Isolation And Identification Of Main Pathogenic Bacteria Of Cow Mastitis In Changji Area Of XinJiang And Establishment Of Multiplex PCR Detection Method

The Dairy cow mastitis is an important reason causing low efficiency in dairy industry.The pathogenic mechanism of dairy cow mastisis is complicated and this is difficult to cure and often leads to the knock out of dairy cows.Pathogenic bacteria infection is the main cause of mastitis in dairy cows.Many pathogenic bacteria reported are mainly Staphylococci and Streptococci,etc.Meanwhile,the pathogenic bacteria isolated from different regions are different,in order to understand the main pathogenic bacteria of cow mastitis in Changji region of Xinjiang,the experiment collected milk samples from the dairy farm of Changji region of Xinjiang,and isolated and identified the pathogenic bacteria of CMT positive samples,combined with the main pathogenic bacteria of cow mastitis reported in different areas,a multiplex PCR method was established to detect the main pathogenic bacteria.the following results were obtained.1.In this experiment,160 milk samples were collected from 3 large-scale farms in Changji,Xinjiang,among which 84 milk samples were positive for CMT detection,and 84 positive milk samples were selected for isolation of pathogenic bacteria.49 kinds of bacteria were isolated from 84 positive samples,with a total of 156 bacteria,including 45 strains of Escherichia,24 strains of Staphylococcus,19 strains of Streptococcus,15 strains of Enterococcus,10 strains of Bacillus,10 strains of Enterobacter,9 strains of Klebseria,4 strains of Shigella,Acinetobacter and 16 strains of other species.2.Do drug sensitive test to the separated Escherichia coli,Staphylococcus aureus,Enterococcus faecalis 12 kinds of main pathogenic bacterias,The 12 main pathogenic bacteria showed serious drug resistance to Oxacillin,Penicillin and Cephalothin,and the drug resistance rate of Staphylococcus haemolyticus was 78%,while 11 of the 12 pathogenic bacteria showed sensitivity to Florfenicol,which could be used as the priority choice for antibiotic treatment of dairy mastitis in Changji area.3.A multiplex PCR method was developed for simultaneous and rapid detection of 6 pathogenic bacteria.According to the isolation of bovine mastitis bacteria in different regions of the world,China,Xinjiang and this experiment,six kinds of bacteria including Klebsiella pneumoniae,Staphylococcus aureus,Enterococcus faecalis,Escherichia coli,Streptococcus agalactiae and Proteus mirabilis were selected to construct multiplex PCR method,it was found that the best reaction system was 0.9 μl of primer for Klebsiella pneumoniae by primer design,single PCR and multiplex PCR,a total of 0.9 μl of Staphylococcus aureus primer(20 mol),0.9 μl of Enterococcus faecalis primer,0.9 μl of Streptococcus agalactiae primer,0.9 μl of Escherichia coli primer,a total of 0.9 ΜL Primer,4.1 μl dd H2 O and 12.5 μl r Taq(RR901A)were used in the reaction system.The optimal annealing temperature was 52°C and the optimal cycles were 30 times.The coincidence rate between the detection results and the identification results of bacteria was 72%.