flask, bottle, test
tube,serum,cell culture flask,microscope.

PROCEDURE :-
To prepare cell cultures, tissue fragments are first dissociated, usually with the aid of trypsin or
collagenase.
Either primary or continuous cell lines known to be susceptible to suspected virus may be
inoculated
1. Select a monolayer cell culture grown on cover slips in bottles and pour growth medium.
2. Wash the monolayer twice with HBSS.
3. Inoculate 0.1 ml of virus suspension and incubated bottle at 370C for 60 minutes to allow
the virus to absorb into the cells.
4. Add 1 ml maintenance medium or medium without serum and incubate bottle at 370 C for
3-7 days. Also keep control bottle without any virus inoculums.

STUDY OF CYTOPATHIC EFFECT

Cytopathic effect or cytopathogenic effect ( CPE) refers to structural changes in host cells
that are caused by viral invasion. The infecting virus causes lysis of the host cell or when
the cell dies without lysis due to an inability to reproduce. Both of these effects occur due
to CPEs
. If a virus causes these morphological changes in the host cell, it is said to be
cytopathogenic.
Common examples of CPE include rounding of the infected cell, fusion with adjacent
cells to form syncytia, and the appearance of nuclear or cytoplasmic inclusion bodies.
CPEs and other changes in cell morphology are only a few of the many effects by
cytocidal viruses. When a cytocidal virus infects a permissive cell, the viruses kill the
host cell through changes in cell morphology, in cell physiology, and the biosynthetic
 events that follow. These changes are necessary for efficient virus replication but at the
expense of the host cell.

Principle
Primary cultures are usually prepared from large tissue masses. Thus, these cultures
contains
a variety of differentiated cells. Embryonic tissues are preferred for primary cultures due
to that the embryonic cells can be disaggregated easily and yield more viable cells. The
quantity of cells used in the primary culture should be higher since their survival rate is
substantially lower.

Materials Required:
Non sterile: 8-10 days old embryonic eggs,70% alcohol, Swab

Sterile: 100 ml beaker 1 ,2 pairs of scissors ,A pair of bent scissors ,2 big forceps
,Petriplates,100mlconical flasks Small funnel covered with cheese cloth 1 ,10 ml test
tubes cotton plugged 4 ,Trypsinization flask 1

Reagents: Dissection BSS, 2.5% Trypsin crude in D-PBS ,Growth medium (EMEM with
10% fetal bovine serum)
Calcium, Magnesium free phosphate buffered saline (PBS).

Instruments: CO2 incubator, magnetic stirrer, Haemo-cytometer, Microscope

PROTOCOL:
Chick embryos of age 9-12 days are needed for culture.
1.Take 8-10 days old chick embryo.
2.Break the shell with the use of forceps carefully.
3.Resterilize the forceps and then use the forceps to peel off the white shell membrane to
reveal the chorioallontoic (CAM) membrane below with its blood vessel.
4. Pierce the CAM with sterile curved forceps , and lift out embryo by grasping it gently under
the head . Do not close forceps completely ,or else the neck will severe ; place the middle digit
under the forceps and use the finger pad to restrict the pressure of the forefinger.
5.Using two sterile forceps ,remove the head,limbs ,and viscera.Be sure to remove the entire
limb by pulling at the proximal end. Move the remaining tissues of the embryo to yet another
dish and wash with DBSS.
6.mince the embryo finely with scissors. Transfer the tissue to DBSS and rinse,repeat it for 2
times.
Chopped with cross scalpel into about 3mm cubes. transfer the pieces in preweighted vial and
allow pieces to settle.
Trypsinization: there are three methods of trypsinization.
Warm Trypsinization- 37o C for 30 min, less viability of cells.
Cold Trypsinization- 4Oc for 6-18 hrs, viability of cells are very good.
Combination of Warm and Cold Trypsinization.
Transfer all the pieces to empty trypsinization flask,flushing the vial or tube with DBSS.
Remove most of the residual fluid and add 80 ml of 0.25 % trypsine solution.
Add sterile magnetic bar inside the flask.
Cap the flask, and place it on the magnetic stirrer in an incubator or hot room 37o C.
Stirr at about 100 rpm for 25-30 min .at 370 c.
After 30 min , collect disaggregated cells as follow:
a.Allow the pieces to settle.

b.Pour off the supernatant into centrifuge 1ml of foetal calf serum and place it on ice.

c. Add 50 ml fresh trypsin to the pieces remaining in the flask and continue to stir and
incubate for further 30 min.

d. Collect the 2nd collection in a tube containing 1 ml FBS.

e. Pole the both collection and centrifuge the harvested cells at 1200 rpm for 10 min.

f. Discard the supernatant and resuspend the pellet in 10ml of EMEM (Growth Medium).
7. Centrifuge the cells@ 1200 rpm for 10 min.
8. Discard the supernatant and resuspend the pellet in 2-3 ml of EMEM (Growth Medium).
9. The total cell count and viable count was calculated and the cells were adjusted to 1x 10 5 cells
per ml.
10. The cells were plated in T25 and T75 Tissue culture flask (as per required).
11. Incubate the flask at 37o C in incubator supplied with 5% CO2 .
12. On next day ,check the flask for cell growth . Unattached cell debris will be floating , and
then change the media with GM.
13.Change the medium at regular intervals (2-4) days as dictated by decrease of pH.
 Practical No.7-8
Isolation of Blue tongue virus in BHK21/Vero cell line,Study the CPE
Bluetongue (BT) is an infectious, non contagious arthropod-borne disease of ruminants caused
by Bluetongue virus (BTV), prototype species of the genus Orbivirus, within the family
Reoviridae
Isolation of virus:

