Isolation,Drug Sensitivity Test and Construction of Prokaryotic Expression Vector of Virulence Genes of Enterococcus from Dairy Cow with Mastitis in Gansu Province

The aim of this study was to isolate Enterococcus in clinical dairy cow mastitis,detect its drug resistance and virulence genes,a total of 93 milk samples were collected from 41 dairy cosw with clinical mastitis in eastern,central and western regions of Gansu province,and then construct a prokaryotic expression vector for virulence genes.This experiment used selective medium to isolate and purify bacteria.16S rRNA and biochemical experiments combined method to identify the isolated strains.16 antibiotics were selected for drug sensitivity test,and conventional PCR method was used to detect the carrying of 11 virulence genes.Finally,the detected virulence genes with immunogenicity were selected for the construction of prokaryotic expression vectors.The separation and identification results showed that 18 strains of Enterococcus were isolated and identified from the 93 milk samples,which were divided into 9 species.Drug susceptibility results showed that most of the isolates were multiple resistant to bacteria,accounting for 88.89%.No vancomycin-resistant Enterococcus was found,vancomycin sensitivity rate was 94.12%,and all isolates were resistant to at least one antibiotic.Virulence gene test results demonstrated that 11 virulence genes were detected,the detection rate of cob gene (44.44%) was the highest,the detection rates of efaA,hyl,ccf and esp genes were 33.33%,27.78%,27.78% and 22.22% respectively,the detection rates of Asa1,cylA,EF3314 and gelE genes were all 16.67%,while Ace and cylM genes had the lowest detection rate (11.11%).The virulence genes combination was different due to different strains.Ace and gelE genes with immunogenicity were selected and the prokaryotic expression vector pET32a-Ace and pET32a-gelE were successfully constructed.The results provided basic data and biological materials for subsequent mapping of regional epidemiology of cow mastitis in three regions and preparation of corresponding antibodies and subunit vaccines.