Targeted Interventions to Reduce Antibiotic Use and Antimicrobial Resistance in Cattle Production

Non-Technical Summary:<br/>
There are increasing public concerns about antimicrobial use in animals and the development, persistence, accumulation and dissemination of resistance in enteric bacteria of livestock origin and its implications for human health. These concerns have lead regulatory organizations around the world to promulgate rules to protect public health by either reducing the number and/or formulations of antimicrobial drugs available for use in food animal agriculture (e.g., the ban of antimicrobial growth promoters in the European Union) or by tightening the approval and monitoring processes for new antimicrobial drugs intended for food animal use (e.g., FDA's Guidance for Industry #152 in the United States). Furthermore, the availability of currently efficacious therapeutic antibiotics may be curtailed, as evidenced by the FDA proposed ban on all extralabel use of cephalosporins in food animals in 2008. The future costs to animal agriculture (and potentially to consumers and other stakeholders) will be tremendous if certain classes or uses of antibiotics are no longer available. The use of antibiotics for treatment and prevention of bacterial infections in beef and dairy cattle is essential for sustaining profitability in these two sectors, for producing safe and wholesome food for consumers, and for ensuring the maximum welfare of the animals. Discovering and sharing proven, responsible and prudent ways to make better use of both existing and new antibiotics - with minimal risk to human health - will not only reduce the costs associated with antibiotic resistance, but also promote a profitable and sustainable agriculture in the future. Additionally, the development of safe and efficacious alternatives to antibiotic treatments may help slow accumulation and dissemination of antimicrobial resistance in food animals. The proposed project addresses gaps in the knowledge of antimicrobial resistance ecology of enteric bacteria in food animals, effective interventions to reduce antimicrobial resistance in enteric bacteria in livestock, and alternatives to antimicrobial uses for treatment of mastitis in dairy cattle. Specifically, we will describe temporal changes in antimicrobial resistance in dairy cattle, determine if probiotics can be used to reduce antimicrobial resistance in pre-weaned dairy calves, and determine if Manuka honey is a practical and safe alternative to non-antibiotic treatment for mastitis in dairy cattle. At the conclusion of the proposed studies we expect to have developed applied knowledge that dairy farmers can use to reduce their use of antimicrobial drugs and reduce current levels of antimicrobial resistance, and hence address the mounting public concern about use of antimicrobial drugs in livestock production.
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Approach:<br/>
This project is primarily field based and applied research. We will use the latest technology in quantifying phenotypic resistance and save samples for subsequent genotypic evaluations. To complete the 3 main objectives of the proposed research, we will enroll 50 dairy cows (obj. 1) for continued sampling biweekly for a full production cycle (13 to 14 months), one-hundred and eight neonatal dairy calves (obj. 2), and a total of 176 dairy cows for objective 3. For objective 1, fecal samples will be used to quantify the total E. coli counts and E. coli resistant to four commonly used antimicrobial drugs using a spiral plating technique. Diluted feces will be grown on MacConkey agar and MacConkey agar containing the four antimicrobial drugs at their resistance breakpoint as determined by the Clinical Laboratory and Standards Institute. Temporal changes in distributions of total and resistant E. coli counts will be described and the impact of different management practices and health events will be evaluated.
<br/>For objective 2, calves will be enrolled after birth and followed for 4 weeks. Calves will be enrolled shortly after birth and divided into four groups. Two groups will receive a probiotic at feeding and one of these groups will be treated with a ceftiofur antibiotic for 3 days (days 7-9), one group will only be treated with ceftiofur on days 7-9, and one group will be the negative control. Feces will be collected from calves every other day. The microbiological approach and methods will be identical to what is described for objective 1.