Material required:
1) Media
2) Infected tissue inoculum
3) TC flasks
4) Serological pipettes etc
Procedure:
Either primary or continuous cell lines known to be susceptible to suspected virus may be
inoculated
i) The susceptible cell line, based on the permissiveness of cell lines / primary cell line
from the host can be utilized . e.g. BTV virus ,BHK 21 cell line are used
ii) A monolayer cell culture was grown prior to a day of infection (so that the on the day
of infection cell will be in the exponential growth condition) grown on 25cc TC
flask.
iii) On the day of infection, the growth media from the TC flasks was decanted and
wash twice with HBSS.
iv) To the TC flask , one TC flask was lebeled as infected and other kept as control .
v) In the labeled infected , 0.1 ml of virus suspension/ ( inoculums extreacted from the
infected tissue material) and 400ul HBSS was inoculated. And to the control flask
500 ul of HBSS was added.
vi) Both the flask were incubated at 37C in CO2 incubator with 5% CO2 tension,
with 15 min intermittent shaking to avoid the drying of the monolayer.
vii) Incubtion was carried out for 60 minutes to allow the virus to adsorb onto the cells
viii) Then the inoculums from infected / control HBSS incoclum was decanted, then four
ml of maintenance medium was added and flasks were incubated at 370C for 3-7
days until the CPE observed, as observed twice daily.
The pathological evidence of viral infection as a cytopathic effect (CPE) were observed .
It is essential to passage each specimen or virus suspension in cell culture at least 3-5 time before
declaring any specimen as negative.

Cytopathic effects (CPE)

Morphological changes securing in cell culture due to multiplication virus is known as

CPE. It occurs due to certain biochemical changes such as shut down of cellular DNA , RNA and
protein synthesis which follows synthesis of virus coded proteins. The viral protein form a large
crystalline aggregates or inclusion which visibly destroy the cell. Secondly as high concentration
of virus protein or capsid protein has toxic effect on cell and produces CPE. Some viruses
produce CPE by altering cellular microtubules. Alteration of mesosomal membrane may
activate release hydrolytic enzymes which cause CPE. Cell fusion or syncytia formation occurs
when newly synthesized budding virus from cell fuses with plasma membrane of an adjacent cell
or there is fusion of virus specific areas of two adjacent cells.

BlueTongue virus- After 48 hours the tissue culture bottle were observed for cytopathic
effect, like cytopathic changes/morphological changes using inverted microscope. CPE occurs in
a uniform manner in the tissue culture bottles, infected cells became swollen and well defined.
Rounding of cells, syncytia formation, giant cell formation and grouping of cells may also be
observed.
 Practical No.9

Isolation of IBR/Swine fever virus in cell cultures ,study of CPE

Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis (IBR/IPV)

IBRcaused by bovine herpesvirus 1 (BoHV-1), is a disease of domestic and wild cattle. The
disease is characterised by clinical signs of the upper respiratory tract, such as a (muco)purulent nasal
discharge, hyperaemia of the muzzle (red nose disease) and by conjunctivitis. Signs of general illness are
fever, depression, inappetence, abortions and reduced milk yield. The virus can also infect the genital
tract and cause pustular vulvovaginitis and balanoposthitis.

The virus can be isolated from nasal or genital swabs from animals with respiratory signs, vulvovaginitis
or balanoposthitis.

Virus Isolation

For virus isolation, bovine cells of various origins can be used. Primary or secondary bovine kidney, lung
or testis cells, cell strains derived from bovine fetal lung, turbinate or trachea, and established cell lines,
such as the MadinDarby bovine kidney cell line (MDBK), are suitable for BoHV-1 propagation. Cell
cultures can be grown in glass or plastic tubes, plates or dishes.

To identify the recovered virus as BoHV-1, the supernatant of the culture should be neutralised with a
monospecific BoHV-1 antiserum or neutralising monoclonal antibody (MAb).

An alternative method of virus identification is the direct verification of BoHV-1 antigen in cells around
the CPE by an immunofluorescence or immunoperoxidase test . The virus produces a cytopathic effect in
24 days.

Cytopathic effect of the IBR virus in the cell line MDBK (Fig. 1) was characterized by appearance of
rounded cells detach from the surface and moved into the maintenance medium. Previously tight cell
monolayer formed area free from cell window in the monolayer, islands of cells that remained
attached in a state of strands joined together.

Monolayer of MDBK cell line: a not infected cells; b infected cells after 24 h of incubation; c
infected cells after 40 h of incubation.
Swine fever virus

Classical swine fever is a highly contagious disease of pigs caused by agent classical swine fever virus
(CSFV)[1]. CSFV belongs to genus Pestivirus within family Flaviviridae.Generally no CPE can be
observed when CSFV is cultured on host cells in vitro whereas serious tissue damages occur at infection
on pig body.
 Practical No.10

New Castle Disease Virus:Heamagglutination and Heamagglutination Inhibition test

HAEMAGGLUTINATION (HA) TEST

Object:
To detect the presence of Ranikhet disease virus in a given suspension and to find out
heamagglutination titer of virus.

