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Previous Article | Next Article 
Clinical Microbiology Reviews, April 2004, p. 434-464, Vol. 17, No. 2
0893-8512/04/$08.00+0 DOI: 10.1128/CMR.17.2.434-464.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Intraspecific Diversity of Yersinia pestis
Andrey P. Anisimov,1 Luther E. Lindler,2 and Gerald B. Pier3*
Department
of Infectious Diseases, State Research Center for Applied Microbiology,
142279 Obolensk, Serpukhov District, Moscow Region,
Russia,1
Department of Bacterial
Diseases, Walter Reed Army Institute of Research, Silver Spring,
Maryland 20910,2
Channing Laboratory,
Department of Medicine, Brigham and Women's Hospital, Harvard
Medical School, Boston, Massachusetts
021153
Increased interest in the pathogenic potential of Yersinia
pestis has emerged because of the potential threats from
bioterrorism. Pathogenic potential is based on genetic factors present
in a population of microbes, yet most studies evaluating the role of
specific genes in virulence have used a limited number of strains. For
Y. pestis this issue is complicated by the fact that most
strains available for study in the Americas are clonally derived and
thus genetically restricted, emanating from a strain of Y.
pestis introduced into the United States in 1902 via marine
shipping and subsequent spread of this strain throughout North and
South America. In countries from the former Soviet Union (FSU),
Mongolia, and China there are large areas of enzootic foci of Y.
pestis infection containing genetically diverse strains that have
been intensely studied by scientists in these countries. However, the
results of these investigations are not generally known outside of
these countries. Here we describe the variety of methods used in the
FSU to classify Y. pestis strains based on genetic and
phenotypic variation and show that there is a high level of diversity
in these strains not reflected by ones obtained from sylvatic areas and
patients in the Americas.
Plague is a zoonotic infection that is spread to humans from natural
rodent reservoirs, commonly via the bite of an infected flea.
Yersinia pestis, the causative agent of bubonic, septicemic,
pneumonic, pharyngeal, cutaneous, and enteric plague as well as plague
meningitis, can be found in populations of more than 200 species of
wild rodents which inhabit natural plague foci in all the continents
save Australia (Fig.
1). Over 80 species of fleas are proven vectors of plague
(7-9,
11,
13,
28,
30,
31,
34,
49,
55,
56,
104,
132,
135,
141,
146,
160). Utilizing such a
broad host and vector range provides a large opportunity for genetic
diversity and natural selective forces to generate considerable
variability in the Y. pestis genome. Yet much of what is known
about the genetic and phenotypic properties of Y. pestis comes
from studies of a limited number of strains commonly found in the
Americas, wherein there is very restricted genetic diversity. Thus,
much of the pathogenic potential of Y. pestis for humans
remains largely unknown, locked away in the multitude of strains
circulating in natural foci, many of which are found in isolated
regions of Russia and Asia and are not easily accessible to many
researchers.
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FIG. 1. Global
distribution of plague. Reprinted with permission from K. L.
Gage and J. A. Montenieri, Centers for Disease Control and
Prevention, Fort Collins,
Colo.
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Enzootic circulation of Y. pestis in
natural plague foci requires active infection of host rodents and
growth in the fleas to produce blockage by a large mass of bacilli in
the proventriculus, a sphincter-like organ that separates the stomach
and esophagus. These factors are essential for continued transmission
of the bacilli to new hosts and thus for maintenance of the infectious
focus in a natural environment. The organism must be able to resist
host defense systems, multiply, and cause bacteremia for further
transmission by fleas to a new host. Each of these stages of the Y.
pestis life cycle is dependent on elaboration of specific
bacterial virulence factors that may act in concert or separately
(7-9,
11,
13,
28,
30,
31,
34,
49,
55,
56,
104,
132,
135,
141,
146,
160,
179). Many of the
natural plague foci are geographically not connected, resulting in
considerable ecological differences needed for Y. pestis to
survive and be transmitted in these different environments. This
results in considerable diversity in genotype and phenotype among
plague isolates from different natural foci
(1,
2,
9,
11,
16-18,
23-25,
31,
33,
52,
55,
56,
60,
67-69,
104,
107,
109,
120-122,
127,
130,
132,
135,
140,
141,
169,
172,
177,
179,
181,
184,
198; M. I.
Levi, Abstr. Sci. Conf. Natur. Focality Prophyl. Plague Tularemia, p.
72-74, 1962; F. Zhenya, Z. Xiang, L. Yunheng, L. Jun, W.
Shenrong, Z. Yaoxing, J. Lingling, and L. Feng, Abstr. 7th Int. Symp.
Yersinia, abstr. P-127, Med. Microbiol. [Ned. N.
Voor] 6[Suppl. II]: S42, 1998).
As the
current concern with use of microbes as agents of bioterrorism grows,
it will be essential to gain a fuller understanding of the variety of
strains of Y. pestis that can be found in the world and the
effectiveness of countermeasures taken to control, prevent, and treat
plague infections. Much of what is known is not available in English
language publications, since many of the studies are published in
Russian language journals. Therefore, we have compiled information from
around the world to summarize what is known about diversity in isolates
of Y. pestis. Given the natural genetic and phenotypic
diversity in an organism that has very high pathogenic potential for
humans, it is essential to understand how genetic and phenotypic
variation impacts the pathogenesis, diagnosis, treatment, and
development of immunologic therapies for
plague.
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GLOBAL DISTRIBUTION OF YERSINIA PESTIS STRAINS
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In spite of being present on
all continents save Australia, Y. pestis is not widespread
throughout all of the world. Natural plague foci cover 6 to 7%
of the dry land of the Earth. While notorious as a cause of disease in
Europe for a long period, it is now notably absent from Western and
Central Europe as well as Canada and parts of North and South America
(Fig. 1). In the most
northerly and southerly parts of the world, winter temperatures may
preclude maintenance of the plague transmission cycle of flea to
rodent. Obviously vigorous quarantine and public health measures in
places such as Australia can effectively prevent plague from taking
hold in that continent, although the island nation of Madagascar
nonetheless now has endemic foci of plague and human plague outbreaks
due to introduction of the organism via marine shipping. Quarantine and
public health measures probably limit the occurrence of
human plague in the more developed countries of the world,
but it is not clear how this prevents endemic foci of plague from
becoming established in wild rodents. There is obviously much
complexity to plague, flea, and rodent biology and the ecology of
Y. pestis transmission that would impact the sustainability of
the organism in sylvatic foci in different parts of the
world.
Geographical Distribution of Plague in the Former Soviet Union
Forty-three natural foci are
found in the southern and southeastern regions of the former Soviet
Union (FSU) (Fig.
2; Table
1). They cover more than 216 x 106 hectares, or
8.6% of its territory
(132). Separation and
subsequent classification of these natural plague foci was performed
first on the basis of their geographical distribution and then on
the basis of the primary infected host found in
each focus. Primary hosts from different foci have several ecological
characteristics in common with each other, such as a large and steady
quantity of the rodent hosts, infectious bacteremia as one of the
stages of the disease, flea parasites that serve as active plague
vectors, and fleas that can easily survive within rodent's burrows
and nests. As a rule, these features are not necessary for infection of
secondary hosts. Adaptation for continued transmission of the plague
pathogen within different rodent species is assumed to contribute
strongly to the emergence of variant Y. pestis subspecies,
differentiated by fermentative activity, nutritional requirements, and
ability to cause infectious bacteremia and death in different animal
species
(157).
