Oxytetra Cycline Resistant Gram-negative Bacteria in Dairy Cattle: Risk Factors and Implications on Food Safety

The first objective of the study is to determine the outcome of feeding or withdrawing milk replacers to calves supplemented with subtherapeutic concentrations of oxytetracycline to calves. A prospective longitudinal study will be conducted on 8 dairy farms. Feces from calves and lactating cattle will be collected. Environmental swabs and bulk tank milk will also be collected. Samples will be collected periodically from each farm for duration of 300 days. The samples will be examined for oxytetracycline gram-negative bacteria. (OXY-GNB). Samples will be plated on Monkey´s agar supplemented with oxytetracycline. Bacteria that grow on the plates will be speciated using API-20E identification system. OXY-GNB bacteria will be subjected to antimicrobial susceptibility testing using the methods described for dilution antimicrobial susceptibility tests for aerobic bacteria by National Committee for Clinical Laboratory Standards. The OXY-GNB bacteria will be subtyped using the pulsed field gel electrophoresis (PFGE) technique. The PFGE protocol followed in our laboratory is based on Centers for Disease Control and Prevention recommended procedures. The genetic determinants of tetracycline resistance will be done using standardized protocols developed for tetracycline resistance analyses. In our laboratory, we have successfully analyzed tetracycline determinants in Escherichia coli and Salmonella isolates. The second objective of the study is to determine the prevalence and distribution of OXY-GNB, shiga toxin producing Escherichia coli and Salmonella in feces of lactating cattle and bulk tank milk. A survey will be conducted using the guidelines established by the National Animal Health Monitoring System ´98 study. Under this objective, dairy herds located in 67 counties in Pennsylvania will be surveyed. The questionnaire will be administered to the dairy producer during the farm visit. The survey instrument has been designed to elicit both quantitative and or qualitative responses, which can be scored and used to assess farm management practices including use of antimicrobial agents. A total of 160 farms will be surveyed. Bulk tank milk and fecal samples from approximately 10% of the animals on each herd will be collected and examined for OXY-GNB, shiga-toxin producing F. coli and Salmonella. Isolation, identification and characterization of OXY-GNB. Shiga-toxin producing E. coli and Salmonella will be done as described under objective 1. Isolates identified as belonging to the genus. Escherichia will be tested for shiga-toxins stx(1) and stx(2) by a specific PCR assay. The third objective of the study is to utilize the data collected from objective 1 and 2 to determine the risk factors for OXY-GNB, shiga toxin producing E. coli and Salmonella in dairy herds. Data will be entered and maintained on Microsoft Excel. Appropriate statistical analysis will be done to answer the research questions listed under objective I and 2. A scheme termed as interactive Dichotomizer 3 will be used to induce decision trees for classification tasks to develop the neural network model. The results of this study will be the development of a prototype software.
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The development of resistance to tetracyclines in the context of human and animal use has raised great concern. Tetracyclines have been used in humans and animals for more than four decades. This has resulted in the emergence of resistant bacterial variants, particularly those containing tetracycline resistant genes, observed most frequently in multi-drug resistant gram-negative bacteria. In recent years the use of oxytetracyclines as animal growth promoters and the implications for human health has been questioned. The purpose of this study is to gather relevant and accurate information on the epidemiology (descriptive and molecular) of oxytetracycline resistant gram-negative bacteria in dairy cattle. This study will provide much needed scientific information with reference to prevalence, trends of antimicrobial resistance on the farm with respect to oxytetracycline and other antimicrobial agents, identify risk factors for resistance development, and develop and implement interventions to reduce risk for resistance. A clear understanding of critical farm management practices will allow us in the long term to: (1) develop and implement hazard analysis critical control point-based programs to address issues related to antimicrobial-resistant bacteria, and food borne pathogens on dairy herds, and (2) provide a strong and pertinent knowledge base to undertake risk analysis investigations.