Sample collection:
Live birds- tracheal and cloacal swabs, the latter should be visibly coated with faecal
material. The samples should be placed in isotonic phosphate buffered saline (PBS),
Dead birds- oro-nasal swabs, as well as samples collected from lung, kidney, intestine
(including contents), spleen, brain, liver and heart tissues.
Embryonated egg inoculation by allantoic route:
The supernatant fluids of faeces or tissue suspensions obtained through clarification by
centrifugation at 1000 g for about 10 minutes at a temperature not exceeding 25C are inoculated
in 0.2 ml volumes into the allantoic cavity of each of at least five embryonated SPF fowl eggs of
911 days incubation.
After inoculation, these are incubated at 3537C for 47 days. Eggs containing dead or
dying embryos as they arise, and all eggs remaining at the end of the incubation period, are
chilled to 4C and the allantoic fluids tested for haemagglutination (HA) activity.

Principle:

Certain viruses contain in their envelope virus coded glycoproteins (peplomers)

capable of binding to the complementary mucoprotein receptors over the surface of erythrocytes.
Such viruses bridge the gap between the red cells leading to format of lattice. This reaction is
seen in the polypropylene plates as mat formation. This property of virus may be employed for
detection of concentration of virus in a given suspension.

Material required:
Laxbro plates, N.S.S., dropping pipettes (0.25/0.50 ml). test suspension, 1% fowl RBC
suspension.

Preparation of 1% Fowl RBCs:

Take 5 ml of citrated fowl blood in test tube and centrifuge it to separate plasma and
cells. Discard the plasma and add equal volume of NSS. With the help of pipette agitate RBCs.
Then centrifuge tube at 1500 rpm for 10 min. Discard the supernatant and add fresh NSS. Repeat
this procedure 3 times. After last centrifugation discard the supernatant. Then take 1 ml of this
packed cell volume and add to 99 ml of NSS to give 1% washed fowl RBCs or 0.1 ml + 9.9 ml
of NSS to give 1% FRBCs.

Text procedure:

1) Mark first row of wells with dilutions 1:2, 1:4. 1:8, 1:16... and mark any last two other wells
as virus control (VC) and fowl cell control (FCC).

2) Add 0.25 ml of NSS in the wells of first row and in FRBC control (FCC) & virus control
(VC).

3) Add 0.25 ml of test virus suspension to the first well. Mix and transfer 0.25 ml to next well
and so on. From last well discard 0.25 ml.

4) Add 0. 25 ml of test suspension to virus control

5) Add 0.25 ml of FRBCs to all the well including virus and FRBC control.

6) Rotate the plate gently to mix the contents and incubate at room temperature for 30 min

7) The titer is read when the FRBC control shows a button formation appearance.

Result:
Reciprocal of the highest dilution of virus showing complete haemagglutination or
lattice formation is taken as virus titer. It is expressed as H.A. units.

Advice to farmers:
Depending on the mortality pattern in the flock, lesions observed on post mortem, age of
birds and the symptoms shown, the farmer should be advised as follows.

In case of positive haemagglutination test:

The possibility of presence of Ranikhet disease infection. Therefore collection of
serum from unaffected and affected birds is must for confirmation of heamagglutination
inhibition test. Farmer should be advised to do vaccination of healthy birds with live or killed
Ranikhet disease vaccines, culling of the affected birds, disinfecting the sheds, waterers. feeders
and other equipment.
PROTOCOL FOR HAEMAGGLUTINATION TEST (HAT):

Sr.No. 1 2 3 4 5 6 7 8 VC FCC

NSS 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Virus 0.25 Mix and transfer Discard from last well

Virus 0.25

1%FRBC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Dilution 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256

Incubate at room temperature for 30 min

M M M M M B B B M B

M Mat formation B Button formation

VC Virus Control FCC-Fowl Cell Control

HAEMAGGLUTINATION INHIBITION (HI) TEST

BETA PROCEDURE (DECREASING SERUM CONSTANT VIRUS)

Object: To determine HI titre units in given serum.

Principle:
In this test there is an antigen-antibody reaction in which the antibody attaches to the
specific virus present and the reaction is indicated by sealing down of the RBCs in the well to
give button formation (HI positive). If the serum contains antibodies specific to the virus, such
reaction will be seen at the lower dilutions. As the dilution of antibodies increases, a stage arises
when there is very little or no antibody to attach to virus and virus combines with receptors on
RBCs since it is free in the solution, to give a lattice formation at higher dilution of the serum(
HI Negative).
Requirements:
Laxbro plates, N S S., Test unknown serum, 8 HA units Ranikhet disease virus
suspension, dropping pipettes

Preparation of 8 HA units of virus:

The neat Lasota vaccine virus is titrated using a HA test and the number of HA is
determined. For example, if the Lasota vaccine contains 1024 HA units, then 8 HA units can be
calculated as 1024/8=128 which is a dilution factor to be used in order to obtain 8 HA units.
Therefore 1 ml of neat virus added to 127 ml of N.S.S. gives 8 HA units of Ranikhet disease
virus.

