| Pasteurella multocida(P.multocida)and Mannheimia hemolytica(M.haemolytica)are two common bacteria that inhabit the respiratory tract of cattle,which can cause respiratory diseases in cattle and lead to certain economic losses to the breeding industry.Therefore,it is significance to establish a detection method for simultaneously detecting the two bacteria.At present,bovinederived P.multocida is widely distributed in China,and studying its drug resistance,resistance genes and virulence genes can provide a theoretical reference for understanding the epidemic characteristics,prevention and treatment of this bacterium.(1)In order to improve the efficiency of clinical diagnosis of diseases caused by these two bacteria,the reseach designed specific primers based on the sequence of the Kmt1 gene of P.multocida and the Lkt gene of M.haemolytica,and established a duplex PCR assay for detection of the two bacteria.The results showed that the established duplex PCR assay was negative for other bovine bacteria and viruses,and the detection limits of P.multocida and M.haemolytica pathogenic genomic DNA were 1.57×105 copies/μL and 1.98×106 copies/μL,respectively,indicated that the method has high specificity and adequate sensitivity.A total of 118 clinical samples of diseased cattle with respiratory symptoms in ningxia were detected by the duplex PCR method,the results showed that the detection rate of P.multocida was 57.63%(68/118),the detection rate of M.haemolytica was 30.51%(36/118),the detection rate of co-infection was 24.58%(29/118).Compared with the classical single PCR detection method,the total coincidence rates of P.multocida 91.53%and M.haemolytica 94.07%,indicating that this method can be used for clinical detection.(2)The positive P.multocida identified by the double PCR method above was isolated and identified.P.multocida was identified by observing the morphology of bacterial colonies on blood plate medium,gram staining microscopy and PCR-specific amplification.As a result,14 P.multocida strains were identified and the isolation rate of 20.59%(14/68).(3)In order to based on capsular serotype,lipopolysaccharide genotype and MLST typing for 14 P.multocida isolates by PCR.The results showed that 11 isolates of P.multocida were A:L3,and 3 isolates of P.multocida were B:L2.One serum type A P.multocida was ST 188 and one type serum B P.multocida was ST 1 87.(4)The results of drug sensitivity test of 14 isolates of P.multocida showed that the all to ampicillin with a resistance rate of 100%;there were more resistant isolates to cefazolin and cefotaxime,with the resistance rate of 78.57%;there were more intermediate isolates to tetracycline,with the sensitivity to gentamicin and norfloxacin were 100%.(5)The detection of 10 resistance gene and 27 virulence genes in 14 isolates of P.multocida was carried out by PCR.The results showed that only one drug resistance gene,β-blaTEM drug resistance gene,was detected was 100%.The results of 27 virulence genes tested showed that they carried a total of 20 virulence genes,with the carrying rate of 74%.(6)The virulence gene ptfA of the isolated serum type A P.multocida was sequenced to analyze the sequence consistency,evolutionary relationship and bioinformatics analysis.The results showed that the sequence identity of ptfA gene of this isolate with other strains included in NCBI was 98.6%～100%,isolate from the same branch as Iranian bovine derived P.multocida(KX679397.1).The ptfA gene sequence was 435 bp in length and encoded 144 amino acids.The protein encoded by this gene was a hydrophobic protein with one transmembrane region,no signal peptide,12 potential phosphorylation sites,and subcellular localization mainly in endoplasmic reticulum.And there were a large number of α-helix and irregular coil in the secondarystructure of ptfA proteinand had five antigenic epitopes in total. |
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