- Animal Products: dairy
- Animal Production: herbal medicines
- Education and Training: focus group, on-farm/ranch research
- Production Systems: organic agriculture
The use of antibiotics may result in selective pressure for antibiotic resistance in foodborne pathogens such as Salmonella and E. coli, as well as resistance to other pathogens that cause disease in cattle and people. Drug residue avoidance in both meat and milk must also be considered when pharmaceuticals are administered to dairy cows. It is important to note that organic dairy producers do not use antibiotics at all, and mastitis is the most frequent disease condition in dairy cattle affecting animal health, longevity in the herd, and the production of quality milk. While non-antibiotic products for mastitis have been marketed, limited data is available regarding the safety and efficacy of these products. The overall purpose of our proposed research is to assess the efficacy and safety of two traditional therapies, guava and honey, for use in treating bovine mastitis. First, in-vitro work will establish antimicrobial activity of these compounds against common mastitis pathogens. Once candidate formulations of honey and/or guava are identified, limited intra-mammary infusions will be used in healthy cows to evaluate safety and pharmacokinetics. Finally, on-farm field trials will assess the efficacy of candidate formulations for naturally-occurring mastitis. Key opinion leaders in the Michigan dairy and organic industries will be recruited for the field-based trials. Dissemination of the findings will be through seminars in sustainable agriculture at the MSU Kellogg Biological Station pasture-based dairy, publication in the Michigan Dairy Review Extension newsletter, and through the AVMA Guidance on Complementary and Alternative Veterinary Medicine. Approval for use by the National Organic Program will be pursued for safe and efficacious formulations. In summary, non-antibiotic formulations for the treatment of bovine mastitis will have value not only to organic dairy farmers but also conventional dairy farmers.
Project objectives from proposal:
Phase I: Establishing in vitro efficacy of honey and guava and development of formulation candidates.
Objective: To determine the minimum inhibitory concentration (MIC) of Manuka honey and guava leaf extract to bacteria commonly associated with bovine mastitis.
The Clinical Laboratories Standards Institute (CLSI) manual M31-A3 will be use as primary guidance for determining the susceptibility of common mastitis pathogens (CLSI, 2008) to the non-antibiotic extracts of interest in this study. Broth dilution based on mL/dL% will be used to determine the minimum inhibitory concentration (MIC) for honey, while mg/dL will be used to determine MIC for purified guava leaf extract.
In assessing the MIC for honey, guava, and honey/guava, we will use American Type Culture Collection (ATCC) and clinical mastitis bacterial strains. Per CLSI M31-A3, cephalothin will be used as a 1st generation cephalosporin class representative for cephaparin, using ATCC strains S. aureus 29213, E coli 25922 and Streptococcus pneumonia 49619 as control organisms. For each “drug and bug” combination, we will assess up to eight different treatment concentrations in order to determine the MIC for each combination. The formulation to be selected for use in the safety and clinical mastitis trials will be the highest MIC obtained for each combination of drug and bug/pathogen. The final formulations will be tested for pH to ensure that pH is within a tissue friendly range. If that is not the case, the pH will be adjusted using an acidic or basic buffer.
Phase II: Establishing safety of honey and guava candidate formulations.
Objective: To establish in-animal safety of candidate formulations and in-milk inhibition metabolites residue depletion.