Test procedure:

1) Mark first row of wells with dilutions 1:2,1:4,1:8,1:16, and mark last three wells as virus
control (VC) and fowl cell control (FCC) and serum control (SC).
2) Add 0.25 ml of NSS in the wells of first row and in all the controls.

3) Add 0.25 m! of test serum to the first well Mix and transfer 0.25 ml to next well and so on.
From last well discard 0.25 ml.

4) Add 0. 25 ml of scrum to serum control.

5) Add 0.25 ml of 8 HA units of RDV to all the wells and virus control. (Not to the serum
control and FRBC control).

6) Incubate the plate at room temperature for 10 min.

7) Add 0.25 ml of 1% FRBCs to all the wells including all the controls.

8) Incubate the plate at the room temperature for 30 min. and read the first results as soon as in
Fowl cell control button formation occurs. After another 15 min read the final result i.e. after 45
min.

Results:

The reciprocal of the highest dilution of serum which shows complete inhibition of
haemagglutination or it shows button formation is taken as serum antibody titer and is expressed
in HI units
Advice to farmers:

In case of serum titer between 4-8 HI units, it indicates that flock is having a normal
antibody response. Titer above 16 or 32 indicates a recently vaccinated flock and in case
unvaccinated flock with titre 16 or 32 indicates an active infection of Ranikhet disease. In case of
low titers the farmer should he advice lo revaccinate the flock. In case of unvaccinated flock
showing high titers the effected birds should be culled. Vaccination of healthy birds along with
disinfection of shed and other preventive measures should be undertaken.

PROTOCOL FOR HAEM AGGLUTINATION INHIBITION TEST

(BETA PROCEDURE)
(SERUM UNKNOWN-STANDARD VIRUS)

Sr.No. 1 2 3 4 5 6 7 8 VC FCC SC

NSS 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Test 0.25 Mix and transfer Discard from last well

serum
Test -- -- -- -- -- -- -- -- -- -- 0.25
serum
8HA unit 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 -- --
virus
Incubate at room temperature for 10 min
1%FRBC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Dilution 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256

Incubate at room temperature for 30 min

B B B B B M M M M B B

M Mat formation B Button formation

VC Virus Control FCC-Fowl Cell Control
SC Serum Control
HAEMAGGLUTINATION INHIBITION (HI) TEST
ALPHA PROCEDURE (CONSTANT SERUM DECREASING VIRUS)

Object:
To find out whether birds is actively suffering from Ranikhet disease.

Principle:
By this test bird is confirmed for the presence of Ranikhet disease virus. The test can also
be used for checking potency of Ranikhet disease vaccine. It can be used as indirect test to find
out antibody level to Ranikhet disease virus. An unknown Ranikhet disease virus previously
titrated by heamagglutnation test is diluted to serial dilutions of 1:4, 1:8. etc. in N.S.S. and
constant quantity of 1:5 dilution of antiserum is added to it. After which FRBCs are added. In
first few wells there is heamagglutination i.e. lattice formation, whereas in higher dilution of
virus, inhibition or button formation is observed.
This is because in first few wells concentration of virus is higher than dilution of antibody and
therefore virus attaches to receptors on RBCs to give a lattice formation. As dilution of virus
increases, a stage and mark three other wells as serum control, virus control (VC) and fowl cell
control (FCC).

2) Add 0.25 ml of NSS in all the wells of first row and in all the controls.

3) Add 0.25 ml of virus to the first well, mix and transfer 0.25 ml to next well and so on. From
last well discard 0.25 ml

4} Add 0.25 ml of virus to virus control

5) Add 0.25 ml 1: 5 diluted antiserum to all the wells of first row and to the serum control

6) Incubate the plate at room temperature for 10 min.

7) Add 0.25 ml of 1% FRBCs to all wells in first row and to all the control

8) Rotate the plate to mix the contents and incubate the plate at room temperature 40 to 60 min.
As soon as button formation is observed in FRBC control and serum control the test is read.

Result:
The reciprocal of the highest dilution of virus which shows complete inhibition of
heamagglutination or it shows button formation is taken as serum end point i.e. reciprocal of
dilution of first wells showing button formation after lattice
formation is taken as serum end point.

The serum antibody titre can be calculated as

Virus end point

HI units = _____________ X Dilution factor of serum
Serum end point

Advice to the farmer:

Titer between 5-10 HI units indicate normal birds on farm and above 40 HI units
suspicious for Ranikhet infection in unvaccinated birds and above 80HI units indicates a positive
response to infection therefore accordingly a farmer should be advised to cull infected birds in
case presence of Ranikhet is confirmed by above titer.

PROTOCOL FOR HAEM AGGLUTINATION INHIBITION TEST

(ALPHA PROCEDURE)
(CONSTANT SERUM DECREASING VIRUS)
Sr.No. 1 2 3 4 5 6 7 8 VC FCC SC

NSS 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Test virus 0.25 Mix and transfer Discard from last well

Test virus -- -- -- -- -- -- -- -- 0.25 -- --

Standards 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
erum
Incubate at room temperature for 10 min

1%FRBC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Dilution 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256

Incubate at room temperature for 45 min

M M M M B B B B M B B

M Mat formation B Button formation

VC Virus Control FCC-Fowl Cell Control
SC Serum Contro
 Practical No:12

Infectious bursal disease: detection of antigen/ antibody by agar gel immunodiffusion test
(AGID), detection of antibodies by neutralization test.

