Yersinia pestis

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36
36.1 INTRODUCTION
Plague, caused by Yersinia pestis, is one of the most dev-
astating bacterial infections in human history. Plagues have
claimed millions of lives during the three recorded pandem-
ics.1 Plague circulates naturally among rodent reservoirs
and ea vectors in various enzootic foci.2 Humans typically
acquire plague infections via the bite of an infected ea or
via direct contact with infected rodents or plague patients.
Enzootic plague foci exist throughout the world, especially
in Eurasia, Africa, and America. Although the occurrence
of large-scale human plague epidemics is minimally prob-
able at present, human cases of plague are reported annually.
Moreover, Y. pestis can be used as a biowarfare or bioter-
rorism agent.3 It was employed during warfare in the four-
teenth century. The notorious Japanese Unit 731 conducted
plague warfare from 1937 to 1945 in China.4 This nefarious
pathogen was classied as a category A bioterrorism agent
by the Centers for Disease Control and Prevention (CDC),
United States.5
36.2 CLASSIFICATION AND MORPHOLOGY
Y. pestis is a Gram-negative bacterium that was rst isolated
and correctly described by Dr. Alexandre Émile Jean Yersin
from the Pasteur Institute, Vietnam, in 1894 during a plague
outbreak in Hong Kong, although Dr. Shibasaburo Kitasato
from Japan also contributed to the rst isolation of this patho-
genic bacterium.6 The previously reported synonyms for
Y. pestis include Bacterium pestis Lehmann and Neumann
1896, Bacillus pestis (Lehmann and Neumann 1896) Migula
1900, Pasteurella pestis (Lehmann and Neumann 1896)
Bergey et al. 1923, and Pestisella pestis (Lehmann and
Neumann 1896) Dorofeev 1947.7,8 According to the current
prokaryote classication, the genus Yersinia belongs to the
family Enterobacteriaceae, order Enterobacteriales, class
Gammaproteobacteria, division/phylum Proteobacteria,
and domain/empire Bacteria (http://www.bacterio.cict.fr/
classicationsz.html).
The genus Yersinia has 18 species and 3 subspecies
(http://www.bacterio.cict.fr/xz/yersinia.html), including
three human pathogenic species, namely, Y. pestis,
Y. enterocolitica, and Y. pseudotuberculosis. The high DNA
similarity (83%) of Y. pestis to Y. pseudotuberculosis led to
its reclassication as a subspecies of the latter.9 However,
the two bacteria cause very distinct diseases; thus, the name
Y. pseudotuberculosis subsp. pestis was rejected to avoid
confusion.10
36.2.1 Biotyping
Y. pestis is a homogenous species with only one serotype and
one phage type.1 Only a few antibiotic-resistant strains were
found.11,12 Biotyping systems based on biochemical features
are widely used in plague research. The nature of plague
and its causative agent hinders exchanging bacterial strains
between plague researchers. Other typing systems, such as
Yersinia pestis
Dongsheng Zhou and Ruifu Yang
CONTENTS
36.1 Introduction ..................................................................................................................................................................... 403
36.2 Classication and Morphology ........................................................................................................................................ 403
36.2.1 Biotyping .............................................................................................................................................................403
36.2.2 Ecotyping .............................................................................................................................................................404
36.2.3 Subspecies Classication ..................................................................................................................................... 404
36.2.4 Genotyping ..........................................................................................................................................................404
36.3 Biology and Epidemiology .............................................................................................................................................. 405
36.4 Clinical Features, Pathogenesis, and Transmission ......................................................................................................... 405
36.4.1 Major Clinical Features ....................................................................................................................................... 405
36.4.2 Pathogenesis .........................................................................................................................................................406
36.4.3 Transmission ........................................................................................................................................................ 407
36.5 Identication and Diagnosis ............................................................................................................................................407
36.6 Treatment and Prevention ................................................................................................................................................ 408
36.6.1 Antimicrobial Therapy ........................................................................................................................................408
36.6.2 Immunization and Prevention ..............................................................................................................................409
36.7 Conclusions and Future Perspectives...............................................................................................................................409
Acknowledgments ......................................................................................................................................................................410
References ..................................................................................................................................................................................410
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404 Manual of Security Sensitive Microbes and Toxins
ecotyping and subspecies classication system, are only
studied and used in certain countries.2,13,14 Although these
traditional methods have insufcient discriminatory power,
show poor reproducibility, and are affected by physiologic
factors, and their specic reagents lack of availability, they
have greatly contributed to plague prevention and control and
to our understanding of Y. pestis.
