Source: NORTH CAROLINA STATE UNIV submitted to
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Jul 1, 2012
Project End Date
Sep 30, 2013
Grant Year
Project Director
Jacob, M.
Recipient Organization
Performing Department
College of Vet Medicine
Non Technical Summary
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.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
To better understand the food safety threat posed by Shiga Toxin-Producing E. coli (STEC) in North Carolina meat goats, we aim to determine the prevalence of E. coli O157 in the feces and carcasses of North Carolina meat goats at harvest, compare the Pulsed-Field Gel Electrophoresis (PFGE) profile of isolates obtained in the feces and on carcasses to determine their relationship, and screen the feces of goats for the presence of six non-O157 STEC serotypes (O26, O45, O103, O111, O121 and O145). Information from these results may influence the pre- and post-harvest intervention strategies of meat goat processing and provide preliminary data on the public health risk of meat goats in North Carolina. It is expected that the results generated from this project will be presented at one state or national conference and the results will be published in a peer-reviewed scientific journal.
Project Methods
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.

Progress 07/01/12 to 09/30/13

Target Audience: Public Health industry and Goat Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This provided an opportunity for an undergraduate student to wrok in a research environment at the College of Veterinary Medicine and prepare for a decision as to attend a veterinary college. The student contributed in sample collection and laboratory processing. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

What was accomplished under these goals? We evaluated the prevalence and genetic relatedness of STEC in goats at a regional processing plant. Fecal, hide and carcass samples were collected from 300 meat goats presented for harvest. All samples were evaluated by routine culture techniques to determine the presence of E. coli O157:H7. Samples were collected over nine collection days during a twelve week period beginning in August 2012. Prevalence of E. coli O157:H7 was 11.1%, 2.7%, and 2.7%, in feces, hides, and carcasses respectively. All E. coli o157:H7 isolates were evaluated by Pulsed Field Gel Electrophoresis (PFGE) to determine their genetic relatedness, and to make observations about the epidemiology of the organisms within the processing plant. Sixteen PFGE subtypes were identified among 49 E. coli O157:H7 isolates, some of which were present on multiple sample types or collection days. This was the first report of E. coli O157:H7 on goat meat products in the United States, and there was evidence for contamination within the processing plant. Genotyping of E. coli O157:H7 isolates also indicated that these isolates may be important human pathogens and work to assess mitigation strategies after processing is warranted. In addition, feces from the goats were used as a template for a non-O157 multiplex PCR capable of detecting other STEC serogroups and virulence genes of public health importance. All six major non-O157 serogroups were detected by PCR in our samples, and 14.5% of samples were positive for at least one serogroup. Prevalence of O26 was highest with 6.4% of goat fecal samples positive. The prevalence of O45 was 3.4%, O103 was 4.4%, O111 was 4.1%, O121 was 1.4% and O145 was 3.0%. Twenty-two of 296 fecal samples (7.4%) had more than one non-O157 serogroup detected in the feces. Two samples had evidence of three non-O157 STEC serogroups. This was the first report in the U.S. of goats shedding non-O157 STEC. Goats appear to be an important reservoir for STEC.