Agar gel immunodiffusion test:-

The presence of virus (antigen) in the tissue of affected animal pr presence of antibodies in
serum of affected animal can be detected by agar gel immunodiffusion test

Principle:

When soluble antigens combine with its specific antibodies in presence of electrolytes at a
suitable temperature and pH, the antigens combine with antibodies to form antigenantibody
complexes in the form of insoluble white precipitate. The precipitation can take place in gels
such as agar, agarose, polyacrylamide, etc in form of white line. The soluble antigens and
antibodies placed in the wells cut in the gel medium diffuse through gel leading to formation of
precipitation bands at their equivalence points.

The precipitation by gel diffusion technique is useful in finding out number of specific antigen
and antibody system present in the reacting antigen and antiserum. The individual reactants
(antigen and antibody) react without interfering the reactions of other antigen and antibody
systems, producing separate lines of precipitation. A single antigen in presence of homologous
antibody gives rise to single precipitation line. Antigenic preparations of bacteria may consist of
several somatic, flagellar and capsular antigens each of which give rise to a separate line of
precipitation. This method is thus useful in antigenic analysis of bacteria.

Requirements:

Agarose, PBS (0.01 M, pH=7.2), soluble antigen, known positive and negative sera, test sera
samples, bores Pasteur pipettes, rubber bulb, spirit lamp, needle, test tube, etc.
Procedure:

1. Wash the microscopic glass slide, make it grease free and let it dry.
2. Prepare 2 % suspension of agarose (1 ml) in PBS (0.01 M, pH=7.2), melt it and spread on the
slide.
3. Keep this slide in hot air oven for drying.
4. Prepare 1 % suspension of agarose (3 ml) in NSS and melt it.
5. Pour the melted agarose on dried gel in such aaway that it shall form a uniform agar gel layer
on the slide of thickness 3 mm. Let the gel solidify.
6. Keep this gel at 4 0C for 10 minutes.
7. Punch the wells of 2 mm diameter in agar gel using borer as shown in the template, leaving 2
mm space in between two adjacent wells.

8. Removes the gel from wells using needle.

9. Fill up the central well with known standard soluble antigen using a Pasteur pipette and
rubber bulb.
10. Add known positive and negative control sera and test sera in separate peripheral wells in a
similar fashion.
11. Do never overflow the wells with either antigen or sera samples. Let the contents of wells
diffuse in the agar gel.
12. Cover the plate with lid and incubate the plate at 370C temperature in humid atmosphere for
24 hours.

Observations:

Observe the plate for development of line of precipitation for known positive and negative
control sera samples. The known positive sample shows the line of precipitation while known
negative sample do not show line of precipitation. Depending upon presence of specific
antibodies against antigen in central well, the test sera samples develop the lines of precipitation.

Interpretation:

When two similar antigens placed in adjacent peripheral wells reacts with the homologous
antiserum in the central well, the lines produced by them joins producing a continuous line in
form an angle. This is also called as lines of complete identity.

When two different antigenic preparations are allowed to react with a serum containing
specificities for both, precipitation bands formed will cross each other. This is called as lines of
non identity.

If only one of the two antigenic preparations possess specificity with the serum, and other posses
two antigens (one specific and other nonspecific), precipitation will be formed in the form of
spur. This is called as lines of partial identity.

Thus this method is useful in simultaneous comparison of different antigenic preparations against
an antiserum.

Result:

A positive reaction between antigen antibody is indicated by apperence of line of precipitation

between antigen and antibody wells.

Detection of antibodies by neutralization test

The serum virus neutralization (SVN) assay is a serological test to detect the presence
and magnitude of functional systemic antibodies that prevent infectivity of a virus. The SVN
assay is a highly sensitive and specific test that may be applied to influenza A viruses in swine to
measure the titer of neutralizing antibodies post-infection or after vaccination.
SERUM NEUTRALIZATION TEST

A test in which a patients serum, which may contain a neutralising antibody and a
microorganism of interest (e.g., an adenovirus or enterovirus), is either placed in a cell culture or
injected into a host organism so as to evaluate levels of protective antibodies present within the
serum.

Cells :
The Madin-Darby Bovine Kidney (MDBK) cell lineage, originally obtained from ATCC
(CCL-22), was multiplied in Eagles minimal essential medium (MEM; Gibco) supplemented
with 10% fetal bovine serum and antibiotics (penicillin and streptomycin) at usual
concentrations. All cells, sera and media were tested free of bovine viral diarrhea virus (BVDV),
bovine herpesviruses or antibodies to such viruses. Cells were tripsinized every three to four
days following standard procedures.

Viruses:
Strains of viruses have been used for serum neutralization test.

Cattle serum samples:

Sera from animal e.g. cattle were examined in the present study; All sera were obtained
from adult cattle with no history of vaccination to bovine herpesviruses. All sera were heat
inactivated and stored at -20oC.

Neutralization tests:

1. Neutralizing antibodies of strain of viruses were detected in a serum-virus neutralization test

(SN) with the constant virus, varying serum method. SN tests were carried out in 96-well cell
culture plates.

