| Dairy mastitis is one of the widely spread infectious diseases in dairy farming.In addition to the economic loss caused by the reduction of milk yield caused by mastitis,the contamination of food by microbial metabolites,especially toxins,is also a worrying problem.The incidence of mastitis varies from farm to farm,so much so that the primary cause of mastitis may vary significantly.For the treatment of mastitis,antibiotics are usually the first choice.However,the effectiveness of antibiotic treatment is limited due to the increase in bacterial resistance,and the level transfer of microorganisms from animal populations to humans may also be a public health hazard due to antibiotic resistance.In order to develop effective mastitis control measures,it is important to identify the major mastitis pathogens and drug resistance.In this study,88 samples of mastitis milk from 16 dairy farms in Shaanxi were isolated and identified,and a multiplex PCR method was established for the detection of the main pathogens.The drug resistance of coagulase-negative staphylococci(CNS)was analyzed.The main test results are as follows:1.A total of 143 strains of bacteria were isolated from 88 samples of mastitis milk.The main pathogens accounted for 67.83% of the total isolates,including Staphylococcus(26.57%),Enterobacter(19.58%),Streptococcus(13.29%)and Klebsiella(8.39%).S.aureus(6.29%),E.coli(14.69%),S.agalactiae(5.59%),K.pneumoniae(7.69%)and CNS(20.28%)accounted for the largest proportion.2.Using genomic DNA of E.coli,K.pneumoniae,S.aureus and S.agalactiae as templates,corresponding primers were designed and synthesized for pho A gene of E.coli,khe gene of K.pneumoniae,nuc gene of S.aureus and tuf gene of S.agalactiae.By optimizing the reaction conditions,a quadruple PCR method was established.Multiple PCR reaction conditions were optimized and specificity and sensitivity tests were performed.The results showed that the four pathogens were specifically detected by multiplex PCR.Sensitivity test showed that the minimum detection amounts of genomic DNA for E.coli,S.aureus,S.agalactiae and K.pneumoniae were respectively 17.11,19.16,109.6 and 20.89 pg/μL.Clinical tests showed that the positive detection rate of this method was higher than that of bacterial separation,and the coincidence rate of the two methods was as high as 94.3%.3.Drug sensitivity test and drug resistance gene detection were performed for isolated coagulase-negative staphylococci.The results showed that 29 CNS strains showed different degrees of resistance to 19 antibiotics and multiple drug resistance.The sensitivity to ciprofloxacin,norfloxacin and flfenicol was 69.0%,86.2% and 86.2%,respectively.The drug resistance rate of lincomycin was 100%,followed by the drug resistance rate of macrocyclic lipids,sulfonamides and β-lactam antibiotics were 86.2%,75.8% and 48.3%,respectively.The positive rates of dfr G,Blaz,Inu A and mec A were 27.6%-55.2%,and the positive rates of msr B and erm C were less than 20.7%.The positive rate of drug resistance phenotype was not consistent with the detection of drug resistance genes,indicating that CNS drug resistance mechanisms are complex and diverse,and phenotypic resistance of all antibacterial drugs cannot be inferred based on the presence or absence of drug resistance genes.In conclusion,this study based on the analysis of the main pathogens causing mastitis in dairy cows in some areas of Shaanxi Province,a quadruple PCR method for detecting the main pathogens was established,and the relationship between the drug-resistant phenotype and drug-resistant genes of coagulase-negative staphylococci was compared and analyzed,providing a reference for the prevention and treatment of cow mastitis in this area. |
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