Defective Innate Cell Response and Lymph Node
Infiltration Specify Yersinia pestis Infection
Franc ¸oise Guinet1*, Patrick Ave ´2, Louis Jones3, Michel Huerre2, Elisabeth Carniel1
1Unite ´ des Yersinia, Institut Pasteur, Paris, France, 2Unite ´ de Recherche et d’Expertise d’Histotechnologie et Pathologie, Institut Pasteur, Paris, France, 3Groupe Logiciels
et Banques de Donne ´es, Institut Pasteur, Paris, France
Since its recent emergence from the enteropathogen Yersinia pseudotuberculosis, Y. pestis, the plague agent, has acquired
an intradermal (id) route of entry and an extreme virulence. To identify pathophysiological events associated with the Y.
pestis high degree of pathogenicity, we compared disease progression and evolution in mice after id inoculation of the two
Yersinia species. Mortality studies showed that the id portal was not in itself sufficient to provide Y. pseudotuberculosis with
the high virulence power of its descendant. Surprisingly, Y. pseudotuberculosis multiplied even more efficiently than Y. pestis
in the dermis, and generated comparable histological lesions. Likewise, Y. pseudotuberculosis translocated to the draining
lymph node (DLN) and similar numbers of the two bacterial species were found at 24 h post infection (pi) in this organ.
However, on day 2 pi, bacterial loads were higher in Y. pestis-infected than in Y. pseudotuberculosis-infected DLNs. Clustering
and multiple correspondence analyses showed that the DLN pathologies induced by the two species were statistically
significantly different and identified the most discriminating elementary lesions. Y. pseudotuberculosis infection was
accompanied by abscess-type polymorphonuclear cell infiltrates containing the infection, while Y. pestis-infected DLNs
exhibited an altered tissue density and a vascular congestion, and were typified by an invasion of the tissue by free floating
bacteria. Therefore, Y. pestis exceptional virulence is not due to its recently acquired portal of entry into the host, but is
associated with a distinct ability to massively infiltrate the DLN, without inducing in this organ an organized
polymorphonuclear cell reaction. These results shed light on pathophysiological processes that draw the line between a
virulent and a hypervirulent pathogen.
Citation: Guinet F, Ave ´ P, Jones L, Huerre M, Carniel E (2008) Defective Innate Cell Response and Lymph Node Infiltration Specify Yersinia pestis Infection. PLoS
ONE 3(2): e1688. doi:10.1371/journal.pone.0001688
Editor: Kirsten Nielsen, University of Minnesota, United States of America
Received October 26, 2007; Accepted January 22, 2008; Published February 27, 2008
Copyright: ? 2008 Guinet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was from the Pasteur Institute, which had no role in study design, data collection and analysis, decision to publish or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
Bubonic plague is an acute bacterial disease which, if untreated,
leads to death in 50–90% of the cases  in generally less than
5 days . In spite of major improvements in the management
and control of the disease since the ravaging pandemics of the past,
the disease is far from eradicated and trends show that it is
expanding or re-emerging in many countries [3,4].
Plague is an anthropozoonosis affecting primarily rodents [2,5]
and the disease has a host-vector-host transmission cycle. The
vectors are fleas which transmit the disease by intradermal (id)
biting. Humans are generally contaminated through the bite of an
infected rodent flea. Experimental id or subcutaneous (sc) injection
of the plague agent into laboratory rodents, e.g. guinea pigs, mice
and rats, causes a disease similar to naturally acquired bubonic
plague [6–12]. From the inoculation site the infection proceeds to
the draining lymph node (DLN) via lymphatic channels. The
infected node increases in size and, within a few days, gives rise to
the so-called bubo, hallmark of the disease. Clinically, the bubo is
a voluminous and exceedingly painful lymphadenitis, whose
sudden appearance coincides with the onset of fever. Histologi-
cally, buboes are typically heavily infected and display over-
whelming inflammatory and necrotic alterations [6,8,13,14].
Septicemia and hematogenous spread to distal lymph nodes and
deep organs appear to follow an initial phase of containment of the
infection in the proximal lymph node [11,15]. The septicemia-
induced shock is believed to be the proximal cause of death.
The plague agent is an Enterobacteriaceae named Yersinia pestis. All
other Yersiniae are transmitted through the fecal-oral route and
they are either non-pathogenic or responsible for generally self-
subsiding digestive symptoms [16,17]. Among the enteropatho-
genic Yersinia species is Y. pseudotuberculosis, from which Y. pestis
originated less than 20 000 years ago . Hence, in a short time
frame, Y. pestis has developed both an extreme virulence and a
mode of transmission unique among the Enterobacteriaceae family.
The two species are genetically nearly identical . Based on
their close relatedness it has been proposed that the two species
should be reclassified as a single one, a proposition that was
subsequently rejected in consideration of their extreme divergence
in pathogenicity, life cycle and public health impact [20,21]. The
Y. pestis/Y. pseudotuberculosis pair thus provides a unique opportunity
to explore, by comparative analysis, the pathophysiological
processes associated with a high degree of pathogenicity.
Indeed, the specific mode of action whereby Y. pestis rapidly kills
its host is still elusive. Little is known of the sequence of events
mediating the exceptional severity of plague, including the time
and place where these events appear during the course of disease
and the impact of the newly acquired dermal portal in plague
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pathogenesis. To address these questions, we undertook a study of
the disease progression of bubonic plague in a mouse model. To
highlight pathophysiological events likely to be critical to plague-
specific pathogenesis and to identify histological lesions that
differentiate plague from other bacterial infections, the findings
were contrasted with the disease induced by id inoculation of Y.
pseudotuberculosis, the closely related and less virulent ancestor of Y.