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FIG. 2. Distribution
of natural plague foci in the FSU. Identifications of the plague foci
are given in Table 1 and
the text. The red line indicates the FSU frontier; green lines indicate
the frontiers of the states of the FSU; brown lines indicate boundaries
of the plague foci; black lines indicate frontiers of other countries.
Plague foci are yellow (for foci containing the main Y. pestis
subspecies) or light brown (for foci containing non-main Y.
pestis subspecies). This figure is based on references 17, 104,
132, 135, and
169.
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Plague in the Americas
Y. pestis was first introduced into
the United States at the turn of the 20th century through the port of
San Francisco during the third pandemic, which is still ongoing. There
were hundreds of deaths from plague in the first quarter of the 20th
century, with the last major outbreak of pneumonic plague occurring in
Los Angeles in 1924 to 1925. Since then, most cases in the United
States have been found in individuals living in sylvatic areas close to
foci of plague circulation in rodents. The fact that Y. pestis
is essentially a recently introduced pathogen into the Americas
indicates that the genetic and phenotypic diversity of American
isolates is relatively restricted, particularly compared to those from
Central and East Asia. Thus, strains of Y. pestis from
patients and collections in the Americas that are readily available to
investigators are likely to be much less genetically and phenotypically
diverse than strains from other parts of the
world.
Current Assessment of Plague Diversity
A major study by Achtman et al.
(2) proposed that Y.
pestis is a recently emerged clone of Yersinia
pseudotuberculosis, since the authors found that within five
housekeeping and one lipopolysaccharide (LPS) biosynthesis gene there
was essentially no genetic diversity among 36 globally diverse Y.
pestis strains. The Y. pestis alleles were identical or
nearly identical to those in 12 strains of Y.
pseudotuberculosis. By taxonomic standards, Y. pestis
might be considered to be Y. pseudotuberculosis, but because
of large differences in disease manifestations and the role of Y.
pestis in human disease and history, this grouping has not been
pursued. Plasmid content and perhaps other small genetic differences
are thought to account for the different diseases caused by Y.
pestis and Y. pseudotuberculosis. Obviously if plasmid
content and small genetic differences can account for the differences
in the host range and virulence of Y. pestis and Y.
pseudotuberculosis, it might be reasonable to assume that similar
small differences could arise is the multitude of Y. pestis
strains found in natural foci of infection and that this could
potentially have important impacts on the pathogenesis of Y.
pestis and the manifestations of plague.
Achtman et al.
(2) also performed a
restriction fragment length polymorphism analysis of 44 strains of
Y. pestis, using a probe for the IS100 element, and
found by constructing a neighbor-joining phylogenetic tree that three
major biovars of Y. pestis, which have been proposed as a
basis for further differentiation of Y. pestis strains and
have been designated Antiqua, Medievalis, and Oreintalis, were each
composed of closely related strains and that all of the strains were
derived from a common ancestor. However, a closer analysis of the
strains used by Achtman et al.
(2) shows that only six of
the strains were from European Russia or Central Asia
(Kurdistan) and that five of the six were all of the highly
related Medievalis biovar. No data were reported for the sixth strain.
The remainder of the strains were from non-Central Asian parts of the
world. Achtman et al. (2)
also noted that Y. pestis, like Mycobacterium
tuberculosis, has a relatively uniform genetic structure, but
endonuclease restriction analysis suggests more diversity
(85,
119). Overall, whether
the Y. pestis isolates from diverse natural foci in European
Russia and Central Asia share a similar close genetic relatedness has
not been addressed, leaving open the possibility that these strains may
have virulence properties or pathogenic potential distinct from the
more closely related isolates of Y. pestis that have been
found to date to have limited genetic
diversity.
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HISTORICAL METHODS OF DISTINCTION OF YERSINIA PESTIS STRAINS IN EUROPEAN RUSSIA AND CENTRAL ASIA
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Intraspecific Taxonomy
Many studies have been carried out over the years by
investigators in the FSU to attempt to classify Y. pestis and
understand the diversity of phenotypic traits in this species. Early
studies (1928) by Bezsonova
(23) divided Y.
pestis strains into two varieties on the basis of their ability to
ferment glycerol (i.e., glycerol positive and glycerol negative).
Glycerol-negative strains were reclassified in 1938 by Berlin and
Borzenkov (22) into the
oceanic variety, since they were usually isolated from rats in
seaports, and glycerol-positive strains, termed the continental
variety, since such strains were isolated from "wild"
rodents, susliks (ground squirrels), gerbils, etc., from natural plague
foci. The designation of glycerol-negative plague strains as the
oceanic variety reflected their predominant distribution at the time,
but it also appears that the source of these strains may have been the
Yunnan interior region in China, which is close to the border of
present-day Myanmar (Burma), suggesting that glycerol-negative strains
were present in southeastern Asia before the outbreak of the third
pandemic in the late 19th century. However, a number of investigators
have found in the southern part of Vietnam only anthropogenic plague
foci in inhabited localities but no natural foci
(176). The main flea
vector in Vietnam was Xenopsylla cheopis, while the main
rodent host was Rattus exulans. The lack of sylvatic plague
foci in Vietnam indicates there were not widespread foci of plague
throughout Southeast Asia. Nowadays, glycerol-negative strains are
found in natural plague foci located in the United States, South
Africa, and Southeast Asia as such strains spread to these regions via
marine shipping from Hong Kong starting in 1894 and during the
following years of the third pandemic
(2,
141).
Devignat in
1951 (52) and Tumanskii
in 1957 (181) used
glycerol fermentation, nitrate reduction, and ammonia oxidation to
classify Y. pestis into three intraspecific groups that were
named Orientalis, Antiqua, and Medievalis by Devignat (Table
2). The Devignat classification is currently widely used, referring to
strains as belonging to biovars, although some Y. pestis
strains cannot be classified into any of these three biovars
(3,
130,
155,
156,
174). Melibiose
fermentation was found by Mollaret and Mollaret
(127) to distinguish
biovar Orientalis and biovar Antiqua strains, neither of which ferments
this sugar, from most biovar Medievalis strains, which do ferment it.
However, it should be noted that biovar characteristics are unstable
and that one strain can undergo spontaneous phenotypic variation which
would cause it to be classified into another biovar
(96,
106). Additionally,
strains identical in essentially all of their studied characteristics
but differing in their biovars may circulate within one rodent
population (106;
S. V. Balakhonov, personal
communication).
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TABLE 2. Subgroups
of Y. pestis identified on the basis of glycerol fermentation,
nitrate reduction, and ammonia oxidationa
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Subsequently, new natural plague foci containing
Y. pestis variants with some additional characteristics
differentiating them from previously described strains were discovered.
