Seminar in Nucleic Acids

Dr. Geoffrey Zubay

March 23, 2004
 Bubonic
Septicemic
Primary
Secondary
Pneumonic
Primary
Secondary
Bubonic Septicemic Pneumonic
Most common form fatality rate of 30- 100% death rate if
(~85% of all cases) 50% in treated cases, not treated within first
causes swollen 50-90% in untreated 24 hrs
lymph nodes (buboes) cases can be transmitted
can only spread from causes severe blood via direct inhalation of
person to person via infection throughout the the germs
direct contact with bubo body and gangrene of least common, yet
drainage acral regions (nose and most dangerous form
1%-15% death rate if digits) if untreated Primary:
treated; if not treated, Primary: occurs via inhalation
40%-60% death rate occurs when a flea of pneumonic
the backbone of the inserts y. pestis directly respiratory droplets
survival of y. pestis into the bloodstream Secondary:
because it can develop Secondary: occurs when bubonic
into both secondary occurs as a severe or septicemic plagues
septicemic and development from spread to the lungs
pneumonic types bubonic or pneumonic
(when y pestis migrates
to bloodstream)
Lungs of a pneumonic
plague patient
 Reservoirs Vectors Incidental Hosts
Urban and domestic Xenopsylla cheopis (the
oriental rat flea; nearly
humans
rats
worldwide in moderate Domestic and feral
Ground squirrels cats
climates)
Rock squirrels Oropsylla montanus (United Dogs
Prairie dogs States)
Lagomorphs (rabbits
Deer mice Nosopsyllus fasciatus and
(nearly worldwide in
Field mice hares)
temperate climates)
Gerbils Xenopsylla brasiliensis Coyotes
Voles (Africa, India, South Camels
Chipmunks America)
Goats
Marmots Xenopsylla astia (Indonesia
and Southeast Asia) Deer
Guinea pigs Antelope
Xenopsylla vexabilis (Pacific
Kangaroo rats Islands)
over 200 identified ~30 identified flea vectors *http://www.cidrap.umn.edu/cidrap/con
tent/bt/plague/biofacts/plaguefactsheet.
reservoirs html#_Reservoirs/Vectors/Modes_of_T
ransmissio
 *BITES FROM FLEA VECTORS*
Direct contact with infectious body
fluids or tissues while handling an
infected animal (which can be dead or
alive)
Ingestion of raw or uncooked meat
from an infected animal (marmots,
prairie dogs, goats, camel)
Inhalation of infectious droplets
 BUBONIC SEPTICEMIC PNEUMONIC
Bites from flea vectors Bites from flea vectors Inhalation of respiratory
Bites or scratches from where Y pestis is inserted droplets from infected
infected animals, such as directly into bloodstream animals such as cats
cats no discernible bubo Inhalation of respiratory
Direct contact with present droplets from a person with
1 primary or secondary
infected animal carcasses,
such as rodents (especially pneumonic plague
marmots), rabbits, hares, Handling Y pestis cultures in
carnivores (eg, wild cats, the laboratory setting
coyotes), and goats
Develops as a complication Bubonic and 1
of bubonic or 1 pneumonic septicemic spread plague
plague bacilli hematogenously to the
When Y pestis enters lungs
the bloodstream
2
To 2
only

1
Or only
Septicemic
Plague patients in the U.S.
Medieval doctor cutting off a
bubo from a plague victim
*The first record of plague was an
outbreak among the Philistines in
1320 B.C. when God enacted his
vengeance upon them for capturing
his Ark, which belonged to Israel:
He [the Lord] brought devastation upon them and afflicted them with
tumors. And rats appeared in their land, and death and destruction
were throughout the cityThe LORDS hand was against that city
throwing it into great panic. He afflicted the people of the city, both
young and old, with an outbreak of tumors. I Samuel 5:6-12

The Philistines asked, What guilt offering should we send to Him?

