西伯利亚鲟海豚链球菌的分离鉴定及毒力基因检测

Isolation,identification and detection of virulence genes of Streptococcus iniae from Acipenser baerii

2013年8—9月,四川雅安汉源湖养殖的西伯利亚鲟发生一种以体表溃疡、内脏出血和心外膜囊肿为特征的传染病。为明确其病因,本研究从自然发病鱼的肝、脾和肾进行了病原菌的分离、人工感染、分离菌的表型特征和分子生物学特征的检测。结果从患病鱼体内分离到一株G+链状球菌(Ab130920),人工感染实验证实了其病原性,生理生化特性与海豚链球菌(ATCC29178)基本一致;16S r DNA序列(Gen Bank登录号:KJ162337)与Gen Bank中S.iniae16S r DNA序列同源性最高,在以16S r DNA序列及其Gen Bank中同源性较高的相关序列构建的系统发育树上,分离菌与海豚链球菌聚为一支;同时,在基于S.iniae lct O基因的特异性PCR检测中,从分离株基因组DNA扩增出预期大小的870 bp条带,进而鉴定分离菌Ab130920为S.iniae。在对cps D、sim A、sag A、pdi和scp I等5种S.iniae毒力基因的多重PCR检测中,分离菌均扩增出相应大小的特异性片段,表明其为一毒力较强的菌株,与人工感染实验的高致病性结果相佐证;药物敏感性检测发现其对阿莫西林、强力霉素、氟苯尼考等抗菌药物敏感,但对新生霉素、利福平、氧氟沙星耐药。

During August and September of 2013, a serious infectious disease characterized by body ulcer, internal organs haemorrhage and nodules showing on epicardium occurred in Acipenser baerii in Hanyuan Lake,Ya＇ an, Sichuan Province. To elucidate the aetiology, the bacteria isolated from the livers, spleens and kidneys, and artificial infection, physiological and biochemical analysis and molecular analysis of the isolate were conducted. The results showed that a Gram-positive, chain-forming coccus （Ab130920）was isolated and the strain was lethal to A. baerii. Physiological and biochemical characteristics were consistent with Streptococcus iniae（ATCC29178）;the 16S rRNA gene sequencing analysis by Blast in GenBank indicated that the isolate （KJ162337） showed high levels of similarity to S. iniae （99%） ; A phylogenetic tree was constructed based on the 16S rDNA sequences of the isolate and the homologous sequences of other strains of Streptococcus, and the strain and other S. iniae strains formed a tight cluster; Meanwhile, expected segment of 870 bp was yielded in the specific PCR detection of S. iniae based on S. iniae lctO gene. As expected,the strain Ab130920 was positive by multiplex PCR for all five virulence genes（ cpsD,simA ,sagA, pdi and scpI）test, showing amplification products of the expected sizes, which was further identical the isolate to S. iniae and confirmed the pathogenicity of it, and consistent with the result of artificial infection. The drug sensitivity results showed the isolate was sensitive to amoxicillin, doxycycline and florfenicol, etc. but resistant to novobiocin, ofloxacin, rifampicin.