This led to changes in the intraspecific classification of Y.
pestis. The differential biochemical characteristics used in these
groupings were nitrate reduction and ammonia oxidation; fermentation of
sugars such as rhamnose, arabinose, melibiose, melezitose, maltose,
mannose, and trehalose; pesticin-fibrinolytic-coagulase activities;
nutritional requirements; susceptibility to pesticin 1; and virulence
for mice and guinea pigs
(11). All of these
classification schemes were published in Russian based on the recovery
of plague isolates from around the FSU. While newer, more specific
classifications are now available, the diversity in phenotypic
characteristics of Y. pestis isolates from the FSU highlights
the importance of expanded studies to include such strains in
evaluations of factors of plague pathogenesis and host
immunity.
A major question concerning these diverse Y.
pestis isolates is that of their pathogenic potential for
different hosts, including humans. Levi (Abstr. Sci. Conf. Nat.
Focality Prophyl. Plague Tularemia, 1962) supplemented Y.
pestis intraspecific differentiation with several additional tests
that allowed him to designate two additional Y. pestis
subgroups, gerbil and vole (Table
3), using such specific characteristics as host-parasite interactions and
selective virulence in laboratory animals, including development of
bacteremia. However, the induction of bacteremia was not very
reproducible, nor could it be routinely applied for diagnosis, and so
the use of this test in the taxonomic classification of Y.
pestis was discontinued
(11).
Numerical Taxonomy and Standardization of Yersinia pestis Classification in the Former Soviet Union
Using numerical taxonomy,
I. L. Martinevskii
(120) classified Y.
pestis into three varieties: mediaasiatica montana
(corresponding to biovar Antiqua), mediaasiatica deserta
(corresponding to biovar Medievalis), and oceanica
(corresponding to biovar Orientalis). He also concluded that strains
isolated from common voles in natural foci of infection in the
Transcaucasian highlands (Fig.
2, foci 4, 5, and 6) or
from Mongolian pikas in the Mountain Altai and Transbaikalian regions
(foci 36 and 38) were a different species, Yersinia pestoides,
and included three varieties: Yersinia pestoides
parvocaucasica, Yersinia pestoides altaica, and
Yersinia pestoides transbaicalica, respectively. Surprisingly,
after three decades, "Pestoides" reappeared in
publications emanating from the United States as a strain designation
and as part of the nomenclature used to classify strains imported from
the FSU (3,
103,
130,
155,
156; P. L.
Worsham and C. Roy, Abstr. 8th Int. Symp. Yersinia, abstr.
P-41, 2002), although this classification was practically forgotten in
the FSU. While not in use in the FSU, this classification scheme
nonetheless further indicates the amount of genetic and phenotypic
diversity in plague isolates related to the sylvatic areas in which
they are circulating as epizootic pathogens.
Timofeeva
(177) proposed a new
classification of Y. pestis into subgroups, which was based on
numerical taxonomy, and used subspecies as a taxon designator (Table
4). This classification was formulated subject to the International Code of
Bacterial Taxonomy and used the nomenclature indicating the main
species and subspecies, (i.e., Yersinia pestis subsp.
pestis). She further divided the main Yersinia pestis
subspecies into two more groups, continental and oceanic. Also, with
the help of numerical taxonomy, Peisakhis and Stepanov
(140) proposed a
classification of Y. pestis strains which were isolated in the
FSU into groups based on 25 phenotypic features, some of which are
shown in Table
5. Since these numerical taxonomic classifications were constructed to
include the Y. pestis isolates that were found in the
territory of the FSU and also not generally available outside of the
FSU, additional assessments of non-FSU strains were not
included.
To bring some standardization to the system of
classification of Y. pestis isolates, the conference of
experts of the Anti-Plague Establishments of the Soviet Union (Saratov,
1985) recommended classifying all of the variants of the plague
pathogen that were isolated from the territory of the FSU and Mongolia
(Fig. 2 and
3A) into the "subspecies" Y. pestis subsp.
pestis (sometimes referred to as the "main"
subspecies), Y. pestis subsp. altaica, Y.
pestis subsp. caucasica, Y. pestis subsp.
hissarica, and Y. pestis subsp. ulegeica on
the basis of the numerical analysis of 60 phenotypic features. In 1998,
Sludskii (169) proposed
one more intraspecific group, Y. pestis subsp.
talassica. The last five subspecies are sometimes referred to
as the "nonmain" subspecies (Table
6) and have also been referred to as the "pestoides" group
of Y. pestis isolates. The numerical analysis was based on a
similarity index (SI), calculated from the formula
where
a is the number of coincident signs and b is the
number of unmatched classification features. In comparing the
properties of strains belonging to the main subspecies,
pestis, with strains of other subspecies, the SI was found to
be within the range of 0.82 to 0.95. The SI is also sometimes expressed
as percent similarity (i.e., SI x 100%). The most
significant differences were found between the pestis and
caucasica subspecies (SI = 0.82), whereas subspecies
altaica and hissarica were found to be closely
related (SI = 0.95)
(11,
12). It was also found
that in general among the five non-pestis subspecies (i.e.,
the "pestoides" subgroup), subspecies
caucasica was classified as biovar Antiqua and subspecies
altaica, hissarica, ulegeica, and
talassica were biovar
Medievalis.
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FIG. 3. Distribution
of Y. pestis subspecies (A) and plasmidovars
(B) in Mongolia. Symbols for provinces (aymags): 1,
Bayanölgie; 2, Uvs; 3, Hovd; 4, Dzavham; 5, Govï-Altay; 6,
Hovsgol; 7, Arhangay; 8, Bayanhongor; 9, Bulgan; 10,
Övörhangay; 11, Ömnögovï; 12, Selenge;
13, Töv; 14, Dundgovï; 15, Hentiy; 16, Dornogovï; 17,
Dornod; 18, Sühbaatar. Panel A reprinted with permission from
S. V. Balakhonov
(15), Antiplague Research
Institute of Siberia and Far East, Irkutsk, Russia. Panel B reprinted
with permission from A. Erdenebat
(62), Centre for Control
and Research of Natural Infectious Diseases, Ulaanbaatar,
Mongolia.
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TABLE 6. Taxonomic
characters of strains which distinguish different Y. pestis
subspecies isolated in the territory of the FSU and Mongolia, and
compliance of subspecies with biovarsa
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Current Use and Utility of Classification of Yersinia pestis Strains in the Former Soviet Union
The classification of Y. pestis strains into the
different subspecies, including the main subspecies pestis and
the nonmain subspecies altaica, caucasica,
hissarica, ulegeica, and talassica, is
currently widely used in the work of the Anti-Plague Establishments of
the FSU. Chinese plague experts use their own classification, utilizing
the term "ecotype" for different Y. pestis
subspecies groups, which differ in some phenotypic properties from the
Russian nonmain subspecies
(175). However, these
classification systems are not currently included in the International
Bacterial Nomenclature. Derived from the best studies produced in the
Soviet Union over 50 years, the Russian system currently stands up to
repeated use in classifying strains isolated in the FSU and Mongolia.
New proposals for its improvement appear periodically
(67,
78,
109), but none have been
felt to be good enough to take the place of the original one. Thus, the
phenotypic characteristics listed in Table
6 should be a useful basis
for identification and classification of Y. pestis strains
until other systems are found to be more useful, practical, or amenable
to classification of this genetically diverse group of otherwise
closely related bacterial
strains.
Limitations of the Classification in Identifying Potentially Virulent Strains of Yersinia pestis
Atypical strains.
