一株凡纳滨对虾病原弧菌的分离鉴定与毒力分析

Isolation and identification of a bacterial pathogen,Vibrio parahaemolyticus,for vibriosis of Litopenaeus vannamei

2012年6∽9月福建省沿海城市凡纳滨对虾养殖高位池发生病害，主要症状表现为肝胰腺发白肿大，空肠，死亡率50％以上．本研究对发病现场患病对虾的肝胰腺进行了微生物分离鉴定， BOX-PCR 鉴定所分离的菌株为一株优势细菌，编号为1937．通过16S rRNA 基因分析确定该菌株为弧菌属（Vibrio sp．）．进一步基于 rpoD、topA 和 toxR 3个基因的多位点序列分析（MLSA），表明该菌株为副溶血弧菌（Vibro parahaemolyticus）．从该菌株中还扩增获得副溶血弧菌特异性的溶血素基因．人工浸浴感染试验表明，菌株1937对凡纳滨对虾后期幼体表现出较强致病性，半致死浓度为1．33×106 cfu ／cm3．肌肉注射感染试验表明该菌株对凡纳滨对虾成虾也具有致病性，半致死浓度为1．42×106 cfu ／cm3，并且注射后成虾肌肉白浊，肝胰腺发白，致死率高于其他已报道的致病性弧菌，该菌株极有可能为此次发病的主要病原菌．抗生素试验表明，该菌株对诺氟沙星等29种抗生素中的20种比较敏感．这些研究结果对于该病原菌导致的虾病暴发的防控有参考价值．

Mass mortality of shrimp Litopenaeus vannamei occurred in shrimp farms in a coastal city,Zhangpu Coun-ty,Fujian Province,from June to September,2012.The disease outbreak caused heavily economic loss to shrimp culture industry.The symptom of infected shrimps showed a swelled and whitened hepatopancreas and empty intes-tine,with mortality over 50%.Bacteria were isolated from the hepatopancreas of infected shrimps,with a dominant one designated as strain 1937.Based on 16S rRNA and multilocus sequence analysis （rpoD,toxR and rpoA gene）, strain 1937 was identified as Vibrio parahaemolyticus.What’s more,the haemolysin gene （tlh）specific to V.para-haemolyticus was also detected in strain 1937.Pathogenicity test by immersion infection confirmed that strain 1937 was virulent to Litopenaeus vannamei post larvae and adults,and the LC50 value was 1.33 ×106 cfu /cm3 and 1.42 ×106 cfu /cm3 ,respectively.The symptom of the shrimps challenged with bacterial strain 1937 demonstrated the same as the ones in shrimp farms.Thus,V.parahaemolyticus strain 1937 was proposed as the causative agent for this disease outbreak of shrimp Litopenaeus vannamei.