Isolation And Identification Of Main Pathogens And Establishment Of Triple PCR Method For Bovine Mastitis Milk From Some Dairy Farms In Shaanxi Province

Bovine mastitis is caused by injury and infection of mammary gland tissue which is responsible for the decrease of milk quality and yield.The pathogenic bacteria of dairy farms in different regions showed great difference due to the influence of climate,feeding and management condition as well as dairy cows themselves.At present,antibiotics were widespread overused when the main pathogenic are not clear in clinical treatment.It caused a great degree of drug residues and increased drug resistance.In this study,126 milk samples of mastitis were collected from 3 dairy farms in Shaanxi province,isolated and identified the main pathogens and carried out biochemical and suseptibility detection for Bacillus cereus,Staphylococcus chromogenes and Klebsiella pneumoniae.A triple PCR assay was established for these 3 pathogens.The main results were as follows:1.Isolation,identification and susceptibility detection of the main pathogens from milk samples of mastitisA total of 126 milk samples of mastitis were aseptically collected and 366 strains were isolated,the percentage of main pathogenics were 56.24%,including 12.02% of Staphylococcus,10.92% of Streptococcus,10.65% of Enterobacteria,6.83% of Bacillus and6.55% of Klebsiella.Among them,B.cereus(6.83%),K.pneumoniae(6.28%)and S.chromogenic(4.92%)accounted for a large proportion,and the biochemical identification of 3 pathogens was consistent with the 16 S r DNA sequencing results.The susceptibility detection indicated that 3 pathogens showed a certain degree of drug resistance while 100%B.cereus had quadruple-drug resistance for furazolidone,kanamycin,gentamicin and penicillin.2.Establishment of triple PCR assay for 3 main pathogens of bovine mastitisUsing genome DNA of B.cereus,S.chromogenes and K.pneumoniae as templates,primer pairs were selected and synthesized for the hemolytic gene(hbl A),the hemolytic enzyme gene(khe),and the superoxide dismutase(sod A)of these 3 target pathogenics.The triple PCR assay was developed by optimizing the amplification condition,it showed the optimized annealing temperature was 54℃ while the optimized concentration of BC-hbl A、KP-khe、SC-sod A primer pairs were 0.6 μmol/L,0.1 μmol/L and 0.2 μmol/L,respectively.Specific experiments showed that E.coli,Pasteurella,S.aureus,S.treptococcus,C.perfringens,C.septicum were all negative except the 3 pathogens.The thresholds of detection of the triple assay were 35.76 pg/μL,40.2 pg/μL and 6.28 pg/μL of genomic DNA of B.cereus,K.pneumoniae and S.chromogenes,respectively.To detect 16 milk samples from mastitis,all culture-positive samples were detected positive by the triple PCR for the corresponding bacteria.In summary,this study understanding the main pathogens of bovine mastitis and drug resistance in part dairy farms of Shaanxi province,established a triple PCR assay for 3 main pathogens,which could provide scientific data as well as quick detection methods for the prevention and treatment of bovine mastitis.