In studies of the pathogenesis of Y. pestis
infection workers have used a number of different animal models (4
). Currently, there is no consensus concerning which model system is best. However, similar results are often obtained when terminal stages of infection are observed using a variety of animal models. A reevaluation of the mouse models of primary pneumonic plague was warranted because many of the older studies (32
) predate our modern understanding of both the host immune response and the ability of Y. pestis
to modulate this response. In this study we examined the early pathogenesis (0 to 72 h) of pulmonary infection with Y. pestis
using two different infectious doses with both inbred and outbred strains of mice to determine if genotype-based differences could be observed.
Our studies revealed an early quiescent phase with respect to bacterial growth at the site of infection, followed by strong outgrowth of bacteria after 24 h postinfection. These findings agree with the findings of previous studies in which the workers looked at the kinetics of Y. pestis
infection in both mouse and rat models (19
). We also observed that the kinetics of infection were essentially the same for both an outbred mouse strain (CD1) and an inbred strain (C57BL/6). The estimated intranasal LD50
in our studies was consistent with that reported by Lathem et al., and the LD50
s were determined to be similar for the two strains of mice. These data indicate that there were no major differences in the outcome and course of bacterial spread in pulmonary Y. pestis
infection for these two strains of mice.
Members of the genus Yersinia
which are able to cause disease in humans have a common 70-kb virulence plasmid. It is well established that the 70-kb virulence plasmid (pCD1) contains genes that code for proteins (Yops and LcrV) that downmodulate the host immune response to infection (6
). It has been hypothesized that these virulence factors enable the bacteria to avoid setting off major innate immune warning signals early during infection that would trigger a strong host response. Certainly, it is possible that the virulence factors encoded on the 70-kb plasmid contribute to the phenotypes which we observed in the first 24 h of infection.
One of the initial steps in mounting an effective immune response to gram-negative bacterial pathogens like Y. pestis
is the recruitment of polymorphonuclear cells to the area of infection. These cells are part of the innate immune system and are very efficient at phagocytosing bacteria and releasing proinflammatory signals to help orchestrate the immune response. Indeed, with bacterial burdens ranging from 106
CFU/g tissue in the lungs one would expect a robust proinflammatory response. However, our studies revealed a distinct delay in recruitment of PMNs to the alveolar spaces and lung tissue compared to both our observations of pulmonary infection with other gram-negative pathogens, such as K. pneumoniae
, and the observations of other workers (20
). Importantly, mice from the same shipment as the mice used in the Y. pestis
infections that were infected with a lower number of LD50
s of K. pneumoniae
had a strong inflammatory response as early as 24 h postinfection. While the initial dose of K. pneumoniae
was larger than that of Y. pestis
, when the LD50
s were compared, the K. pneumoniae
group was inoculated with ~30 times the LD50
(1 × 103
]) rather than the nearly 100 times the LD50
(40 to 250 CFU [19
]) of Y. pestis
that was given to the high-dose Y. pestis
group. Despite the higher dose given to the K. pneumoniae
mice, we believe that given the difference in the LD50
s of the two bacteria, this is a valid comparison. Furthermore, the results of a recent study by Lawlor et al. describing pulmonary K. pneumoniae
infection suggested that there was a relative increase in lung bacterial burdens similar to the increase which we observed with Y. pestis
). Together, these data suggest that the mice were capable of generating an inflammatory response to infection and link the lack of a response to Y. pestis
The delayed arrival of PMNs to the site of infection in a pneumonic plague model is consistent with recent observations reported for C57BL/6 mice (19
) and the observations reported for a bubonic plague model using rats (31
). The fact that this delayed arrival was evident in both outbred and inbred mice suggests that it was linked to the pathogen and not the host. A recent study revealed evidence that neutrophils are important in the phagocytosis and control of Y. pestis
infection in the spleens of mice infected subcutaneously with wild-type Y. pestis
strain GB (23
). Together, these data suggest that controlling PMN responses during the initial phases of pulmonary Y. pestis
infection is an important aspect of the pathogenesis of disease and may partially explain the extraordinary virulence of Y. pestis
The bronchoalveolar lavage fluid from the animals that received a high dose of Y. pestis was analyzed for the presence of multiple cytokines and chemokines. The overall theme of what we found was that there was a delay in the detection of proinflammatory cytokines and chemokines in the BALF. A small increase in the level of IL-10 was observed at 24 h postinfection, and the level of IL-10 decreased by 48 h postinfection. However, the IL-10 levels were close to the level of detection for the assay, and it remains to be determined if this finding is biologically relevant; nevertheless, IL-10 has potent anti-inflammatory effects, and it is possible that it plays a role in modulating inflammation early in infection. Often proinflammatory cytokines and chemokines were undetectable until 48 h after infection, which is further evidence for a delay in the inflammatory response to infection. The delay in cytokine and chemokine expression is consistent with the delay in PMN recruitment to the lung, which requires both a chemokine gradient and inflammatory signals (cytokines) to activate the endothelium and promote efficient transmigration from the circulation to the tissue.
