Veterinary and Biomedical Sciences, Department of

 

Date of this Version

Fall 12-1-2015

Document Type

Article

Comments

A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Integrative Biomedical Sciences, Under the Supervision of Professor Rodney A. Moxley. Lincoln, Nebraska: December, 2015

Copyright (c) 2015 Zachary R. Stromberg

Abstract

Shiga toxin-producing Escherichia coli (STEC) are enteric pathogens of humans. Cattle serve as a reservoir and harbor STEC in their intestines. Intimin-positive STEC are referred to as enterohemorrhagic E.coli (EHEC). Seven serogroups (O26, O45, O103, O111, O121, O145, and O157) account for the majority of illness due to STEC and are hereafter referred to as STEC/EHEC-7. To improve detection, enrichment broths were compared for supporting growth of STEC-7 and STEC O104:H4 (STEC-8). In pure culture, STEC enriched in trypticase soy broth (TSB) had significantly greater growth compared to TSB containing antimicrobials. In fecal samples, E. coli broth enrichment yielded growth of STEC-8 that was significantly greater than in TSB. Optimized culture conditions allow for greater detection of EHEC-7 in cattle. To determine the prevalence of EHEC-7 in feedlot cattle, culture-based methods and molecular screening assays were used. In 576 feedlot cattle, EHEC-7 prevalence in hide samples as detected by NeoSEEK (NS) was 80.7% compared to 1.2% by culture. The prevalence of EHEC-7 on carcasses was 6.0% detected by NS. Additionally, EHEC-7 prevalence was determined in 100 culled dairy cows. The EHEC-7 prevalence in feces, hides, and carcasses, respectively, was 6.5%, 15.6%, and 1.0% by culture, and 25.9%, 64.9%, and 7.0% by NS. These studies provide evidence that EHEC are ubiquitous on cattle hides and to a lesser extent feces and carcasses. Given the discordant results, continued improvement in EHEC-7 detection methods is needed. Comparison studies were performed using CHROMagar STEC, Possé differential agar (Possé), Possé with modified antimicrobials, STEC heart infusion washed blood agar with mitomycin C (SHIBAM), and SHIBAM with modified antimicrobials. CHROMagar STEC performed better than Possé or SHIBAM for detection of EHEC-7 in cattle feces, but modifications of the antimicrobials in the latter two media resulted in significant improvements. STEC attachment facilitates colonization of the intestine. All STEC-8 strains tested adhered to bovine colonic explants and Caco-2 cells. One strain invaded both bovine colonic epithelial cells and Caco-2 cells. STEC O104:H4 had significantly higher levels of adherence on Caco-2 cells compared to most STEC. Interventions which block adherence may be effective for pre-harvest control.

Advisor: Rodney A. Moxley

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