一株芽孢杆菌PC024的鉴定及其抗WSSV感染效果的研究  

Isolation and identification of Bacillus sp.and evaluation of its effect on WSSV disease resistance in Litopenaeus vannamei

作　　者：孙艳[1] 宋晓玲[1] 刘飞[1,2] 李玉宏[1,3] 黄倢[1] 

机构地区：[1]中国水产科学研究院黄海水产研究所,山东青岛266071 [2]中国海洋大学海洋生命学院,山东青岛266003 [3]上海海洋大学水产与生命学院,上海201306

出　　处：《水产学报》2013年第4期574-583,共10页Journal of Fisheries of China

基　　金：公益性行业(农业)科研专项(201103034);国家虾现代产业技术体系专项(nycytx-46)

摘　　要：为了筛选WSSV的防病益生菌株,从健康中国明对虾消化道分离纯化一株芽孢杆菌PC024,经Biolog碳源利用反应、ATB微生物自动鉴定系统、脂肪酸气相色谱分析得出该菌株与坚强芽孢杆菌的生理生化特性最为相似,该菌株为革兰氏阳性菌,有一根端极鞭毛;细胞呈球杆状,有椭圆芽孢;单个菌落呈圆形,中间略微凸起;16S rRNA序列分析表明,该菌株与坚强芽孢杆菌进化地位最接近,同源性均达到100%,综合以上4种方法的鉴定结果,该菌株被鉴定为坚强芽孢杆菌。将已鉴定的PC024菌株粘附于对虾饲料表面投喂给凡纳滨对虾20 d后,进行WSSV肌肉注射感染,测定投喂和感染后对虾血淋巴上清和肝胰腺的免疫相关酶活性,并对对虾肠道总菌数和添加菌PC024进行计数及鉴定。结果表明:添加该菌的实验组对虾相对存活率高,相对保护率达33.7%;投喂含该菌饲料的实验组对虾血清和肝胰腺的相关免疫酶活性较对照组显著提高,对虾肠道总细菌数始终显著高于对照组,并在实验组能够分离得到坚强芽孢杆菌,坚强芽孢杆菌PC024可作为WSSV的防病益生菌株应用于对虾养殖生产。In order to select WSSV disease-resistant strains,a marine Bacillus sp.was isolated and purified from digestive tract from the healthy of Chinese shrimp（Fenneropenaeus chinensis）.The morphology and gram stain indicated that the strain is a gram-positive and rod-shaped bacterium,with a single polar flagellum and oval spores.The colony is circular and slightly raised.Identification analyses by the Biolog Carbon Source Utilization,ATB Microbial Identification System,and the fatty acid gas chromatography indicated that the most similar strain in physiological and biochemical characteristics is Bacillus firmus.Phylogenetic analysis with 16S rRNA sequence showed that it has 100% homology with the previously reported Bacillus firmus.The cultured strain PC024 was added to feed by conglutinating to the surface of the pellets and fed to Litopenaeus vannamei.After feeding for 20 d,the shrimp was challenged with WSSV by intramuscular injection to observe the cumulative mortality in 14 d post-challenge.The results showed that the experimental group fed with the strain PC024 had a relative survival rate of 33.7% in comparison with the control group.The immune-related enzyme activity in the serum and hepatopancreas of shrimp in the experimental group was significantly increased than the control group.And the total number of bacteria of the intestine of the experimental group is always significantly higher than that of the control group and Bacillus firmus can be isolated from the experimental group.This study suggests that the Bacillus firmus PC024 can be used as the WSSV disease prevention probiotic strains and can further be used in shrimp farming.

关 键 词：凡纳滨对虾 坚强芽孢杆菌 鉴定 免疫酶活性 抗病力 

分 类 号：S943[农业科学—水产养殖]