1. Y. pseudotuberculosis is less virulent than Y. pestis upon
Comparison of the reported lethal doses 50 (LD50) of various
strains of Y. pestis and Y. pseudotuberculosis injected sc shows a 105–
106fold higher LD50 for Y. pseudotuberculosis [5,22–25]. To test
whether a similar difference would appear after id inoculation,
which most closely mimics the natural transmission mode of
bubonic plague, serial dilutions of Y. pestis CO92 and Y.
pseudotuberculosis IP32953 were injected id into the ear of outbred
mice and LD50s were calculated from the mortality rates after a
3 week follow-up. Y. pseudotuberculosis LD50 was found to be
,56105colony forming units (cfu), and Y. pestis LD50 was
,20 cfu. Thus, the mortality studies showed that in the id
injection model Y. pestis CO92 was more virulent than Y.
Further experiments were carried out to determine the
minimum Y. pestis infective dose that would cause death of
100% mice and the maximum Y. pseudotuberculosis dose to which
100% of the animals would survive. Infective loads between
,30 cfu to ,36107were tested on a total of 225 mice followed up
for a minimum of 7 days. The lowest Y. pestis dose to which all
mice succumbed within 4 days was between ,1,000 and
,2,000 cfu. All mice having received ,1,300 to ,17,000 Y.
pseudotuberculosis cfu were still alive on day 4 post-inoculation (pi)
and 2% of them died between day 4 and day 7 pi.
2. Bacterial loads at the injection site and in the draining
To determine whether the different degrees of severity of the Y.
pestis and Y. pseudotuberculosis infections were associated with distinct
bacterial dynamics within the host, bacterial loads at the injection
site (IS) and in the DLN of Y. pestis- and Y. pseudotuberculosis-infected
mice were quantified. Mice received intradermally (mean6SEM)
42006427 cfu of Y. pseudotuberculosis or 40006299 cfu of Y. pestis.
Enumeration of cfu recovered from the IS at time 0 was performed
in a subset of experiments to confirm, as previously published ,
that it matched the injected load. Because all of the mice used in
the experiments were naive, there were no bacteria in the DLN at
time 0. Cfu enumerations from the IS and the DLN were
performed 24 h (two groups of 16 mice infected with either Y.
pseudotuberculosis or Y. pestis, 48 h (13 Y. pseudotuberculosis- and 18 Y.
pestis–infected mice), and 72 h and 96 h (2 groups of 7 Y.
pseudotuberculosis-infected animals each) pi. On day 3 pi, the
dramatic decrease in the number of available Y. pestis-infected
mice, due to around ,80% mortality, precluded valid statistical
analysis of the cfu counts at this time point.
Y. pseudotuberculosis was able to survive and to proliferate in the
dermis during the first two days pi. Its growth rate over the first
24 h pi was even higher than that of Y. pestis (Figure 1).
Furthermore, Y. pseudotuberculosis was found in the proximal lymph
node, and similar numbers of both Yersinia species were present in
the DLN at 24 h (p=0.39). On the following day, however, Y.
pestis numbers in the DLN were significantly higher than those of
Y. pseudotuberculosis. Over extended observation periods of 7 days,
DLN Y. pseudotuberculosis loads reached a plateau from day 3 on
(Fig. 1 and data not shown). Therefore, both species were able to
multiply at the inoculation site and to reach the proximal lymph
node but while, in the DLN, Y. pseudotuberculosis growth was
contained, Y. pestis multiplication was not controlled and the
animals started to die between days 2 and 3.
3. Y. pestis and Y. pseudotuberculosis induce similar
lesions at the injection site
In a separate series of experiments, the gross and microscopical
pathology at the IS was examined to compare the lesions induced
locally by the two Yersinia species at 24 h, 48 h and 72 h (for the
surviving mice) pi.
At the gross pathology level, the infected ear dorsum appeared
normal or exhibited slight inflammatory changes such as a diffuse
moderate hyperemia or a red inflammatory dot at the needle entry
point irrespective of the injected species. On microscopical
examination, edema and polymorphonuclear leukocyte (PMN)
influx were observed at the IS of most infected mice. Figure 2
shows examples of PMN reactions at the IS accompanying Y. pestis
and Y. pseudotuberculosis infections. Semi-quantitative scoring of the
intensity of the inflammatory response, performed independently
by two investigators on a set of 29 infected dermis sections,
indicated a slide to slide variation in the intensity of inflammation
but no significant impact of the injected species. Thus, Y. pestis and
Y. pseudotuberculosis induced lesions of similar type and intensity at
the injection site.
4. Y. pestis and Y. pseudotuberculosis induce different
lesions in the draining lymph node
To know whether Y. pseudotuberculosis would induce in the
proximal lymph node lesions comparable to those observed in the
Figure 1. Bacterial loads at injection site and in draining lymph
node. Data are means of log10of cfu numbers recovered from 77 mice
infected over six independent experiments. Bars=Standard Errors. Stars
indicate that the data are significantly different between the two
Yersinia species (p#0.0001). The dashed line shows the detection limit
(10 cfu). The cross symbol indicates that most Y. pestis-infected mice
died between days 2 and 3.
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plague bubo, DLNs collected on day 1, 2 and 3 from mice infected
with comparable numbers (3,500–6,500 cfu) of either Y. pestis or Y.
pseudotuberculosis were examined. A total of 36 DLNs were
4.1. Gross pathology.
While DLNs infected with either
Yersinia species were macroscopically subnormal on day 1 pi,
DLNs collected on days 2 and 3 pi consistently displayed
distinctive features of Y. pestis or Y. pseudotuberculosis infections. Y.
pseudotuberculosis infected DLNs were frankly purulent, whereas Y.
pestis infected DLNs were hemorrhagic, sometimes partly purulent,
and their consistency was dense and firm (Fig. 3). Therefore, the
DLN is the first organ to display species-specific alterations at the
gross pathology level during the course of infection, and this
difference appears on day 2 pi.
