| Cow mastitis is one of the most common diseases in dairy cattle breeding.Cows suffering from mastitis would have a decrease in milk production and the quality of milk produced.Cows would also experience fever and weight loss.In severe cases,it could also cause the death of cows.Dairy cow mastitis often brings huge economic losses to the breeding industry,and also restricts the development of dairy cow breeding industry.The most common cause of dairy cow mastitis is microbial infection,especially Staphylococcus sp.Once the cow’s mammary glands are infected with pathogenic bacteria,it is very difficult to cure.And cross-infection may occur,causing serious economic losses to the pasture.At present,the treatment of dairy cow mastitis mainly relies on antibiotics.Due to problems such as serious residues of veterinary drugs and the emergence of drug-resistant strains,scholars are actively looking for new treatment methods that could replace antibiotics.In this study,samples were collected from the milk,nipples,fur and other parts of cows with mastitis in the process of dairy cow breeding,milking workshops and related environments.The pathogenic bacteria of dairy cow mastitis were isolated and identified.Staphylococcus aureus was the representative pathogen and was most difficult to cure as the research selected object.Its epidemiology was investigated.A bacteriophage of lytic S.aureus was isolated from the dairy farming environment.The antibacterial ability and characteristics of the selected bacteriophage against the pathogenic bacteria of dairy cow mastitis were explored.The main findings are as follows:(1)A total of 1,000 samples were collected from dairy farms,and 494 strains of bacteria were isolated,with a total isolation rate of 49.40%.The isolated strains were identified by 16 S r DNA sequencing and were divided into 13 categories,including: Staphylococcus,Enterococcus faecalis,Proteus,Escherichia coli,Bacillus cereus,Corynebacterium glutamicum,Enterococcus haresi,Corynebacterium,Klebsiella,Aerococcus faecalis,Swine fecal bacteria,Streptococcus urinary,Lactobacillus gasseri,including 18 different types of Staphylococcus species.(2)16 strains of S.aureus were further identified and isolated from the above-mentioned Staphylococcus genus,and their epidemiological characteristics were studied and tested.The analys is found that all isolated strains carried virulence genes and drug resistance genes.A total of 13 enterotoxin genes,5 other virulence genes,2 disinfectant resistance genes and 12 drug resistance genes were detected.The traditional enterotoxin only detected sec gene,with a carrier rate of 56.25%.The carrier rate of 12 new enterotoxins genes was between 12.50% and 87.50%.12 strains of S.aureus carried the nuc gene,with a carrier rate of 75.00%,of which 6 strains carrying mec A gene(37.50%).All nuc-positive strains carried both hlα and hlβ genes(75.00%),with tsst-1 gene carrying rate(68.75%),eta gene carrying rate(12.50%).Among the tested 5 disinfectant-resistant genes,10 strains of S.aureus carried both qac A/B and qac G genes(62.50%).A total of 12 drug resistance genes were detected,with grl A gene carrying the highest rate(87.50%),with van A and msr A genes the lowest(6.25%),and the other drug resistance gene carrying rates were between 12.50% and 81.25%.(3)16 isolates of S.aureus showed resistance to 19 kinds of antibiotic drugs.And 50.00% of the strains were multi-drug resistant(3-15 resistant).There were 6 strains with the broadest spectrum of resistance reaching 15 resistant,12 resistant and above.The strains had the strongest tolerance to teicoplanin(75.00%),followed by penicillin(50.00%),and also showed varying degrees of tolerance to other antibiotics such as levofloxacin,ampicillin,trimethoprim,norfloxacin.All 16 strains were completely sensitive to ceftazidime,imipenem,oxacillin,and streptomycin.(4)A strain of lytic S.aureus phage(named SP-CmSa-11)was selected.And its titer was 7.6×109 PFU/m L;the best MOI is 0.1;the incubation period is 60 min,the burst period is 40 min;the burst size is approximately 130 PFU/cell.The lysis profile of the phage includes 13 strains of S.aureus from cows with mastitis,7 strains of MRSA,1 strain of Xylococcus,1 strain of S.epidermidis,and 1 strain of Enterococcus faecalis.The p H tolerance range is 4.0-10.0;it can survive normally at 40 ℃,and its growth was inhibited to some extent at 50 ℃.It can survive at 60 ℃ for 40 min and 70 ℃ for 20 min.It is not sensitive to chloroform and ultraviolet.(5)SP-Cm Sa-11 showed excellent antibacterial effect in LB medium and pasteurized milk.In LB medium,SP-Cm Sa-11 could be fully lysed S.aureus in the matrix in about four hours.In pasteurized milk,compared to the control group without phage,the concentration of S.aureus in pasteurized milk could be reduced by up to 106 CFU /m L.It could continuously inhibit up to 36 h. |
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