奶牛乳房炎性无乳链球菌药敏试验及毒力基因检测

Drug sensitivity test and virulence gene test of Streptococcus agalactiae in cow breast

旨在调查山东某奶牛养殖场无乳链球菌的感染状况、药敏性及毒力基因携带情况。采集该牛场临床型乳房炎奶样200份,分别用5%绵羊血琼脂平板、MHA药敏琼脂培养基、16S rRNA基因引物、12种药敏片和多重PCR进行无乳链球菌的药敏性及毒力基因的检测。从中分离出42株无乳链球菌,分离率为21%。所分离出的无乳链球菌培养菌落特征符合该菌特性;依据GenBank数据库中无乳链球菌16S rRNA基因序列设计相关引物扩增所得PCR产物在400 bp处出现单一条带,经测序比对符合GenBank数据库中的无乳链球菌序列。而药物敏感试验结果显示,链球菌对临床上常用的β-内酰胺类(青霉素)、大环内酯类(红霉素)、大观霉素及喹诺酮类(氧氟沙星)类药物较敏感,耐药性分布并不严重。通过多重PCR法对分离出的无乳链球菌进行毒力基因检测,分离出的无乳链球菌毒力基因中pavA,cylE,hylB,pbplA,cfb和fbsA的基因检出率为100%,lmb基因检出率为97.62%,cspA基因检出率为31%,未检出bac和rib基因。

The purpose of this study was to investigate the infection status, drug sensitivity and virulence gene of Streptococcus agalactiae(S.agalactiae) in a dairy farm in Shandong province.Two hundred milk samples from cows with clinical mastitis were collected, the bacteria were identified using 16 S rRNA analysis, the susceptibility of S.agalactiae was tested using MHA drug-sensitive agar medium and 12 drug-sensitive tablets.The virulence genes were detected using multiplex PCR amplification.Forty-two strains of S.agalactiae were isolated, the isolation rate was 21%.The colony characteristics of the isolated bacteria were consistent with the characteristics of S.agalactiae.The sequence of the 16 S rRNA gene amplified by PCR with the relevant primers was consistent with the sequences of the S.agalactiae in the GenBank database.The drug sensitivity test results showed that S.agalactiae was sensitive to β-lactam(penicillin),macrolide(erythromycin),spectinomycin and quinolone(ofloxacin) drugs commonly used in clinic.The distribution of drug resistance was not serious.The multiplex PCR method was used to detect the virulence genes of the isolated S.agalactiae.The gene detection rate of pavA,cylE,hylB,pbplA,cfb and fbsA in the isolated S.agalactiae was 100%,the gene detection rate of lmb was 97.62%,the detection rate of cspA gene was 31%,and bac and rib genes were not detected.