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United States Department of Agriculture

Agricultural Research Service

2009 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
STEC Colonization: Demonstrated by using in vitro adherence assays that deletion of gene sidA (a homolog of quorum-sensing regulator LuxR) enhanced the ability of O157:H7 to adhere to epithelial cells. Additional experimentation revealed that increases in the expression of surface structures, such as flagella and fimbriae, and factors required for intimate adherence might be responsible for the increased adherence of O157:H7 lacking SdiA. Since the role of SdiA in coordinating bacterial behavior in response to changes in bacterial population densities in the environment is not known, this research will help define the role of SdiA in influencing O157:H7 colonization of cattle intestine in response to factors produced by O157:H7, by other intestinal bacteria, or by bovine intestinal tissues.

Interventions: Evaluated a metabolic inhibitor of E. coli growth for reducing E. coli O157:H7 colonization of cattle intestines. Preliminary animal trials have demonstrated that cattle treated with the metabolic inhibitor showed reductions in the colonization of their intestines by O157:H7 as was evidenced by reductions in both the durations and the levels of O157:H7 shedding in the feces of these animals. These results provide evidence that this metabolic inhibitor may be useful for decreasing intestinal colonization and fecal shedding of E. coli in infected food animals.

Interventions: Demonstrated by preliminary studies that cattle vaccinated with heat-killed bacterins prepared from two mutant strains of O157:H7 resulted in significant increases in the levels of serum antibodies directed towards specific antigens that are critical for the ability of O157:H7 to colonize cattle intestines. These findings provide preliminary evidence that an optimal vaccine might prove effective in reducing 157:H7 colonization of cattle intestines.

1. PCR Assay for Enterotoxigenic E. coli (ETEC). To identify mechanisms of E. coli adherence involved in postweaning colibacillosis in swine. Rapid identification of ETEC strains possessing multiple virulence factors requires an approach capable of detecting several genes simultaneously without the need for laborious and time-consuming microbiological and immunological methods. A multiplex PCR assay capable for detecting nine different virulence factors was developed and validated for rapid and specific detection of ETEC strains responsible for diarrheal and edema diseases in pigs. This rapid diagnostic approach will facilitate immediate removal of infected pigs for treatment and preventing transmission of these pathogenic strains to healthy animals.

6.Technology Transfer

Number of the New/Active MTAs (providing only)1
Number of New Patent Applications Filed1

Review Publications
Casey, T., Bosworth, B.T. 2009. Design and Evaluation of a Multiplex PCR Assay for the Simultaneous Identification of Genes for Nine Different Virulence Factors Associated with Escherichia coli that Cause Diarrhea and Edema Disease in Swine. Journal of Veterinary Diagnostic Investigation. 21(1):25-30.

Nystrom, E.A., Stoffregen, W.C., Bosworth, B.T., Moon, H.W., Pohlenz, J.F. 2008. Early Attachment Sites for Shiga-toxigenic Escherichia coli O157:H7 in Experimentally Inoculated Weaned Calves. Applied and Environmental Microbiology. 74(20):6378-6384.

Stamm, I., Mohr, M., Bridger, P., Schropfer, E., Konig, M., Stoffregen, W.C., Nystrom, E.A., Baljer, G., Menge, C. 2008. Epithelial and Mesenchymal Cells in the Bovine Colonic Mucosa Differ in Their Responsiveness to Escherichia coli Shiga Toxin 1. Infection and Immunity. 76(11):5381-5391.

Last Modified: 9/4/2015
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