Diversity in genotype and
phenotype is found even among plague isolates from the same natural
focus. Thus, no system of classification is likely to be perfect,
having nearly 100% specificity and sensitivity for classifying
plague isolates. Some idea of what is already known about atypical
strains and how this might impact the classification and virulence of
Y. pestis isolates is warranted, particularly with the
potential that atypical or engineered strains with a variety of unusual
characteristics might reasonably be expected to be a cause of human
infection, either accidentally or deliberately.
Most of what is
known about the appearance of atypical strains comes from field studies
in endemic foci of infection. In the field, the appearance of atypical
Y. pestis strains can correlate with different phases of the
epizootic cycle (105,
173,
179). So-called atypical
strains differ in some of the principal features found in the
predominant Y. pestis variant isolated from a given plague
focus. Thus, the ecological and other changes that occur during the
epizootic cycle can allow nondominant Y. pestis strains to
emerge in more consequential numbers in newly infected rodents,
potentially providing the opportunity for the generation of new
genotypes with altered virulence properties and for the exchange of
potential virulence genes among different clones of Y.
pestis.
An analysis of the frequency of the appearance of
variant forms of Y. pestis isolated from diverse natural
plague foci found a low level of variation in the Volga-Ural steppe
focus (1.58%) and the Trans-Ural focus (3.3%) (Fig.
2, foci 15 and 17,
respectively). In the Gissar (focus 34) and Central Asian Desert (foci
18 to 30 and 42) foci, this index was 6.59 and 6.55%,
respectively. In the Volga-Ural sandy focus (focus 16), variant forms
were not found. In the Trans-Ural focus (focus 17), variant strains
were isolated during all of the phases of the epizootic process with an
identical frequency. In the Volga-Ural steppe and Gissar foci (foci 15
and 34, respectively), such strains were recovered only during the
height of an acute epizootic spread of Y. pestis. The
frequency of strain variation in the early and later stages of an acute
epizootic was uniform in the Central Asian Desert foci (foci 18 to 30
and 42). During the phase when the epizootic started to wane, the
number of variant isolates increased
(173).
In the
Ural-Emba focus (focus 18), the greatest number of atypical strains
(6.5%) was isolated at the height of the acute epizootic spread.
It was found that 24.4% of atypical strains had modified
biochemical activities, 17.6% were lysogenized with
bacteriophages, 13.5% had modified requirements for growth
factors, 14.27% had mutations in the hms (for
"hemin storage system") locus, 16.75% displayed
reduced virulence, 9.93% were F1–,
7.14% were Lcr–, 2.36% were pesticin
deficient, and 0.4% were resistant to the lytic action of
diagnostic phages. A lower percentage of atypical strains was detected
during the persistent phase of the epizootic process
(195). According to
other data (183),
different foci were characterized by the presence of high proportions
(up to 48%) of strains with reduced virulence in laboratory
animals, with 10% of these isolates being essentially avirulent.
A total of 0.2 to 1.2% and 0.5 to 2% of the variants from
different foci had no autonomous pFra or pPst plasmid; 0.2 to
8.4% of the isolates from different foci did not display
Ca2+-deficient growth cessation; and 0.04 to
29.2% were Hms–. These results reinforce the
idea that there is a lot of genetic diversity among Y. pestis
strains circulating in natural plague foci, with the consequent
opportunities for genetic exchange and rapid emergence of new
phenotypes.
One problem for making determinations about what
represents an atypical strain of Y. pestis is that bacterial
systematics has not yet reached a consensus for defining the
fundamental unit of biological diversity, the species, let alone the
subspecies. Cohan (43)
thought that for bacteria, the fundamental unit of biological diversity
is not the species but the ecotype, representing the population of
organisms occupying the same ecological niche, whose genetic diversity
is affected primarily by natural selection and whose diversity can be
defined by sequence-based approaches. A typically named bacterial
species could contain many ecotypes. One major question of relevance is
that of the diversity in the properties of ecotypes that are associated
with virulence for humans and animals of economic importance and
whether these can be defined such that comprehensive tests for
diagnosing plague can be developed, treatments can be validated against
the range of strains pathogenic for humans, and active and passive
immunotherapies can be deemed to be comprehensive enough to cover the
range of pathogenic plague
strains.
Phenotypic Variation and Virulence for Laboratory Animals
Unfortunately, many of the
phenotypic signs that are listed in Table
6 and used for
intraspecific differentiation of Y. pestis strains in the FSU
are not absolute for a given subspecies, and thus the variability in
the pathogenic potential of these strains can be large, even for
strains from the same subspecies. One likely indicator of potential
virulence for humans is a high level of virulence in animals. The
guinea pig has been the animal of choice for virulence studies in the
FSU. However, when tested for virulence in guinea pigs, most Y.
pestis subspecies pestis strains are lethal whereas
strains of subspecies altaica, caucasica,
hissarica, ulegeica, and talassica as a rule
exhibit dramatic reductions in virulence or even the complete absence
of virulence for these animals (Tables
3 to
6). However, a few
isolates belonging to these non-pestis subspecies and
circulating in the same geographical region as the poorly virulent
strains can kill guinea pigs, with 50% lethal dose
(LD50) between 10 and >109 CFU per animal
(1,
60,
61,
110,
169,
184). When 40 Y.
pestis subspecies caucasica strains isolated within
several years from the Transcaucasian highland (Fig.
2, foci 4 to 6) were
examined for virulence in guinea pigs, it was shown that Y.
pestis isolates recovered from one focus in this region, the
Leninakan focus (focus 4), were more virulent for guinea pigs than were
strains obtained from another focus in this region, the
Zanzegur-Karabakh focus (focus 6)
(61). Other investigators
also noticed the high variability in guinea pig virulence of individual
Y. pestis strains isolated in the Armenian highland focus
(foci 4 to 6) (1), the
Dagestan-highland focus (focus 39)
(60), the Gissar focus
(focus 34), the Talas focus (focus 40)
(169), and Mongolia
(Fig. 3A, provinces no. 1
and 11) (110). Thus,
even though there may be considerable homogeneity of different Y.
pestis isolates circulating within a natural focus of infection,
as determined by membership in a specific taxonomic group, differences
in properties, such as lethality for guinea pigs, that may impact their
potential virulence for humans is also found.
While many of these
strains are not readily classifiable into one of the subspecies and may
not be lethal for mammals, they still have the potential to cause
considerable morbidity, involving such conditions as pneumonia and
bacteremia, that would make them serious pathogens of humans.
Intraspecific heterogeneity in virulence, host specificity, and
biochemical and physiological traits is also found among isolates
outside of the FSU and related Asian areas such as Mongolia. For
example, 24 Y. pestis strains from different natural foci in
Africa showed significant variability in their phenotypic
characteristics and virulence for mice and guinea pigs (Table
7). Given that many studies of Y. pestis pathogenesis performed
outside of the FSU and Asian countries have been carried out with a
limited number of closely related strains, it cannot be concluded with
complete confidence that the virulence factors identified in these
strains are essential factors for all strains of Y. pestis or
that lethality is necessarily the only outcome in experimentally
infected animals that would be predictive of virulence for
humans.