Early investigators utilized its capacity to ferment glycerol
to classify Y. pestis strains into glycerol-positive and glyc-
erol-negative strains.2 The negative strains were called the
oceanic type because they were usually isolated from rats in
seaports; the positive ones were called continental because
they were isolated from “wild” rodents, susliks (ground
squirrels), gerbils, and natural plague foci.2
Y. pestis can be classied into four biovars: Antiqua
(glycerol positive, arabinose positive, and nitrate posi-
tive), Medievalis (glycerol positive, arabinose positive,
and nitrate negative), Orientalis (glycerol negative, arabi-
nose positive, and nitrate positive), and Microtus (glycerol
positive, arabinose negative, and nitrate negative).15 The
rst three biovars were linked to the rst, second, and
third pandemic of human plague, respectively,16 whereas
the fourth is avirulent to humans, naturally causing only
Microtus plague and its epidemic. Some natural plague
foci have been found in Russia, where no human cases of
plague have been reported. It is possible that additional
new biovars may be found in the future. However, biovar
characteristics are unstable and one strain can undergo
spontaneous phenotypic variation that may cause it to be
classied into another biovar.2 For example, the Nicholisk
51 strain, an isolate from Manchuria, was classied into
biovar Orientalis via the IS100 genotype and the presence
of specic a phage remains, but it is glycerol positive and
should be classied into biovar Antiqua.17 The authors con-
sidered Nicholisk 51 as an ancestor of biovar Orientalis
or a variant of this biovar that had undergone phenotypic
reversion to the glycerol-positive phenotype.17
36.2.2 Ecotyping
Researchers have developed an ecotyping system that
exploits several biochemical features such as glycerin,
rhamnose, maltose, melihimose, and arabinose fermenta-
tion; nitrate reduction; amino acid utilization; mutation rate
from Pgm+ to Pgm−; and water-soluble protein patterns on
SDS-PAGE to group Chinese isolates of Y. pestis into 18
ecotypes.13,14,18,19 Each of the ecotypes is located in a par-
ticular geographic region. Most of the plague foci with dif-
ferent primary reservoirs have unique ecotypes.19 For the
other plague foci, more than one ecotype occurs in a sin-
gle focus with a single primary reservoir, and each of the
ecotypes corresponds to a unique set of natural landscapes
and primary vector(s). The ecotyping system has been used
as a framework for ecological and epidemiological analy-
sis of plague in China. It provides a preliminary explana-
tion of the relationship of the ecotypes of Y. pestis, natural
environment, reservoirs, and vectors.
36.2.3 SuBSpEciES claSSification
The subspecies classication system was proposed by
Russian scientists, and the following description about this
classication scheme was derived from Anisimov’s review.2
Y. pestis isolated from the FSU and Mongolia were classied
into six “subspecies,” including Y. pestis subsp. pestis (some-
times referred to as the “main” subspecies), Y. pestis subsp.
altaica, Y. pestis subsp. caucasica, Y. pestis subsp. hissarica,
Y. pestis subsp. ulegeica, and Y. pestis subsp. talassica based
on the numerical analysis of 60 phenotypic features. The last
ve subspecies are sometimes referred to as the “nonmain”
subspecies and have been referred to as the “pestoides”
group of Y. pestis isolates (Table 36.1). All pestoides strains
ferment rhamnose and are less virulent to guinea pigs,
but they are highly virulent to mice. These strains cause
occasional human and animal plague cases, but they have
rarely been associated with epizootics of plague.2,20 Recent
studies indicate that Y. pestis strains from Microtus in
Inner Mongolia and Qinghai Province of China should be
classied into Y. pestis subsp. xilingolensis and qinghaiensis
in accordance with the previous nomenclature.21,22
36.2.4 gEnotyping
Several genotyping methods have been described for
Y. pestis, including ribotyping,23 different region (DFR)
analysis,24 multilocus variable number tandem repeat
analysis (MLVA),21 repetitive DNA,25 clustered regularly
interspaced short palindromic repeats (CRISPRs),22 and
single-nucleotide polymorphisms (SNPs).26,27 They have
all been successfully used for genotyping Y. pestis in spite
of their profound differences in discriminatory power. For
a specic investigation of a plague outbreak, they are all
applicable. However, for effective source tracing, MLVA
and SNP analysis demonstrate a higher discriminatory
power. MLVA was successfully employed to trace the source
of a primary pneumonic outbreak in Qinghai Province of
China in 2009.28 SNP analysis and whole genome sequenc-
ing have also been used to conrm that the ancient Black
Death in Europe in the fourteenth century was caused by
Y. pestis.29,30
AQ1
AQ2
TABLE 36.1
Relationship between Y. pestis Biovar and Subspecies
Biovar Subspecies Other Nomenclatures
Antiqua pestis Main subspecies
caucasica Nonmain subspecies
Medievalis altaica Pestoides
hissarica
ulegeica
talassica
Orientalis pestis
Microtus xilingolensis Once classied as
biovar Medievalis
qinghaiensis
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405Yersinia pestis
36.3 BIOLOGY AND EPIDEMIOLOGY
Y. pestis is a Gram-negative, rod-shaped, nonmotile, non-
spore-forming, bipolar-staining coccobacillus (0.5–0.8 μm in
diameter and 1–3 μm in length) (giving it a safety pin appear-
ance).1 Y. pestis grows optimally at 28°C. Biochemically,
Y. pestis is unable to ferment lactose, sucrose, rhamnose, and
melibiose; it does not produce hemolysin, urease, and indole,
but it is catalase positive. Y. pestis can survive for hours after
drying on different environmental surfaces and for at least
5 days with the addition of nutrient media onto the surfaces.31
Thus, public areas could be contaminated with Y. pestis even
after the infected individual or source has left.