The lesions present in the infected
DLNs were much more complex and varied than in the dermis, so
that an objective evaluation of the impact of the species and of the
infecting dose on the DLN alterations was not possible without a
detailed statistical analysis, using tools suitable for large categorical
data sets. Cluster analysis and multiple correspondence analysis
were employed, after decomposition of the histological patterns
into elementary lesions.
a) Cluster analysis identifies histology patterns associated with either Y. pestis or Y.
Criteria aimed at assessing the PMN inflammatory reaction, the
extent and organization of bacterial involvement, and changes in
DLN morphology and density were defined (Table 1 and Fig. 4),
and DLN sections were scored as «+» or «2» for each criterion so
that to each DLN was associated a ‘‘+/2’’ sequence representing
its histopathological profile and allowing clustering analysis of the
data. The scoring was done blinded and repeated at least twice by
the same observer for each lymph node.
The cluster analysis delineated three main types of histopath-
ological patterns (Fig. 5 and Fig. 6). Type 1 patterns corresponded
to both Y. pestis and Y. pseudotuberculosis infected DLNs collected
shortly after the bacterial injection. Therefore, this type, which was
characterized by no or limited alterations of the lymph nodes,
represented early, non species-specific lesions. The type 2 group
comprised only Y. pseudotuberculosis-infected DLNs, all from day 2
or 3 pi. Hence, type 2 corresponded to advanced Y. pseudotuber-
culosis lesions. Type 3 comprised only profiles associated with Y.
pestis infection, and all of them but one were from DLNs taken on
day 2 or 3 pi. Type 3 thus represented advanced Y. pestis lesions.
Type 3 DLNs appeared to be generally more severely altered than
type 2 DLNs and four of them were totally destroyed, the normal
DLN tissue being entirely replaced by bacteria or by PMNs.
Therefore, the cluster analysis strengthened our preliminary
observation that a differential evolution of the DLN lesions
appears on day 2.
b) Exploration of an extended infecting dose range
An extended bacterial dose range (500–14,000 cfu) was
explored, to know whether a higher infecting dose of Y.
pseudotuberculosis could generate destructive lesions similar to those
induced by Y. pestis, and if, after infection with lower Y. pestis loads,
histopathological profiles similar to type 2 would appear. As seen
in Fig. 7, this did not modify the relative grouping of infected
DLNs, as all DLNs from mice infected with Y. pestis segregated in
group 1 or 3, while all DLNs but one from mice infected with Y.
pseudotuberculosis clustered in group 1 or 2. Therefore, the bacterial
load does not appear to influence the type of lesions, which is
strictly associated with the species type.
c) Identification of the criteria most discriminating between types 2 and 3
Thus, the 42 criteria together efficiently discriminated between
advanced Y. pestis and Y. pseudotuberculosis DLN histopathology
Figure 2. Inflammatory reaction at the injection site. Examples of
edema and PMN influx 48 h following injection of ,4500 Y. pestis
(panel B) or Y. pseudotuberculosis (panel C) cfu, compared to a saline-
injected control (panel A). Immunostained PMNs are brown-orange on
the preparations. Bar=20 mm.
Figure 3. Draining lymph node gross pathology. Macroscopical aspect of lymph nodes two days after inoculation of either saline, ,5500 Y.
pseudotuberculosis cfu or ,5500 Y. pestis cfu. Lymph node from Y. pseudotuberculosis-infected mice is purulent whereas Y. pestis-infected lymph node
is reddish and adherent to neighboring tissues. Bar=2 mm.
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Table 1. Criteria used to score sections of lymph nodes infected with Y. pestis or Y. pseudotuberculosis, and their discriminating
General features explored
by criteria Description of criteriaCriterion Nu
Similar to saline-injected controls1
Extent and organisation
of lesions and of
PMNs,50% of surface area of the section2
PMNs.50% of surface area of the section3
Wedge shaped abscess4 4A
Abscess involve one pole of the DLN5
Abscess.25% of section surface6
Destructive non-abscess lesions.50% of surface7
+3.95 Lesion=100% of surface8
Peripheral band of PMNs containing bacterial foci9 4B
Layer of PMNs bordering a peripheral band of bacteria+PMNs+cell debris10 4C
Blunt border of inflammatory front 11 4B
Extent of bacterial
colonization of the LN
Bacteria in sub-capsular sinus, ,1/3 of circumference 12
Bacteria: small peripheral focus or foci 13
Patches of densely packed bacteria, bordered by PMNs14 4D
Atypical bact. patches (low density, undefined boundery, weak PMN border)15 4E
Bacteria in $ 1/3 of sub-capsular sinus 16
Bacteria involve large areas of the DLN 17 4F
Invasiveness in the LN tissueInward bacterial projections from the subcapsular sinus (like flames)18 4I
Isolated host cells within a bacterial zone194G
At high magnification: bacterial infiltration around host cells 20 4H
At high magnification: free floating bacteria21 4H
Breaching of the PMN barrier Inward bacterial infiltration beyond the PMN line 22
Bacterial clusters in direct contact with lymphocytes 23
Tissue density alterations Normal LN tissue density24
Area of reduced host cell density within bacterial areas25
Area of reduced host cell density outside bacterial areas26 4I
Area of reduced host cell density with a reticular pattern27 4J
Area of reduced host cell density without a reticular pattern 28
Area of reduced host cell density in a purulent zone 29
Area of reduced host cell density central to the inflammatory front30
Area of reduced host cell density peripheral to the inflammatory front31
Presence of numerous pycnotic cells 32 4J
«Moth eaten» appearance33 4K
Islet of apparently normal lymphocytes in an otherwise altered DLN 34
Part of the DLN is missing35
Final stages of LN destructionTotally destroyed LN massively infiltrated with bacteria 36
Totally destroyed LN massively infiltrated with PMNs 37
Enlarged blood vessels packed with RBCs40 4L
Surrounding, extranodal tissue, adhering to the LN41
More than 1 LN on the slide 42
Discriminating indexes are the test values in the right column. Only significant test values (i.e. .1.96 or ,21.96) are given. Positive and negative test-values are
distinctive of, respectively, Y. pestis and Y. pseudotuberculosis infections.