They replied, Five gold tumors and five gold rats because the same
plague has struck both you and your rulers. I Samuel 6:4
 Justinians Plague (~540-700AD)
The Black Death (1346-1350)
The China Epidemic (~1855-1908)
AKA The Modern Pandemic
 The first recorded and
confirmed pandemic
Occurred during the reign
of the Roman Emperor
Justinian
It spread from Egypt
through the known world
Population losses of 50-
60% occurred in North
Africa, Europe, and
central and southern Asia
for an approximate total of
100 million deaths Justinian the Roman Emperor
 The 2nd pandemic is thought to have originated in the Gobi desert in the 1330s
where the bacillus was active and abundant
China was a major player in trade, thus it took less than a
decade for the plague to spread across western Asia and into
Europe
In Oct 1347, Italian merchant ships returned to Sicily from a trip
to the Black Sea, and by the time they docked, many of the
merchants were already dying from the plague Lists of the dead were
It is believed to have been spread via fleas embedded in the fur they traded published regularly
By the following August (1348), it spread all the way north to England
The disease lay dormant in winter (due to flea inactivity), but within 5 years of its
onset, 25 million people were killed in Europe (1347-1352)
25 million = 1/3 of the population; however, these numbers could be askew due to
the presence of other diseases that may have assisted in depopulation of Europe
After the first 5 yr cycle, the death toll lessened; however, for the next 130 years,
outbreaks occurred in 2-5 year cycles
A period of rest was seen from 1480-17th C., then the Great Plague struck killing
~100,000 more people in London
The plague caused major political, cultural, and religious ramifications
It is also responsible for the introduction of hospitals as care centers rather than
quarantine locations as it catalyzed the movement toward more effective health
care and a cleaner, healthier style of living
17th Century London (plague locations + death toll in London)
 Beginning in the Yunnan province of China in 1885, the
3rd pandemic spread to all inhabited continents
excluding Australia
It spread to Canton and Hong Kong in 1894 and
Bombay in 1898
By 1900, it had spread via steamship to the rest of the
world
By 1903, India was losing an average of 1 million
people per year
Ultimately, it killed more than 12 million people in India
and China alone in the period from 1898-1918
Small outbreaks of plague continue to occur as a result
of the stable enzootic foci found round the world from
the 3rd pandemic (except, of course, in Australia)
This pandemic was the least severe of the three due to
understanding its nature and the advent of effective
public health measures (1002512 million)
Most importantly, antibiotics were discovered during
this outbreak, and some patients were actually cured,
which is why it is sometimes referred to as the modern
pandemic
 June 1894: Alexandre Yersin successfully
isolates the plague organism which he calls
Bacterium Pestis. Kitasoto makes the same
discovery independent of Yersin; however, his
data shows discrepancies, thus Yersin is
credited.
He develops a treatment (an antiserum) to
combat the disease and cures a plague
patient in 1896
He is the first to suggest that it may be
caused by the rodent/flea pathway, and he
also identifies the black rat as the reservoir
for the Manchurian outbreak
1897: Masanori Ogata and Paul-Louis
Simond independently discover the role of
the flea in plague transmission
1910: W.M. Haffkine demonstrates the
efficacy of his vaccine
Post-1911: L-T. Wu recognizes the
Manchurian outbreak as the pneumonic form
and establishes measures to prevent the
spread of disease via aerosolization
1970: After 3 name changes, plague
bacterium is renamed Yesinia Pestis in honor
of Alexandre Yersin Costume worn by plague doctor to protect
against 'miasmas' of poisonous air
 World Health Organization reports ~ 1000-3000
cases/year worldwide w/ an average of ~1700/yr
For the period of 1954-1997, a total of 80,613 cases
were reported with 6,587 deaths
The maximum number of reported plague cases
(6004) occurred in 1967 and the minimum (200)
occurred in 1981
This is vastly underreported due to lack of proper
surveillance and laboratory capabilities in many
countries
There are only 38 countries that report plague activity
Generally occurs in rural areas where enzootic foci
and rodent populations abound (L.A. outbreak in 1924
was last urban outbreak)
 There are 7 countries that have been affected by plague every year: Brazil,
Democratic Republic of the Congo, USA (with the exception of 1955, 64,
68), Madagascar, Myanmar, Peru, and Vietnam
There have been three periods of increased plague activity from 1954-97:
1) During the mid-60s
2) Between 1973-1978
3) mid-80s-present
The rise of reported plague morbidity has increased worldwide in the 90s,
especially in Africa
GEOGRAPHICAL SHIFTS: ASIAAMERICASAFRICA
In the 1950s, plague was a problem primarily in Asia, with little activity in
the Americas
By the early 60s, plague activity in the Americas increased, and in Africa, it
began to play a role
The mid-60s-early 70s show a magnificent increase in plague activity in
Asia which is primarily due to the Vietnam War
For the past 15-20 years (since~1982), a dramatic rise in activity can be
seen for Africa
Number of cases of plague reported to World Health
Organization, 1954-1997
A plague epidemic in Vietnam from 1966 to 1972 was largely responsible for the increased
plague activity during the mid-sixties
This epidemic is largely a result of the defoliation of vast areas (which contained the enzootic
foci for plague) during military operations, as well as the disruption of the economy, ecosystem
and infrastructure as a result of prolonged armed conflict
The number of reported cases of plague with data from Viet Nam excluded:

