Isolation And Application Of Bacteriophage In Prevention Of Mastitis In Dairy Cows

Bovine mastitis is one of the main bacterial disease which caused substantial economic losses, including lower milk yields, reduced milk quality and higher production costs, etc. Currently there was no effective treatment other than antibiotics to control mastitis in dairy cow. However, with antibiotics widely used to treatment bacterial infectious diseases, dependence is marked first by an increased tolerance, with more and more of the substance required to produce the desired effect, and then by the appearance of unpleasant withdrawal symptoms when the substance is discontinued, therefore the incidence of antibiotic-resistant bacteria had increased. As a result, growing concerns on antibiotic resistance have spurred researchers into looking for alternative treatment methods. Bacteriophages, one of the most numerous life forms on earth, are viruses infecting bacteria, re-emerged as an alternative or supplementary treatment in bacterial infectious diseases. Bacteriophages are obligate intracellular parasites and lack their own metabolism and characterized by their specific ability to selectively infect bacterial cells. Based on the features and advantages of phage, phage therapy once again become a hot topic in life science research.To further explore the bacteriophage as a biological agent of prevention and control of mastitis. In this study, a novel virulent bacteriophage was isolated from the waste water sample in a local dairy farm after three times enrichment in LB liquid medium co-cultured with Staphylococcus aureus (S. aureus) isolates and it biological properties was determined.The prophylaxis experiments for bovine mastitis with the isolated phage was also conducted. Phages were isolated, identified and purified by double-layer agar plate method with the strain of S. aureus, one of the main pathogens caused mastitis of dairy cows, as host cells. Bacteriophage enuneration was conducted by soft agar overlay proceduers. The newly isolated virulent phage was named SWP in recognition of its source, being a phage of S. aureus and isolated from waste water samples. The plaques of the phage SWP were formed transparent uniformed, clear, round zones and their sizes were 2 mm in diameter. The titer of phage SWP toward S. aureus isolates was more than 109 PFU/mL. Its therapeutic effect was examined in in vitro experiments.The morphology of phage SWP was examined by transmission electron microscope (TEM) after the particles were amplified and purified. Phage thermostability, pH stability, genome size and restriction endonuclease digestion pattern, optimal multiplicity of infection (MOI), one growth curve of phage SWP was determined. TEM observation of the isolated phage SWP had an icosahedral capsid of 62.5 nm in diameter and a long (187.5 nm) tail, there was a connector linked the head and tail. Its morphological examination indicated that the bacteriophage belongs to the Myoviridae families. The phage SWP was a dsDNA viruse, the DNA could be cut by EcoRI, HindIII and NdeI into distinct bands, visualized with an agarose gel. The optimal multiplicity of infection (MOI) of the phage SWP was determined to be 0.01. One-step growth kinetics of the pahge SWP showed that the latent period was 40 min, the rise period was 50 min, and the average burst size was 44 phages particles/infected cell. At 4℃, the phage can be stored 1 month, -80℃can be stored less than 3 months. The phage SWP still remained high lysis activities after incubated at 60°C for 60 min or 70°C for 30 min and lysis activities disappeared entirely after incubated at 70°C for 40 min. The phage SWP could be alive at the range of pH value from 4 to 8 and slightly affected in the presence of chloroform and the treatment of ultraviolet light.The results of prevention of mastitis in animal experiments were showed as follows: In the control group, positive test for subclinical mastitis in udder areas increased by 10, the incidence of disease increased by two head, however, in the experimental group, positive subclinical mastitis reduced by 7 udder areas, the incidence of disease decreased by over three heads. In the experimental group, the disease incidence decreased 1.97%, 3.33%; in the control group, increased by 2.8%, 2.2%. Knowledge from a serials of experiments and data of the SWP suggests that the isolated bacteriophage can be used as a promising candidate for phage preventive or therapeutic agents applied to the dairy farm of bovine mastitis.