2007 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.
Infection studies to evaluate STEC virulence factors and the role of leukocytes in Stx-mediated disease: Showed that the rectal anal mucosal swab (RAMS) sampling technique (a sensitive sampling technique for bacteriologic detection of STEC O157:H7-infected cattle) can also be used to obtain rectal squamous epithelial cells for use in vivo and in vitro STEC adherence studies. This identified a readily available source of bovine rectal squamous epithelial cells which will facilitate identification of host and bacterial factors involved in STEC binding to squamous epithelial cells. Showed that Shiga toxins promote STEC O157:H7 infections in cattle and provided first evidence that STEC infections suppress development of an antigen-specific cellular immune response in cattle. These findings extend the evidence that Stx modulation of the mucosal immune response in cattle contributes to the persistence of STEC in cattle. This information is critical for developing vaccines for reducing STEC in cattle.
Genomic techniques to assess gene expression in vivo: Showed that aroA mutants of E. coli were avirulent and attenuated for growth in vivo in neonatal pigs. These results demonstrate that metabolic genes expressed in vivo are valid targets for intervention strategies against E. coli infections. Constructed a vector containing a plasmid expressing ampicillin resistance and encoding green fluorescence protein. This vector will be used for analysis of in vivo gene expression (IVET) to identify genes that promote in vivo colonization of cattle intestine by STEC O157:H7. Also obtained custom-designed microarrays for detection of STEC O157:H7 gene expression and measurement of RNA transcription of these genes in bacteria isolated directly from the intestinal tract of cattle. IVET and microarrays will help identify genes involved in STEC O157:H7 adherence, colonization and shedding in cattle.
Interventions: Established dose ranges and safety of a non-antibiotic metabolic inhibitor in neonatal pigs and provided preliminary evidence that this inhibitor is effective in reducing the incidence and severity of ETEC diarrhea in neonatal pigs. Showed that candidate O157:H7 vaccines are immunogenic in cattle and that, following experimental challenge with STEC O157:H7, vaccinated calves have fewer O157:H7 bacteria in their feces than do non-vaccinated calves. These findings extended evidence that these candidate vaccines have potential for reducing STEC O157:H7 in cattle, and consequently, reducing STEC infections and disease in humans.
Provided new tools for studying interactions of E. coli O157:H7 with bovine epithelial cells. Shiga toxin-producing E. coli bacteria that cause disease in humans are carried in the gastrointestinal tracts of healthy cattle and other ruminants. E. coli O157:H7 are frequently found on epithelial cells located on the mucosal surface at the junction between the rectum and the anus in infected cattle. We developed techniques for collecting epithelial cells from cattle and assays for studying E. coli O157:H7 binding to these cells. These tools will facilitate examination of microbial and host-microbe interactions affecting food safety and animal health. This work aligns with Component 1.1 of the ARS NP 108 Food Safety Action Plan and addresses Problem Statements: 1.1.1 (Methodology), 1.1.3 (Ecology, Host Pathogen, and Chemical Contaminants Relationships), and 1.1.4 (Intervention Strategies).
Identified species-specific effects of Shiga toxin on E. coli O157:H7 colonization in ruminants. Shiga toxin-producing E. coli bacteria that cause disease in humans are carried in the gastrointestinal tracts of healthy cattle and other ruminants. We showed that Shiga toxins promote E. coli O157:H7 infections in cattle, but not in sheep. We also provided the first evidence that Shiga toxin-producing E. coli infections suppress immune responses in cattle. These results extend the evidence that Shiga toxins have different effects on different species and provide evidence that Shiga toxins help E. coli O157:H7 establish infections and persist in cattle. Species-specific effects of Shiga toxins such as those identified in this research must be addressed in designing and evaluating vaccines for reducing Shiga toxin-producing E. coli infections in cattle. This information critically impacts food safety, animal health, and human health. This work aligns with Component 1.1 of the ARS NP 108 Food Safety Action Plan and addresses Problem Statements: 1.1.1 (Methodology), 1.1.3 (Ecology, Host Pathogen, and Chemical Contaminants Relationships), and 1.1.4 (Intervention Strategies).
5.Significant Activities that Support Special Target Populations
|Number of active CRADAs and MTAs||1|
|Number of invention disclosures submitted||1|
|Number of non-peer reviewed presentations and proceedings||4|
Cornick, N.A., Helgerson, A.F., Sharma, V.K. 2007. Shiga toxin and shiga toxin-encoding phage do not facilitate E. coli O157:H7 colonization in sheep. Applied and Environmental Microbiology. 73(1):344-346.
Helgerson, A.F., Sharma, V.K., Dow, A.M., Schroeder, R., Post, K., Cornick, N.A. 2006. Edema disease caused by a clone of Escherichia coli O147. Journal of Clinical Microbiology. 44(9):3074-7.
Hoffman, M.A., Menge, C., Casey, T., Laegreid, W.W., Bosworth, B.T., Nystrom, E.A. 2006. Bovine immune response to Shiga-toxigenic Escherichia coli O157:H7. Clinical and Vaccine Immunology. 13(12):1322-1327.