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characteristics. In order to assess the relative contribution of each
criterion to this discrimination, lymph node histology patterns
were further analyzed by Multiple Correspondence Analysis
(MCA), a multivariate statistical method designed to describe
objects characterized by categorical variables [27–29]. The
method reduces the complexity of the initial data set while
retaining most of the information on their relationships, and
provides graphical outputs so that the above relationships can be
visualized. This is achieved through the fitting of axes onto which
objects and variables are projected. Here, the «objects» are the
DLNs and the «variables» are the criteria. The first axis is
calculated as the best fit linear model of the dispersed data, and
each successive axis is orthogonal with the preceding axis. For
representation of the data, the minimum number of axes are
selected that maximizes this representation. All DLNs correspond-
ing to advanced lesions (types 2 and 3) were included in the
analysis, except those that were totally destroyed.
The first axis, the first two axes together and the first three axes
together represented 93.9%, 96.8% and 99.1% of the inertia,
respectively, indicating that almost the totality of the information
was contained within the first three axes and that the first axis
alone contributed most of it. Figure 8 shows a projection on the
Figure 4. Illustrations of some of the criteria used for scoring. Panels A–E, G, H and J–L display HE stained sections, panels F and I show
sections immunostained with Y. pestis specific antibodies. In the text below, the criteria are referred to according to the numbering in Table 1. A:
Wedge shaped abscess (criterion 4) indicated by arrows. Within the abscess bacterial colonies are visible as pink patches. B: Peripheral layer of PMNs
containing bacterial foci (criterion 9), with a blunt demarcation (arrows) from the lymph node tissue (criterion 11). Inset: higher magnification to show
the characteristic horseshoe shaped nuclei of the PMNs, and a bacterial focus. C: Layer of PMNs bordering a peripheral band of bacteria, cell debris
and PMNs (criterion 10). The inset shows the typical PMN morphology of the cells within the layer. Behind this layer, bacterial aggregates are seen as
pink areas (arrowheads) containing purple dots that are, as seen as higher magnification (not shown here) PMNs and cell debris. D: Patch of densely
packed bacteria, bordered by PMNs (criterion14). Packed bacteria form a pink 8-shaped area at the centre of the picture (star). E: Atypical bacterial
patch (criterion 15). At the center of the picture an aggregate of bacterial rods, not as densely packed as the preceding one, is loosely surrounded by
inflammatory cells. F: A large bacterial zone (criterion 17), stained brownish on the preparation. G: Isolated host cells within a bacterial zone (criterion
19). Isolated host cells and cell remnants are seen amid a sea of bacteria which gives a ‘‘ground glass’’ appearance to this part of the LN section. H,
left: bacterial infiltration around host cells (criterion 20). A bacterial strand (arrow), that seems to originate from a nearby colony (star), passes between
host cells. H, right: Free floating bacterium, indicated by an arrow (criterion 21). I: Zone of reduced tissular density (arrows) outside bacterial areas,
which are brownish on this preparation (criterion 26). This image also shows flame-like inward bacterial projections (criterion 18). J: Area of reduced
host cell density with a reticular pattern (criterion 27) and containing numerous pycnotic cells (criterion 32). K: Moth eaten appearance (criterion 33)
of a lymph node with areas of contrasting tissular densities. L: Vascular congestion (criterion 40), showing bright red on this preparation.
Magnifications: Panels A–C, F, I–L : bar=200 mm. Insets of panels B and C: bar=50 mm. Panels D, E, G and H: bar=10 mm.
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first two axes of the DLNs with advanced-Y. pestis and advanced-Y.
pseudotuberculosis lesions. It can be seen on the figure that the Y. pestis
and the Y. pseudotuberculosis groups are clearly separated from each
other, thus confirming by another method that the two Yersinia
species induce different alterations of the DLN. Figure 8 also
displays the 42 criteria. Criteria located on the left- and right-hand
side of the graph are those that characterize Y. pseudotuberculosis-
and Y. pestis-induced lesions, respectively.
To quantify the contribution of each criterion in discriminating
between the advanced Y. pestis and Y. pseudotuberculosis lesions, the
‘‘test-value’’ function of the MCA software was used (see Materials
and Methods). The test attributes to each criterion and for each
projection axis a numerical value which indicates the probability
that the criterion is discriminating between the two groups along
this axis. A test-value .1.96 or ,21.96 indicates a .95%
probability that the criterion is significantly associated with one of
the two groups . Significant test-values for the first axis are
shown in Table 1. Positive and negative test-values corresponded
to criteria associated with the Y. pestis and Y. pseudotuberculosis
The criteria most discriminative for Y. pseudotuberculosis infection
were the patches of packed bacteria in close contact with
surrounding PMNs (Table 1, criterion 14) and the peripheral
band of PMNs clearly delineated from the DLN parenchyma
(criteria 9 and 11). An example of a type 2 DLN exhibiting these
features is shown in Fig. 6. Also highly discriminative were images
of large size wedge-shaped or polar abscesses (criteria 4, 5 and 6)
and a normal tissue density of the organ (criterion 24).
Twenty-three out of 42 criteria were significantly associated
with Y. pestis infection, reflecting the profound modification
induced in the DLN by this bacterium. The highest test-value
was that of criterion 19, i.e. images of isolated host cells within
bacterial areas. This characteristic might be the result of the ability
of bacteria to infiltrate between neighboring host cells, a property
corresponding to the second most discriminative criterion for Y.
pestis infection (criterion 20). A moth eaten appearance, resulting
from areas of reduced cell density, was the next discriminating
criterion (Nu33), followed by vascular congestion (criterion 40).