Enzootic plague foci exist throughout the world, espe-
cially in Eurasia, Africa, and America. Plague is primarily
an enzootic disease transmitted among rodents by the bite of
infected eas. Y. pestis is a multihost and multivector patho-
gen that involves more than 200 wild rodent species as reser-
voirs and over 80 ea species as vectors.2 The maintenance of
plague in nature is almost absolutely dependent on the cyclic
transmission between eas and rodents in various enzootic
foci,32 although Y. pestis has a limited ability to live in envi-
ronments such as soil.33,34 In this case, an enzootic plague
focus can be considered as a well-balanced terrestrial eco-
system composed of spatial contacts and food chains among
Y. pestis, its animal hosts, and the environment.
The natural environment, including biotic and abi-
otic factors, in enzootic plague foci is a crucial habitat for
Y. pestis and its rodent reservoirs and ea vectors.35 Biotic
factors include animals, plants, microorganisms, soil, and
vegetation, whereas abiotic factors consist of longitude and
latitude, topography, geomorphology, geology, and climate
(temperature, humidity, sunlight, and environmental chemi-
cal factors). Compared with biotic factors, abiotic ones are
relatively stable. The natural environment determines geo-
graphic zoning, regional distribution, habitats, and popula-
tion abundance of both rodent reservoirs and ea vectors.
The natural environment in enzootic plague foci determines
the geographic distribution, habitats, and population abun-
dance of both rodent reservoirs and ea vectors.
The rodent reservoirs of Y. pestis are usually classied
as major, minor, and accidental reservoirs.19 Major reser-
voirs are typically the predominant rodent species in the
plague focus. They are the primary carriers of Y. pestis
and are essential to the long-term survival of Y. pestis in
nature. Major reservoirs are generally highly susceptible
to Y. pestis. However, the infected individuals that are
somewhat tolerant to the pathogen challenge will not die
within a short period. The mammalian bacteremia contin-
ues for a certain period with high numbers of Y. pestis in
the blood, which facilitates pathogen transmission by ea-
bites. Moreover, some of the rodents may be highly tolerant
to Y. pestis in nature, even with asymptomatic infections.
The dynamics of population abundance in major reservoirs
coincides with the epidemic situation of animal plague in
the enzootic focus.36 Major reservoirs determine the type
and geographic distribution of a plague focus, as well as
the epidemiological pattern of the plague in the focus. The
enzootic properties of plague disappear without major res-
ervoirs. Minor plague reservoirs have a very high mortality
and usually die shortly after infection without bacteremia
or with temporary bacteremia. Minor reservoirs are insuf-
cient for the long-term survival of Y. pestis in nature,
although plague epidemics occasionally occur violently in
their populations. Accidental reservoirs are highly resistant
to the pathogen, but they usually catch Y. pestis because of
frequent contact with major and minor reservoirs. Human
contact with any of the Y. pestis–infected reservoirs is
likely to cause the incidence of human plague.
Although Y. pestis is highly pathogenic to rodent res-
ervoirs, it has a limited effect on the entire rodent popula-
tion. Even during the period of animal plague epidemics,
the abundance of rodents remains high. In specic plague
foci or parts of foci, the unique natural environment ulti-
mately molds a distinct food chain-based relationship among
Y. pestis, reservoirs, and vectors. The resulting specic com-
plex interaction among the environment, hosts, and Y. pestis,
termed as “host–niche interaction,” determines the presence
and types of Y. pestis.37 The survival of Y. pestis in nature
primarily depends on rodents and eas, whereas eas para-
sitize rodents and act as vectors for bacterial transmission.37,38
Natural environments in various plague foci remodel distinct
sets of rodents and eas. The accumulation of functional
genetic variations promotes the parallel diversication of
Y. pestis in different plague foci, as reected by the expansion
of various plague foci in nature.37,38 This adaptive evolution
is likely determined by the complex interaction among the
environment, hosts, and pathogen.