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TABLE 7. Relevant
characteristics of Y. pestis strains isolated in northern,
northwestern, western, and equatorial
Africaa
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Attempts To Predict the Pathogenic Potential of Variant Yersinia pestis Clones
Overall, in spite of trying to standardize classification of Y.
pestis by using the large variety of isolates available in the
FSU, it is still not feasible to really know which particular set of
genetic and phenotypic traits are indicative of virulence for
laboratory animals or humans. Kuklev had attempted to use epidemiologic
data to determine whether certain strains from specific foci are more
likely to cause human illness and/or be transmitted to humans
(108). As one might
expect, the question of epidemic potential for humans of strains within
a given natural focus is more complex than just the issue of the
virulence of an individual strain for humans. It obviously also
involves such factors as intensity of human contact, population
densities of host rodents and fleas, and other factors that are
sometimes difficult to quantify. Kuklev
(108) proposed the
following formula to calculate epidemic potential (EP) of strains of
Y. pestis from natural foci:
where
A represents the diffusion and intensity of epizootic
manifestations of plague and B represents the intensity of
human contact with the environment of the natural focus. The first
term, A can be defined as
where
S is the physical area of epizootic manifestation, Y
is the intensity of the epizootic in terms of infected animals,
K is the proportion of the focus area populated by the main
rodent host of the Y. pestis strain, P is the number
of rodents within 1 Ha, M is the number of fleas within 1 Ha,
and V represents the virulence of Y. pestis strains.
Obviously, many of these values must be determined by field and
laboratory studies.
The index V is of especial interest
since it is an important modifier of the overall equation. It takes
into account two findings, laboratory studies measuring virulence for
guinea pigs and the ability to ferment rhamnose. The V index
runs across a scale of 0.1 to 1.0 and can thus cause up to 10-fold
decrease of the total EP index (Table
8).
The second term, B, can be defined as
where
B1 represents the potential human contact with the
fleas of the wild rodents in the field, B2
quantifies the presence of rodents and fleas in human habitations,
B3 quantifies the presence of camels and their
number (which can transmit plague to humans
[40,
63]),
B4 quantifies the use of hunting for animals likely
to be carrying fleas infected with Y. pestis,
B5 quantifies the closeness of the place of
residence of rodent hosts to human habitations and contact of human
children with rodents, and B6 quantifies the
presence of cats and dogs in human habitations. Each of the indexes has
its own numerical range and technique for calculation, but in total the
EP cannot be more than 100. As with the qualitative estimation of the
epidemic potential, more than 50 is high, 25.1 to 50 is intermediate, 5
to 25 is low, and less than 5 is very low.
Kuklev
(108) used these
formulas to determine the epidemic potential of Y. pestis
spread from four different plague foci: Kara-Kum focus (Fig.
2, focus 25), EP =
70.1; Upper-Naryn focus (focus 32), EP = 54.0;
Central-Caucasian focus (focus 1), EP = 15.6; and
Transcaucasian-highland foci (foci 4 to 6), EP = 2.8. While
this type of investigation provides one framework for evaluating the
potential virulence of Y. pestis strains found in natural
foci, many of these factors would not be relevant to the virulence
potential of strains that can be encountered outside of these rural
environments. At the moment, there is no one definitive criterion for
stating that a given strain of Y. pestis has high or low
pathogenic potential for humans, but guinea pig virulence and rhamnose
fermentation are the two traits currently known to be the best
predictors of likely virulence for
humans.
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PHENOTYPIC AND GENOTYPIC DIVERSITY
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Obviously, with large areas of European Russia and
Central Asia sites of endemic foci of zoonotic plague, there is a
tremendous opportunity for vast genetic and consequent phenotypic
diversity. Some have referred to this as the "metastability of
phenotype" (27).
Mechanisms giving rise to the metastability of phenotypes include the
overall plasmid content, diverse reversible intergenomic realignments
that include displacement of IS elements, rearrangements of
variable-number tandem repeats (VNTRs) or changes generated during
their mismatch repair, integration of plasmids and bacteriophages into
the bacterial chromosome, and frameshift mutations in regulatory genes.
These intergenomic realignments can be detected by different
approaches, and it will be essential in the future to try to determine
the effect of genetic variability on the virulence of Y.
pestis for humans.
Diversity and Virulence
When virulence is measured in the context of animal
infections, the intensity and manifestations of pathogenicity of an
individual microbial strain are dependent on many conditions: animal
species used for evaluation, immune status of the host, conditions of
animal care and feeding, route of infection, and potentially even
subtle variations such as the time of day or season when the infectious
dose is given
(8-10,
100,
141,
144). Since bacteremia
in an infected rodent is a necessary condition for Y. pestis
transmission to a new host via fleas, strains of Y. pestis
maintained in endemic foci must be sufficiently virulent to establish a
bacteremic state conducive to transmission
(7-9,
30,
31,
34,
89,
104,
141). Long-term studies
of isolates from different natural foci indicate that the majority of
strains possessed sufficient virulence to cause bacteremia in the main
rodent host, although isolates from different foci differ from each
other in terms of their virulence for different animal species
(1,
11,
12,
15,
22,
33,
55,
56,
101,
104,
106,
107,
118,
123,
151,
166,
167,
169,
177,
179,
184,
198).
Kozlov
(106) and Kokushkin
(104) discriminated two
main variants of glycerol-positive Y. pestis strains obtained
from natural plague foci within the FSU which differed in their
epidemic potential. (i) The first are "rhamnose
fermentation-negative" strains, which were highly virulent for
guinea pigs and were isolated from foci with different potentials for
epidemic spread into humans (i.e., associated or not with reported
cases of human to human plague transmission). These were Y.
pestis subsp. pestis. (ii) The second are
"rhamnose fermentation-positive" strains, which were of
low virulence or avirulent for guinea pigs but caused occasional
disease in humans that was not accompanied by outbreaks of
human-to-human transmission of infection (Y. pestis subspp.
altaica, caucasica, hissarica,
ulegeica, and talassica).
Kudinova
(107), studying 107
Y. pestis subsp. pestis strains isolated from great
gerbils and their fleas in the Ili-Karatal interriver region of the
Pre-Balkhash focus (Fig.
2, focus 30), studied the
differences in the virulence of strains isolated from two foci
separated by 40 km. The strains isolated near the Karoi settlement were
virulent not only for great gerbils but also for other laboratory
animals (mice and guinea pigs), while the strains isolated from the
Bosugen tract (focus 30) could cause death in great gerbils but were
avirulent for mice and guinea pigs (LD50 >
108CFU). While the genetic or physiologic basis for this
difference was not reported, one might consider differential
susceptibility to host defenses, particularly susceptibility to
complement-mediated killing, as a potential explanation for the
differences in virulence in different rodent species.
Studying
121 Y. pestis subsp. pestis strains isolated from
1971 to 1988 in the Central-Caucasian natural plague focus (Fig.
2, focus 1), Serdyukova
(167) found that all of
the tested strains were highly virulent (LD50 = 1 to
104 CFU) for their main host, the mountain suslik.
Interestingly, Y. pestis strains isolated from the right bank
of the river Baksan in this focus (focus 1) that were also found not to
require proline for their growth displayed high virulence for both mice
and guinea pigs (LD50 = 1 to 104 CFU). On
the other hand, only 22% of strains auxotrophic for proline and
isolated from the left bank of the river Baksan (focus 1) were virulent
for these laboratory animals. Other strains auxotrophic for proline and
with high virulence for mountain susliks had low virulence
(LD50 = 104 to 108 CFU) or
even avirulence (LD50 > 108CFU) for mice
or guinea pigs, either singly or in some cases for both of these animal
species. Spontaneous proline prototrophs obtained from initially
proline-requiring strains maintained the virulence selectivity of their
parent strains, indicating that proline requirements were not
determinants of Y. pestis virulence for laboratory animals.