The other criteria having test-values .5 were: large number of
bacteria in the sub-capsular sinus, free floating bacteria separated
from nearby colonies, the presence of bacteria located centrally to
the PMNs, and inward bacterial projections from the subcapsular
sinus (criteria 16, 21, 22, 18). Other criteria with lower test values
but still strongly characteristic of Y. pestis infection corresponded to
poorly organized PMN reaction (criterion 10), altered DLN
structure and density (criteria 25–28, 32, 34), large extension of the
lymph node alterations (criteria 7, 8), breaching of the PMN
barrier (criterion 23), high amounts of bacteria (criterion 17) and
intranodal hemorrhages (criterion 39). An example of type 3 DLN
is shown in Fig. 6. Of note, the two criteria (Nu2 and 3) related to
the amount of PMNs in the DLN did not discriminate between the
advanced lesions induced by the two Yersinia species.
Two major steps in the evolution from Y. pseudotuberculosis to Y.
pestis have been the acquisition of an id portal of infection and a
sharp increase in virulence, raising the question of a possible causal
link between the two events. However, in our system this route of
entry did not endow Y. pseudotuberculosis with the killing power of its
descendant, indicating that the high virulence of Y. pestis is a
genuine property of this species.
Previous images of dermis sections of human and animal plague
cases have indicated that Y. pestis penetration into the dermis is
followed by local bacterial expansion [8,31]. This finding is here
confirmed and quantified. However, Y. pestis dermal expansion is
not superior to that of the less virulent species, indicating it does
not account for the exceptional severity of plague. In fact,
unexpectedly, there were even higher numbers of Y. pseudotuber-
culosis than Y. pestis within the IS at 24 h and 48 h pi. This
difference does not seem to be linked to a lower in situ
inflammatory reaction to Y. pseudotuberculosis because similar
inflammatory lesions were observed in the dermis of mice injected
with either Yersinia species. At 24 h pi, similar numbers of Y. pestis
and Y. pseudotuberculosis were present in the DLN. Altogether these
observations show that the first steps of the disease, including a
Figure 5. Dendrogram of the draining lymph node histopath-
ological profiles. Each label indicates (in order): infecting species (P
for Y. pestis, T for Y. pseudotuberculosis), delay between mouse infection
and lymph node collection (in days), amount of injected cfu. Top
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phase of bacterial multiplication in the DLN within the first 24 h,
proceeded similarly during the two Yersinia infections, indicating
that the expression of Y. pestis unique pathogenicity is delayed until
later stages of the infectious process.
In contrast, Y. pestis loads in the DLN were higher than those of
Y. pseudotuberculosis on day 2, indicating that, in this organ, Y. pestis
growth was not controlled as efficiently as that of Y. pseudotuber-
culosis past the first 24 h of infection. Our findings show that the
large accumulation of bacteria in the draining lymph node
previously reported in descriptions of human and animal plague
buboes [6,8,13,14] is specific to Y. pestis compared to its sister
species. As has been suggested, accumulation of Y. pestis in the
extravascular lymph node reservoir, followed by brutal release into
the blood stream, may be important for the generation of the high
level septicemia necessary for transmission to a new host by the
blood sucking vector . Therefore, the difference in the
intranodal bacterial accumulation of the two species might be
critical to their different pathogenic potentials.
Significantly different histopathological patterns were associated
in the DLN with Y. pestis and Y. pseudotuberculosis infections. This
conclusion was reached through decomposition of images of the
histological sections into elementary lesions and subsequent
analysis by statistical tools able to deal with large sets of data
with no assumed theoretical distribution of the variables or the
individuals [27–29]. To our knowledge the approach used here,
which combines the use of clustering analysis methods, MCA and
test-value calculations, has never been reported so far for
comparative analysis of histological images. While this study
Figure 6. Examples of the three histopathological types of the draining lymph nodes. Lymph nodes were taken 24 h (type 1) and 72 h
(type 2 and 3) after inoculation of ,4500 cfu of Y. pseudotuberculosis (types 1 and 2) or Y. pestis (type 3). Sections were stained by Hematoxylin-Eosin
(HE), and by antibodies specific to Y. pseudotuberculosis or to Y. pestis (Bact), to PMNs and to B lymphocytes (BL). Immunostainings give a brownish
color. Arrowheads on the HE-stained sections indicate the border of the inflammatory front. Bacteria (arrows) are present as tiny bacterial foci at the
periphery (type 1), well delimited patches (type 2) or flame-like formations (type 3). The BL staining shows that the overall structure of the type 1, but
not of the type 3, lymph node is preserved, while, in the type 2 lymph node, the inflammatory reaction forced B lymphocytes to the central part of
the organ. Bar=100 mm.
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focused on the host response to Yersinia infection, another potential
application of this approach is the comparison of the lesions
induced by Y. pestis mutants in order to determine the role of
specific genes to plague pathogenesis. These mutants would likely
express phenotypes less distant from Y. pestis than does Y.
pseudotuberculosis. However, the technique was developped to detect
small differences in complex data sets so it could probably detect
differences more subtle than between the two Yersinia species,
although it would probably give lower percentages of inertia for
The MCA analysis yielded a list of elementary lesions that
specified the two types of infection with respect to one another. All
Figure 7. Extended dendrogram of the DLN histopathological profiles. Each label indicates (in order): Infecting species (P for Y. pestis, T for
Y. pseudotuberculosis), time post-injection (in days), amount of injected cfu. Top scale=% similarity. As compared to Figure 5, an extended range of
infective-loads was used, and hence more DLNs were included in the analysis (see the Result section). The seven last profiles in the dendrogram
correspond to completely destroyed lymph nodes.