As an enzootic pathogen, Y. pestis has the potential to
infect humans. Humans typically acquire plague infections
via the bite of infected eas or through direct contact with
infected rodents, yet this deadly disease can be transmitted
from person to person by the respiratory route.32 In most
cases, humans are a biological dead end for Y. pestis. Thus,
the long-term maintenance of Y. pestis in nature is generally
independent of humans.
Three major global plague pandemics have been recorded
in history, namely, the Justinian plague, the Black Death,
and the modern plague.1 The third plague pandemic was
believed to have originated from Yunnan, China, in 1855
and then spread around the world through modern trans-
portation.37 A recent study revealed that the Black Death
was caused by Y. pestis29 and might have spread along the
ancient Silk Road.26
36.4 CLINICAL FEATURES, PATHOGENESIS,
AND TRANSMISSION
36.4.1 Major clinical fEaturES
Clinically, plague has different forms. The three common
forms, namely, bubonic plague, septicemic plague, and pneu-
monic plague, are mentioned in textbooks and the literature.
Other kinds of plague have been reported, including skin
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406 Manual of Security Sensitive Microbes and Toxins
plague, plague encephalitis, tonsillar plague, eye plague, and
intestinal plague. In epidemic areas, asymptomatic plague
has also been detected.39
Bubonic plague: This is the most common form clini-
cally. After a bite from an infected ea, the patient usually
develops swollen lymph nodes, which become buboes, the
classic sign of bubonic plague. The affected lymph node
depends on the bite site. Inguinal nodes are the most fre-
quently involved because the legs are the most common
site of eabites. The incubation period is 2–6 days. Flu-like
symptoms, such as a sudden onset of fever, weakness, head-
ache, and chills, are the primary clinical manifestations at
the beginning of the disease. When buboes develop, the
local subcutaneous tissues swell with the enlarged node(s),
which range from 1 cm × 1 cm for small ones to 5 cm × 7 cm
for larger ones. The enlarged nodes coalesce and adhere to
the surrounding tissues with unclear edges. The nodes are
rm and the patient feels severe pain when touched, which
constrains posture.
Pneumonic plague: It is the most contagious form of
plague because Y. pestis spreads from person to person by
respiratory droplets. Pneumonic plague is classied as pri-
mary or secondary. Primary pneumonic plague is caused
by direct inhalation of Y. pestis, and secondary pneumonic
plague is caused by blood spread from bubonic or septice-
mic plague. Patients develop other kinds of plague before
they develop secondary pneumonic plague. Primary pneu-
monic plague usually presents with severe pulmonary signs
in addition to u-like symptoms, with a very short incubation
period (2–4 days).28 The patients generally display high fever
(39°C–41°C), chills, bloody and suppurative coughing, weak-
ness, chest pain, dyspnea, hemoptysis, lethargy, hypotension,
and shock. Due to the severe respiratory distress, the patients
develop hypoxia, which manifests as lip or even body cya-
nosis. Thus, the disease plague epidemics were called Black
Death in medieval times.
Septicemic plague: It is the most severe form of plague
clinically. A large quantity of Y. pestis is present in the
patient’s blood and could become new sources of patho-
gens for eabites. If bubonic plague and other kinds of
plague are not treated properly, the disease worsens to
septicemic plague. Patients have systemic symptoms,
including high fever, severe headaches, hypotension, sei-
zures in children, hepatosplenomegaly, severe arrhythmia
or extremely weak pulse, delirium, and shock, with sub-
cutaneous or mucus membrane bleeding, bloody diarrhea,
and vomiting. The patient usually dies within 1–3 days
after the onset of these symptoms if specic treatment is
not administered on time. The patient may even die before
any symptoms appear.
36.4.2 pathogEnESiS
The transmission of Y. pestis relies primarily on the bite of
ea vectors, although infections can occur through direct
contact or inhalation. The development of heavy bacteremia
in rodents is crucial to reliably infect eas, which can then
transmit the disease by biting new animal hosts.40 The
infected eas regurgitate about 25,000–100,000 Y. pestis into
the host’s skin when they bite people.41
Y. pestis synthesizes biolms to attach onto the surface of
proventricular spines, and the heavy bacterial proliferation
in the biolms promotes the blockage of the gut of eas.42
Blockage of eas inhibits feeding and makes them feel hun-
gry and repeatedly attempt to feed, during which the ingested
blood is regurgitated back into the bite sites, causing Y. pestis
to infect the new hosts.