Nonetheless, these findings point out that endemic strains of Y.
pestis maintaining their transmission cycle by infecting
comparable animal species can still have marked differences in their
virulence for other mammalian hosts. Clearly, then, some of these other
hosts could potentially include humans.
Although these studies
indicate that there is host specificity for some Y. pestis
subsp. pestis strains
(33,
107,
167), many of these
strains also represent atypical strains even for this subspecies. As
with the Y. pestis strains of subspp. altaica,
caucasica, hissarica, ulegeica, and
talassica which are circulating within diverse populations of
voles and Ochotona pricei (a type of pika, which is classified
as a lagomorph and thus is related to rabbits and hares), there is more
of a tendency to find selective virulence for different animals.
Rhamnose fermentation-positive strains tend to be virulent for mice and
for some species of wild rodents, but as a rule they have low
virulence, or are even avirulent, for guinea pigs, other species of
wild rodents (1,
11,
12,
55,
104,
118,
169,
177,
184), and human
volunteers in one study (Zhenya et al., Abstr. 7th Int. Symp.
Yersinia, 1998).
This large amount of strain
heterogeneity in Y. pestis can obviously have a major impact
on virulence studies and provides a major challenge for investigators
trying to define essential factors involved in Y. pestis
virulence. As with any pathogen, choosing a set of strains for
virulence studies by using such techniques as directed inactivation of
genes relies on choosing properly representative parental strains for
the investigations. This standard method for studying microbial
pathogenesis can readily determine the contribution of gene products,
either singly or in combination, to a virulence phenotype, but the
outcome of the experiments is obviously dependent on the overall
genetic makeup of the parental strain being studied. Since microbial
pathogenesis is complex and multifactorial, with several virulence
factors usually acting in concert to produce infection
(71), the large array of
potential genotypic diversity that strains of Y. pestis can
draw upon due to the extensive occurrence of this pathogen in natural
foci may explain, in part, the conflicting experimental data on the
role in virulence of some Y. pestis pathogenicity factors
(Table
9). Use of different parental strains that can possess unidentified allelic
variations in genes that are not directly under study but whose
products are not necessary for survival within their natural host
undoubtedly underlies some of the variability in virulence study
outcomes. Elimination or inactivation of one or more genes from such
strains could lead to a significant decrease in virulence when tested
in laboratory animals, but the parental strain may or may not be
particularly representative of strains of Y. pestis with
pathogenic potential for humans. Thus, in another strain background,
the specific virulence factor may make a relatively minor contribution
to pathogenesis. Since many studies of plague pathogenesis have focused
on strains isolated from the Americas that possess much less genetic
diversity than strains in Russia and Asia, the conclusions from these
studies may not be entirely applicable across the board for all strains
of Y. pestis with high virulence for
humans.
Impact of Diversity on the Interrelationship of Host Immune Factors and Virulence
The
ability of pathogenic bacteria to survive in the face of host defense
systems is intimately linked to virulence
(8,
9,
71). The ability of
Y. pestis to maintain its transmission cycle in rodents, as
well as to infect incidental hosts such as humans, is highly dependent
on both rapid growth in the host and effective resistance to host
innate immune effectors including bactericidal cationic peptides as
well as opsonic and lytic complement proteins. These innate immune
factors can rapidly kill bacteria and prevent acute infections
(82,
83,
131). Thus, Y.
pestis must survive exposure to bactericidal complement conditions
within the blood and bactericidal cationic peptides conditions within
the phagocytes and must therefore have evolved or acquired complex
systems to counteract host defenses
(8,
9,
20,
74,
93,
141,
142,
149,
182; A. P.
Anisimov and S. V. Dentovskaya, unpublished data). In
contrast to other strains of Y. pestis, strains of subsp.
caucasica are highly susceptible to the bactericidal activity
of 80% human serum
(87; Anisimov and
Dentovskaya, unpublished), while all Y. pestis strains are
able to grow in heat-inactivated human serum or in 80% normal
mouse serum (Anisimov and Dentovskaya, unpublished). The lcrV
virulence gene leads to immunosuppression by inducing the
anti-inflammatory cytokine interleukin-10 via interactions with CD14
and toll-like receptor-2 (169a). It seems likely that similar factors
exist in many of the strains of Y. pestis found in natural
foci that have comparable effects on the inflammatory responses of
their native hosts. Since host innate immune factors such as CD14 and
toll-like receptors are fairly conserved across species, there is clear
potential for variant LcrV-like proteins to be present in non-American
strains of Y. pestis, and such proteins may not be amenable to
neutralization by antibodies raised to LcrV vaccines prepared from
strains found in the Americas.
Another factor with potential for
high variability in chemical and antigenic activity involved in
providing resistance to host defenses is LPS, with the oligosaccharides
mediating resistance to the bactericidal effect of complement and,
along with features of the lipid A, mediating resistance to the effects
of antimicrobial peptides. Investigation of the responses to polymyxin
B of Y. pestis strains isolated from various foci showed that
many are usually highly resistant to polymyxin B (MIC, 200 to 3000
µg/ml) (74,
122). However, Y.
pestis strains of subspp. hissarica
(122; S. V.
Balakhonov, personal communication) and caucasica
(74,
122; S. V. Balakhonov,
personal communication) and fresh isolates of subsp. altaica
are highly sensitive to polymyxin B (MIC, 10 to 25 µg/ml),
suggesting differences in the aminoarabinose content in the lipid A
(180) or in heptose
content of the LPS core in bacteria from subspecies pestis and
some non-pestis subspecies (Y. A. Knirel,
E. V. Vinogradov, S. N. Senchenkova, N.
A. Kocharova, B. Lindner, O. Holst, R. Z. Shaikhutdinova,
A. P. Anisimov, and T. A. Gremyakova, Abstr.
Carbohydr. Workshop, Güstrow-Rostock, Germany, 2003). The
recently published structure of the core oligosaccharide of one strain
of Y. pestis
(185) has already
indicated that variability in structure occurs in relation to growth
temperature. Given the high mobility of genetic loci containing genes
whose products are enzymes involved in the synthesis of LPS
polysaccharie components, there is clearly some potential for
variability in this structure in Y. pestis that could impact
virulence and host immune capabilities. Overall, it appears that
adaptations made by the plague pathogen that allow it to infect
specific mammalian species are based, in part, on quantitative and
qualitative changes in Y. pestis factors that counteract host
immune components, with some of these virulence factors likely to be
effective against innate immune resistance in
humans.
Plasmid Content
The
discovery of Y. pestis plasmids of 9.5 kb (termed pPst, pPla,
pPCP1, or pYP), 70 to 75 kb (termed pCD1, pCad, pVW, pYV, or pLcr), and
100 to 110 kb (termed pFra/Tox, pFra, pTox, pMT1, or pYT)
(21,
66,
148,
150,
154) made it possible to
compare plasmid profiles between strains isolated from different
natural plague foci and determine if there was an association of
plasmid content with virulence. Plasmids are known to be key factors
that determine the virulence of Y. pestis, and the potential
for these genetic elements to move among different strains could
readily underlie the acquisition and loss of virulence potential.