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PLoS ONE | www.plosone.org8 February 2008 | Volume 3 | Issue 2 | e1688
the abscess-type lesions, whether wedge-shaped, polar or orga-
nized as a peripheral band, were characteristic of Y. pseudotuber-
culosis infection and, in these structures, the infectious foci were
kept separate from the normal lymph node tissue. Thus, an
organized PMN response able to contain the bacteria was a
hallmark of Y. pseudotuberculosis infection, which implies, conversely,
that one specificity of plague lesions was the lack of an organized
innate cell reaction. Indeed, PMNs in buboes were often disposed
as a thin, irregular and fragmented layer, a feature highly
discriminatory towards plague. Abscesses are the expression of
an acute cellular response to bacterial invasions and play a
major role in the control of bacterial infections . Therefore the
lack of an abscess-type structure is certainly a major impediment to
the control of Y. pestis proliferation in the DLN and probably
accounts largely for the high representation of plague bacilli in this
Figure 8. Multiple Correspondence Analysis of type 2 and type 3 histopathological profiles. The types 2 and 3 histopathological profiles
represented in the dendrogram of Figure 7, excepted the completely destroyed profiles, were included in the analysis. Here is shown a graphical
display of the results of the Multiple Correspondence Analysis, on the first two dimensions. Criteria, numbered as in Table 1, are represented by
arrows. Lymph nodes are represented by dots and denoted by alpha-numerical labels which are listed in the accompanying table, at the top right of
the figure. In this table are indicated, for each DLN, first, the time interval (in days) between mouse infection and lymph node collection, then, the
number of injected cfu. It can be seen that Y. pseudotuberculosis- and Y. pestis-infected lymph nodes are grouped on the left and right sides of the
figure, respectively. Therefore, criteria on the left side of the figure are associated with Y. pseudotuberculosis profiles and criteria on the right side of
the figure are associated with Y. pestis profiles. Criteria that are closest to the first (horizontal) axis are the most discriminating between the two
Specific Plague Pathogenesis
PLoS ONE | www.plosone.org9 February 2008 | Volume 3 | Issue 2 | e1688
The molecular and cellular mechanisms underlying abscess
formation remain poorly defined [33,34], except for the step of
PMN recruitment from the blood stream about which several
inflammatory cues and molecular mechanisms have been
uncovered [35,36]. The Y. pestis-associated deficit in abscess
formation is not the result of a massive defect of PMN recruitment
to the DLN, because, from the morphological data, there was no
gross difference in the amount of PMNs in the DLN during the
two Yersinia infections. Therefore it is possible that functional
alterations of the PMNs, or of other cells involved in abscess
formation, are induced by the plague agent.
Bacterial extensions from the subcapsular sinus or from
colonies, seen at low magnifications as «flames» and at high
magnification as bacterial strands streaking between host cells, and
free floating isolated bacteria, all reflected an infiltrating character
of the plague infection. The images of isolated host cells within
bacterial areas might be the result of this infiltrating process
around host cells. The above criteria all had discriminatory values
above 5, making the infiltrative character of the infection the most
specific feature of plague in the comparison with Y. pseudotuberculosis
infection. It remains to be determined whether specific mecha-
nisms, such as selective tissue destructions, are responsible for this
infiltrating behavior, in addition to the failure of the inflammatory
cells to contain the infection.
In conclusion, no major differences were noted between the two
Yersinia infections during the progression to DLN, and in the DLN
before day 2 pi. At this point, Y. pseudotuberculosis infection had
induced an organized PMN reaction and was contained, unlike Y.
pestis growth which was not controlled, leading to death within
hours. These findings point to the population of PMNs recruited to
the DLN as a likely primary or secondary target of the Y. pestis
specific strategy. More work directed at characterizing the
mechanisms by which Y. pestis specifically prevents the formation
of an effective abscess-type defense should further our under-
standing of plague pathogenesis.
Materials and Methods
Animals and bacteria
Eight-week old female OF1 mice (Charles River, l’Arbresle,
France) were maintained under specific pathogen-free conditions
at the Institut Pasteur in compliance with European animal
welfare regulations. Bacterial strains were Y. pestis CO92  and
Y. pseudotuberculosis IP32953 . Cultures were carried out at
28uC on LB agar medium supplemented with 0.002% (w/v)
hemin. Prior to animal infections, bacteria were resuspended in
saline and concentrations of the resulting suspensions were verified
by plating on agar medium. Mice were anaesthetized by
intraperitoneal injection of 0.5 ml Avertin (1.25% Tribromoeth-
anol+2.5% tert-amyl alcohol) and the ear dorsum was spread on
several layers of adhesive tape applied on the experimentator
thumb, so as to facilitate the injection and protect the
experimentator from accidental self-injection. In preparatory
studies, it was verified by id injection of 1% methylene blue that
the draining lymph node of the ear dorsum was the superficial
parotid lymph node, according to the nomenclature defined in
. For infections, ten microliters of bacterial suspensions were
injected id with a 0.3 ml insulin syringe (Becton Dickinson, New
Jersey, USA). At various time points, infected ears and ipsilateral
draining lymph nodes were collected and crushed, and recovered
bacteria were enumerated by plating on agar medium. LD50were
estimated according to the method of Reed and Muench ,
with groups of 5 mice followed up for 21 days. Protocols for
animal experiments were prepared according to the guidelines of
the Institut Pasteur safety committee.