2. Sera were diluted in twofold dilutions ( and ) and the serumvirus mixtures (equal volumes
of 50 L) were incubated at 37C for 1 hour before the addition of MDBK cells (3 to 5 x 104
cells/ well/50L).

3. Tests were performed separately against each of the six different virus strains, with about 100
fifty per cent tissue culture infectious doses (TCID50) in every case.

4. Plates were incubated at 37oC in a moist atmosphere with 5% CO2 for up to 120 hours until
the final reading, which was performed with basis on the presence or absence of a cytopathic
effect.
5. Results were calculated by the method and expressed as positive (neutralizing antibody titre
2 or negative neutralizing antibody titre <2).

6.The sensitivity of the test was here defined as the capacity to detect antibodypositive sera in
relation to the maximum number of positive sera detected in samples from either of the two
geographic regions or over the total number of positive sera and was calculated with a 95%
confidence interval.

7. Sera which reacted positively at SN with at least one of the viruses were considered positive;
sera which reacted negatively with all six viruses were considered negative. All tests were
repeated at least twice by different operators. Sera that gave rise to discrepant results (that is,
disagreeing results to different SN challenge viruses) were tested at least three times against the
different viruses.
 Practical No:-13

Markess disease: AGID , CIEP

Agar gel immunodiffusion test:-

The presence of virus (antigen) in the tissue of affected animal pr presence of antibodies in
serum of affected animal can be detected by agar gel immunodiffusion test

Principle:

When soluble antigens combine with its specific antibodies in presence of electrolytes at a
suitable temperature and pH, the antigens combine with antibodies to form antigenantibody
complexes in the form of insoluble white precipitate. The precipitation can take place in gels
such as agar, agarose, polyacrylamide, etc in form of white line. The soluble antigens and
antibodies placed in the wells cut in the gel medium diffuse through gel leading to formation of
precipitation bands at their equivalence points.

The precipitation by gel diffusion technique is useful in finding out number of specific antigen
and antibody system present in the reacting antigen and antiserum. The individual reactants
(antigen and antibody) react without interfering the reactions of other antigen and antibody
systems, producing separate lines of precipitation. A single antigen in presence of homologous
antibody gives rise to single precipitation line. Antigenic preparations of bacteria may consist of
several somatic, flagellar and capsular antigens each of which give rise to a separate line of
precipitation. This method is thus useful in antigenic analysis of bacteria.

Requirements:

Agarose, PBS (0.01 M, pH=7.2), soluble antigen, known positive and negative sera, test sera
samples, bores Pasteur pipettes, rubber bulb, spirit lamp, needle, test tube, etc.
Procedure:

13. Wash the microscopic glass slide, make it grease free and let it dry.
14. Prepare 2 % suspension of agarose (1 ml) in PBS (0.01 M, pH=7.2), melt it and spread on the
slide.
15. Keep this slide in hot air oven for drying.
16. Prepare 1 % suspension of agarose (3 ml) in NSS and melt it.
17. Pour the melted agarose on dried gel in such aaway that it shall form a uniform agar gel layer
on the slide of thickness 3 mm. Let the gel solidify.
18. Keep this gel at 4 0C for 10 minutes.
19. Punch the wells of 2 mm diameter in agar gel using borer as shown in the template, leaving 2
mm space in between two adjacent wells.

20. Removes the gel from wells using needle.

21. Fill up the central well with known standard soluble antigen using a Pasteur pipette and
rubber bulb.
22. Add known positive and negative control sera and test sera in separate peripheral wells in a
similar fashion.
23. Do never overflow the wells with either antigen or sera samples. Let the contents of wells
diffuse in the agar gel.
24. Cover the plate with lid and incubate the plate at 370C temperature in humid atmosphere for
24 hours.

Observations:

Observe the plate for development of line of precipitation for known positive and negative
control sera samples. The known positive sample shows the line of precipitation while known
negative sample do not show line of precipitation. Depending upon presence of specific
antibodies against antigen in central well, the test sera samples develop the lines of precipitation.

Interpretation:

When two similar antigens placed in adjacent peripheral wells reacts with the homologous
antiserum in the central well, the lines produced by them joins producing a continuous line in
form an angle. This is also called as lines of complete identity.

When two different antigenic preparations are allowed to react with a serum containing
specificities for both, precipitation bands formed will cross each other. This is called as lines of
non identity.

If only one of the two antigenic preparations possess specificity with the serum, and other posses
two antigens (one specific and other nonspecific), precipitation will be formed in the form of
spur. This is called as lines of partial identity.

Thus this method is useful in simultaneous comparison of different antigenic preparations against
an antiserum.

Result:

A positive reaction between antigen antibody is indicated by apperence of line of precipitation

between antigen and antibody wells.

