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Brandon Luedtke

Comparison of methods for the enumeration of enterohemorrhagic Escherichia coli from veal hides and carcasses
, PhD
Research Microbiologist
Agricultural Research Service
Roman L. Hruska U.S. Meat Animal Research Center, USDA
Clay Center, Nebraska 68933-0166, USA

Brandon E. Luedtke and Joseph M. Bosilevac

The increased association of veal calves with enterohemorrhagic Escherichia coli (EHEC) has led the United States Department of Agriculture Food Safety and Inspection Service to require the reporting of veal meat contaminated with O157:H7 and/or the Top 6 serogroups (O26, O45, O103, O111, O121, and O145) separately from beef cattle. This contamination likely occurs via the transmission of STEC from the veal hide to the carcass. However, detection and enumeration methods for determining the prevalence and abundance of STEC are lacking. Here we compared the ability of a molecular based most probable number assay (MPN) and qPCR to detect and enumerate EHEC from veal hides at that abattoir and the resulting pre-intervention carcasses. Digital PCR (dPCR) was used to validate the MPN and qPCR results for select samples. For each enumeration method, 95 carcass and hide pre-enrichment samples were utilized in parallel assays targeting the EHEC associated E. coli attaching and effacing gene-positive conserved fragment 1 (ecf1). Using the MPN assay, total EHEC was enumerable in 46 (48%) of the hide samples and ranged from approximately 4 to greater than 4,255 CFUs/mL (95% CI 0.6-18,000 CFUs/mL). The qPCR assay was able to enumerate total EHEC in 23 of the samples enumerated by the MPN assay and 7 (7%) samples not enumerable by the MPN assay and had a range of approximately 8 to 22,853 CFUs/mL. The carcass samples had lower amounts of EHEC with a range of approximately 0.5 to 3 CFUs/mL (95% CI 0.05-14 CFUs/mL) in 26 (29%) of the samples enumerable by MPN. For the qPCR assay, the carcass samples ranged from approximately 12 to 826 CFUs/mL for 16 (17%) samples with an enumerable amount of EHEC. The correlation coefficient between all of the enumeration methods indicated a weak relation, which was likely due to the samples having an amount of total EHEC below the reliable limit of detection for the qPCR and dPCR assays. Interestingly, after enrichment, 82% of the hide samples and 94% of the carcass samples had a detectable amount of total EHEC by qPCR. The average Cq value for the ecf1 target was 30.9 (Cq range 24.1-37.6) and 34.7 (Cq range 31.1-38.9) in the hide and carcass enrichments, respectively. From our analysis, the MPN assay provided a higher percentage of enumerable hide and carcass samples, however determining an appropriate dilution range and the limited throughput offers additional challenges to large sample sets.