Interventions for Controlling Antimicrobial Resistance of Salmonella and Campylobacter in Dairy Cattle

As public health problems, Campylobacter, Salmonella, and E. coli are the most common infectious causes of foodborne illness in the U.S., and the presence of antimicrobial resistance in these bacteria has wide-ranging implications for human health. Foods of animal origin (meat, milk, and eggs) are a common source of these bacteria: while these organisms can cause severe disease in humans, food animals are able to carry these bacteria with no ill effects. On dairy farms, bacteria may acquire resistance to antimicrobial drugs in three major ways: 1) the use of antimicrobial drugs only allows drug-resistant bacteria to survive and proliferate (selection pressure); 2) introduction of cattle that already carry drug-resistant bacteria; and 3) animals on the farm being exposed to drug-resistant bacteria introduced by other means, such as from wildlife or farm workers. If selection pressure from antimicrobial drug use on the farm occurs, then stopping antimicrobial drug use should increase bacterial susceptibility to drugs, which would result in a decrease in bacterial drug resistance.
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Half of the farms will continue feeding medicated milk replacer, while the other half will begin feeding milk replacer without tetracycline. Samples will be taken from calves (fecal samples) and their housing environments (calving pen and calf housing). Data about herd management and antimicrobial use will be collected. These samples will be processed to identify Campylobacter, Salmonella, and E. coli, and antimicrobial susceptibility testing for tetracycline and 16 other drugs will be conducted using an automated system to determine the minimum level of a given drug needed to inhibit bacterial growth (the minimum inhibitory concentration, or MIC). A subset of Campylobacter isolates will be tested by agar dilution, an alternative method for susceptibility testing, to compare the results between tests. The median MIC for tetracycline in Salmonella, Campylobacter, and E. coli will be determined for each herd, and the effect of not feeding medicated milk replacer on changes in median MICs over time will be analyzed. Regression models will be used to determine the likelihood of bacterial isolates becoming resistant to tetracycline. The findings of this study will provide information about herd management practices that may influence the levels of antimicrobial resistance on dairy farms, which will be invaluable in the dairy industry's efforts to ensure a safer food supply. These data can be used to develop herd management practices targeted at reducing antimicrobial resistance on the farm, and will be provided to dairy farmers, agricultural extension agents, and veterinarians through conferences, informational pamphlets, and a project website. Dairy farmers enrolled in the study will be provided with results of bacterial testing for their herds and for the rest of the project herds.
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Ten dairy farms (with tetracycline-resistant Salmonella and Campylobacter in calves and a history of using milk replacers containing tetracycline) from an ongoing study will be used. One farm of each pair will continue feeding medicated milk replacer, and the other farm will begin feeding a milk replacer that does not contain tetracycline. Farms will be visited every month for one year. Calves currently being fed a milk diet will be randomly selected at each sampling visit. Ten gram fecal samples will be obtained directly from the rectum of each animal. Environmental samples from the floors of calving pens and calf housing, will be collected to assess calf exposure to resistant bacteria in their housing environment. Questionnaires will be used to collect data on farm management practices and calf age, health and medications. Management practices will focus on farm use of antimicrobials and risk factors associated with the introduction of antimicrobial resistance onto the farm and transmission across animal groups. Standard bacterial isolation techniques will be used, and isolates will be banked in support media and 65% glycerol and frozen at -80C. Since Salmonella and Campylobacter are shed intermittently by cattle, E. coli, a common bacteria, will also be cultured and subjected to antimicrobial susceptibility testing. The SensiTitre system, accepted for use by the CDC National Antimicrobial Resistance Monitoring System for surveillance of enteric pathogens, will be used for antimicrobial susceptibility testing. Standard SensiTitre protocols will be followed for Salmonella and E. coli, while the protocol for Campylobacter will add the use of Haemophilus testing media while growing banked isolates, to improve recovery. Customized SensiTitre plates with azithromycin, amoxicillin/clavulanic acid, ampicillin, ceftiofur, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, florfenicol, gentamicin, kanamycin, metronidazole, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim/sulfamethoxazole will be used for Campylobacter. SensiTitre plates with amikacin, amoxicillin/clavulanic acid, ampicillin, apramycin, ceftiofur, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, florfenicol, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim/sulfamethoxazole will be used for Salmonella and E. coli. Analyses will be conducted separately for Campylobacter, Salmonella, and E. coli, and for each individual antimicrobial agent. The median MIC for tetracycline will be determined for each herd at each visit. The effect of intervention on the change in MICs over time will be analyzed using repeated measures ANOVA, and median MIC values for tetracycline in Salmonella and Campylobacter will be compared to the tetracycline MICs for the same herds from our current study. Each calf will be classified as having resistant or non-resistant Campylobacter, Salmonella, or E. coli. Multivariable logistic regression models with random effects for calf resistance status will be generated, including herd (as the random effect term), treatment group, season of sample, and other risk factors.
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Since the use of antibiotic drugs on the farm may select for drug-resistant bacteria, stopping antibiotic use should decrease bacterial drug resistance. Dairies which fed calves milk replacer with antibiotics will switch to replacers without antibiotics, to determine whether eliminating antibiotics results in increasing susceptibility to antibiotic drugs by Campylobacter and Salmonella.