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E. coli O157:H7 and super-shedding. E. coli O157:H7 is an important food-borne pathogen which poses a serious public health concern and financial burden. Cattle are the principal animal reservoir of E. coli O157:H7 and while rumen has been shown to harbor this pathogen on occasion, the rectal-anal junction (RAJ) has been shown to be the predominant colonization site. Once colonized, an animal can shed various amounts of E. coli O157:H7 in the feces. Super-shedders (RAJ colonized at greater than 10,000 CFU/g) have a significant effect on contamination of the cattle hide and carcass and are reported to be responsible for increased transmission of E. coli O157:H7 within production environments. Therefore, it is critical to identify, minimize, or eliminate super-shedders from the cattle population in order to reduce E. coli O157:H7 transmission and beef carcass contamination for enhancing food safety. Several approaches for reducing E. coli O157:H7 colonization of the cattle gastrointestinal tract (GIT) have been experimentally tested, such as different feeding regiments, feed additives, probiotics, and vaccines. To date, most of these studies have either been inconclusive or treatments have only modestly affected colonization. More importantly, none of these interventions have tested whether the prevalence of super-shedding is reduced in cattle populations. This is significant, as modeling studies suggest that possibly as high as 96% of E. coli O157:H7 isolates originate from super-shedding animals. It is evident that a more thorough understanding of the factors promoting super-shedding is needed before we can design effective evidence-based methods of reducing transmission of STEC from cattle populations to the food supply. The super-shedding phenomenon can be broken down into three principle components: (1) phylogenetic lineage of the colonizing O157:H7 strain, (2) the community composition of the microflora of the rectal-anal junction, and (3) the innate and adaptive immune response of the host. This proposal is designed to determine the contribution of strain type in super-shedding. <P> The project objectives are: 1. Identify cattle shedding E. coli O157:H7 at levels (> 10,000 CFU/g or swab). 2. Isolate E. coli O157:H7 strains from super-shedders. 3. Characterize strains to identify super-shedder specific traits.
Approach:
Isolation of E. coli O157:H7. We propose to collect fecal swab samples from cattle at slaughter to obtain samples from 100 super-shedders. As super-shedders typically constitute only a small fraction of cattle (2-5%), over 2000 samples will be needed. Samples will be collected by swabbing the rectal-anal junction, then placing the swabs in 15 ml conical tubes containing 4 ml of tryptic soy broth. Tubes will be stored on ice for shipment to the laboratory. Upon arrival at the lab, the culture sample tubes will be vortexed vigorously for 30 sec then 50 ul aliquots will be plated for enumeration of STEC using a spiral plater. The limit of detection for this method of enumeration is 80 CFU/swab and the spiral plating system allows for a 6-log dynamic range in detection, therefore, the levels of super-shedding (> 10,000 CFU/swab) will be accommodated. Samples will be plated onto CHROMAgar O157 with 5 mg/L novobiocin and 2.5 mg/L potassium tellurite (ntCHROMAgar O157). When detected, counts of O157:H7 will be reported as colony forming units (CFU) per swab. Animals will be classified as super-shedders when counts are greater than 10,000 CFU/swab, shedders when counts are between 80 (the limit of detection) and 10,000 CFU/swab. Strain characterization. E. coli O157:H7 strains from supershedder samples will be subjected to PFGE subtyping, lineage-specific polymorphism assay (LSPA), lineage analysis based on tir gene SNPs, Shiga toxin subtyping, determination of attachment factors, and quantitation of Shiga toxin expression by qPCR.