It appears that the bacteria take advantage of the time when they seem to remain largely undetected by the host immune system, replicating and producing large numbers of cells in host tissues. Although the host does mount a response to the bacteria in the lungs between 24 and 48 h postinfection through increased production of proinflammatory cytokines and chemokines and increased neutrophil recruitment to the site of infection, it appears that this response is effectively too late to prevent a morbid outcome of the infection.
Currently, it is unclear whether the initiation of an inflammatory response is a result of bacterial products or a result of damage to host tissues. One finding addressing this issue is the observation that at 48 h postinfection, when bacterial burdens commonly reached 109 CFU/g of liver tissue, multiple microcolonies were present in the liver without an appreciable inflammatory response. This suggests that the inflammatory response in the lung, observed late in infection, may be a response to host tissue damage rather than a response to bacterial products.
It has been shown previously that administration of proinflammatory cytokines, namely TNF-α and IFN-γ, prior to bacterial challenge can provide protection for the animals in an intravenous mouse model of Y. pestis
). More recently, Montminy and coworkers demonstrated that Y. pestis
expressing lipopolysaccharide lipid A at 37°C that more closely resembled the more immunogenic lipopolysaccharide lipid A expressed at 26°C enhanced inflammatory responses to Y. pestis
and eliminated the lethality of this modified strain (25
). Based on the information from these studies and our study, it is intriguing to speculate that if the bacteria are unable to deter and delay important proinflammatory proteins, such as TNF-α, MIP-2, and KC, early in the infection, the host may be able to effectively control the infection and recover.
Consistent with the delay in PMN recruitment to the lungs and the delay in cytokine and chemokine expression, substantial changes in histopathology are not evident until late in infection. The pronounced pathology reported for terminal infections of humans and primates is not evident in mice until 36 to 48 h postinfection, when the mice are moribund. In this study we focused on the histopathology that develops during the first 48 h of primary pneumonic plague in both outbred and inbred mice using an infectious dose of 0.8 × 104
to 2.9 × 104
CFU. In contrast to previous studies, we did not observe eosinophilia in either mouse strain at any time, nor did we observe pronounced neutrophil infiltration until after 24 h, which is in contrast to what was reported by Smith (32
). This difference was probably due to differences in mouse strains (it is unclear what strain Smith used in his study) or to the fact that we used specific-pathogen-free animals maintained in barrier facilities that were uncommon in 1959. However, most of our other observations were similar to what Smith reported and to what was recently reported by Latham et al. Liver and spleen lesions were similar to what was described by Brubaker and coworkers, but less severe. This difference may have been due to differences in strains, to the severity of spleen and liver lesions in pneumonic plague models compared to spleen and liver lesions in systemic plague models, or to differences in the duration of disease in the two models. Together, the histopathological findings for the outbred CD1 mice and the inbred C57BL/6 mice were very similar.
Previous studies of a variety of pathogens illustrated that in many cases, the host genotype had a large effect on the outcome of infection (2
). In order to evaluate the influence of host genotype on the outcome of infection, we compared Y. pestis
infection in an outbred strain, CD1, and Y. pestis
infection in an inbred strain, C57BL/6. Our results indicate that in these two host strains the progression and outcome of disease were very similar, suggesting that, at least for CD1 and C57BL/6 animals, the host genotype has little effect on the outcome of Y. pestis
infection. We speculate that a possible reason for this lies in the fact that pneumonic plague is an acute infection, killing the host within a few days, which leaves clearance of the bacteria exclusively to innate immune mechanisms. Many of the phenotypes dependent on the host genotype appear later in infection, when the adaptive response begins. For pneumonic plague, the host does not live long enough to develop an adaptive immune response. Because of this, the general trends in Th1 T-cell responses versus Th2 T-cell responses in different mouse strains would be predicted to have less effect on the outcome of pneumonic plague, which is supported by the results of our study.
Overall, we concluded that there are some subtle differences in pneumonic plague infection between the inbred mouse strain C57BL/6 and the outbred mouse strain CD1. For example, we observed low levels of IFN-γ and IL-12p70 in the BALF of CD1 mice at 48 h after infection, while the levels in C57BL/6 mice were undetectable. However, these subtle differences did not significantly change the outcome or progression of disease in this model, as the LD50, kinetics of infection, and general delays in the proinflammatory response to infection all followed similar courses. More significantly, in both strains of mice, a prolonged delay in detection of proinflammatory cytokines and chemokines, as well as the appearance of inflammatory cells, was evident. We hypothesize that the delayed inflammatory response to infection is beneficial to Y. pestis growth and may be a key virulence strategy of this pathogen.