Mastitis Resistance to Enhance Dairy Food Safety

NON-TECHNICAL SUMMARY: In the United States, the dairy industry contributes in excess of 65 billion dollars per year to the national economy. Cash receipts from the sale of New York milk during 2004 totaled 1.95 billion dollars (USDA, NASS, NYASS, No. 973-5-05). Dairy production is the largest industry in New York State, which is the 3rd highest producer of milk nationally. The single most costly disease of dairy cattle is bovine mastitis, affecting virtually every dairy farm and approximately 38% of all dairy cows. Recent studies indicate that antibiotic resistance is an important risk factor for a failure to cure after antibiotic therapy. More importantly, antibiotic resistance created with the use of antibiotics for the treatment and prevention of cattle mastitis could be considered a public health concern. In the United States alone, 2 million people per year are diagnosed with hospital acquired infections, of which 90,000 die as a result of their infection. The nature of the antibiotic usage in the food animal industry favors the appearance of antibiotic resistance due to subtherapeutic dosage, mass treatment and long term administration. Bacteriophages are viruses that infect bacteria; they are obligate intracellular parasites and lack their own metabolism (cannot survive outside bacteria). Phages are extremely host specific, able only to infect specific species or even strains of bacteria. The concept of combating pathogens by means of phages is obvious, and was proposed shortly after the discovery of phages approximately 90 years ago. Bacteriophages could be considered the perfect antimicrobial agents; they are highly specific to few bacterial species; they are non-toxic to mammals; and they grow in exponential scale while precisely killing pathogenic bacteria. Unfortunately, the discovery of antibiotics basically eliminated research on phage therapy. Furthermore, the pharmaceutical companies are not attracted by the potential use of phage therapy due to the difficulties associated with patenting the rights of phage therapy use. The objectives of this project are therefore to 1) isolate lytic environmental bacteriophages from dairy farms that are specific and efficacious against the most common mastitis bacterial pathogens, 2) evaluate the in vitro antibacterial efficacy of all bacteriophage isolates, 3) assemble a cocktail of highly effective bacteriophages, and evaluate its efficacy as a treatment of clinical mastitis of dairy cattle. At our laboratory at Cornell University we have already isolated several bacteriophages from environmental samples of commercial dairy farms. The in vitro antimicrobial efficacy of our bacteriophages isolates was assessed and found to be comparable with the following antibiotics: florfenicol, tetracycline, ceftiofur, ampicillin, spectinomycin, and streptomycin. By the end of this project we expect to have developed and tested a novel organic antimicrobial treatment that will be effective against bacterial mastitis of dairy cattle.

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APPROACH: 1) To assemble a cocktail of environmental phages and perform in vitro antimicrobial efficacy evaluation the following will be performed: a. Phages will be isolated from environmental samples collected from commercial dairy farms; we will use Streptococcus agalactiae, Streptococcus spp., Staphylococcus aureus, Staphylococcus spp., Escherichia coli, Klebsiella spp., and Pseudomonas spp. isolated from the milk of cows diagnosed with clinical mastitis as target hosts for phage isolation. b. The antimicrobial efficacy of all isolated phages will be tested by assessing the effect of phage inoculation at several different concentrations on the growth curve of the host bacteria. c. We will select the 2 most efficacious phages from each host bacteria for the final phage cocktail. Electron microscopy and genetic characterization will be performed in all 14 selected bacteriophages. d. Evaluation and optimization of different phage lysate purification methods such as cesium chloride density centrifugation and size exclusion chromatography. 2) To evaluate the efficacy of the treatment of clinical mastitis with the phage cocktail as compared with positive controls the following will be performed: a. A prospective, double-blinded clinical trial will be conducted. 160 lactating dairy cows from a large commercial dairy farm diagnosed with clinical mastitis will be randomly assigned to one of two treatments: control (antibiotic of choice) or treatment (intramammary administration of purified phage cocktail). Milk cultures will be repeatedly performed to assess bacteriologic cure.