Microbial Ecology of Shiga-Toxigenic Escherichia Coli in a Cattle Herd in California

Non-Technical Summary: Shiga-toxigenic Escherichia coli (STEC) are a significant public health threat in many countries. Cattle (and other ruminants) are the primary reservoirs for STEC and human disease is most often associated with consumption of foods contaminated by cattle manure. Outbreak cases associated with food have been linked to hamburger, unpasteurized apple cider and apple juice, hotdogs, raw milk, raw potatoes, and salad bar items such as ranch dressing, pea salad, and cantaloupe. Recent outbreaks associated with consumption of lettuce grown in California has resulted in heightened interest from California Department of Food and Agriculture and California Department of Health Services to develop research programs designed to investigate the microbial ecology of STEC and E. coli O157:H7 in cattle. Given that there are approximately 720,000 adult beef cows in California (18) grazing on approximately 38 million acres of rangelands, knowledge of the ecology of these important human pathogens is essential. This study is designed to follow one herd of beef cattle throughout a period to determine signifiant associatons with shedding of STEC and E. coli O157-H7. <P> Approach: With the assistance of UC Extension livestock advisors and the CA Cattlemen's Association, a list of cow-calf producers in the Salinas Valley will be created. An informational meeting will present details of the study and solicit cooperation and 12 farms will be included in the project. Each identified herd will be sampled 12 times and up to 50 samples will be collected from each farm at each sampling: 40 fecal samples (freshly passed manure) and 10 environmental samples (water from troughs and natural waterways, feeds, soils). One gram of fresh feces will be mixed with 25 ml of trypticase soy broth (TSB) containing novobiocin (20 ug/ml) and vancomycin (40 ug/ml) and incubated for 18-24 hr at 37 C. Serial dilutions of 10-5 to 10-7 of fecal suspension are then plated onto sorbitol-MacConkey with novobiocin and vancomycin as before, incubated for 18-24 hr at 37 C, with up to 10 non-sorbitol-fermenting colonies subcultured onto MUG plates if present. Following 18 hr incubation at 37 C, colonies will be screened for UV fluorescence. MUG+ isolates will confirmed as E. coli using standard biochemical tests, then subjected to PCR for detection of Stx-1 and 2 (20). SLTEC+ isolates will be serotyped at the Escherichia coli Reference Center at Pennsylvania State University. A standardized questionnaire will be developed and applied to the herd managers at the initial visits. The questionnaire will consist of the following sections: a) demographic information, such as location, herd size; b) management practices, such as calving management, pasture stocking density, etc; c) biosecurity protocols, including perimeter control, personnel management, and vaccination programs; d) presence and estimated density of wildlife. At subsequent visits, changes in pasture or stocking density or feed supplementation will be noted. During the year, date and amount of rainfall and daily minimum and maximum temperatures will be recorded. The primary outcome variable will be prevalence of samples positive for both STEC and E. coli O157:H7 at each sample collection. Ninety-five percent confidence intervals will be calculated. Predictor variables, including season, temperature and rainfall within the previous 7 days of sampling, pasture location and type, stocking density, presence of calves, and feed supplements, will be summarized and descriptive statistics calculated. A generalized linear model will be constructed to determine significant associations that may exist between shedding prevalence and potential risk factors. A further study will be performed to study the effects of various management practices on prevalence of STEC in the herds. There is evidence that low rumen pH may decrease survival of STEC within the rumen. Range cattle eating primarily forages typically have higher rumen pH values than cattle on grain-based diets, so addition of supplements that will reduce rumen pH may be a management technique that could help reduce environmental loading of STEC. New vaccine technology, utilizing purified extracts of siderophore receptors and porins (SRPs) has been shown to decrease the fecal shedding of E. coli O157:H7 in a challenge situation.