Indeed, characterizing the plasmid content and variations in plasmid
sizes has been instrumental in identifying genetic diversity among
Y. pestis strains within the FSU and China. There are quite a
number of these studies, which demonstrate different types of changes
in plasmid content and/or plasmid size, and their results emphasize one
of the major mechanisms of generation of genetic diversity in Y.
pestis and the potential of this diversity to impact virulence,
antigen expression, and susceptibility of strains to diagnostic
reagents. Importantly, an understanding of the natural variation in the
content and size of plasmids found in Y. pestis will be
critical for distinguishing which plasmids are present in given strains
from different regions of the world, the contribution made by the
different plasmids to the pathogenic potential of strains carrying
them, and the question whether newly engineered plasmids with added or
rearranged virulence factors have been made and introduced into strains
of this organism. Also, knowing something about where different Y.
pestis strains carrying different types of plasmids are typically
found will be instrumental in epidemiologic identification of sources
of outbreaks of disease.
Variation in plasmid content.
One striking finding from
research into enzootic strains of Y. pestis in the FSU is that
in some populations the plasmid content is quite stable whereas in
other populations there is considerable variation in plasmid content
among strains isolated from rodents living in close proximity. For
example, plasmids in strains isolated from four autonomous foci on the
northern border of the Central Asian zone (Fig.
2, foci 36 to 38 and 41)
were characterized as stable and independent of the source and time of
strain isolation. In contrast, by using agarose gel electrophoresis to
determine the plasmid content in strains of Y. pestis subsp.
caucasica isolated from common voles and their fleas in
Leninakan, Pre-Sevan, Zanzegur-Karabakh, and Dagestan-highland natural
plague foci (foci 4, 5, 6, and 39, respectively), it was found that
this group was missing the pPst plasmid that carries the genes for
pesticin-fibrinolytic-coagulase activities and showed susceptibility to
pesticin 1. However, in the nearest plague foci involving primarily
Y. pestis subsp. pestis the organisms isolated from
susliks (focus 3), gerbils (foci 7, 8, and 11 to 13), or their flea
vectors contained, as a rule, the three classical plague plasmids
(12,
15,
67-69,
73,
99,
168,
186; L. Bakanidze, D.
Tsereteli, M. Kekelidze, I. Velijanashvili, L. Beridze, E. Zangaladze,
M. Zakalashvili, and P. Imnadze, Abstr. 8th Int. Symp.
Yersinia, abstr. P-63, 2002; Worsham and Roy, Abstr. 8th Int.
Symp. Yersinia, 2002; P. L. Worsham and M. Hunter,
Abstr. 7th Int. Symp. Yersinia, abstr. P-88, Med. Microbiol.
[Ned. N. Voor] 6[Suppl.
II]:S34-S35, 1998). Similarly, strains of Y.
pestis isolated from mountain susliks and their fleas in the
Central-Caucasian natural focus on the left bank of the river Baksan
(focus 1) showed that the strains that were auxotrophic for proline
carried an additional 3- to 4-MDa plasmid
(67,
79,
114,
167,
197) while the majority
of strains from the right bank (focus 1) had only three classic plague
plasmids and did not require proline for growth. Spontaneous proline
prototroph derivatives of initially proline-requiring strains retained
the 3- to 4-MDa plasmid
(79,
167). Hence, the plasmid
was associated with a phenotype but was not responsible for it and was
not readily transmissible to nearby Y. pestis strains. The
role of the 3- to 4-MDa plasmid in virulence or its potential to be
modified is not known.
In Mongolia it was found that the plasmid
content of 894 Y. pestis strains isolated from patients, wild
mammals, and arthropods were divisible into three distinct populations
of strains based on different plasmid contents (Fig.
3B and
4). These were divided into plasmidovars. The first plasmidovar harbors
three plasmids with molecular masses of 6 MDa (pPst), 45 to 47 MDa
(pCad), and 62 to 65 MDa (pFra). The second plasmidovar contained
plasmids with molecular masses of 6 MDa (pPst), 16 MDa (cryptic), 45 to
47 MDa (pCad), and 62 to 65 MDa (pFra). The third plasmidovar harbored
plasmids of 8 MDa (pPst), 45 to 47 MDa (pCad)
(18,
62), and 62 MDa (pFra)
(according to reference
62) or 75 to 80 MDa
(pFra) (according to reference
18).
View larger version (39K):
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FIG. 4. Distribution
of Y. pestis plasmidovars in some Central Asian natural foci.
Reprinted with permission from S. V. Balakhonov
(17), Antiplague Research
Institute of Siberia and Far East, Irkutsk,
Russia.
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Plasmidovar
classification was associated, to a high but not exclusive degree, with
strain source and phenotype, documenting the potential utility of this
method for epidemiologic investigations and for contributing to the
determination of the pathogenic potential of a given isolate. All of
the strains bearing the combination of plasmids designated as the third
plasmidovar were isolated from Microtus brandti (a type of
vole); 80.9% of these isolates were classified as Y.
pestis subsp. altaica. Strains carrying plasmids
representative of either the first or second plasmidovar groups were
isolated from different rodents (58.6% of the strains, including
2.7% of the strains isolated from M. brandti), fleas
(33.1% of the strains), and humans (8.1% of the strains).
In this series, 95.2% of the strains with the plasmidovar 2
plasmid profile were Y. pestis subsp. pestis while
4.8% were classified as Y. pestis subsp.
ulegeica. Although Y. pestis subsp.
pestis can express the plasmidovar 2 profile, expression of
the plasmidovar 1 profile appears to be largely, but not exclusively,
confined to Y. pestis subsp. pestis (91.6 to
99.3% of strains, depending on the region of isolation). The
remainder of the strains expressing the plasmidovar 1 profile were
classified as Y. pestis subsp. ulegeica (0.7 to
8.4% of strains) and Y. pestis subsp. altaica
(0 to 5.6% of strains).
Plasmid content is sometimes
associated with other phenotypic properties of the strains, suggesting
some clonality and also providing another tool for diagnostic and
epidemiologic investigations. Strains with different plasmid contents
can vary in their carbohydrate fermentation activity. The strains of
plasmidovar 2 did not metabolize rhamnose and melibiose. The strains of
plasmidovar 3 did not ferment arabinose. Amino acid requirements for
growth also varied among the strains. Isolates of plasmidovars 1 and 2
required methionine for their growth, while those of plasmidovar 3
required arginine and leucine.
It was also mentioned that during
long-term laboratory storage some strains maintained their initial
plasmid profiles but nonetheless had changes in other phenotypic
characteristics, which caused their reclassification into other
subspecies (62). Hence,
even storage can lead to genetic and phenotypic changes in Y.
pestis.
