2008 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.
Shiga toxin-producing Escherichia coli (STEC) infection studies: Showed that Shiga toxins (Stx) bind to and have functional affects on porcine blood PMN and eosinophils in vitro. These results extend the evidence that Stx binding to polymorphonuclear leukocytes (PMN) plays a role in the pathogenesis of STEC disease. These findings extend the evidence that pigs, which like humans are susceptible to Stx-mediated disease, are a suitable model for evaluating the role of granulocytes in STEC disease.
Identified and characterized cells in the large intestines of cattle that have Shiga toxins (Stx) receptors (molecules that bind Stx). Identified Stx receptor-positive epithelial cells which were not responsive to Stx and novel Stx receptor-positive non-epithelial cells which were responsive to Stx. The identification of cells with varying Stx receptor expression patterns and Stx responsiveness will help elucidate the roles of different types of host cells during STEC colonization of the bovine intestine and determination of how Stx promote STEC persistence in cattle.
Constructed QseA and SdiA mutants of O157:H7 and are using them to test the hypothesis that O157:H7 mutants lacking the QseA mutant will not colonize and persist in cattle intestines as well as the SdiA mutant. Quorum sensing (QS) signaling molecules help bacteria coordinate their behavior based on bacterial densities in their environment. This research will help define the role QS molecules (produced by O157:H7, other intestinal bacteria, or bovine intestinal tissues) play in STEC colonization of cattle.
In vivo gene expression: Used a novel genetic selection procedure, along with bacteriophage-mediated transduction, to transfer specific mutations to wild type pathogenic E. coli strains which are normally resistant to this gene transfer system. The generalized procedure, which can be adapted to transfer any desired mutation to wild type E. coli, facilitates the construction of isogenic (i.e., differ only by desired mutation) mutants. We will use these mutants to identify nutritional requirements of E. coli within the intestinal tract.
Interventions: Evaluated a metabolic inhibitor of E. coli growth for use as a treatment of E. coli infections in animals. This inhibitor reduced the incidence of diarrhea and the numbers of bacteria in the intestines of neonatal pigs experimentally inoculated with pathogenic E. coli. The results provide evidence that this metabolic inhibitor may be useful for decreasing intestinal colonization and fecal shedding of E. coli in infected food animals.
Postweaning colibacillosis: Developed a simple, rapid multiplex PCR assay for simultaneous identification of 9 major virulence factors associated with postweaning colibacillosis (5 adhesins, 3 enterotoxins and Shiga toxin 2e). This simple, rapid, comprehensive test will facilitate diagnosis of colibacillosis and rapid characterization of pathogenic E. coli from swine. This project addresses National Program 108 Food Safety Action Plan Sections 1.1.1 Methodology, 1.1.3 Ecology, Host Pathogen, and Chemical Contaminants Relationships, and 1.1.4 Intervention Strategies.
Early STEC O157:H7 Colonization Sites in Cattle.
Cattle are an important source of Shiga toxin-producing Escherichia coli (STEC) O157:H7 bacteria, foodborne pathogens that cause severe diarrhea and sometimes kidney failure and death in humans. One way to reduce the risk of STEC O157:H7 infections in humans is to reduce the level of STEC O157:H7 in cattle. We described early STEC O157:H7 colonization sites in experimentally-inoculated weaned calves and the effects of dexamethasone (used to simulate stress) on STEC colonization in calves. We showed:.
Provided New Tools for Tracking STEC O157:H7 Infections in Cattle.
Cattle are a major source of Shiga toxin-producing E. coli O157:H7 and can secrete E. coli O157:H7 in their feces for extended periods. Biological tools for easy monitoring and enumeration of E. coli O157:H7 in cattle feces will facilitate identification of bacterial and host factors that promote persistence of E. coli O157:H7 in cattle. We described and genetically modified a small plasmid harbored by STEC O157:H7 for stable expression of green fluorescence and resistance to antibiotic ampicillin under conditions simulating the environment of cattle intestine. This modified plasmid will facilitate monitoring of fecal shedding of genetic variants of E. coli O157:H7 (containing this modified plasmid) relative to that of an unmodified parent strain of E. coli O157:H7 (lacking the modified plasmid) in infection studies to identify STEC colonization mechanisms in cattle. Strains containing this modified plasmid will also facilitate monitoring the efficacy of vaccines aimed at reducing persistence and fecal STEC O157:H7 shedding of in experimentally inoculated cattle. This project addresses National Program 108 Food Safety Action Plan Sections 1.1.1 Methodology, 1.1.3 Ecology, Host Pathogen, and Chemical Contaminants Relationships, and 1.1.4 Intervention Strategies.
1)calves treated with dexamethasone were more susceptible to STEC O157:H7 colonization;.
2)colon and cecum were sites from which inoculum type bacteria were most often recovered;.
3)rectum, ileo-cecal valve and distal colon were sites most likely to have O157-associated tissue damage;.
4)ileum and gall bladder were other sites with STEC O157:H7. Identification of sites where STEC O157:H7 initially colonize in experimentally inoculated weaned calves will facilitate identification of bacterial and host factors that promote STEC infections in cattle. Knowing where STEC O157:H7 initially colonize in cattle is critical for designing and evaluating strategies aimed at reducing STEC infections in cattle. This project addresses National Program 108 Food Safety Action Plan Sections 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 the New MTAs (providing only)||1|
|Number of Non-Peer Reviewed Presentations and Proceedings||2|
Menge, C., Nystrom, E.A. 2008. Dexamethasone depletes gamma-delta T cells and alters the activation state and responsiveness of bovine peripheral blood lymphocyte subpopulations. Journal of Dairy Science. 91(6):2284-2298.
Sharma, V.K., Stanton, T.B. 2008. Characterization of a 3.3-kb plasmid of Escherichia coli O157:H7 and evaluation of stability of genetically engineered derivatives of this plasmid expressing green fluorescence. Veterinary Microbiology. Available: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD6-4SKB3CT-2&_user=6956098&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000052423&_version=1&_urlVersion=0&_userid=6956098&md5=4acc7d9e43e9b59307dc8e59dc8022c6.