The molecular pathogenesis of fully virulent, wild-type Y. pestis in relevant animal models has been relatively neglected because of the scarcity of secure BSL-3 facilities and trained personnel certified to work with this Class A select agent. The threat of bioterrorism and the emergence of multiply-antibiotic resistant strains of Y. pestis increases the urgency for a more detailed understanding of the host-pathogen relationship at the molecular level that may lead to the design of improved medical countermeasures and diagnostics. We have established mouse and rat models of bubonic plague that incorporate flea-to-rodent transmission to investigate the role of specific Y. pestis virulence factors and to characterize the host response to naturally acquired infection. We have characterized the kinetics, microbiology, and histopathology of bubonic plague in rats following intradermal injection of Y. pestis, and used this model to characterize the gene expression profile of Yersinia pestis in the infected lymph node during bubonic plague using whole-genome microarray technology. Our previous work has shown that three important Y. pestis virulence factors, Ail (a Y. pestis outer surface protein), the Type III secretion system (T3SS) encoded on the Yersinia virulence plasmid, and the plasminogen activator (Pla) encoded on the 9.5-kb Y. pestis-specific plasmid all act to limit the polymorphonuclear leukocyte response to bubonic plague infection in vivo (polymorphonuclear leukocytes, also referred to as PMNs or neutrophils, are phagocytic cells that are an important innate defense against infection). Thus, we now have several lines of evidence that the PMN response correlates with successful outcome to infection, and this aspect of host-pathogen interaction has become a focus of our lab. During the past year, we characterized the interaction of Y. pestis with PMNs and their fate following phagocytosis. We reported that a small but significant percentage of Y. pestis, even T3SS-negative attenuated strains, survive and eventually replicate within phagosomes after being ingested by neutrophils. Infected neutrophils were also demonstrated to be taken up by macrophages in vitro, where Y. pestis could continue to replicate. The results indicate that neutrophil phagocytosis is not invariably fatal to Y. pestis, and that virulence factors other than the T3SS that counteract the strongly bactericidal environment of the neutrophil phagosome are important to plague pathogenesis. We have developed intravital imaging methodologies to examine the early events that occur in the dermis after transmission by flea bite. These studies revealed that the initial prominent response to flea-borne plague is a focused, rapid recruitment of neutrophils and macrophages that interact with the bacteria. These initial findings have guided the implementation of new experimental strategies to identify and characterize mechanisms of Y. pestis dissemination from the dermis and resistance to the innate immune response. Wild rodents typically sustain a permanent ectoparasitic population of fleas in their coats and in their burrows, and experience fleabites regularly throughout their lives. In most arthropod-borne disease systems, vector saliva influences infectivity due to its immunomodulatory properties. For example, exposure of mice to sandfly bites results in a delayed-type hypersensitivity response to salivary components that inhibits the transmissibility of Leishmania parasites from these vectors. We investigated whether similar phenomena have a role in the ecology of plague. We found that prolonged exposure of mice to uninfected flea bites induces very little inflammation and no hypersensitivity. An IgG response is generated, directed primarily to the major component of flea saliva, a family of phosphatase-like proteins. In contrast to most vector-borne disease systems, prolonged prior exposure to uninfected flea bites did not affect the incidence or progression of bubonic plague in mice challenged by infected blocked fleas. This study is now in press. Convalescent serum from human plague patients is virtually impossible to come by. However, a small percentage of mice and rats that we challenge by flea bite or by low-dose intradermal injection develop initial signs of disease, but then spontaneously recover. We have slowly accumulated a bank of these convalescent sera. We are using these sera in immunoblots of Y. pestis membrane proteins separated by 2D gel electrophoresis, and immunoreactive proteins are being identified by mass spectrometry at NIAIDs Research Technologies Branch. The objective of this strategy is to identify correlates of immune protection and candidate components of a third-generation plague vaccine. Antigens detected only in animals infected by flea bite would be of particular interest.