Counter immunoelctrophoresis (CIEP)

Principle:

The immunodiffusion test allows the antigen and antibody to react and precipitate purely by
diffusion, thus this reaction usually occurs slowly. It is possible to enhance the speed of the
reaction by driving antigen and antibody together by creating an electrical field. This technique
is known as Counter Immnoelectrophoresis (CIEP) and may be employed in diagnosis of
infectious diseases. The basic principle of the reaction is electrophoresis of antigen and antibody
in a gel medium in opposite direction simultaneously from separate wells resulting in
precipitation at a point intermediate between their origins.
Procedure:

1. Preparate 1% agar gel in Veronal buffer (0.05 M, pH=8.6) on CIEP slides.

2. Cut wells in the gel as shown in template.

3. The larger wells represent antibody wells and smaller wells are antigen wells.
4. If we are detecting antigen fill the antigen wells with different dilutions of antigen and
antibody wells with fixed quantity of known positive serum.
5. If we are detecting antibodies fill the antigen wells with known antigen and antibody wells
with sera samples.
6. Keep one well as positive control and one as negative control.
7. Apply a current of 10 mA using electrophoresis apparatus for 21/2 hrs.
8. Dry the gel in gel drier and stain with 0.1% brilliant blue for 12 hrs followed by distaining.
9. Observe the line of precipitation in positive, negative controls and test samples.

Interpretation:

The line of precipitation between antigen and antibody wells indicates positive result.
 Practical No:- 14

Bluetongue: detection of antibodies by AGID

Agar gel immunodiffusion test:-

The presence of virus (antigen) in the tissue of affected animal pr presence of antibodies in
serum of affected animal can be detected by agar gel immunodiffusion test

Principle:

When soluble antigens combine with its specific antibodies in presence of electrolytes at a
suitable temperature and pH, the antigens combine with antibodies to form antigenantibody
complexes in the form of insoluble white precipitate. The precipitation can take place in gels
such as agar, agarose, polyacrylamide, etc in form of white line. The soluble antigens and
antibodies placed in the wells cut in the gel medium diffuse through gel leading to formation of
precipitation bands at their equivalence points.

The precipitation by gel diffusion technique is useful in finding out number of specific antigen
and antibody system present in the reacting antigen and antiserum. The individual reactants
(antigen and antibody) react without interfering the reactions of other antigen and antibody
systems, producing separate lines of precipitation. A single antigen in presence of homologous
antibody gives rise to single precipitation line. Antigenic preparations of bacteria may consist of
several somatic, flagellar and capsular antigens each of which give rise to a separate line of
precipitation. This method is thus useful in antigenic analysis of bacteria.

Requirements:

Agarose, PBS (0.01 M, pH=7.2), soluble antigen, known positive and negative sera, test sera
samples, bores Pasteur pipettes, rubber bulb, spirit lamp, needle, test tube, etc.

Procedure:

25. Wash the microscopic glass slide, make it grease free and let it dry.
26. Prepare 2 % suspension of agarose (1 ml) in PBS (0.01 M, pH=7.2), melt it and spread on the
slide.
27. Keep this slide in hot air oven for drying.
28. Prepare 1 % suspension of agarose (3 ml) in NSS and melt it.
29. Pour the melted agarose on dried gel in such a away that it shall form a uniform agar gel
layer on the slide of thickness 3 mm. Let the gel solidify.
30. Keep this gel at 4 0C for 10 minutes.
31. Punch the wells of 2 mm diameter in agar gel using borer as shown in the template, leaving 2
mm space in between two adjacent wells.

32. Removes the gel from wells using needle.

33. Fill up the central well with known standard soluble antigen using a Pasteur pipette and
rubber bulb.
34. Add known positive and negative control sera and test sera in separate peripheral wells in a
similar fashion.
35. Do never overflow the wells with either antigen or sera samples. Let the contents of wells
diffuse in the agar gel.
36. Cover the plate with lid and incubate the plate at 370C temperature in humid atmosphere for
24 hours.

Observations:

Observe the plate for development of line of precipitation for known positive and negative
control sera samples. The known positive sample shows the line of precipitation while known
negative sample do not show line of precipitation. Depending upon presence of specific
antibodies against antigen in central well, the test sera samples develop the lines of precipitation.
Interpretation:

When two similar antigens placed in adjacent peripheral wells reacts with the homologous
antiserum in the central well, the lines produced by them joins producing a continuous line in
form an angle. This is also called as lines of complete identity.

When two different antigenic preparations are allowed to react with a serum containing
specificities for both, precipitation bands formed will cross each other. This is called as lines of
non identity.

If only one of the two antigenic preparations possess specificity with the serum, and other posses
two antigens (one specific and other nonspecific), precipitation will be formed in the form of
spur. This is called as lines of partial identity.

Thus this method is useful in simultaneous comparison of different antigenic preparations against
an antiserum.

Result:

A positive reaction between antigen antibody is indicated by apperence of line of precipitation

between antigen and antibody wells.
 Practical No:15

Rotavirus: Latex Agglutination test , Electropherotyping

Principle

Latex agglutination is observed when a sample containing the specific antigen (or
antibody) is mixed with an antibody (or antigen) which is coated on the surface of latex
particles.
Test reagent are coated with rabbit antibodies raised against a pool of different rotavirus isolates.
Whrn a fecal extract is mixed with the test reagent any rotavirus antigens present will react with
the sensitizing antibodies, resulting in visible agglutination of the latex particles.

Material required:

LK08a Rotavirus latex reagent

LK08b Rotavirus control reagent
LK08c positive control
LK08d Extraction Buffer
Additional requirement:
Centrifuge, stoppered, bacteriological loop, micropipettes and tips

Specimens collection and handling.