Informed consent from Michigan State University’s Institutional Animal Care and Use Committee (IACUC; approval of animal use in research), Institutional Review Board (IRB), and approval of MSU Site Use will be obtained to proceed through phases II and III;
IUCAC and MSU Dairy Field Laboratory site approval has been received for this objective. Twenty late lactation and healthy cull cows at the MSU Dairy Teaching and Research Field Laboratory will be selected for enrollment. Enrollment criteria will consist of reviews of cows’ udder health history. Cows with teat injuries, elevated SCC or repeated treatments for mastitis will not be eligible. The formulations of honey and guava determined in phase 1 of the study will be suspended in peanut oil. Four replicates of each formulation candidate (honey, guava, honey-guava, carrier only and a negative control [no carrier or formulation]) will be evaluated for animal safety following dermal exposure and intra-mammary infusion. Prior to intra-mammary infusion of any candidate formulations, dermal sensitivity will be ascertained by applying a gauze pad impregnated with the candidate formulation (plus carrier) to the udder by means of an adhesive patch. Following 24 hours of contact, the patch will be removed and the area assessed for any signs of irritation or inflammation (swelling and redness). Following successful assessment of dermal sensitivity, the four replicates of five treatments will be injected into a randomly selected quarter of the twenty enrolled cows. Prior to intra-mammary injections, the cows’ baseline health parameters will be ascertained, and the teats will be cleaned to reduce the possibility injecting bacteria into the quarter at the time the test product is infused. Following intra-mammary infusion of candidate formulations, the cows’ health will be assessed for 10 days according to the daily assessment of the following parameters (outcomes): Physical appearance of the cow and the infused quarter (i.e. signs of inflammation), temperature, heart rate, and respiration, California mastitis test, somatic cell count (SCC), and dry-matter intake (DMI).
Cows will be housed in stanchion barn and fed a total mixed ration (TMR) according to MSU Dairy Field Laboratory standard operating procedures. The cost of feeding cows at the MSU Dairy and Filed Laboratory and discards of milk for the 10 day period is included in the budget. Although unlikely, we have also included the cost of loss of two cull cows.
Secure stand-alone and networked computers will be used to enter recorded outcomes from the study. We do not expect adverse outcomes for treated cows, however if adverse outcomes are encountered, the formulations causing the adverse outcome will be re-evaluated. Because of the relative low number of cows receiving each of the five treatments, every abnormal outcome will precipitate a formulation re-evaluation.
Personnel evaluating the cows after treatments will be blinded as to the treatments that the cows received.
Phase III: Controlled field-based clinical trials
For the third phase of the proposed research, we will enroll organic and conventional dairy farms in Michigan. Formulations to be evaluated in the field phase will contain non-GMO and organically-sourced honey, guava, and carrier/excipient. This will ensure that no privately-owned animals lose their organic status. A total of 80 cows on conventional and 80 cows on organic dairies will be enrolled. To be eligible for inclusion in the study, the herds must have individual cow identification, written records and perform fore-stripping of quarters as part of their milking procedure. Cows exhibiting clinical signs of mastitis (changes in appearance of milk such as flakes or clots, positive CMT test, localized inflammation of the quarter) will be considered for enrollment. Cows exhibiting systemic signs of fever, depression, off-feed, or hard/hot quarters with watery milk will NOT be eligible for enrollment. Our intent is to select routine uncomplicated cases of clinical mastitis. Individual quarter milk samples will be collected for bacteriological culture, California mastitis test, and SCC pre-treatment (day 0) and 7, 14 and 28 days post-treatment. Date of return to normal appearing milk will also be an outcome of interest. Upon identification of a case of clinical mastitis meeting the above criteria, the enrolled producer will contact an MSU researcher who will tell them which of the four experimental treatment should be given to that particular cow. This assignment will be made via a randomization schedule established at the beginning of the field trial. This method insures that only eligible cases are enrolled, and that there is strict adherence to the pre-determined randomization plan within each herd. Additionally, the farmer will be blind to which treatment an identified cow will get. Four treatments will be used in this study: 1) honey, 2) guava, 3) honey and guava, and 4) Phyto-MAST™ for organic dairies or cephapirin sodium (Cefa-LAK) for conventional dairies. Assignment of one of the four treatments will be assigned in blocks of four in order to reduce bias from weather and other changes in the cows’ environment. Cows will be housed and fed according to the participating farms’ standard operating procedures. The cost of feeding cows at the participating dairies for 28 days and discards of milk for 8 days following treatment is included in the budget.
The study design is a randomized, blinded multi-site clinical trial, which is considered the best and least biased experimental study design, and the “gold standard” of scientific rigor.