Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Cattle are a reservoir for major Shiga toxin-producing Escherichia coli (STEC), which includes STEC O157 and the top six non-O157 serogroups (STEC-6; O26, O45, O103, O111, O121, O145). Collectively known as the STEC-7, these organisms are harbored in the hindgut and shed in the feces of cattle, which can contaminate hides. The de-hiding step during beef cattle processing can introduce fecal contaminants from the hide onto the carcass surface, creating the potential for contaminated beef products. The STEC-7 have been declared by the USDA-Food Safety and Inspection Service as adulterants in ground beef and non-intact beef products, and are monitored during beef cattle processing. However, many of the culture- and PCR-based tests for detection and/or quantification of the STEC, particularly of the STEC-6, are not established or require improvement and also virulence characteristics of STEC strains from cattle have not been fully analyzed. Therefore, the following studies were conducted: 1. Immunomagnetic separation (IMS)-based culture-method for detection of STEC-6 in cattle feces was developed and compared to a PCR-based method; 2. Detection sensitivity of pooled vs. individual IMS beads for isolation STEC-6 from cattle feces was evaluated; 3. Real-time PCR assay, based on the clustered regularly interspaced short palindromic repeat sequence polymorphisms (CRISPR), was developed and validated for serotype-specific detection and quantification of STEC O157:H7 in cattle feces; 4. Virulence gene profiles of bovine enterohemorrhagic (EHEC), enteropathogenic (EPEC) and putative non-pathotype E. coli O103 strains were examined with whole genome sequence (WGS)-based comparative analysis; 5. Prevalence and concentration of STEC-7 of fed-beef, cull beef and cull dairy cattle were determined. The culture and PCR methods detected all six serogroups in samples negative by the other method. Based on noninferiority tests, detection with pooled IMS beads was not inferior to detection with individual beads. Detection limits of the CRISPR-based qPCR assay for cattle feces spiked with pure cultures were 2.1 x 10³ and 2.3 x 10⁰ colony-forming units/g before and after enrichment, respectively. WGS-based analysis of E. coli O103 strains revealed key differences in the virulomes and mobilomes of EHEC, EPEC, and putative non-pathotype strains. The prevalence study revealed that a significantly higher (P < 0.01) proportion of hide samples from fed beef cattle (4.8%) were positive for STEC O157:H7, compared to samples from cull beef (1.6%) or cull dairy (0.2%); the majority of quantifiable STEC O157:H7 from each cattle type was at concentrations between 3 to 4 log CFU/100 cm². These data contribute to a knowledge gap on prevalence and concentration of STEC-7 and surrogate bacteria on cattle hides and carcasses, respectively. Furthermore, the development and refinement of culture- and PCR-based screening assays may lead to increased surveillance of major STEC serogroups, especially if the potential of WGS-based comparative genomics in identifying novel gene targets can be harnessed.

Master of Science / Department of Diagnostic Medicine/Pathobiology / T. G. Nagaraja / Shiga toxin-producing E. coli O157 is a major foodborne pathogen. The organism colonizes the hindgut of cattle and is shed in the feces, which serves as a source of contamination of food. Generally, cattle shed E. coli O157 at low concentrations (≤ 10[superscript]2 CFU/g), but a subset of cattle, known as “super-shedders”, shed high concentrations (>10[superscript]3 CFU/g) and are responsible for increased transmission between animals and subsequent hide and carcass contamination. Therefore, concentration data are an important component of quantitative microbial risk assessment. A four-plex quantitative PCR (mqPCR) targeting rfbE[subscript]O157, stx1, stx2 and eae was developed and validated to detect and quantify E. coli O157 in cattle feces. Additionally, the applicability of the assay to detect E. coli O157 was compared to conventional PCR (cPCR) targeting the same four genes, and a culture method. Specificity of the assay to differentially detect the four genes was confirmed. In cattle feces spiked with pure cultures, detection limits were 2.8 x 10[superscript]4 and 2.8 x 10[superscript]0 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. Of the 100 samples that were randomly picked from the 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from the 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar’s chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. Applicability of the assay to quantify E. coli O157 was determined with feedlot cattle fecal samples (n=576) and compared to spiral plate method. Fecal samples that were quantifiable for O157 by mqPCR (62/576; 10.8%) were at concentrations of ≥ 10[superscript]4 CFU/g of feces. Only 4.5% (26/576) of samples were positive by spiral plate method, with the majority (17/26; 65.4%) at below 10[superscript]3 CFU/g. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect and quantify E. coli O157 in cattle feces.