Fecal specimens should be tested as soon as after collection as possible and stored overnight at
2-8C for longer periods at -200Cor below.
Prepare an 10% suspension of the fecal specimens by transferring 0.1 ml of sample into 1.0 ml of
extraction buffer in a stoppered tube. Mix the content well. Allow to stand at rom temp. for 1-2
min. before proceeding with the test.

Procedure :-

Process the specimens as mentioned in specimens storage and preparation.

Allow the reagent to reach at room temperature.
Centrifuge specimens extract at approximately 1000g for 10 mins.
Pipette 0ul of clear supernatant onto each of two wells on the reaction card.
Add 20 ul of well mixed test reagent (LK08a) to one well and 20ul of well mixed control
reagent (LK08b)to the other.
Mixed the content of each well using a seprate mixing stick foe each sample, covering the
entire area of the well.
Gently rock the card and observe the agglutination for up to two mins.
Interpretation and result:-

A positive test is indicated by agglutination of the rotavirus latex reagent(LK08a) with no

agglutination of the rotavirus control reagent(LK08b).

The result is negative if there is no agglutination of either the rotavirus latex reagent(LK08a) or
the rotavirus control reagent (LK08b) is observed within the 2 min test periods.

Electropherotyping:-
Serotyping and subgrouping with monoclonal antibodies
: Serotypic determination of rotavirus G-type8
Reagents and equipment
Tris-HCl
CaCl2
NaCl
NaHCl 3
Na2CO3
KCl
Na2HPO4-12H2O KH2PO4
Tween 2
Casein
Tetramethyl benzidine (TMB)
Dimethyl sulphoxide (DMSO)
Sodium acetate
Citric acid
Hydrogen peroxide
Sulphuric acid
Horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin
Rabbit anti-rotavirus polyclonal antiserum
Serotype and subgroup-specific anti-rotavirus monoclonal antibodies (see
Table 1) 96-well microtiter plates
Plate sealers
Water bath at 37oC
Microtitre plate reader (spectrophotometer)
Refrigerator
Rotavirus positive control antigens from cell culture
Procedure Day 1
Before starting the ELISA, prepare 10% fecal suspensions in 0.01 M Tris solution, 1) pH
7.5, with NaCl and CaCl2.

Coat a 96-well immunoplate with 100 l/well of rabbit polyclonal antisera diluted
in PBS, pH 7.2. Coat an entire column with each polyclonal antisera as detailed in the
plate
o Determine the appropriate dilution of each polyclonal antisera (1:1000 to
1:10,000).
o Prepare new stock for each assay.
o Polyclonal antiserum should represent each of the standard rotavirus serotypes .

Seal plates with Linbro plate sealer (ICN-Flow), and incubate for 1.5 h at 37 oC in a
water bath.
Make a 0.5% casein solution in PBS-T (requires ~200 ml per 5 plates). Make the 4)
solution fresh daily.
Dilute 10% fecal extracts prepared previously 1:4 in 0.5% casein solution. 6) Similarly
dilute positive and negative control fecal specimens. Dilute tissue culture control antigens
1:2.
Add 100 l of diluted fecal extracts or tissue culture controls to each well in 7) a specific
row.
Each plate should have positive and negative fecal specimens and one tissue culture
strain. Use positive control tissue culture antigens representing the five common rotavirus
G serotypes in each assay.
Control virus strains should represent the specific G serotype of each Mab included in
the assays:
Cultivate virus strains in MA104 cells, aliquot into 1ml stocks, and store at 70oC until
required.
Seal plates with plate sealer, and incubate overnight at 40C.

Day 2
1) Aspirate off fecal supernatant, and wash plates five times with PBS-T wash buffer,
allowing ~3 min for each wash. Blot plates onto an absorbent towel between each
wash to remove excess buffer.
2) Add 100 l/well of mouse Mabs diluted in 0.5% casein solution. Add Mabs to an
appropriate polyclonal-coated column as detailed in the plate layout (Table 2).
Determine each dilution by titration against control antigens, and prepare fresh
before use..
3) Seal plates with plate sealer, and incubate for 2.5 h at 370C in a water bath.
4) Wash plates with PBS-T wash buffer 5 times, allowing ~3 min between each
wash. Blot plates onto an absorbent towel between each wash to remove excess
buffer.
5) Add 100 l/well of diluted horseradish peroxidase conjugated sheep anti-mouse
immunoglobin (Silenus, DAH) to all wells (diluted in 0.5% casein solution).
Determine the appropriate dilution by checkerboard titration; screen a range
between 1:5,000 and 1:50,000.
6) Seal plates with plate sealer, and incubate for 1.5 h at 37oC in a water bath.
7) Wash plates with PBS-T wash buffer 5 times, allowing 3 min between each wash.
Blot onto an absorbent towel between each wash.

Detection procedure
1) Make up TMB substrate buffer, and add 100 l of buffer to all wells.
2) Incubate plates for 10 min at room temperature; positive wells turn blue.
3) Stop the reaction by adding 50 l/well of 2M H2
SO4
; positive wells turn yellow.
4) Read plates within 30 min on an EIA plate reader (e.g., Titertek Multiscan
MCC/340 MKII) using a 450-nm filter.

Interpretation of results
Specimens are positive if the absorbance exceeds 0.2 and is at least twice the absorbance
of the background control.