Asia is primary The trend Africa shows a

location for shifts in the drastic increase
plague activity 60s and the in the 80s-
in the 50s Americas present
become more
dominant
Africa
Beginning in the 1980s, there has been a steep upward trend in the number of plague cases in Africa
A total of 19,349 cases and 1,781 deaths in Africa from 1980 to 1997, comprising 66.8% and 75.8% of the
world's total with an average case fatality rate of 9.2%
From 1980-1997, human plague was reported from 13 countries in Africa (Angola, Botswana, Democratic
Republic of the Congo, Kenya, Libya, Madagascar, Malawi, Mozambique, South Africa, Uganda, United
Republic of Tanzania, Zambia, Zimbabwe)
Madagascar and the United Republic of Tanzania accounted for 62.5% of the total plague cases reported in
Africa during 1982-1997
Asia
Most cases of plague worldwide were reported from Asia from 1954-80s
There were outbreaks in Tanzania and Madagascar in the 1990s
Outbreaks occurred in India in 1954, 1963, and then again 30 years later in 1994
The plague outbreak in India in 1994 is a result of the earthquake in September 1993 that disturbed the
equilibrium density of domestic rodents and their fleas
A holiday that brought crowds together is also thought to have facilitated the spread of human plague
The Americas
Human plague was reported from five countries (Bolivia, Brazil, Ecuador, Peru and the United States
of America)
Three of these countries have notified some cases of human plague every year (Brazil, Peru, and the
United States of America)
Brazil and Peru accounted for 82% of the total cases reported in the Americas during the last 15 years
Totals for the period from 1980-1997 were 3,137 cases with 194 deaths
The mean case fatality rate was 6.2% during the period
 Human plague has been
reported most often from the
four western states of New
Mexico, Arizona, Colorado
and California

341 cases of human

plague were reported during
1970-1995

The overwhelming
majority of cases were
bubonic plague
For the last 45 years, the mean perennial plague case fatality for the
world (i.e. the average over the past 45 years of the annual reported
number of plague deaths divided by the annual reported number of
plague cases) has been 11.8%.
There is wide variation in reported case fatality rates by continent and
by year
There is also considerable variation from country to country and from
epidemic to epidemic
Despite the availability of a number of highly effective therapeutic
agents, mortality due to plague in many countries was high during the
period 1954-1997 which can most likely be attributed to the fact that it
often went unrecognized until too late
also, the majority of the countries dont have the capital to afford the
proper health care required
Outbreaks from the 20th Century-
present
1900
San Francisco
>Arrival of plague in the United States when
Chinese
laborer is found dead in a hotel basement
1924
Los Angeles
>the last US urban outbreak
>Mexican male is misdiagnosed with STD
>31 of the 33 total cases die before proper
health
measures are taken
1967-72
Outbreaks in Vietnam
1992
Arizona
>case results in death due to misdiagnosis of
pneumonia
> Source believed to have been from household
cat
 1994
India
>induces widespread panic
>causes more than 600,000 people to flee Surat
>110 of these people are plague victims
> Source is rats found in grain stockpiles
>Begins in bubonic form and develops into pneumonic
>~5150 cases suspected from 26 states; 53 confirmed fatalities
with 300 more suspected
January 1997
Zambia
>90 cases with 22 fatalities
>Outbreak possibly linked to heavy rains and flooding which
force rodents into populated areas
August 1997
Mozambique
>115 cases reported between June 7th and July 4th ; No fatalities
reported
Plague outbreak in India
October 1997 reported in Newsweek,
Malawi 1996
>43 cases reported between September 29th and October 23rd
>582 total cases reported
>Over 60% of cases are children under 5 years
>No fatalities reported
November 1997
Mozambique
>update-335 total cases since outbreak began in June
>No fatalities reported
 1998
Uganda
>49 cases reported, no fatalities recorded
May 1999
Namibia
>39 confirmed cases
>8 recorded fatalities
July 1999
Malawi
>74 suspected cases, no confirmed deaths
March 2001
Zambia
>436 cases, 14 deaths
February 2002
India
>16 cases reported, 4 deaths
May 2002
Malawi
>71 cases of bubonic reported, no deaths
June-July 2003 Depiction of death carts in London
Algeria carrying victims of plague
>10 cases reported; 8 bubonic, 2 septicemic
>1 fatality reported
Nov 2003
New York
>2 cases; couple contracted plague in Santa Fe
>No deaths, but the male had to get his foot amputated
 Bubonic 1 Septicemic 1 Pneumonic
Flea Flea Inhaled infected
respiratory
Skin
Blood vessels droplets (from
either cat or
Lymphatic vessels human)
Organs
Forms buboes

Lungs
Blood Blood

Organs Lungs
2septicemic
2pneumonic
Molecular Biology
of Yersinia Pestis

Characteristics
Biovars of Y. pestis
Evolution of Y. pestis
Pathogeneisis of plague
 Yersinia Pestis
Gram negative
coccobacillus
Non-motile
Enterobacteriaceae family
Non-spore forming
(unlike Anthrax)
Facultative anaerobe
Obligate parasite
 Yersinia Pestis
0.5-0.8 m in diameter

