Evaluation of Cattle for Naturally Colonized Shiga Toxin-Producing Requires Combinatorial Strategies.

Shiga toxin-producing (STEC) serogroups O157, O26, O103, O111, O121, O145, and O45 are designated as food adulterants by the U.S. Department of Agriculture-Food Safety and Inspection Service. Cattle are the primary reservoir of these human pathogens. In this study, 59 Angus crossbred heifers were tested specifically for these seven STEC serogroups using a combination of standard culture, serological, PCR, and cell cytotoxicity methods to determine if comparable results would be obtained. At the time of fecal sampling, the animals were approximately 2 years old and weighed 1000-1200 lbs. The diet comprised of 37% ground alfalfa hay, 25% ground Sudan hay, and 38% ground corn supplemented with trace minerals and rumensin with ad libitum access to water. Non-O157 STEC were isolated from 25% (15/59) of the animals tested using a combination of EC broth, CHROMagar STEC, and Rainbow Agar O157. Interestingly, the O157 serogroup was not isolated from any of the animals. Non-O157 STEC isolates were confirmed to be one of the six adulterant serogroups by serology and/or colony PCR in 10/15 animals with the predominant viable, serogroup being O103. PCR using DNA extracted from feces verified most of the colony PCR results but also identified additional virulence and O-antigen genes from samples with no correlating culture results. Shiga toxin- (Stx-) related cytopathic effects on Vero cells with fecal extracts from 55/59 animals could only be associated with the Stx gene profiles obtained by fecal DNA PCR and not culture results. The differences between culture versus fecal DNA PCR and cytotoxicity assay results suggest that the latter two assays reflect the presence of nonviable STEC or infection with STEC not belonging to the seven adulterant serogroups. This study further supports the use of combinatorial culture, serology, and PCR methods to isolate viable STEC that pose a greater food safety threat.