Histology and immunohistochemistry
Skin samples at the inoculation site and draining lymph nodes
were fixed in 4% neutral buffered paraformaldehyde, and
processed by usual methods with standard hematoxylin-eosin
(HE) staining . To identify inflammatory and immune cells,
samples were collected in a Zinc-based preservative [41,42] and
then in alcohol before being embedded in low melting-point
paraffin (Poly(ethylenglycol) diesterate, Aldrich). Sections were
treated for endogenous peroxidase activity by incubation for
20 min in 0.3% (v/v) H2O2and blocked for 20 min in normal
serum from the appropriate animal host (dilution 1:10 in PBS
(pH 7.4) containing 1% (w/v) milk powder) prior to incubation
for 1 h with one of the following antibodies: a rabbit anti
huCD3, which cross reacts with mouse CD3 antigen (pre-
diluted; NeoMarkers), a rat anti-mouse CD45R (B220 clone,
dilution 1:40; Caltag), a rat anti-mouse F4/80, (dilution 1:50;
Caltag), a rat anti-mouse GR1 (dilution 1:200, Caltag), a
hamster biotinylated anti-mouse CD11c (dilution 1:40; BD
Pharmingen), and rabbit polyclonal antisera specific for the Y.
pestis F1 Ag, and the Y. pseudotuberculosis type I O-Ag (produced
by the French Reference Center for Yersinia). After three washes
in PBS-1% (w/v) milk powder, sections were incubated for 1 h
with appropriate secondary antibodies: EnVision+System-HRP
Anti-Rabbit (undiluted, Dako), streptavidin-peroxidase conjugate
(diluted 1:600, Dako), or rat-specific biotinylated Ig (diluted
1:400) followed by streptavidin-peroxidase conjugate. Bound
peroxidase activity was detected using 3-amino-9- ethylcarbazole
(AEC) substrate (Sigma) [41,43]. Tissues were counterstained
with Harris’ hematoxylin.
Slides were examined through an E800 Nikon microscope
equipped with 26to 1006Plan Apochromat objectives. Determi-
nation of the criteria for scoring was made upon examination of
preparations stained by HE and with all the above antibodies;
scoring was made on preparations stained by HE and with anti-Y.
pestis or anti-Y. pseudotuberculosis antibodies. Sections were identified
by a code that did not reveal which Yersinia species had been
Significance tests for bacteria enumeration data.
of injected Y. pestis and Y. pseudotuberculosis cfu numbers and
bacterial loads in organs were compared using the InStat software
(GraphPad Software,San Diego
Kolmogorov-Smirnov and Fisher tests to verify the normality of
the data distribution andthe homogeneity
Depending on the results of these tests, unpaired t-tests or Man-
Whitney tests were performed.
Cluster analysis of the histology
patterns was performed with the BioNumerics software, version
4.0 (Applied Maths, Kortrijk, Belgium) using the unweighted pair
group method with average linkages (UPGMA) and the Dice
coefficient to analyze the similarities of the patterns.
Multiple Correspondence Analysis (MCA).
performed on a complete disjunctive form table, using the software
SPAD  and the ade4  package for the R statistics system.
The SPAD software was also used to calculate test-values ,
which are numerical indicators of the influence of a variable for
discriminating defined subgroups of objects. Calculation of the
relative contribution of each axis to the total inertia was done
according to the formula adapted by Benze ´cri for use on complete
disjunctive form tables .
The MCA was
Specific Plague Pathogenesis
PLoS ONE | www.plosone.org10February 2008 | Volume 3 | Issue 2 | e1688
We are grateful to Marie-Agne `s Dillies for her help and advices for the
Conceived and designed the experiments: EC FG. Performed the
experiments: PA FG. Analyzed the data: EC LJ MH FG. Contributed
reagents/materials/analysis tools: LJ MH. Wrote the paper: EC FG.
1. Prentice MB, Rahalison L (2007) Plague. Lancet 369: 1196–1207.
2. Pollitzer R (1954) Plague. Geneva: World Heath Organization.
3. (2006) International meeting on preventing and controlling plague: the old
calamity still has a future. Wkly Epidemiol Rec 81: 278–284.
4. Crook LD, Tempest B (1992) Plague. A clinical review of 27 cases. Arch Intern
Med 152: 1253–1256.
5. Perry RD, Fetherston JD (1997) Yersinia pestis–etiologic agent of plague. Clin
Microbiol Rev 10: 35–66.
6. Flexner S (1901) The pathology of bubonic plague. The American Journal of the
Medical Sciences 122: 396–416.
7. Eastwood A, Griffith F (1914) Report to the local government board on an
inquiry into rat plague in East Anglia during the period July–October, 1911.
Journal of Hygiene 14: 285–316.
8. Crowell BC (1915) Pathologic anatomy of bubonic plague. The Philippine
Journal of Science 10 B: 249–307.
9. Petrie GF (1992) In: Council MR, ed. A system of bacteriology in relation to
medicine. London: His Majesty’s Stationary Office.
10. Jawetz E, Meyer KF (1944) The behaviour of virulent and avirulent P. pestis in
normal and immune experimental animals. J Infect Dis 74: 1–13.
11. Sebbane F, Gardner D, Long D, Gowen BB, Hinnebusch BJ (2005) Kinetics of
disease progression and host response in a rat model of bubonic plague.
Am J Pathol 166: 1427–1439.
12. Sebbane F, Jarrett CO, Gardner D, Long D, Hinnebusch BJ (2006) Role of the
Yersinia pestis plasminogen activator in the incidence of distinct septicemic and
bubonic forms of flea-borne plague. Proc Natl Acad Sci U S A 103: 5526–5530.
13. Smith JH, Reisner BS (1997) Plague. In: Connor DH, ed. Pathology of infectious
diseases. Stamford, CT: Prentice-Hall. pp 729–738.
14. Dennis DT, Meier FA (1997) Plague. In: Horsburgh RC Jr, ed. Pathology of
Emerging Infections. Washington, DC: American Society for Microbiology. pp
15. Herzog M (1904) The Plague, Bacteriology, Morbid Anatomy, and Histopa-
thology. Including a consideration of insects as plague carriers; Labora-
tories BoG, ed. Manila: Bureau of public printing. pp 1–149.