1-3 m long

Grows optimally at 28C

and a pH of 7.2-7.6

Bacterial cell wall

F1 Protein Envelope
http://www.nature.com/genomics/papers/y_pestis.html
 Biovars of Y. Pestis
There are 3 biovars of Y. pestis, each named for the
pandemic that it is thought to have caused
They are named based on their ability to convert nitrate to
nitrite and ferment glycerol
Glycerol Nitrite
Antiqua (1st pandemic) + +
Medievalis (2nd pandemic) + -
Orientalis (3rd pandemic) - +

The 3 biovars exhibit no difference in their virulence or

pathology in animals or humans.
 Genome
1 chromosome 4.65Mb
Orientalis and

Medievalis strains
have been sequenced

3 plasmids
pMT1 96.3kb

pYV 70.3kb

pPla 9.6kb

The plasmids are

crucial to the virulence
of Y. pestis
 Evolution of Y. pestis
There are 11 species of Yersinia

3 pathogenic species of Yersinia

Yersinia enterocolitis enteropathogen

Yersinia pseudotuberculosis enteropathogen

Yersinia pestis systemic pathogen

Y. pestis evolved from Y. pseudotuberculosis 1500-15,000

years ago
Evolution of Y. pestis
 Y. pseudotuberculosis vs. Y. pestis
Disease: Enteric infection Disease: Bubonic Plague

Transmission: enters mammals Transmission: rodent to humans

through food and water through flea vector

Chromosomal DNA 4.74Mb Chromosomal DNA 4.65Mb

90% chromosomal DNA
relatedness with Y.
pseudotuberculosis
Extrachromosomal DNA Extrachromosomal DNA
pYV
pYV
pPla
pMT1

Two thousand years ago, it only

gave you a tummy ache Within a few hundred years - an
- Brendan Wren, evolutionary eye blink- Y. pestis
geneticist learned to leap between fleas and
mammals, to live in the blood
instead of the intestine, and to
cause the swelling, coughing and
hemorrhaging of medieval
nightmares.
http://www.nature.com/nsu/011004/011004-12.html
 Plasmids crucial to virulence of Y. pestis
Plasmid Name Size (kb) Virulence determinants Role in disease

pMT1* 96.2 F1 capsule antigen Bacterial

transmission by
Fleas

pYV 70.3 Several Yops, Type III Toxicity. Avoidance

secretion system of immune system

pPla* 9.6 Plasminogen activator Dissemination

from intra-dermal
site of infection

*unique to Y. pestis only

Pathogenesis of plague
(focus on Bubonic plague)
 Manner in which fleas transmit plague

Flea feeds on Y. pestis-infected blood

Y. Pestis enters fleas midgut & multiplies logarithmically

Clump of Y. pestis forms in the midgut, blocking fleas foregut

During next meal, blood cannot enter the midgut & flea gets very hungry

Flea bites vigorously & regurgitates the contents of its midgut into the
next wound
 Importance of flea blockage

http://www.asm.org/ASM/files/CCLIBRARYFILES/FILENAME/0000000467/nw20030086p.pdf
Unblocked, uninfected flea on the left (A) and blocked, infected flea on the
right (B).
After flea feeds on Y. pestis infected blood, the bacteria enter the midgut of the
flea, where it will grow and multiply, eventually forming a large mass that can lodge
in the fleas foregut. During next meal, blood cannot enter midgut.
The ensuing blockage causes the starving flea to go into a blood-feeding
frenzy, in which it regurgitates the mass of Y. pestis and transmits it to a
mammalian host.
Experiments indicate that only blocked fleas effectively transmit plague to
mammals.
 Y. pestis mechanisms that contribute
to flea blockage
Hemin storage proteins (Hms)
Genes located on chromosome

Necessary for flea blockage, which is essential for efficient

transmission of plague from flea to mammals
Hms play a very important role in the transmission of plague,
changing the Y. pestis from a harmless inhabitant in the flea vectors
midgut to one that amasses in the foregut, causing the blockage.

In the flea, the Hms proteins alter the hydrophobicity of the bacterial
cell, thereby promoting aggregation and clumping of bacteria within
the blood meal. This is one of the main mechanisms by which
blocking of fleas occur.
 Hms is temperature dependent
Experiments indicate that fleas do not become blocked at higher
temperatures (above 28C = 82.4F)

It is not known however whether it is the expression of the Hms gene that
is affected by temperature, or rather its protein product is affected by
temperature.

For instance, if held at 30C, fleas survive Y. pestis infections in an

unblocked state, perhaps explaining why human bubonic plague
epidemics often end after the onset of warmer temperatures.

