Detection of Shiga Toxin-Producing Escherichia Coli in North Carolina Meat Goats at Harvest

Non-Technical Summary:<br/>
Shiga toxin-producing E. coli(STEC)are a subset of E. coli that produce Shigella-like toxin and often associated with foodborne illness. Over 100 STEC serotypes are known to cause human disease with symptoms ranging from mild diarrhea to hemolytic uremic syndrome. In 2009, an incidence of 0.99 per 100,000 U.S. people was reported for E. coli O157 by the CDC. Non-O157 STEC serotypes including O26, O45, O103, O111, O121 and O145 are becoming increasingly acknowledged as a public health concern with a CDC reported incidence of 0.6 per 100,000 U.S. people in 2009; this estimate likely understates the importance of non-O157 STEC, and should increase as we improve our clinical awareness and diagnostic capabilities. Ruminants are considered the primary reservoir for E. coli O157, and non-O157 STEC are frequently found in animal feces. These organisms colonize or transiently pass thru the gastrointestinal tract and shed in the feces where they can contaminate animal hides, carcasses or other food and water products. Direct transmission of STEC can also occur, as highlighted locally with two E. coli O157 outbreaks associated with animal contact at the North Carolina State Fair. The epidemiology and ecology of E. coli O157 is well studied in cattle; prevalence of E. coli O157 in the feces has been established and correlated to prevalence on beef carcasses at slaughter. Although goats are known reservoirs for E. coli O157, the epidemiology has not been as well described, particularly in the United States. In addition, newer studies evaluating the prevalence and importance of specific non-O157 STEC in the feces and on the carcasses of cattle have not been conducted for meat goats. There is, however, evidence that up to 56% of goats are positive for at least one STEC, so identifying which serotypes are most prevalent is relevant and important for assessment of goats as reservoirs of STEC, and future intervention strategies. The meat goat industry is important to American and North Carolina agriculture. As of January 2012, the meat and other goat inventory for the United States was 2.36 million. In North Carolina, the meat goat industry continues to expand as the state becomes more multicultural. Better understanding of the risks associated with goat meat has implications on the health and safety of U.S., and specifically North Carolina, citizens. Commonly reported, previously published techniques for assessing O157 and non-O157 prevalence in meat goats will be used in all three objectives of this study. Achieving the three objectives will demonstrate the extent of E. coli O157 in the feces of North Carolina goats, evaluate the potential risk of goat meat products, and allow us to identify potential non-O157 STEC serotypes to target in future work.
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Approach:<br/>
Three hundred meat goats presented for harvest at a North Carolina processing facility will be selected for inclusion in this study. Sampling will occur over 6 weeks, with 50 animals selected for sampling on one day each week. Fecal samples will be collected from each animal at the abattoir prior to processing by rectal grab. Each sample will be placed into a bag and transported on ice to the laboratory for processing. Carcass samples will be collected by swabbing a 500-cm2 area on the right side of each goat carcass with a Speci-Sponge soaked with 15-ml of buffered peptone water. After collection, carcass samples will be placed back into a bag and sent to the laboratory. All sample collectors will wear nitrile gloves, with a new glove used for each fecal and carcass sample to avoid cross-contamination. All animals will be identified by a distinguishing number so fecal and carcass samples can be matched after collection. For isolation of E. coli O157 from fecal samples, approximately 1 g of feces will be placed into an enrichment tube containing 9 ml of Gram Negative broth with antibiotics. The sample will be vortexed and incubated for 6 h. After incubation, immunomagnetic bead separation (IMS) will be performed on 1 ml of enrichment, followed by plating onto sorbitol MacConkey agar with antibiotics (CT-SMAC). Plates will be incubated for 16-18 h. After incubation, up to six sorbitol negative colonies will be evaluated for indole production and latex agglutination for the O157 antigen, and confirmed by multiplex PCR. All isolates will be frozen for future analysis. For isolation of E. coli O157 from carcass swabs, sponge samples will be enriched in 90 ml of brilliant green bile broth for 10 h. IMS will be performed on 1 ml of enrichment and plated onto CT-SMAC. Further isolation, identification, and storage of isolates will be as described for fecal samples. Descriptive statistics will be used to report prevalence estimates, and statistical models will be developed to evaluate differences in fecal or carcass prevalence between collection weeks. Statistical significance will be P less than 0.05. To compare the PFGE profile of E. coli O157 isolates obtained from the feces and on carcasses of meat goats will be grown on blood agar plates for 24 h. Pulsed-field gel electrophoresis analysis with XbaI restriction enzyme digestion will follow the standardized PulseNet PFGE protocol. Descriptive statistics describing the proportion of isolates representing different PFGE subtypes will be reported. In addition, the feces of all animals will be screened for the presence of 6 non-O157, STEC serogroups using multiplex PCR. One gram of feces will be enriched in 9 ml of EC broth and incubated for 6 h. After enrichment, DNA will be extracted from fecal suspensions using a commercially available extraction kit. Extracted DNA will be stored frozen until it can be sent on ice to Kansas State University for analysis. The multiplex PCR assay will be performed as previously described. Results will be reported back for each sample for analysis and interpretation. Statistics describing the prevalence of each non-O157 STEC serotype detected will be reported.