Yersinia biolms contain bacterial colonies that are
embedded in the self-synthesized extracellular matrix, and
the matrix is primarily composed of exopolysaccharides,
homopolymers of N-acetyl--glucosamine.43 The hmsHFRS
operon is responsible for the synthesis and translocation of
biolm exopolysaccharides across the cell envelope.43,44
The signaling molecule 3′,5′-cyclic diguanylic acid (c-di-
GMP) is a central positive allosteric activator of the enzymes
that catalyzes the production of biolm exopolysaccha-
rides.45 c-di-GMP is produced from GTP by diguanylate
cyclases and is degraded by phosphodiesterases. HmsT46,47
and YPO044948,49 are the only two diguanylate cyclases in
Y. pestis that synthesize c-di-GMP. The predominant effect
of HmsT is in vitro biolm formation, whereas the role of
YPO0449 in biolm production is much greater in eas than
in vitro.48 HmsP exhibits c-di-GMP-specic phosphodiester-
ase activity and is involved in c-di-GMP degradation; there-
fore, it negatively affects biolm formation.46,50
In contrast to its progenitor, Y. pestis, Y. pseudotuber-
culosis is transmitted via the food-borne route. Y. pseu-
dotuberculosis harbors all of the known structural genes
required for biolm formation, but it typically cannot syn-
thesize adhesive biolms and create blockages in eas.51
The action of multiple antibiolm factors such as NghA52
and RcsAB53 produces a tight biolm-negative phenotype
in typical Y. pseudotuberculosis. By contrast, Y. pestis has
lost the function of NghA52 and RcsA53 during its evolution.
Moreover, Y. pestis has acquired an additional factor Ymt,
which promotes bacterial survival in eas.54 The aforemen-
tioned evolutionary events enable Y. pestis to survive in eas
and to synthesize adhesive biolms in ea proventriculi to
cause blockage, which results in efcient arthropod-borne
transmission.55
After a bite from a blocked ea, the organisms migrate
through cutaneous lymphatic vessels to regional lymph
nodes. Once in the lymph nodes, most of the organisms are
phagocytosed and killed by polymorphonuclear leukocytes
that are attracted to the infection site in large numbers.56
However, a few bacilli are taken up by macrophages that are
unable to kill them. After the Y. pestis bacilli grow inside
phagocytes, they develop the ability to resist subsequent
phagocytosis.57 Based on in vitro experiments and in vivo
experiments in rodent peritoneal cavities, the infected mac-
rophages provide a protected environment for the pathogens
to proliferate intracellularly and synthesize their capsule
and other virulence determinants, thereby enabling the
AQ3
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407Yersinia pestis
bacteria to resist phagocytes55,56 and to annihilate the host
immune response.58 After initial subcutaneous and intrader-
mal colonization, the bacteria migrate into regional lymph
nodes, resulting in inammation and cellulitis, and occasion-
ally large carbuncles developing around the bubo (bubonic
plague).59 Without timely and effective treatment, the bac-
teria rapidly escape from containment in the lymph nodes
and systemically spread through the blood to various organs,
including the spleen, liver, lungs, and brain, which initiates
an immunologic cascade that leads to disseminated intravas-
cular coagulation, which in turn results in bleeding, as well
as skin and tissue necrosis.56 High-density bacteremia is
characteristic of moribund patients with plague.
Secondary pneumonic plague could result from hematog-
enous spread from the buboes to the lungs, which presents
in patients as severe bronchopneumonia, cavitation, or con-
solidation with production of bloody or purulent sputum.56
Primary pneumonic plague could be directly caused by the
inhalation of infectious droplets or aerosols, with symp-
toms such as acute pneumonia, intra-alveolar hemorrhage
and edema, profound lobular exudation, brin deposition,
and bacillary aggregation.60 The pneumonic form of the dis-
ease is the most feared because of the rapidity of its develop-
ment (1–3 days) and its high mortality rate among infected
individuals (approaching 100%) without timely effective
treatment. Coughing results in the production of airborne
droplets that contain bacteria, which can be inhaled by sus-
ceptible individuals and lead to the rapid airborne transmis-
sion of diseases among close contacts.