Many studies based on plague pathogenesis have
focused on the role of the three well-characterized Y. pestis
plasmids associated with virulence, with molecular massesof about 6 MDa (pPst), 45 to 50 MDa (pCad), and 60 MDa (pFra). Assuming
that these are representative of at least one class of virulent plague
strains, it would be important to know something about the variation in
plasmid content in strains in the FSU, Mongolia, and China
harboring these plasmids and how such variation would impact virulence
and diagnosis. One study of the plasmid composition of 257 Y.
pestis strains from 31 natural foci in the FSU and other countries
revealed that 68% of the strains carried the three classic
plasmids. In 10% of the strains obtained from different sources,
additional cryptic plasmids were detected. A total of 18% of the
isolates from the Volga-Ural sandy focus (Fig.
2, focus 16) and
43% of the isolates from the Talas focus (focus 40) also
contained cryptic plasmids, usually of 20 MDa. In some strains from
Talas, Central-Caucasian, and Pre-Balkhash foci (foci 40, 1, and 30,
respectively), as well as from Senegal, Sri Lanka, and Indonesia,
cryptic plasmids with molecular masses from 1.6 to 31 MDa were found.
In some cases, the absence of one or two typical plasmids was
observed.
In some geographic areas in the FSU, Mongolia, and
China, plasmids not found in Y. pestis isolates from different
parts of the world were identified. For example, 1,020 strains of
Y. pestis isolated from 44 counties of Yunnan province in
China and the border of China-Myanmar that were analyzed by agarose gel
electrophoresis were found to carry plasmids of nine different sizes,
having molecular masses of about 3.93 MDa (pYC) 6.05 MDa (pPst), 22.97
MDa (cryptic), 35.65 MDa (cryptic), 45.35 MDa (pCad), 64.82 MDa (pFra),
74.59 MDa, 111.36 MDa, and 129.55 MDa
(58). Y. pestis
strains carrying the specific combination of plasmids of 3.93, 35.65,
and 111.36 MDa were isolated only from Yunnan province. The plasmid
content of these 1,020 strains allowed for their division into 10
plasmidovars. These findings emphasize that in the strains isolated
from Russia and Asia there is marked variability in plasmid content,
which can potentially affect virulence and the effectiveness of host
defense mechanisms.
Other variant plasmids have been identified
in strains of Y. pestis. Strains isolated in Tuva (Fig.
2, foci 37 and 41)
contained an additional 21.5-MDa (cryptic) plasmid
(16,
67). Testing of this
plasmid using PCR with a set of 10 pairs of primers based on different
genes usually found on Y. pestis classical plasmids and in the
chromosome indicated that this plasmid contained nucleotide sequences
derived from the structural genes for plasminogen activator
(pla) and the bacteriocin pesticin (pst) (from pPst)
as well as from the structural gene for capsular antigen fraction I
(caf1) derived from pFra (S. V. Balakhonov, Abstr.
8th Int. Symp. Yersinia, abstr. P-64, 2002). Overall, Y.
pestis clearly has the capacity to harbor a large amount of
genetic information in its plasmids, making these structures potential
targets of genetic engineering to change virulence properties and
diagnostic antigens useful for intervention in an outbreak of
plague.
Variation in plasmid size.
Because the classical Y.
pestis plasmids can vary in size, this property can be useful for
determining the relatedness of strains isolated from different
geographic areas. Also, plasmid size has the potential to be easily
changed, which could be confusing with regard to whether a plasmid
identified in a clinical isolate is a natural variant or potentially an
engineered variant with possible additional virulence factors added in.
Therefore, understanding the variation in plasmid size found among
different Y. pestis isolates is important for differentiating
natural variation from engineered variation leading to potentially
greater virulence in strains harboring such theoretical constructs.
Hence, the pPst plasmid isolated from strains circulating in several
natural foci have been found to have additional sequences enlarging it
from 6 up to 8 MDa (18,
62) or can even harbor a
dimer of the plasmid
(42). pCad and especially
pFra may also vary in size depending on the geographical origin of the
isolate (18,
38,
58,
62,
67-69,
112,
141; Worsham and Roy,
Abstr. 8th Int. Symp. Yersinia, 2002; Worsham and Hunter,
Abstr. 7th Int. Symp. Yersinia, 1998). The larger variants of
plasmid pFra (69 to 190 MDa) were characteristic of the
non-pestis subspecies of Y. pestis
(15,
67-69,
109; V.
Kutyrev, O. Protsenko, G. Smirnov, E. Bulgakova, L.
Kukleva, I. Zudina, N. Vidyaeva, and I. Kuzmichenko, Abstr. 8th Int.
Symp. Yersinia, abstr. P-9, 2002; Worsham and Roy, Abstr. 8th
Int. Symp. Yersinia, 2002; Worsham and Hunter, Abstr. 7th Int.
Symp. Yersinia, 1988). Specific plasmid rearrangements in
different Y. pestis strains which distinguish Y.
pestis biovar Orientalis strains from other biovars have also been
demonstrated (147,
152). A more thorough
determination of plasmid sizes among a large collection of Y.
pestis strains allowed Filippov
(67) to distinguish up to
20 plasmidovars, many of which were characteristic of strains
circulating in certain natural foci (Table
10). Such natural variation could make it difficult to distinguish highly
pathogenic Y. pestis from low-pathogenicity enzootic isolates
without a thorough understanding of the implications of size
variation.
Plasmid variation in American strains of Y. pestis.
In
contrast, in the Americas there is much less variability observed in
the plasmid content of Y. pestis isolates, indicating the much
more restricted nature of plague diversity in this part of the world.
In Brazil, a profile of four plasmids with molecular masses of 6.4 MDa
(pPst), 14.9 MDa (cryptic), 44 MDa (pCad), and 60 MDa (pFra) was found
in all of the 26 strains which were isolated from Paraiba State. DNA
cleavage with EcoRI further demonstrated the uniform
plasmid content of these Y. pestis isolates
(112). When 250
Brazilian Y. pestis strains stored in bacterial collections
for different periods were analyzed for plasmid content, it was
confirmed that the majority of the strains contained a homogeneous
pattern composed of the three classic Y. pestis plasmids:
pPst, pCad, and pFra. More recent analysis of the plasmid patterns of
53 Y. pestis isolates from three plague foci from the state of
Ceara, Brazil, showed that 39 strains had the three classical plasmids
while seven isolates had an additional plasmid that was longer than 90
kb. The other seven lacked all or some of the plasmids
(38). Fourteen of these
strains with different plasmid content were analyzed by Southern
blotting with probes for the genes pla (from pPst),
lcrV (from pCad), and caf1 (from pFra). The
pla probe reacted with pPst and with bands of about 35 kb. The
lcrV probe reacted with pCad and also with bands that migrated
slower than the pFra plasmid itself, as well as with bands that
migrated between pFra and pCad. The caf1 probe hybridized with
the pFra plasmid and to bands that migrated slower than pFra
(113; N. C.
Leal, M. S. B. da Silva, T. C.
A. Leal, and A. M. P. de Almeida, Abstr. 7th Int.
Symp. Yersinia, abstr. O-14, Med. Microbiol. [Ned. N.
Voor] 6[Suppl. II]:S9, 1998). Ultimately,
the Brazilian researchers came to the conclusion that "the
Brazilian Yersinia pestis strains displaying atypical plasmid
profiles could not represent true wild type spontaneous
variants" (113;
Leal et al., Abstr. 7th Int. Symp. Yersinia, 1998), since 
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