Ecology of E. Coli O157:H7 in Beef Cow-Calf Operations from Ranch to Feedlot

NON-TECHNICAL SUMMARY: The long term goal of this project is to develop effective on?farm strategies for E. coli O157. Our current research goals are to investigate the genetic diversity of E. coli O157, validate new diagnostic methods, and investigate survival mechanisms in the gut. We will investigate the role of flies and cattle feed in the survival and transmission of E. coli O157 in cattle herds.

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APPROACH: <BR> 1. To determine the genetic diversity of E. coli O157 within individual fecal samples. <BR> 2. To compare the genetic subtypes of E. coli O157 among isolates from cattle feces, water, and cattle feed within and between feedlots. <BR> 3. To compare methods for phenotypic and genotypic characterization of E. coli O157. <BR> <BR> We will use pulse-field gel electrophoresis to genetically characterize isolates collected from the feces of cattle on commercial operations (specific objective 1) and isolates from feedlot cattle feces, feed, and water which have been categorized and stored from our previous field studies (specific objectives 2). Toxin profiles and antibiotic resistance patterns will be compared within and between genotypes. Field samples collected as part of our ongoing studies will be cultured as per our standard diagnostic methods. Additionally, we will perform PCR diagnostics on 400 of the samples to compare the sensitivity and specificity of the two methods. <BR> <BR> 1. To compare growth of E. coli O157:H7 in an in vitro ruminal and hindgut (cecal and or colonic) fermentation systems. <BR> 2. To relate fermentation characteristics of the rumen and the hindgut (cecum and colon) on survival and growth of E. coli O157:H7. <BR> 3. To study the influence of dietary ingredients (fiber, starch, protein, NPN) and feed additives (antibiotics, probiotics, buffering compounds such as Sodium bicarbonate, MgO, bentonite) on growth of E. coli O157:H7.<BR> <BR> In vitro ruminal and hindgut fermentation systems will be used to study ruminal or hindgut factors that influence the survival and growth of E. coli O157:H7. Nalidixic acid-resistant strains of E. coli O157:H7 will be used to facilitate quantification of the organisms. The fermentation characteristics of the in vitro systems will be manipulated to identify factors that inhibit, reduce or promote growth of E. coli O157:H7. Feed samples will be collected from feed bunks of 40 commercial dairy farms and cultured for E. coli O157. Based on our research on culture methods for feed samples of feedlot cattle, we will incorporate two enrichment methods into our culture technique and interpret the results in parallel. Flies will be sampled from within the cattle pens on 40 commercial operations. The exterior of the flies will be sterilized, allowing culture of the intestinal contents for E. coli O157.

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PROGRESS: 2003/07 TO 2005/06<BR>
The mucosa in the rectoanal junction of the gastrointestinal tract has been suggested as the principal colonization site for E. coli O157 in cattle. It has been theorized that E. coli O157 in the feces was derived from the rectoanal junction as feces passed through this region. Therefore, we conducted a study to determine the genetic relatedness of E. coli 0157 in the mucosal region of the rectoanal junction in cattle to isolates from colonic contents or feces. To eliminate or minimize fecal contamination of the sample, we collected intact rectums from slaughtered cattle and the rectum was then cleansed free of visible feces. The region, 2 to 5 cm proximal to the rectoanal junction, was swabbed with an applicator. Incisions of the mucosa in the same region were made to swab the submucosa. Colonic contents were also sampled. Prevalence of E. coli 0157 in the colonic contents, feces, rectal mucosal surface, and rectal submucosal region was 21, 29, 54, and 34 percent, respectively. Sixty-two percent of the cattle were positive for E. coli 0157 in at least one sampling site and was higher (P < 0.05) than prevalence in any single site. Pulsed-field gel electrophoresis was used to compare clonal similarity among isolates. Sixty-seven animals had O157 isolates in the rectal mucosa and the feces of which 55 (82 percent) were identical (clonal similarity > 95 percent). Comparison of isolates in animals with O157 isolates in the colon contents and RAMS revealed that 61 percent had > 95 percent clonal similarity and there was 76 percent similarity between colonic and fecal isolates. Our results suggest that rectal mucosal swabs were more sensitive in detecting E. coli 0157 than fecal culture and that the majority of the rectoanal mucosal isolates were of similar genetic type to fecal or colonic content isolates. Also, a study was conducted to describe the prevalence and longitudinal distribution of E. coli O157 in feedlot cattle and feedlot environment. The pen floors, water tanks, other cattle in the feedyard, feed and bird feces in the feedlot environment were sampled for two weeks prior to entry of the study cattle. Twelve pens of cattle were sampled twice weekly in a single feedyard. At each sample time cattle feces, water from tanks in each pen, bunk feed, feed components, bird feces and flies were collected. Bunk feed samples were collected before and after cattle had access to the feed. Overall 28 percent of cattle fecal samples, 3.9 percent of bird fecal samples 25 percent of water samples 2.9 percent of fly samples, 1.25 percent of bunk feed before calf access and 3.25 percent of bunk feed samples after cattle had access to the feed were positive for E. coli O157. Genetic analysis of E. coli O157 isolates was done using PFGE of XbaI digested bacterial DNA. PFGE types identified in sampling of the feedlot prior to calf entry were different than the majority of types identified following calf entry. A single strain type predominated in the samples collected. It was first identified 5 days after entry of the first pen of cattle, and was subsequently identified in all pens.
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IMPACT: 2003/07 TO 2005/06<BR>
Escherichia coli O157 is a major cause of food borne illnesses in humans. Food products contaminated with cattle feces are a major source of E. coli O157 in outbreaks. Reducing the level and duration of fecal shedding in cattle will reduce the potential contamination of carcasses. The long term goal of our research is to identify means to control E. coli O157 at the farm level.