<br/>There are three phases of objective 3. The first phase is in vitro determination of minimum inhibitory concentration of Manuka honey for mastitis pathogens. The second phase is a safety study of intra-mammary infusion of honey and the chosen carrier in 12 adult cull dairy cows. The third phase is an efficacy trial using 82 cows on conventional and 82 cows on organic dairy farms. In the third phase, we will use cows with naturally occurring mastitis. On organic and conventional farms, half the cows will be treated with Manuka honey and the other half with PhytoMASTTM (an organic mastitis treatment) and cephapirin, respectively. Treatments will be assigned at random. The outcomes for objective 3 will be bacteriological culture, California mastitis test, and somatic cell counts pre-treatment (day 0) and 7, 14 and 28 days post-treatment. Time to return to normal appearing milk will also be an outcome of interest. A combination of descriptive and linear and generalized linear mixed models will be used to visualize and determine relationships between outcomes and explanatory variables for all three objectives. The results from the proposed research will be disseminated through national and international meeting, peer-reviewed journal articles and dairy producer meetings.
<P>Progress:<br/>
2011/12 TO 2012/11<br/>
OUTPUTS: Objective 1, Temporal changes in antimicrobial resistance in dairy cattle: 50 dairy cows, 10 in each of 5 different production stages were enrolled in this study in the month of November 2011. Fecal samples were collected from the cows every 2 weeks for 12 months. Production data, health events and movements between barns were recorded. Total coliform counts, and counts of coliforms resistant to ceftiofur, ampicillin, tetracycline and ciprofloxacin in 1g of feces have been determined for the first 19 of 26 sampling dates. Microbiological and statistical analyses have not been completed for this objective. Preliminary descriptive statistics for total coliform counts and coliforms resistant to ceftiofur, ampicillin, and tetracycline have been performed. Additionally, analytical statistics have been conducted for detection of resistance in a sample (dichotomous; yes/no) as compared to the production group the cows was in at the time of sampling. Mixed effect count models and zero-inflated count models are being developed in order to most appropriately analyze our coliform count data. Objective 2, Investigating probiotics as an intervention to reduce antimicrobial resistance in pre-weaned dairy calves: In a pilot randomized controlled clinical trial, we investigated the effect(s) of feeding direct-fed microbials (DFMs) to pre-weaned dairy calves on the counts of total and antimicrobial-resistant fecal coliforms and coliforms resistant to ceftiofur, ampicillin, tetracycline and ciprofloxacin. A total of 42 (6 batches x 7 calves) calves were enrolled in the study. The seven treatment groups consisted of: 1) DFM 1 + Naxcel treatment for three days (days 4, 5, and 6), 2) DFM 1, 3) control (no DFM) + Naxcel treatment for three days, 4) DFM 2 + Naxcel treatment for three days, 5) DFM 2, 6 and 7) control groups (no DFM). To control for the potential effect of ambient temperature and other variables, calves were enrolled into the study in batches of seven. When seven calves were assembled, treatments were assigned at random to the calves. All the calves were fed non-medicated milk-replacer twice daily according to the participating dairy's standard protocol, and they had free access to calf starter grain and fresh water. DFMs were dissolved in 2% milk and administered per os by a catheter tip syringe. Feces was collected by digital stimulation form the rectum of calves every third day after a batch was assembled until day 27 and every 6th day thereafter until weaning at 8 weeks of age. Microbiological and statistical analyses have not been completed for this objective. Preliminary descriptive and analytical statistics for total coliform counts and coliforms resistant to ceftiofur, ampicillin, and tetracycline have been performed. Objective 3, Investigate Manuka honey as an alternative non-antibiotic treatment for mastitis in dairy cows: External funding for this project has been obtained from USDA NCR SARE. We have commenced testing of a batch of Manuka honey for bacterial contamination. One graduate student, two veterinary students (summer students), two undergraduate students and a laboratory technician were tutored as part of these project.
