To comprehensively study the ecology of STEC shedding, we will pursue the following objectives: <OL> <LI> Identify bacterial genotypes and epidemiological factors important for shedding in multiple herds by genotyping >750 strains to examine genotype distributions, genetic diversity and changes in shedding status over time;<LI> Use 454 pyrosequencing to compare the composition, diversity and function of the microbial communities within the rectoanal junction and ruminal fluid of super-shedders and non-shedders; <LI> Determine how STEC affects the bovine immune response by comparing antibody levels, and the expression of cytokine and bovine genes among super-shedders and non-shedders; <LI>Identify STEC inhibitors from the ruminal fluid of non-shedders, and test control strategies aimed at decreasing shedding levels. </ul> The long-term goal of our collaborative research effort is to determine the impact of bacterial, epidemiological, immunological, and ecological factors on STEC shedding in cattle. <P>Through this work, we expect to develop new ideas for direct-fed microbials, vaccines, therapies and other control strategies that can reduce the frequency and level of STEC shedding, an end result that will likely translate to a reduction in food contamination, transmission to humans, and STEC-associated morbidity and mortality.
Non-Technical Summary: STEC is a leading cause of foodborne and waterborne infections. Cattle are the primary reservoir of STEC, but little is known about those factors that impact shedding levels. While some STEC genotypes have been suggested to more effectively colonize cattle, the type and distribution of the gastrointestinal microbiota, cattle immune responses, and the composition of the rumen may also impact shedding. To comprehensively study the ecology of STEC shedding, we will: 1) Identify bacterial genotypes and epidemiological factors important for shedding in multiple herds over time; 2) Compare the composition, diversity and function of the microbial communities within the rectoanal junction and ruminal fluid of STEC shedders and non-shedders; 3) Determine how STEC affects the bovine immune response; and 4) Identify STEC inhibitors from the ruminal fluid of non-shedders and test control strategies aimed at decreasing shedding levels. Our long term goal is to assess how bacterial, epidemiological, immunological, and host factors work together to impact shedding. Multidisciplinary studies of this scope are required to better understand shedding in cattle and aid in the development of enhanced detection methods and control strategies. <P> Approach: To enhance our understanding of STEC colonization of cattle, we will evaluate temporal changes in bacterial genotype distributions as well as the microbial communities and chemical composition of ruminal fluid from shedders, super-shedders and non-shedders. To do this, STEC-positive cattle will be identified from multiple herds with varying management practices in Michigan and Texas. STEC shedders and a subset of non-shedders will be followed over a 6 month period to examine colonization and transmission dynamics by herd and identify epidemiological associations with shedding. Rectoanal junction (RAJ) swabs and tissue, blood, and ruminal fluid will be collected from all super-shedders, matched nonshedders and a subset of low-level shedders to identify differences in the i) genotypic and phenotypic characteristics of STEC; ii) microbiome of the RAJ and rumen via 454 pyrosequencing of 16S rRNA genes and metagenomics; iii) immunological response by examining antibody levels and cytokine expression profiles; iv) composition of the ruminal fluid; and v) genetic makeup of cattle. In vitro studies of super-shedder and nonshedder ruminal fluid will also enable the identification of STEC inhibitors. Based on these results, we will develop control strategies for reducing shedding in vitro, while alternative strategies will be evaluated in the field with naturally infected shedders. These control methods could potentially result in long-range improvements to the agricultural and food industries by reducing STEC colonization in cattle, the subsequent contamination of the food supply, and morbidity and mortality in humans.