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The objectives of our research are to develop improved methods to detect and quantify Escherichia coli O157:H7 and to improve our understanding of the ecology of E. coli O157 in cattle operations.<P> The long term goal of our research efforts is to identify management strategies and production practices which could potentially be manipulated to reduce fecal shedding of E. coli O157 in cattle. <P>The improvement of detection methods, particularly method to quantify fecal concentration of E. coli O157:H7, will broaden our understanding of the ecology of the organism in cattle, which could potentially lead to recognition of animal and management factors influencing fecal shedding of E. coli O157:H7. Specifically, the objectives are to develop a multiplex PCR procedure to detect major virulence genes (eae, fliC, hlyA, rfbE, stx1, and stx2) of E. coli O157:H7, develop a multiplex real-time PCR (RT-PCR) procedure for detection and quantification of E. coli O157:H7 in bovine fecal samples, and to determine the mechanism of the positive association of distiller's grains supplementation and fecal shedding of E. coli O157:H7
Non-Technical Summary: Although the food supply in the US is one of the safest in the world, foodborne illnesses do occur and frequently are associated with foods derived from animals. Escherichia coli O157, a food borne pathogen, is an important public health concern. Cattle are natural reservoirs of this organism and shed the bacteria in their feces, which then serve as a major source of contamination of food and water for human infection. Improvement in detection and quantification of E. coli O157:H7 in feces and further understanding of the ecology of this organism in cattle could potentially lead to mitigation strategies to reduce pathogen prevalence and pathogen load in cattle. <P> Approach: Isolation and identification of E. coli O157 in feces generally involve enrichment, immunomagnetic separation, plating on selective medium, and agglutination for O157 antigen followed by PCR detection of one or more major virulence genes for confirmation. Generally, one or more of the six genes, stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), eae (intimin), hlyA (hemolysin), rfbE (O157 antigen), and fliC (flagellar antigen) are targeted for identifications. In our laboratory and in many other laboratories, two separate PCR reactions are carried out to detect eaeA, stx1, and hlyA in one, and stx2 and fliC in another reaction for E. coli O157:H7 identifications. The type strain of E. coli O157 (ATCC 43894) that possesses all six virulence genes (fliC, stx1, stx2, eae, rfbE, and hlyA) will be used initially for procedure development and optimization. Once the procedure is optimized, validation will be done with bovine fecal and human clinical isolates stored in our collection. Also, non-O157 STEC E. coli strains (O11, O26, O145, etc) will be included. Isolation and identification procedures for E. coli O157 and other STEC in feces or other samples are time consuming and expensive. Multiplex real-time PCR could offer a rapid and sensitive method for detecting multiple genes of E. coli O157 in bovine fecal samples. Target genes for the proposed multiplex real-time PCR include rfbE, a gene that codes for the O157 side chain, and stx1 and stx2 that code for Shiga toxins, which are major virulence factors. We propose to detect these genes to confirm the presence of E. coli O157 in bovine fecal samples and quantify the concentration. The design of the multiplex real-time PCR will be evaluated using pure cultures E. coli O157 (ATCC 43894 and 5 other strains originally obtained from feedlot cattle) and at least one non-O157 isolate. These isolates will also be used to construct a standard curve for the multiplex real-time PCR for pure culture. The procedure will be validated with feces spiked with pure cultures before testing the procedure with feces of cattle that are naturally shedding the organism. The third study is designed to determine whether decreased flow of starch to the hindgut with distiller's grains-supplemented diet may result in relatively more hospitable environment for growth and survival of E. coli O157 in the hindgut. The hypothesis will be tested in a challenge model in cattle fed diets containing the DG with additional starch included to equal the amount of starch in the control diet (No DG). The impact of DG and starch supplemented diet on the fecal shedding of E. coli O157:H7 in cattle will be compared it to a negative control (no DG) and a positive control (distiller's grains). A higher concentration, longer duration of shedding and/or higher percentage of calves remaining positive would indicate a positive relationship between dietary treatments and E. coli O157:H7.