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Identification of Bovine Reservoirs of Human Pathogenic Non-O157 Shiga Toxin-Producing E. Coli

Investigators
Wheeler, Tommy Lee; Shackelford, Steven; Kalchayanand, Norasak; Bosilevac, Joseph; Arthur, Terrance
Institutions
USDA - Agricultural Research Service
Start date
2012
End date
2013
Objective
The overall goal of this project is to determine the prevalence, level, and types of non-O157 Shiga toxin-producing E. coli (STEC) in cattle from different production systems at harvest and determine if production system or regional variations in non-O157 STEC serogroups exist. To accomplish this goal, sample collections will be made at numerous beef processing plants located across the United States in order to generate an extensive set of samples from the most relevant commercial sources. Targeted locations include plants in four regions of the country: West, South, Midwest and East. Samples (n=3,000) will consist of feces collected from colons during processing in order to have samples that accurately reflect the production environment from which the animal originated. Processing plants that harvest fed cattle and cull cows and bulls from dairy and beef sources will be visited for sample collection. Feces will be screened before and after enrichment for the presence of STEC using a semi-quantitative molecular amplification method that detects the presence of Shiga toxin genes. This will not only identify cattle that carry STEC but also identify those that carry higher loads and provide an estimate of the level of STEC that is shed. Samples found positive for STEC will be subjected to culture isolation for the Top-6 (O26, O45, O103, O111, O121, O145) serotypes as well as all other STEC serotypes. All STEC isolates will be serotyped and characterized for genes that are known to contribute to increased virulence.
More information
Approach: Sample collections will be made at numerous beef processing plants located across the United States in order to generate an extensive set of samples from the most relevant commercial sources. Targeted locations include California, Wisconsin, Nebraska, Texas, and Pennsylvania. Samples will consist of feces collected from colons during processing in order to have samples that reflect the production environment from which the animal originated. Processing plants that harvest fed cattle and cull cows and bulls from dairy and beef sources will be visited for sample collection. Feces will be screened before and after enrichment for the presence of STEC using a semi-quantitative molecular amplifiation method that detects the presence of Shiga toxin genes. This will not only identify cattle that carry STEC but also identifying those that carry higher loads and provide an estimate of the level of STEC that is shed. Samples found positive for STEC will be subjected to culture isolation for the Top-6 serotypes as well as all other STEC serotypes. All STEC isolates will be serotyped and characterized for genes that are known to contribute to increased virulence. These genes are Shiga toxin 1 (stx1); Shiga toxin 2 (stx2); intimin (eae); EHEC hemolysin (ehx); non-locus of enterocyte effacement effector (nle) products: nleB, nleC, nleF and nleG-2; subA (the prototype Subtilase cytotoxin); chuA, (an outer membrane heme uptake protein); irp2, (an iron repressible protein carried in a high-pathogenicity island); saa (associated with pathogenic STEC adherence); and other type III secreted effectors of EHEC (espK, and Ibe).

Status: (As of October 2012)
Investigators have made three collection trips to processing plants located in two different portions of the US. There are five more collection trips scheduled to the Texas, California, Wisconsin, Nebraska and Washington state areas. To date, samples from 404 fed cattle, 44 cull beef cattle and 325 dairy cattle have been collected. The differences in the numbers of each type of cattle sampled are due to the types of cattle presenting for slaughter at each location visited so far. It is still anticipated that at least 1,000 head of each type of cattle will be sampled, given the known make up of cattle typically slaughtered at the scheduled processing plants. If proper numbers of samples cannot be obtained within the planned sampling trips, three additional plants have been identified as potential sources.

Investigators are using additional methods to provide STEC snp information. By using this data in addition to the data collected, investigators can more readily identify samples that likely contain a stx and eae positive pathogenic Top 6 STEC, rather than one of many interfering background E. coli. As far as preliminary results go, an initial 176 samples have been fully screened. Of those 87 were identified to be positive for E. coli O157:H7, with 119 identified as potential positive for a Top 6 STEC. 53 samples screened positive for both O157:H7 and potential positive STEC. Interestingly, a number of samples appear to contain E. coli of Top 6 serogroups but lack either stx or eae: Top 6+eae n=20; Top 6+stx n=16. IMS and continued culture isolation from these samples will determine the types of E. coli present and whether they may be pathogens.

Funding Source
Fndn. for Meat and Poultry Research and Education
Project source
View this project
Project number
3040-42000-014-07-T
Accession number
422763
Categories
Escherichia coli