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United States Department of Agriculture

Agricultural Research Service

2011 Annual Report

1a.Objectives (from AD-416)
1) Describe and characterize sites, mechanisms, and Stx-mediated effects of STEC colonization of cattle intestines;.
2)Analyze in vivo gene expression of STEC O157:H7 using genomic techniques to identify genes involved in expression and regulation of adherence, colonization, and shedding;.
3)Develop and test the efficacy of intervention strategies for reducing colonization and shedding of STEC O157:H7; and.
4)Identify mechanisms of E. coli adherence involved in postweaning colibacillosis in pigs.

1b.Approach (from AD-416)
Experimental animal infections and genomic technologies will be used to identify specific STEC genes necessary for colonization and persistence in animals, and to identify and test interventions directed against identified targets. Weaned calves and neonatal pigs will be experimentally inoculated with E. coli. Microbiologic, histologic, and immunologic methods will be used to identify colonization sites and mechanisms of host-pathogen interactions. Biochemical, immunologic, and molecular biologic techniques will be used to identify specific STEC genes necessary for colonization and persistence in animals, and to identify and test interventions (vaccines and antimicrobials) directed against identified targets. Genetic, molecular, and immunologic techniques will be used to identify and characterize bacterial adhesins and other virulence factors of E. coli pathogens which cause diarrhea and edema disease in postweaning swine and evaluate their usefulness as targets for diagnostic assays or immunogens to prevent colibacillosis in swine.

3.Progress Report
This is the final report for project 3625-32000-077-00D terminated in December 2010 and replaced with 3625-32000-100-00D. O157 are Shiga toxin-producing bacteria that colonize cattle intestines without making these animal sick. However, cattle colonized with O157 can shed these bacteria in their feces thus increasing the risk of hide contamination in feedlots and carcass contamination at slaughter plants. The O157-contaminated meat, especially ground beef, is the major cause of human disease outbreaks due to O157. Our research has been directed at understanding mechanisms that promote colonization and persistence of O157 in cattle and to use this scientific knowledge for developing interventions to reduce colonization and shedding of O157 in cattle feces. By using multiple approaches, we have identified specific sites in large intestines of cattle that serve as preferred sites for O157 colonization and adherence. In addition, we have identified specific cell types and molecules present on the surface of these cells that directly promote adherence of O157 to cattle intestines. Development of methods to recover tissues and cells from intestinal sites has enhanced our capability to investigate the details of the mechanisms that promote adherence of O157 to these cells. These discoveries provide the basis for evaluating interventions for blocking interactions and adherence of O157 to sites in cattle intestines. We have identified important genes that affect the production of factors on O157 bacterial cell surfaces that are critical to the adherence of these bacteria to cattle intestines and presumably for the persistence of O157 bacteria in the environment. Identification of these genes and factors have resulted in the development of mutant O157 strains that we have tested as heat-killed vaccines for cattle. We have shown in pilot animal studies that cattle vaccinated with these vaccines shed lower numbers of O157 in their feces compared to unvaccinated cattle. By using a different approach, we have shown that the ability of O157 to synthesize a specific enzyme is critical for 0157 bacteria to produce nutrients that are necessary for their growth and survival in cattle intestines. We have identified a specific chemical that inhibits the function of this particular enzyme and demonstrated that cattle fed with very small amounts of this chemical do not shed O157 in their feces compared to animals not fed this chemical. We have also developed rapid methods for:.
1)transferring specific mutations or genes to O157 bacteria in order to study the effects of transferred genes and mutations on growth characteristics of these bacteria and the ability of these bacteria to produce factors that enhance their utility for use as vaccines; and.
2)rapidly detecting Escherichia coli strains associated with postweaning diarrhea in swine.

Last Modified: 9/4/2015
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