16. Cover TL, Aber RC (1989) Yersinia enterocolitica. New Engl J Med 321: 17–24.
17. Smego RA, Frean J, Koornhof HJ (1999) Yersiniosis I: Microbiological and
clinicoepidemiological aspects of plague and non-plague Yersinia infections
[Review]. Eur J Clin Microbiol Infect Dis 18: 1–15.
18. Achtman M, Zurth K, Morelli G, Torrea G, Guiyoule A, et al. (1999) Yersinia
pestis, the cause of plague, is a recently emerged clone of Yersinia
pseudotuberculosis. Proc Natl Acad Sci U S A 96: 14043–14048.
19. Chain PS, Carniel E, Larimer FW, Lamerdin J, Stoutland PO, et al. (2004)
Insights into the evolution of Yersinia pestis through whole-genome comparison
with Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A 101: 13826–13831.
20. Bercovier H, Mollaret HH, Alonso JM, Brault J, Fanning GR, et al. (1980) Intra-
and Interspecies Relatedness of Yersinia pestis by DNA Hybridization and Its
Relationship to Yersinia pseudotuberculosis. Curr Microbiol 4: 225–229.
21. Judicial Commission of the International Committee on Systematic Bacteriology
(1985) Opinion 60-Rejection of the Name Yersinia pseudotuberculosis subsp. pestis
(van Loghem) Bercovier et al. 1981 and Conservation of the Name Yersinia pestis
(Lehmann and Neumann) van Loghem 1944 for the Plague Bacillus.
International Journal of Systematic Bacteriology 35: 540.
22. Une T, Brubaker RR (1984) In vivo comparison of avirulent Vwa- and Pgm- or
Pstr phenotypes of yersiniae. Infect Immun 43: 895–900.
23. Welkos SL, Friedlander AM, Davis KJ (1997) Studies on the role of plasminogen
activator in systemic infection by virulent Yersinia pestis strain C092. Microb
Pathog 23: 211–223.
24. Brubaker RR, Beesley ED, Surgalla MJ (1965) Pasteurella pestis : Role of Pesticin I
and Iron in Experimental Plague. Science 149: 422–424.
25. Pouillot F, Derbise A, Kukkonen M, Foulon J, Korhonen TK, et al. (2005)
Evaluation of O-antigen inactivation on Pla activity and virulence of Yersinia
pseudotuberculosis harbouring the pPla plasmid. Microbiology 151: 3759–3768.
26. Guinet F, Carniel E (2003) A technique of intradermal injection of Yersinia to
study Y. pestis physiopathology. Adv Exp Med Biol 529: 73–78.
27. Benzecri J-P (1982) L’Analyse des correspondances; Benzecri J-P, ed. Paris:
28. Clausen S-E (1998) Applied Correspondence Analysis: An Introduction; Lewis-
Beck MS, ed. Thousand Oaks, CA: Sage Publications, Inc.
29. Greenacre MJ (1984) Theory and application of correspondence analysis.
London: Academic Press.
30. Morineau A, Aluja-Banet T (1998) Analyse en composantes principales (avec
illustrations SPAD). Paris: Decisia.
31. Sodeinde OA, Subrahmanyam YV, Stark K, Quan T, Bao Y, et al. (1992) A
surface protease and the invasive character of plague. Science 258: 1004–1007.
32. Majno G, Joris I (2004) Cells, Tissues, and Diseases-Principles of General
Pathology: Oxford University Press.
33. Tzianabos AO, Kasper DL (2002) Role of T cells in abscess formation. Curr
Opin Microbiol 5: 92–96.
34. Cobb BA, Kasper DL (2005) Zwitterionic capsular polysaccharides: the new
MHCII-dependent antigens. Cell Microbiol 7: 1398–1403.
35. Phillipson M, Heit B, Colarusso P, Liu L, Ballantyne CM, et al. (2006)
Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct
process from adhesion in the recruitment cascade. J Exp Med 203: 2569–2575.
36. Smith CW (1993) Leukocyte-endothelial cell interactions. Semin Hematol 30:
45–53; discussion 54–45.
37. Parkhill J, Wren BW, Thomson NR, Titball RW, Holden MT, et al. (2001)
Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413:
38. Van den Broeck W, Derore A, Simoens P (2006) Anatomy and nomenclature of
murine lymph nodes: Descriptive study and nomenclatory standardization in
BALB/cAnNCrl mice. J Immunol Methods 312: 12–19.
39. Reed LJ, Muench H (1938) A simple method of estimating fifty per cent
endpoints. American Journal of Hygiene 27: 493–497.
40. Armed Forced Institute of Pathology (1992) Laboratory Methods in Histotech-
nology.; Prophet EB, Mills B, Arrington JB, Sobin LH, eds. Washington, DC.
41. Ferrero RL, Ave P, Radcliff FJ, Labigne A, Huerre MR (2000) Outbred mice
with long-term Helicobacter felis infection develop both gastric lymphoid tissue and
glandular hyperplastic lesions. J Pathol 191: 333–340.
42. Lang T, Ave P, Huerre M, Milon G, Antoine JC (2000) Macrophage subsets
harbouring Leishmania donovani in spleens of infected BALB/c mice: localization
and characterization. Cell Microbiol 2: 415–430.
43. Naish SJ (1989) Handbook Immunochemical Staining Methods Naish S, ed.
Carpentiera, CA: DAKO Corp.
44. Chessel D, Dufour AB, Thioulouse J (2004) The ade4 package-I- One-table
methods. R News 4: 5–10.
45. Benzecri J-P (1992) Correspondence analysis handbook; Owen DB, Cornell RG,
Kennedy WJ, Kshirsagar AM, Schilling EG, eds. Marcel Dekker, Inc.
Specific Plague Pathogenesis
PLoS ONE | www.plosone.org11February 2008 | Volume 3 | Issue 2 | e1688