title: Further development of sample preparation and detection methods for O157 and the top 6 non-O157 STEC serogroups in cattle feces.
abstract: Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens responsible for outbreaks of human infections worldwide. Ruminant livestock harbor STEC in their intestinal tract, and through fecal contamination possess the potential to compromise the safety of food and water. As a human health safety risk, STEC detection methods on beef carcasses and trim are needed as mandated by the USDA-FSIS. In order to monitor STEC prior to harvest and human consumption, our goal was to evaluate and/or improve detection of seven STEC serogroups in cattle feces. In comparison to traditional approaches, sample processing methods in bovine feces were evaluated using a multi-factorial Latin square design which involved freezing or freeze drying feces. Autoclaved versus non-autoclaved feces were spiked with O26:H11 or O157:H7 serotypes in various dilutions and enriched for up to 6h. Each hour, enriched aliquots were compared using traditional culture methods and quantitative polymerase chain reaction (qPCR). Furthermore, a 7-serogroup multiplex PCR (mPCR) was developed to detect O26, O45, O103, O111, O121, O145 and O157 serogroups simultaneously. The diagnostic sensitivity of our mPCR assay following 6h enrichment was superior (10CFU/g across all serogroups) compared to a previously established PCR assay (10CFU/g for O26, and O103; ≥10(4)CFU/g for all other serogroups). Obtaining viable isolates appeared to be limited by the efficiency of current immunomagnetic separation (IMS) methods, which ranged from 20 to 100% effectiveness at retrieving colonies depending on serogroup. After IMS, 70 putative STEC isolates were screened for Shiga toxin and attachment genes by mPCR. Sixty-five isolates contained one or both Shiga toxin genes.
authors.affiliation: University of Lethbridge, Lethbridge T1K 3M4, Alberta, Canada; Alberta Agriculture and Rural Development, Agriculture Centre, Lethbridge T1J 4V6, Alberta, Canada; Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge T1J 4B1, Alberta, Canada.
authors.forename: Cheyenne C
authors.initials: CC
authors.lastname: Conrad
authors.affiliation: Alberta Agriculture and Rural Development, Agriculture Centre, Lethbridge T1J 4V6, Alberta, Canada.
authors.forename: Kim
authors.initials: K
authors.lastname: Stanford
authors.affiliation: Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge T1J 4B1, Alberta, Canada.
authors.forename: Tim A
authors.initials: TA
authors.lastname: McAllister
authors.affiliation: University of Lethbridge, Lethbridge T1K 3M4, Alberta, Canada.
authors.forename: James
authors.initials: J
authors.lastname: Thomas
authors.affiliation: Alberta Agriculture and Rural Development, Agriculture Centre, Lethbridge T1J 4V6, Alberta, Canada. Electronic address: tim.reuter@gov.ab.ca.
authors.forename: Tim
authors.initials: T
authors.lastname: Reuter
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000382:microbiology; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D005243:Feces / Q000382:microbiology*; D005516:Food Microbiology / Q000379:methods; D059022:Food Safety / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D065288:Serogroup; D054323:Shiga-Toxigenic Escherichia coli / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D013997:Time Factors
keywords: Cattle; Detection; Feces; Multiplex PCR; Non-O157; Shiga toxin producing E. coli (STEC)