<br/>PARTICIPANTS: Bo Norby is the PI of the project and he committed approximately 4 month to the project. His role was to oversee the overall management of the project and project development and design. He also participated in sample collection and animal care and data analysis. Lisa Halbert is a research associate who worked approximately 4 months on the project. Her role was to develop and oversee overall management of the project. She also participated in sample collection and animal care. Erik Corbett is a graduate student who worked full time on the project. He participated in study design and was in charge of study conduct, including sample collection, sample processing, sample analysis and data analysis. Jessica Reis is a veterinary student who worked on the project for 3 months. She participated in sample collection and processing, as well as microbial analyses and data entering. Ashley Kwasniewski is a veterinary student who worked on the project for 3 months. She participated in sample collection and processing, as well as microbial analyses and data entering. Kate Elbourne is an undergraduate student who worked approximately 3 months on the project. She helped with all laboratory procedures, including sample processing and analysis. Stephen Carney is an undergraduate student who worked approximately 3 months on the project. She helped with all laboratory procedures, including sample processing and analysis.
<br/>TARGET AUDIENCES: Dairy producers and managers, Veterinarians, Extension and outreach personnel, Nutritionists, Scientists, Consumer groups.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.
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IMPACT: Objective 1, Temporal changes in antimicrobial resistance in dairy cattle: Analyzing the dichotomous outcome of detection of resistance to the tested antimicrobials and comparing dry cows to cows in other stages of production, dry cows had a decreased chance of resistance being detected to one or more antibiotics, as well as detection of resistance to ampicillin and tetracycline (p<0.05). Detection of resistance to ceftiofur was not statistically significant at p<0.05. Results from our preliminary analyses suggest that the stage of production may have an effect on shedding of coliforms and resistant coliforms. Further analysis will assess if stage of production, diet, treatment history, or seasonality affect the levels and patterns of resistance in dairy cows. Understanding the variation in susceptibility patterns of fecal coliforms in dairy cows will assist in the design of potential interventions to reduce levels and patterns of antimicrobial resistance in cull cows entering the food chain. Objective 2, Investigating probiotics as an intervention to reduce antimicrobial resistance in pre-weaned dairy calves: Our preliminary data suggest that feeding of a DFM (DFM2) containing Bifidobacterium animalis ssp. Lactis, Lactobacillus acidophilus, Bacillus licheniformis, Bacillus subtilis and Lactobacillus lactis at certain time-points significantly reduced the total absolute counts of total coliform bacteria, and absolute numbers of coliforms resistant to ampicillin, ceftiofur and tetracycline by more than 2 log10 concentrations. Resistance to ciprofloxacin was not detected. In our models for total coliform counts and coliforms resistant to ceftiofur, ampicillin, and tetracycline, there was a significantly lower counts in absolute numbers of total coliforms (an approximately 2 log10 reduction), and coliforms resistant to ceftiofur, ampicillin and tetracycline (1 to 2 log10 reduction) on day 10 for calves fed DFM 2 as compared to calves fed DFM 1 and the control calves. There was no significant difference between calves fed DFM 1 and the control calves on day 10. Objective 3, Investigate Manuka honey as an alternative non-antibiotic treatment for mastitis in dairy cows: Few Gram positive and Gram negative bacterial strains have been isolated from the Manuka honey. Further microbial analyses of the bacterial isolates will be conducted, and methods for sterilization of the honey will be explored. Three presentations were given to primarily veterinarians as part of outreach activities: Norby, B. (2012) "The Fuss about Food Animal Antibiotic Use, Resistance and Drug Residues: Fact or Folly." Presentation at the Michigan Veterinary Conference, Lansing, January 27, 2012. Halbert, L. (2012) "Regulatory and Residue Insights." Presentation to CVM/MSU Practice-Based Ambulatory Veterinarians, East Lansing, November 1, 2012. Corbett, E.; Norby, B.; Halbert, L.W.; Kaneene, J.B.; Grooms, D.L. (2012) Temporal changes in antimicrobial resistance of fecal coliforms isolated from dairy cows. Presentation to CVM/MSU Practice-Based Ambulatory Veterinarians, East Lansing, November 1, 2012.