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Recent studies show that a few "super-shedders", which result from chronic E. coli O157:H7 colonization at the recto-anal junction, are mainly responsible for E. coli O157:H7 contamination in a large group of beef cattle. However, mechanisms leading to chronic colonization remain poorly defined. Our preliminary studies demonstrated that local inflammation decreased E. coli O157:H7 colonization. <P> We hypothesize that local inflammation reduces E. coli O157:H7 colonization in beef cattle. To test our hypothesis, we will assess the association of local inflammation with E. coli O157:H7 colonization and shedding through longitudinal animal studies. <P>The overall objective of this proposal is to evaluate the role of local inflammatory response in E.coli O157:H7 gut colonization and shedding in beef cattle. The proposed studies are novel, because the role of inflammation in colonization and shedding in beef cattle has not been tested. <P>After completing the proposed studies, we will identify the role of inflammation in E. coli O157:H7 gut colonization and fecal shedding. More importantly, these studies will provide crucial preliminary data for the jointed efforts between Clay Center and UW to apply for further fundings to reduce or eliminate E. coli O157:H7 colonization in beef cattle.
Non-Technical Summary: <BR>E. coli O157:H7 shedding in feces is the major source of contamination in beef. Certain cattle have much higher rates of E. coli O157:H7 shedding (>104 CFU/g feces), so called "super-shedders", which are mainly responsible for beef carcass E. coli O157:H7 contamination. Therefore, eliminating or reducing E. coli O157:H7 colonization in super-shedders is the key to solving problems associated with E. coli O157:H7 contamination and to providing consumers with safer beef. However, the reasons leading to E. coli O157:H7 colonization and "super-shedders" remain unclear. We hypothesize that local inflammation reduces E. coli O157:H7 colonization in the gut of beef cattle. Studies proposed in this project will evaluate the role of local inflammatory response in E.coli O157:H7 gut colonization and shedding in beef cattle. Such knowledge will allow us to design specific strategies to reduce or eliminate E. coli O157:H7 through altering inflammation in the gut in our future studies. <P> Approach: <BR> A cattle study will be conducted at Clay Center. Briefly, more than 300 uniform beef cattle will be used in this study. After weaning, animals will be sorted according to weight and sex into 10 pens. Cattle will be fed a grain-based diet. Three months before slaughter, fecal grab samples will be collected tri-weekly for E. coli O157:H7 enumeration and prevalence analyses. At the same time, a sample of blood will be collected for analyzing pro-inflammatory, anti-inflammatory cytokines and cortisol. During this three month period, 10 super-shedders (>104 CFU/g feces for at least one sampling time) and 10 non-shedders (E. coli O157:H7 is not detected or only transiently positive) will be identified and slaughtered following the standard USDA procedure. The recto-anal section (5 to 10 cm from the anus) will be sampled immediately following exsanguination, and used for collecting mucosal epithelial tissues for immunoblotting and qRT-PCR analyses of inflammatory cytokines and associated inflammatory signaling. The presence of inflammation will be compared between super-shedders and non-shedders. In addition, we will also analyze the location of inflammatory signaling through immunohistochemical staining, and correlate inflammatory loci with E. coli O157:H7 colonization and the presence of A&E lesions. We expect to show that local inflammation is largely absent in the A&E lesions of super-shedders compared to non-shedders. All activities involving live animals will be conducted at the ARS Clay Center by their personnel under their regulations, and that studies conducted at UW involve the use of collected tissues only.