In addition, if you refer to the World Distribution of Plague Map, you

will notice that plague does not occur in the equatorial regions,
evidence which further supports this theory.
 Pathogenesis of plague
(focus on Bubonic plague)

Colonization of Y. pestis in the flea

Transmission of the Y. pestis from flea to mammalian host. A flea bite
transfers 25,000 100,000 organisms to host.
While growing in the flea, Y. pestis loses its antiphagocytic F1 capsular
layer (inactivated at lower temperatures), and so many of the pathogenic
organisms are phagocytosed and killed by mammalian leukocytes.
However, not all engulfed Y. pestis are killed. Bacteria that are
ingested by neutrophils appear to be readily killed, but bacteria within
macrophages are able to survive.
The macrophages provide a protected environment for Y. pestis to
resynthesize their F1 capsular layer and other virulence antigens
(activated by the warm 37C body temperature). The ability of Y. pestis
to survive and grow in macrophages is critical to the early pathogenesis
of plague.
 Pathogenesis of plague
Y. pestis within the
macrophages are then trafficked
to the local draining lymph
node. The massive infiltration
of phagocytic cells within the
lymph nodes cause them to
become hot, swollen and
hemorrhagic.

This gives rise to the

characteristic black buboes
responsible for the name of this
disease.
 Pathogenesis of plague
Within the bubo, by an unknown mechanism, the bacteria then escapes from
the infected macrophages to adopt an extracellular lifestyle, where they further
grow and replicate.
The organisms, with their newly formed antiphagocytic F1 envelope, can now
resist phagocytosis by the leukocytes. In addition, Y. pestis can actually kill
macrophages with an apparatus called the Type III secretion system.

Eventually, the infection can spill out into the bloodstream, leading to
involvement of the liver, spleen, and lungs (which leads to 2 septicemic and 2
pneumonic development).

1 Septicemic
Flea inserts directly into the bloodstream causing migration of y. pestis to
organs

1 Pneumonic
Inhaled Y. pestis bacilli would enter into lungs
Bubonic Plague vs. Pneumonic Plague
 Mechanisms that allow spreading of
Y. pestis in mammalian host
Plasminogen activator protease (Pla)
Genes located on smallest
plasmid pPla
Pla is required for the migration
of Y. pestis from the sub-
cutaneous infection site into the factor X factor Xa
circulation prothrombin thrombin
Pla derives its name from the fact
that it can activate the fibrinogen fibrin
mammalian plasma enzyme transaminase
plasminogen to plasmin.
Plasmin is responsible for the blood clot
breakdown of fibrin plasminogen plasmin
Main virulence role of Pla:
Cleaves fibrin deposits that trap dissolved clot
tissue plasminogen
Y. pestis, thereby promoting activator (TPA)
plague infection http://horizon.unc.edu/projects/monograph/CD/Professional_Schools/MoBy/1
0hrm.doc
 Mechanisms that allow intracellular
lifestyle in the mammalian host
The determinants which allow survival and growth of in the
macrophage are not known

However, Y. pestis has been shown to possess a two-component

regulatory system called Pho/PhoQ which is associated with protection
from macrophage killing mechanisms.

The ability of Y. pestis to survive in macrophages is critical to the early

pathogenesis of the disease.
 Mechanisms that allow extracellular
lifestyle in the mammalian host
F1 antigen
Genes located on largest plasmid pMT1
Exposure to temperatures of around 37C in mammalian host
results in production of large amounts of F1 antigen, which is
exported to Y. pestis surface to assemble into an antiphagocytic
envelope.

Yersiniabactin (Ybt) siderophore

Genes located on chromosome
Used to obtain nutritional iron necessary for bacterial growth from
eukaryotic proteins transferrin and lactoferrin
Bacteria requires iron in order to cause infection

Type III secretion system

Genes located on the middle-sized pYV plasmid
In extracellular environment, this is the weapon used by Y. pestis to
kill macrophages
It is the key virulence mechanism that allows Y. pestis to protect
itself from phagocytosis.
Importance of siderophores

http://gsbs.utmb.edu/microbook/ch007.htm
 Type III secretion system
Type III secretion system is upregulated at 37C,
i.e. within the mammalian host.

This system allows Y. pestis that are in contact

with a macrophage to inject a range of effector
proteins called Yersinia Outer Proteins (Yops)
into the macrophage through a syringe-like
apparatus. The Yops essentially function as a
poison that destroys a macrophages phagocytic
and signalling capabilities, ultimately inducing its
apoptosis.

http://www.rkm.com.au/imagelibrary/index.html
 Yops
When placed in environment that is
around 37C and with a low Calcium
concentration, Y. pestis ceases to grow
and expression of Yops is induced.
Altogether, there are 29 Yops but not all
play a role in Type III secretion system.
There are at least 6 Yops which directly
contribute to the killing of a
macrophage:
Yops E, H, J, O, M

Yops B and D

Required for pore formation in

the macrophage
Low Calcium Response V antigen
(LcrV)
Important for the activation of
the Type III secretion system
 Machinery of this Biological
Syringe
The Type III secretion system
consists of :
The core apparatus for
secretion through two bacterial
membranes
The needle