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PROGRESS: 2004/01/01 TO 2004/12/31<BR>
We evaluated rectoanal mucosal swab (RAMS) sampling technique for prevalence of E. coli O157 in feedlot cattle. The lymphoid tissue in the rectoanal junction of the gastrointestinal tract has been suggested as the principal colonization site for E. coli O157 in cattle. Samples collected by swabbing the rectoanal mucosa have been shown to be superior to fecal samples for detecting prevalence of E. coli O157. We completed a study to compare the two sampling techniques for determining prevalence of E. coli O157 in feedlot cattle (n=747) fed high-grain diets. Escherichia coli O157 was detected in 71 of 747 samples collected by RAMS and 35 of 747 samples tested by fecal culture. Of the 82 animals that tested positive, regardless of collection method, 87 percent were detected by the RAMS, but only 45 percent by the fecal culture. Rectoanal mucosal swab culture was more sensitive than the fecal sampling method for determining prevalence of E. coli O157 in feedlot cattle. Genomic fingerprints of isolates were analyzed by pulse-field electrophoresis (PFGE) to compare clonal similarity between RAMS and fecal isolates from the same animal. Of the 24 pairs of isolates evaluated, 20 had 100 percent similarity and 4 had greater than 95 percent similarity in PFGE banding patterns, suggesting that strains colonizing the rectoanal junction were the same as those from the feces. We determined the prevalence of E. coli O157:H7 in house flies from a cattle feedlot: House flies (HF) (n= 440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4 percent in HF collected from feed bunks and cattle feed storage, respectively. Escherichia coli O157:H7 counts ranged from 30 to 150,000 CFU among the positive flies. Polymerase chain reaction analysis of the E. coli O157:H7 revealed that 90.4, 99.2, 99.2 and 100 percents of isolates (n=125) possessed stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment.
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IMPACT: 2004/01/01 TO 2004/12/31<BR>
Escherichia coli O157 is an emerging cause of food borne illnesses in humans. Food products contaminated with cattle feces are a major source of E. coli O157 in outbreaks. Reducing the level and duration of fecal shedding in cattle will reduce the potential contamination of carcasses. The long term goal of our research is to identify means to control E. coli O157 at the farm level. The goals of our research are to develop and validate improved methods for the detection of E. coli O157 in cattle feces and environmental samples, to improve our understanding of the natural ecology of E. coli O157 in cattle operations, and to identify and test on-farm intervention strategies for control of E. coli O157.

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PROGRESS: 2003/01/01 TO 2003/12/31<BR>
We conducted a study to determine the genetic diversity of E. coli O157 within individual fecal samples. Previous work in our laboratory identified cattle fecal samples with more than one strain of E. coli O157. However, that study was not designed specifically to determine the genetic diversity within samples. Fecal samples were collected from dairy and beef cattle and E. coli O157 was isolated through standard culture techniques. A total of 601 samples were collected and 46 of those were confirmed to be E. coli O157. Samples with no fewer than 7 isolates were selected for subtyping by PFGE using the XbaI digestion enzyme. Twenty-seven samples containing 254 isolates were subtyped. These isolates displayed 19 unique types (=95% homogeneity) with 65 individual subtypes (100% homogeneity). Seven of the 27 samples contained multiple PFGE types. The analytical sensitivity of an improved E. coli O157:H7 culture method with a PCR-based 5' nuclease assay that includes a secondary cultural enrichment to detect the organism in feedlot cattle feces was investigated. A total of 425 fecal samples were collected from feedlot cattle and processed using GN broth as the enrichment broth for both initial and secondary enrichments, before and following IMS, respectively. Three hundred and nine (309/425) samples were negative by culture and PCR detection in initial evaluations, seven (7/425) samples were positive by both procedures, four (4/425) samples were culture negative/PCR positive, and one hundred and five (105/425) were culture positive/PCR negative. This represented a sensitivity of 6.25% (7/112), a specificity of 98.7% (309/313), a predictive value of a positive PCR result as 63.6% (7/11), a predictive value of a negative PCR result as 74.6% (309/414), the PCR test indicated a prevalence of 26.3% (112/425), and an accuracy of 74.4% (316/425). We have conducted a preliminary study to determine whether house flies could be a source of E. coli O157 in a cattle farm. Fifty adult house flies were collected (four times per week) from June till July 2003 from two sites (feed bunks, corn storage) on the Beef Cattle Research Unit at Kansas State University. Individual flies (n=1,600) were homogenized in PBS buffer, serially diluted, and plated onto sorbitol MacConkey agar with cefixime and tellurite using a direct drop plate technique. Sorbitol-negative colonies were screened for O157 antigen by the latex agglutination test, positive colonies were counted, isolated, and identified by the API Rapid 20E test. Number of fecal coliforms in individual flies were determined using mFC agar. Number of fecal coliforms in individual flies were determined using mFC agar. The prevalence of E. coli O157:H7 in house flies from corn storage and feed bunks was 2.6 and 5.4%, respectively, with counts ranging from 3x10 to 3x100,000 CFU/fly.
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IMPACT: 2003/01/01 TO 2003/12/31<BR>
Cattle feces were shown to contain multiple genetic types and subtypes. The genetic diversity present within these bovine fecal samples suggests that multiple isolates should be typed for epidemiological or outbreak investigations. Also, house flies were postive for E. coli O157:H7 and flies may present a potential mechanism for the dissemination of E. coli O157:H7.