The pathogenicity of Y. pestis involves a diverse array of
virulence determinants that are coordinately expressed during
different stages of infection, including colonization and inva-
sion, early intracellular growth, avoidance of host defense,
and extracellular proliferation (Table 36.2). Y. pestis must
survive during infection against different host-responding
milieus that make bacterial living conditions far from opti-
mal through appropriate adaptive/protective responses that
are primarily reected by changes in the expression of spe-
cic sets of genes. Thus, the regulatory networks that govern
a complex cascade of cellular pathways facilitate the Y. pestis
pathogenic mechanisms that operate in a concerted manner.61
36.4.3 tranSMiSSion
Rodents and humans acquire Y. pestis through infected eabites,
contact with infected tissues, or the inhalation of respiratory
droplets or aerosols.1 In addition to acquiring Y. pestis from wild
animals, domestic animals such as cats, dogs, and guinea pigs
have also been reported to be infectious sources of plague.28
Bubonic plague is transmitted by bites from blocked eas.
However, blockage development takes a relatively long time
(about 2 weeks), which might be insufcient for explaining
the rapid spread that typies plague epidemics.32,62 Infected
eas are immediately infectious and efciently transmit the
microorganisms for at least 4 days post infection; the mode of
“early-phase transmission” by unblocked eas has been pro-
posed accordingly.62,63 During the testing of an early-phase
transmission model, defects in Y. pestis biolm formation
did not prevent ea-borne transmission, whereas biolm
overproduction inhibited efcient early-phase transmission.64
Unlike traditional blockage-dependent plague transmission
models, early-phase transmission occurs when a ea takes its
rst blood meal after initial infection by feeding on a bactere-
mic host, which may explain the rapid spread of disease from
eas to mammalian hosts during epizootic outbreak.62,63
Primary or secondary pneumonic plague could be trans-
mitted from person to person by respiratory droplets. Primary
pneumonic plague outbreaks rarely happen in modern
times,28,65–69 but they have been recorded in history. The out-
breaks in Oakland in 1919 and in Los Angeles in 1924,70–72
and in Manchuria from 1910 to 191173,74 are the most cited
instances. According to estimates from data from histori-
cal outbreaks, the basic reproduction number is 2.8–3.5.73,75
R0 measures the transmission potential of a pathogen; it is
the average number of secondary cases that spread from
the introduction of a single primary case into an otherwise
fully susceptible population. However, the transmissibility of
pneumonic plague also depends on the environment where
the patients live. If the patients stay in poorly ventilated rooms
and other people get close contact with the patients without
any preventive measures, more people will be infected.
36.5 IDENTIFICATION AND DIAGNOSIS
The CDC of the United States provides case classications
based on clinical symptoms and laboratory results.76 A
suspected case is dened as a clinically compatible case
that lacks presumptive or conrmatory laboratory diag-
nosis results. A presumptive case is dened as a clinically
compatible case with available presumptive laboratory diag-
nosis results. A conrmed case is a clinically compatible case
with the available conrmatory laboratory diagnosis results.
A suspected case can be diagnosed quickly based on the
symptoms and the epidemiological features, including the
environmental exposure history. When a case of plague is
suspected, clinical specimens can immediately be collected.
Diagnostic samples include blood and appropriate site-
specic samples, such as aspirates from suspected buboes,
pharyngeal swabs, sputum, and endotracheal washings from
patients suspected of plague pharyngitis or pneumonia, and
cerebrospinal uid from those with suspected meningitis.76
Presumptive laboratory diagnosis may also be performed by
observing a fourfold or greater increase in the serum anti-
body titers against the Y. pestis F1 antigen without a history
of plague vaccinations, as determined by enzyme-linked
immunosorbent assays (ELISA) and older, less-sensitive pas-
sive hemagglutination assays. Conrmatory laboratory diag-
noses are traditionally based on the isolation of pure Y. pestis
from clinical specimens together with staining and micros-
copy observation and phage lysis assay with Y. pestis-specic
bacteriophages. Tiny, 1–3 mm “beaten-copper” colonies
appear on blood agar after 48 h of cultivation. Gram’s stain-
ing conrms the presence of Gram-negative rods and, in
some cases, the identication of the double-curved shapes.
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408 Manual of Security Sensitive Microbes and Toxins
However, other Yersinia species may exhibit very similar
appearances. In recent years, numerous rapid tests for detect-
ing Y. pestis based on different variants of polymerase chain
reaction have been developed.77–80 The continued devel-
opment and implementation of DNA-based methods with
increased sensitivity and dened specicity are particularly
useful for detecting residual Y. pestis DNA in situations
where Y. pestis cannot be cultured from clinical specimens.
In addition, immunochromatographic strips and biosensors
have also been developed for plague diagnosis.81–84 Their
applications will greatly help in the rapid diagnosis of plague.