YopB, YopD, YopK, LcrV

Control elements
YopN, TyeA, LcrG
The poison (Anti-host
effector proteins)
YopE, YopH, YopM, YpkA
and YopJ
Yersinias Deadly Kiss
 Plasmids crucial to virulence of Y. pestis
Plasmid Name Size (kb) Virulence determinants Role in disease

pMT1* 96.2 F1 capsule antigen Bacterial

transmission by
Fleas

pYV 70.3 Several Yops, Type III Toxicity. Avoidance

secretion system of immune system

pPla* 9.6 Plasminogen activator Dissemination

from intra-dermal
site of infection

*unique to Y. pestis only

 Clinical Aspects

Signs and Symptoms of Plague

Differential Diagnosis
Laboratory Diagnosis
Treatment
 Initial Signs and Symptoms
Incubation Period: 2 6 days
Bubonic Plague Septicemic Plague
Fever 100% Fever 100%
Headache 85%
Severe exhaustion 75%
Vomiting 25-49% Nausea & Vomiting 50%
Altered mental status 38% Altered mental status common
Abdominal pain 18% Abdominal pain 39%
Cough 25%
Skin rash 23%
2 septicemic plague 23% Diarrhea 39%
2 pneumonic plague 5-15% Chest x-ray Patchy
bilateral
infiltrates
 Signs and Symptoms
(Bubonic Plague)

Pain/tenderness at regional lymph nodes enlarge to become buboes

Extremely painful

occur in groin ,
axilla or cervical
areas

Ulcer or skin lesions at site of

flea bite in <10% of cases
 Septicemic plague
1 septicemic plague is due to spreading of Y. pestis by way
of the bloodstream from the site of inoculation without
bubo formation

Septicemic plague may also follow an initial presentation

of bubonic plague, thereby becoming 2 septicemic plague.

Spreading of Y. pestis to all organs including liver, spleen,

heart, kidneys and CNS occurs, leading to septic shock and
death.
 Signs and Symptoms
(Septicemic Plague)
Complications

Hemorrhagic changes in
skin called purpuric
lesions

Disseminated
intravascular
coagulation (DIC)

Extremity gangrene

It is the blackened gangrene characteristic of advanced

septicemic plague that gave the pandemic of Medieval Europe
the name Black Death.
 Signs and Symptoms

Pneumonic Plague

Incubation period of 1-3 days

Productive cough
Hemoptysis
Rapid, shallow breathing
Cyanosis
Nausea and vomiting
Abdominal pain
Chest x-ray with alveolar infiltrates
 Differential diagnosis of plague
Bubonic Tularemia
Cat Scratch Disease
Chancroid
Lymphogranuloma venereum
Bacterial adenitis
Tuberculosis
Scrub Typhus
Septicemic Septicemia caused by other Gram Negative
bacteria
Meningcoccemia
Rocky Mountain Spotted Fever
Pneumonic Inhalational anthrax
Tularemia
Viral Pneumonia (Influenza, Hantavirus, CMV)
Q fever
 Differential Diagnosis
Pneumonic Plague vs. Inhalational Anthrax

bilateral pulmonary infection, widened mediastinum, resulting

with greater infection in the left in less available space for lungs
lung.
 Diagnosis
Conditions for Suspected Plague:
1. Clinical symptom of Plague, such as fever and buboes, in the person

2. Person resides in or has recently traveled to a plague-endemic region.

Exposure to rodents or fleas in the western U.S.

3. Samples taken from

affected tissues that are
Giemsa stained show the
bacillus to have a bipolar or
safety pin appearance.
Samples are taken from bubo
(bubonic plague), blood
(septicemic plague), or
tracheal/lung aspirate
(pneumonic plague).

http://www.cdc.gov/ncidod/dvbid/plague/p1.htm
 Diagnosis
Conditions for Presumptive Plague:
1. Immunofluorescence stain of sample is positive for the
presence of Y. pestis F1 antigen.

Pro: This test can be done quickly

(less than 2 hours)

Con: Since F1 antigen is expressed

at > 33C, samples that have been
refrigerated or are from culture that
have been incubated at lower http://www.cdc.gov/ncidod/dvbid/plague/p4.htm
temperatures would test negative
 Diagnosis
Conditions for Confirmed Plague:
1. Isolate Y. pestis from the specimen
OR
2. Observe at least a 4 fold elevation in serum
antibody titer to the F1 antigen (smaller
elevations are considered a presumptive
diagnosis).

