Abstracts

Cattle are a reservoir for major Shiga toxin-producingEscherichia coli (STEC), which includes STEC O157 and the top sixnon-O157 serogroups (STEC-6; O26, O45, O103, O111, O121, O145).Collectively known as the STEC-7, these organisms are harbored inthe hindgut and shed in the feces of cattle, which can contaminatehides. The de-hiding step during beef cattle processing canintroduce fecal contaminants from the hide onto the carcasssurface, creating the potential for contaminated beef products. TheSTEC-7 have been declared by the USDA-Food Safety and InspectionService as adulterants in ground beef and non-intact beef products,and are monitored during beef cattle processing. However, many ofthe culture- and PCR-based tests for detection and/orquantification of the STEC, particularly of the STEC-6, are notestablished or require improvement and also virulencecharacteristics of STEC strains from cattle have not been fullyanalyzed. Therefore, the following studies were conducted: 1.Immunomagnetic separation (IMS)-based culture-method for detectionof STEC-6 in cattle feces was developed and compared to a PCR-basedmethod; 2. Detection sensitivity of pooled vs. individual IMS beadsfor isolation STEC-6 from cattle feces was evaluated; 3. Real-timePCR assay, based on the clustered regularly interspaced shortpalindromic repeat sequence polymorphisms (CRISPR), was developedand validated for serotype-specific detection and quantification ofSTEC O157:H7 in cattle feces; 4. Virulence gene profiles of bovineenterohemorrhagic (EHEC), enteropathogenic (EPEC) and putativenon-pathotype E. coli O103 strains were examined with whole genomesequence (WGS)-based comparative analysis; 5. Prevalence andconcentration of STEC-7 of fed-beef, cull beef and cull dairycattle were determined. The culture and PCR methods detected allsix serogroups in samples negative by the other method. Based onnoninferiority tests, detection with pooled IMS beads was notinferior to detection with individual beads. Detection limits ofthe CRISPR-based qPCR assay for cattle feces spiked with purecultures were 2.1 x 10 and 2.3 x 10 colony-forming units/g beforeand after enrichment, respectively. WGS-based analysis of E. coliO103 strains revealed key differences in the virulomes andmobilomes of EHEC, EPEC, and putative non-pathotype strains. Theprevalence study revealed that a significantly higher (P < 0.01)proportion of hide samples from fed beef cattle (4.8%) werepositive for STEC O157:H7, compared to samples from cull beef(1.6%) or cull dairy (0.2%); the majority of quantifiable STECO157:H7 from each cattle type was at concentrations between 3 to 4log CFU/100 cm. These data contribute to a knowledge gap onprevalence and concentration of STEC-7 and surrogate bacteria oncattle hides and carcasses, respectively. Furthermore, thedevelopment and refinement of culture- and PCR-based screeningassays may lead to increased surveillance of major STEC serogroups,especially if the potential of WGS-based comparative genomics inAdvisors/Committee Members: Tiruvoor G. Nagaraja.