application) Investigation of the gastrointestinal disease and renal injury caused by enterohemorrhagic Escherichia coli (EHEC) and the development and testing of interventions to prevent disease following infection have been hampered by the lack of a convenient animal model that effectively reproduces the typical human colonic disease that progresses to HUS. The Principal Investigator proposes to develop the use of a new mouse model of EHEC infection with the long term goal of increasing the ability to study disease pathogenesis and prevention. The new animal model will be compared with prior models in the evaluation of vaccine strategies against EHEC. This will be accomplished through the following two Specific Aims: 1. Evaluation of the use of Citrobacter rodentium expressing Stx in a mouse model of Shiga toxin-producing E. Coli (STEC) infection. C. rodentium, a naturally occurring pathogen of laboratory mice which causes transmissible murine colonic hyperplasia, binds to the mouse enterocyte by a specific attachment and effacement lesion similar to that of EHEC. Strategies have been developed to lysogenize C. rodentium with antibiotic-marked Stx1- and Stx2-expressing bacteriophages as well as to express toxin components from plasmid vectors. Toxin production, phage induction, and lysogen stability will be evaluated in vitro. Mice will be challenged with toxin-producing C. rodentium and evaluated for clinical and pathologic signs of disease. This model has the potential of reproducing both the gastrointestinal and renal injury seen in EHEC infection, allows the use of adult animals and the development of normal immune responses, utilizes the power of mouse genetics to investigate genetic factors in determining gastrointestinal (GI) and systemic disease expression, and provides a significant increase in the ease of identifying and testing new interventions compared to many other animal models. 2. Expression of nontoxic Stx1 and Stx2 antigens using a balanced lethal plasmid system in Vibrio cholerae vaccine strains. Vibrio cholerae will be used as a live oral attenuated vaccine vector to deliver immunogenic antigens of EHEC to stimulate a common mucosal immune response. A balanced lethal plasmid system will be used to provide stable expression of the heterologous antigens from the vaccine strains. The germfree mouse model of V. cholerae colonization will be used to examine mucosal and systemic immune responses to the toxin components expressed by the vector strains. The new mouse challenge model will be compared to prior mouse models in evaluating protection from disease in response to immunization with V. cholerae strains expressing the EHEC antigens.
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