Con: Neither of these 2 techniques is fast Y. pestis grows

very slowly in culture, and antibodies can take a significant
amount of time after disease onset to develop so they are
usually useful only as a retrospective confirmation of plague.
Treatment
Precautions for Dealing with Plague
Victims
Since the only form of human to human spread occurs by
respiratory droplet from a patient with pneumonic or secondary
pneumonic plague, surgical masks, gloves, gowns, and goggles
should be worn at all times
All patients with pneumonic plague should be strictly isolated (as
required by law) until they have received 48 hours of antibiotic
treatment and show signs of improvement
Labs should operate at biosafety level 2, unless they are
performing tests that may aerosol or produce droplets in which
case biosafety level 3 should be observed
No environmental decontamination is necessary as the bacteria is
quite fragile outside of the host environment
 Treatment
In a contained casualty setting, parenteral antibiotic
therapy, especially streptomycin or gentamycin, is
suggested.
In a mass casualty setting, intravenous or intramuscular
therapy may not be possible, so oral therapy, preferably
with doxycycline (or tetracycline) or ciprofloxacin,
should be administered.
Patients with pneumonic plague will suffer from
complications and therefore require substantial
advanced medical supportive care.
 Treatment in a Contained Casualty
Setting
Patient Category Recommended Therapy
Contained Casualty Setting
Adults Preferred choices:
Streptomycin, 1g IM twice daily
Gentamicin, 5 mg/kg IM or IV once daily or 2 mg/kg loading dose followed by 1.7 mg/kg IM or IV
three times daily
Alternative choices:
Doxycycline, 100 mg IV twice daily or 200 mg IV once daily
Ciprofloxacin, 400 mg IV twice daily
Chloramphenicol, 25 mg/kg IV 4 times daily
Children|| Preferred choices:
Streptomycin, 15 mg/kg IM twice daily (maximum daily dose 2 g)
Gentamicin, 2.5 mg/kg IM or IV 3 times daily
Alternative choices:
Doxycycline,
If >= 45 kg, give adult dosage
If < 45 kg, give 2.2 mg/kg IV twice daily (maximum 200 mg/dl)
Ciprofloxacin, 15 mg/kg IV twice daily
Chloramphenicol, 25 mg/kg IV 4 times daily
Pregnant Women Preferred choice:
Gentamicin, 5 mg/kg IM or IV once daily or 2 mg/kg loading dose followed by 1.7 mg/kg IM or IV
three times daily
Alternative choices:
Doxycycline, 100 mg IV twice daily or 200 mg IV once daily
Ciprofloxacin, 400 mg IV twice daily
 Treatment in a Mass Casualty Setting and
Postexposure Prophylaxis
Mass Casualty Setting and Postexposure Prophylaxis#

Adults Preferred choices:

Doxycycline, 100 mg orally twice daily**

Ciprofloxacin, 500 mg orally twice daily

Alternative choices:
Chloramphenicol, 25 mg/kg orally 4 times daily,

Children||
Preferred choices:
Doxycycline,**
If >=45kg give adult dosage
If <45 kg then give 2.2 mg/kg orally twice daily

Ciprofloxacin, 20 mg/kg orally twice daily

Alternative choices:
Chloramphenicol, 25 mg/kg orally 4 times daily,

Pregnant Women Preferred choices:

Doxycycline, 100 mg orally twice daily and

Ciprofloxacin, 500 mg orally twice daily

Alternative choices:
Chloramphenicol, 25 mg/kg orally 4 times daily,
 Benefits of Treatment
Type of Plague Untreated Fatality Rate Fatality Rate with
Treatment

