Seminar in Nucleic Acids
Dr. Geoffrey Zubay
March 23, 2004
• Bubonic
• Septicemic
Primary
Secondary
• Pneumonic
Primary
Secondary
Bubonic
 Most common form
(~85% of all cases)
 causes swollen
lymph nodes (buboes)
 can only spread from
person to person via
direct contact with bubo
drainage
 1%-15% death rate if
treated; if not treated,
40%-60% death rate
 the backbone of the
survival of y. pestis
because it can develop
into both secondary
septicemic and
pneumonic types
Septicemic
 fatality rate of 3050% in treated cases,
50-90% in untreated
cases
 causes severe blood
infection throughout the
body and gangrene of
acral regions (nose and
digits) if untreated
Primary:
 occurs when a flea
inserts y. pestis directly
into the bloodstream
Secondary:
 occurs as a severe
development from
bubonic or pneumonic
(when y pestis migrates
to bloodstream)
Pneumonic
 100% death rate if
not treated within first
24 hrs
 can be transmitted
via direct inhalation of
the germs
 least common, yet
most dangerous form
Primary:
 occurs via inhalation
of pneumonic
respiratory droplets
Secondary:
 occurs when bubonic
or septicemic plagues
spread to the lungs
Lungs of a pneumonic
plague patient
Reservoirs
•Urban and domestic
rats
•Ground squirrels
•Rock squirrels
•Prairie dogs
•Deer mice
•Field mice
•Gerbils
•Voles
•Chipmunks
•Marmots
•Guinea pigs
•Kangaroo rats
…over 200 identified
reservoirs
Vectors
Incidental Hosts
•Xenopsylla cheopis (the
oriental rat flea; nearly
worldwide in moderate
climates)
•Oropsylla montanus (United
States)
•Nosopsyllus fasciatus
(nearly worldwide in
temperate climates)
•Xenopsylla brasiliensis
(Africa, India, South
America)
•Xenopsylla astia (Indonesia
and Southeast Asia)
•Xenopsylla vexabilis (Pacific
Islands)
• humans
~30 identified flea vectors
*http://www.cidrap.umn.edu/cidrap/con
•Domestic and feral
cats
•Dogs
•Lagomorphs (rabbits
and
•hares)
•Coyotes
•Camels
•Goats
•Deer
•Antelope
tent/bt/plague/biofacts/plaguefactsheet.
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
1º
2°
•Bites from flea vectors
•Bites or scratches from
infected animals, such as
cats
•Direct contact with
infected animal carcasses,
such as rodents (especially
marmots), rabbits, hares,
carnivores (eg, wild cats,
coyotes), and goats
SEPTICEMIC
PNEUMONIC
•Bites from flea vectors
where Y pestis is inserted
directly into bloodstream
–no discernible bubo
present
•Inhalation of respiratory
droplets from infected
animals such as cats
•Inhalation of respiratory
droplets from a person with
primary or secondary
pneumonic plague
•Handling Y pestis cultures in
the laboratory setting
• Develops as a complication
of bubonic or 1º pneumonic
plague
–When Y pestis enters
the bloodstream
• Bubonic and 1°
septicemic spread plague
bacilli hematogenously to the
lungs
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 city…The LORD’S 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
• Justinian’s 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 5060% 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 1330’s
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
Lists of the dead were
merchants were already dying from the plague
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)
•
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•
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•
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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 (100→25→12 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
•
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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-60’s
2) Between 1973-1978
3) mid-80’s-present
• The rise of reported plague morbidity has increased worldwide in the 90’s,
especially in Africa
GEOGRAPHICAL SHIFTS: ASIA→AMERICAS→AFRICA
• In the 1950’s, plague was a problem primarily in Asia, with little activity in
the Americas
• By the early 60’s, plague activity in the Americas increased, and in Africa, it
began to play a role
• The mid-60’s-early 70’s 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
location for
plague activity
in the 50’s
The trend
shifts in the
60’s and the
Americas
become more
dominant
Africa shows a
drastic increase
in the 80’spresent
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 1954-80’s
•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 don’t have the capital to afford the
proper health care required
Outbreaks from the 20th Centurypresent
•
•
•
•
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
October 1997
Malawi
>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
Plague outbreak in India
reported in Newsweek,
1996
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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
Depiction of death carts in London
June-July 2003
carrying victims of plague
Algeria
>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
droplets (from
either cat or
human)
Skin
Blood vessels
Lymphatic vessels
Organs
Forms buboes
Blood
Blood
Organs
Lungs
2°septicemic
2°pneumonic
Lungs
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 μm in diameter

1-3 μm long

Grows optimally at 28ºC
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
through food and water

Transmission: rodent to humans
through flea vector

Chromosomal DNA – 4.74Mb

Chromosomal DNA – 4.65Mb
 90% chromosomal DNA
relatedness with Y.
pseudotuberculosis

Extrachromosomal DNA
 pYV

“Two thousand years ago, it only
gave you a tummy ache”
- Brendan Wren,
geneticist

Extrachromosomal DNA
 pYV
 pPla
 pMT1

“Within a few hundred years - an
evolutionary eye blink- Y. pestis
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
secretion system
Toxicity. Avoidance
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 flea’s 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 flea’s 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 vector’s
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 28ºC = 82.4ºF)

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 30ºC, 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 37ºC 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 subcutaneous infection site into the
circulation
 Pla derives its name from the fact
that it can activate the
mammalian plasma enzyme
plasminogen to plasmin.
Plasmin is responsible for the
breakdown of fibrin
 Main virulence role of Pla:
Cleaves fibrin deposits that trap
Y. pestis, thereby promoting
plague infection
factor X
factor Xa
prothrombin
thrombin
fibrinogen
fibrin
transaminase
blood clot
plasminogen
tissue plasminogen
activator (TPA)
plasmin
dissolved clot
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 37ºC 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 37ºC,
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 macrophage’s phagocytic
and signalling capabilities, ultimately inducing its
apoptosis.
http://www.rkm.com.au/imagelibrary/index.html
Yops



When placed in environment that is
around 37ºC 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
Yersinia’s 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
secretion system
Toxicity. Avoidance
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
Fever
100%
Headache
85%
Severe exhaustion
75%
Vomiting
Septicemic Plague
Fever
100%
25-49%
Nausea & Vomiting
50%
Altered mental status
38%
Altered mental status
common
Abdominal pain
18%
Abdominal pain
39%
Cough
25%
Skin rash
23%
2º septicemic plague
23%
Diarrhea
39%
2º 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

1º 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 2º 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,
with greater infection in the left
lung.
• widened mediastinum, resulting
in less available space for lungs
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 > 33ºC, samples that have been
refrigerated or are from culture that
have been incubated at lower
temperatures would test negative
http://www.cdc.gov/ncidod/dvbid/plague/p4.htm
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
rapidly usually within 48
hours of onset
Unknown (too few cases
for accurate results)
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 broadbased 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
mass produce.


It can be delivered in aerosol form


Pneumonic plague causes a rapid
onset of illness with a high fatality
rate
Plague is fragile and dies after
about 1 hr
Manufacturing an effective weapon
using Y. pestis would require
advanced knowledge and

Pneumonic plague has a high
potential for secondary spread of
cases during an epidemic

100-500 bacteria are enough to
cause pneumonic plague
technology
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 antimicrobialresistant 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 primates…The size of the
outbreak would depend on the quantity of biological agent used,
characteristics of the strain, environmental conditions, and methods of
aerosilization…people 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 3060 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.