36.6 TREATMENT AND PREVENTION
36.6.1 antiMicroBial thErapy
If a case of plague is suspected or diagnosed, specic
antimicrobial therapy should be started immediately. The
suspected or diagnosed plague patients and their direct con-
tacts should be arranged individually in separate wards or
isolation rooms. Patients suspected of pneumonic plague
should be managed with respiratory droplet precautions.
All patients should be isolated for the rst 48 h after treat-
ment initiation. If pneumonic plague is present, then respi-
ratory isolation procedures should be enforced strictly and
rigidly, including the use of gowns, gloves, and eye protec-
tion. Patients with pneumonia must be isolated until they
have completed at least 4 days of antibiotic therapy. Timely
antibiotic treatment is effective against plague. When
administered during the early phase of the disease, antibi-
otic treatment effectively reduces overall human mortality
ranging from 5% to 14%. However, when left untreated, the
mortality rate is between 50% and 90%.85 Streptomycin,
chloramphenicol, and tetracycline, alone or in combination,
are the reference drugs for treating plague, and streptomy-
cin is the most preferred.86 ,87 Antibiotics should be given
intramuscularly at a dose of 30 mg/kg/day in two divided
doses. Antibiotics should be administered intramuscularly
for 10 days or until 3 days after the patient’s temperature
returns to normal. Chloramphenicol should be used in cases
of plague meningitis.76 Streptomycin or gentamicin can be
used to treat plague in children. Gentamicin is preferred
TABLE 36.2
Major Virulence Determinants of Y. pestis
Function Gene IDs Description References
Colonization and dissemination
Pla YPPCP1.07 Plasminogen activator
promoting bacterial in vivo
dissemination
[113,114]
Ail YPO2905 Invasin adhesin and invasin [115]
YadBC YPO1387-1388 Invasin adhesin and invasin [116]
YapC YPO2796 Autotransporter adhesin [117]
YapE YPO3984 [118]
Intracellular growth
RipA YPO1926 Putative acetyl coenzyme A
transferase
[119]
Ugd YPO2174 LPS modication [120]
MgtCB YPO1660-1661 Magnesium uptake [120]
Yfe YPO2439-2442 ABC-type iron transporter [121]
FeoBA YPO0132-0133 Ferrous iron transporter [121]
Annihilation of hose immune response
T3SS Annihilation of innate
immune cells
[122]
F1 capsule YPMT1.81c-1.84 Resistance to phagocytosis [123]
pH6 antigen YPO1301-1305 Resistance to phagocytosis [124]
Ail YPO2905 Serum resistance [115]
Tc proteins Toxicity to mammalian cells [125]
O-antigen genes Lack of O-antigen is
essential for Pla function
[126]
Iron uptake
Yfe YPO2439-2442 ABC-type iron transporters [127]
HPI YPO1906-1916 Siderophore yersiniabactin-
based iron acquisition
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409Yersinia pestis
among pregnant women because of its safety.76 Tetracycline
is contraindicated in pregnant women and in children less
than 7 years of age because of possible staining of develop-
ing teeth. Alternatively, antibiotics may be applied.88
The successful treatment of septicemic and pneumonic
plague with antibiotics is less likely because the disease rap-
idly develops and treatment must commence during the early
stages of the infection. Fulminant plague is especially dif-
cult to treat because of the possibility of bacteriolysis, which
subsequently releases large amounts of endotoxin. Early
treatment with sufcient quantities of effective antibiotics is
critical for saving lives from primary pneumonic plague.28
Supportive care is an important part of management, which
may include immediate uid resuscitation, vasopressors,
hemodynamic monitoring, and respiratory care, including
ventilator support. The following symptomatic treatments
may be needed: (1) patients with dysphoria or severe pain
can be given sedatives and painkillers, (2) patients with heart
failure can be given cardiac stimulants, and (3) patients with
toxic shock can be given timely antishock treatment.
36.6.2 iMMunization and prEvEntion
The recent emergence of multiple antibiotic-resistant Y. pestis
strains89,90 and their possible use as bioweapons and bioter-
rorism agents means that the long-term potential for the use
of antibiotics to treat plague is less certain and that a vaccine
effective against plague is urgently needed. The individuals at
high risk of plague should be immunized, including military
troops and other eld personnel working in plague-endemic
areas where exposure to rats and eas cannot be controlled.
Laboratory personnel working with Y. pestis, people who
reside in enzootic or epidemic plague areas, and those whose
vocations bring them into regular contact with wild animals,
particularly rodents and rabbits, should also be vaccinated.