Bubonic 50-90% 5-20%

Septicemic 50-100% 30-50%

Pneumonic 100% and death occurs Unknown (too few cases

rapidly usually within 48 for accurate results)
hours of onset
 How Antibiotics Work
Antibiotics inhibit prokaryote protein synthesis by
preventing the transition from initiation complex to
chain-elongating ribosome and causes miscoding.
 Weaponization
The CDC ranks the plague as a
Category A disease
Agents in Category A have the
greatest potential for adverse
public health impact with mass
casualties, and most require broad-
based public health preparedness
efforts (e.g., improved surveillance
and laboratory diagnosis and
stockpiling of specific
medications). Category A agents
also have a moderate to high
potential for large-scale
dissemination or a heightened
general public awareness that
could cause mass public fear and
civil disruption.
 The Oldest Bioweapon
The plague has a long
history as an agent of
biological warfare
 Yersinia Pestis as a Weapon
Pros Cons
It is relatively easy to obtain and Plague is fragile and dies after
mass produce. about 1 hr
It can be delivered in aerosol form Manufacturing an effective weapon
Pneumonic plague causes a rapid using Y. pestis would require
onset of illness with a high fatality advanced knowledge and
rate technology
Pneumonic plague has a high
potential for secondary spread of
cases during an epidemic
100-500 bacteria are enough to
cause pneumonic plague
 Additional Dangers of Yersinia
Pestis as a Weapon
There is no currently available pre-exposure prophylaxis or
vaccine for plague
Biological attack with plague might employ antimicrobial-
resistant strains that circumvent clinical efforts to deal with the
disease
In 1995 a patient in Madagascar was found who had a Y.
Pestis with a transferable multidrug resistance plasmid
(natural)
Additionally, there are reports that the bioweapons operations
of the former Soviet Union engineered multidrug resistant
and fluoroquinolone resistant Y. Pestis
 Effectiveness of Y. Pestis as a
Weapon
While antibiotic treatment of bubonic plague is usually effective, pneumonic
plague is difficult to treat and often results in death regardless of treatment
Most experts agree that intentional dissemination of plague would most
probably occur via an aerosol of Y pestis, a mechanism that has been shown
to produce [pneumonic] disease in nonhuman primatesThe size of the
outbreak would depend on the quantity of biological agent used,
characteristics of the strain, environmental conditions, and methods of
aerosilizationpeople would die quickly following the onset of symptoms. -
JAMA May 3, 2000 Vol 283, No. 17
In 1970, the WHO estimated that if 50 kg of Y pestis were released as an
aerosol over a city of 5 million, plague could occur in as many as 150,000
persons, 36,000 of whom would be expected to die. And this does not take
into account the people who would die from secondary contraction of the
disease.
According to the CDC, The fatality rate of patients with pneumonic plague
when treatment is delayed more than 24 hours after symptom onset is
extremely high.
 Means of Detection
With its relatively short onset time, the best defense against the
plague is early notification
There are currently two means of detection
Since the disease is passed directly or via a flea vector from
animal reservoirs (such as rats, ground squirrels, etc) to
humans, one mode of detection is observation of these
reservoirs (the bacterium survives by causing chronic disease
in animal reservoirs, so a sudden increase in the death of rats,
will cause the flea vectors to find another source of food)
The employment of Y. Pestis as a weapon would likely
involve aerosilization of the bacteria and direct release upon
humans. Thus new detection methods are required
Autonomous Pathogen Detection
Systems (APDS)
The LLL has just
finished developing a
prototype for a system
capable of detecting
aerosolized bacteria
including Bacillus
Anthracis and Yersinia
Pestis.
 APDS: How It Works
The sample is collected through an aerosol collector
Next the sample is delivered to a fluidics module which reproduces functions
routinely performed by laboratory personnel on the bench, including various mixings,
filterings, and incubations to prepare the sample for detection
Next, the sample is delivered to the immunoassay detector
The detector consists of a column of polystyrene microbeads, which have
antigen-specific capture antibodies attached to them
There are a hundred different bead classes (various beads have different
antibodies resulting in an antibody cocktail)
When the antigen (bacteria) binds to the appropriate antibody, secondary
antibodies labeled with the fluorescent reporter phycoerythrin bind to the now
immobilized antigen from the other side
The fluorescent secondary antibodies can then be detected with classification
lasers and fluorescence detectors.
Most of the sample is discarded as waste, but a small amount is archived, with the
appropriate data
 APDS: How it Detects
Using the previously described fluorescent antibody sandwich detection
system, the system is able to detect the median fluorescent intensity (MFI)
The detection threshold is the background MFI value plus three standard
deviations
For Yersinia Pestis, the background MFI was 155 with a standard
deviation of 25, resulting in a threshold detection limit at an MFI of 230
Establishing this criteria for a detection threshold helps to eliminate false
positives
Because the fluorescence is detected at specific beads, which have specific
antibodies, the system is able to detect the type of antigen in addition to the
concentration of the antigen
 APDS: Primary Testing Results
and Intended Improvements
Primary testing of the APDS has given
excellent results, and proven the APDS
capable of continuous and unattended
operation, and the platform is sensitive
and specific, detecting releases with no
false positives. In addition, the system
is able to detect the simultaneous
release of multiple pathogens.
Scientists are currently developing a
confirmatory nucleic acid-based test to
augment the detection capabilities of
the system
The final prototype is scheduled for
later this year
 APDS: Where and How Can We
Use It
The key functional features of the APDS are that it is autonomous and can
run unattended for 8 day stretches, can detect specific pathogens in a
relatively short period of time (about 1 minute) without false positives
The system is intended for use in office complexes, transportation terminals,
or convention centers where the public is at high risk of the release of
bioagents. In particular, each system can be part of an integrated network of
biosensors for wide-area monitoring of urban areas and major gatherings
The system would need only a source of electricity and a network connection,
to allow it to perform continuous sample collection, immunoassays every 30-
60 minutes, sample archiving, data reporting, and alarming
 Summary
The plague has a long history as a killer, and due
to its notoriety can be used to inspire fear.
Although the plague is a very real threat, the
CDC is taking it seriously, and steps are being
taken to increase our defensive preparedness for
such an attack. Perhaps the best thing that can
be done is to have an early detection system, and
scientists are rapidly making efforts to do just
that.