Both killed and live whole-cell plague vaccines have
saved thousands of human lives in the twentieth century
and have continually been used in a few countries where
the threat of plague is imminent.91,92 A formaldehyde-killed
whole bacilli vaccine is the only US-licensed vaccine for
plague and requires a series of injections. Unfortunately,
the vaccine does not protect against primary pulmonary
plague, and its manufacture was discontinued in 1999.
The EV vaccine was initially developed in the early 1900s
and has been used since then in some parts of the world.
However, the vaccine strain is not avirulent, and its safety
in humans has been questioned. An ideal vaccine candidate
should have an important role in pathogenicity and/or sur-
vival of pathogens during infection. The current interest is
in developing plague vaccines that consist of puried pro-
tein subunits, with improved protection and reduced side
effects.91–93 The subunit vaccines, which consist of a cap-
sular subunit F1 and/or the major virulence V antigen, pro-
vide excellent long-lasting protection in a number of animal
models, including nonhuman primates.94 The efcacy of
this formulation for human use remains to be determined.
The identication of novel protective antigens and their
combined use with F1 and V antigens92,95,9 6 as multicompo-
nent subunit vaccines have been widely considered as one
of the leading strategies for plague vaccine development in
the future.97 Thus, a safe, effective, and licensed vaccine for
plague prevention is still unavailable.
Other measures should be taken to prevent plague dur-
ing outbreaks. For bubonic plague, patients should be iso-
lated for timely medical treatment. The possible site where
the patient acquired the infection should be monitored for
potential infected individuals. If more people are aficted,
the infectious area should be decontaminated by control-
ling eas and mice using pesticides. For primary and sec-
ondary pneumonic plague, aside from patient isolation, all
close contacts should be isolated for medical observation.28
Sometimes, simple preventive measures could effectively
prevent the spread of plague from person to person, includ-
ing the wearing of masks and avoiding close patient contact
(more than 2 m away).28
36.7 CONCLUSIONS AND
FUTURE PERSPECTIVES
Y. pestis has caused tragedies in human history and has
reshaped our civilization. It is one of the most dangerous
bacterial pathogens because it has the capacity to spread
from person to person; thus, it has been listed as a category
A bioterrorism agent. Y. pestis is a typical zoonotic pathogen,
with rodents as reservoirs and eas as vectors. Its life cycle
involves the interaction between among, reservoirs, vectors,
and the environment. Therefore, it could be used as a model
for studying pathogen evolution.98–102
Although Y. pestis was identied more than 100 years
ago, many of its facets remain unclear, including its patho-
genesis, host immune responses, its evolution, and its natural
life cycles. The application of newly developed technologies,
such as omics-based methods,103 to clarify these basic issues,
will help nd targets for developing novel diagnostic proce-
dures, vaccines, and drugs. Considering Y. pestis as a dan-
gerous bioterrorism agent, techniques for the rapid, highly
specic, and sensitive detection of this pathogen on-site that
are easy to use are urgently needed, although a number of
immunologic and nucleic acid-based assays have been devel-
oped to date.77,82–84,104–111 Vaccine development is still a con-
tinuous and difcult task for preventing Y. pestis infections.
Y. pestis could also be employed as a model for developing
strategies for handling bioterrorism events because it is a typi-
cal pathogen with zoonotic and epidemic features, and it has
historically been associated with bioterrorism or biowarfare.
After the “911”anthrax spore letter bioterrorism in the United
States in 2001, a new eld, microbial forensics, was proposed
for the source tracing of Bacillus anthracis.112 A key step for
the success of microbial forensics is the development of an
international database for source-tracing analysis. Y. pestis
represents one of the best model organisms for developing
such a database.112
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410 Manual of Security Sensitive Microbes and Toxins
ACKNOWLEDGMENTS
We thank members of our laboratory for helpful comments
and suggestions. Y. pestis works in our laboratory have
been supported by grants from the National Basic Research
Program of China (2009CB522600), the National Natural
Science Foundation of China (Nos. 30930001, 30430620,
30371284, 30471554, 31000015, and 31071111), and the
National Science Foundation of China for Distinguished
Young Scholars (No. 30525025).
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K15299_C036.indd 412 11/4/2013 12:23:00 PM

AUTHOR QUERIES
[AQ1] Please check “melihimose” for correctness.
[AQ2] Please provide the expansion of the acronym “FSU,” if appropriate.
[AQ3] Please provide the expansion of the acronym “GTP,” if appropriate.
[AQ4] Please provide the expansion of the acronym “EV,” if appropriate.
[AQ5] Please provide publisher location for Ref. [4].
[AQ6] Please provide volume and page numbers for Refs. [42,74,108,118].
[AQ7] Please conrm the inserted publisher name for Ref. [74].
K15299_C036.indd 413 11/4/2013 12:23:00 PM