三疣梭子蟹病原弗氏柠檬酸杆菌的分离鉴定及定居因子抗原基因检测

2009年10月江苏赣榆地区某养殖场养殖的三疣梭子蟹出现大量死亡,症状主要表现为:病蟹行动缓慢、不摄食,蟹体消瘦,打开头胸甲可见肝胰腺、鳃、肌肉等内脏组织水肿,部分肝胰腺和肌肉组织呈腐烂状。从患病梭子蟹肌肉、肝胰脏、体内积液中分离到大量优势生长的细菌。人工感染试验,证明分离菌(JG091120-1)对健康三疣梭子蟹具有很强的致病性。对分离菌进行了形态特征、理化特性等常规表型生物学检验,同时利用分子生物学方法测定了代表菌株的16S rRNA和gyrB基因序列,其中分离菌16S rRNA基因序列长度为1 451 bp(登录号HQ170626),gyrB基因序列长度为1 186 bp(登录号HQ170627),分析了16S rRNA和gyrB两种基因序列的同源性。根据分离菌的表型及分子生物学特性,判定该菌为肠杆菌科枸橼酸属的弗氏柠檬酸杆菌。定居因子抗原cfa是肠杆菌科产肠毒素细菌的一种重要致病因子,利用特异性引物进行cfa基因的PCR扩增,分离菌可以扩增出大小在100 bp的基因片段,表明本次分离的病原弗氏柠檬酸杆菌具有cfa毒力因子。

Detection of cfa gene and isolation and identification of the pathogen Citrobacter freundii isolated from Portunus trituberculatus

Portunus trituberculatus,mainly distributed in East Chins Sea and Yellow Sea,is a large marine economic crabs with rich nutritional and medicinal properties.In recent years,P.trituberculatus aquaculture has developed rapidly along the Jiangsu coastal waters,while some epidemic diseases broke out which was attributed to many factors,for example,pathogenic microorganism.In the late autumn of 2009,the cultured P.trituberculatus suffered from serious disease in one farm of Ganyu County in Jiangsu Province.The main symptom displayed as drumble,not feeding,the tissue oedema of liver,gill and muscle;the liver and muscle were rotten in some crabs.Virtually pure dense cultures were obtained from liver,muscle,and lymph fluid of diseased P.trituberculatus,and strong pathogenicity of the isolate strain(JG091120-1)to the P.trituberculatus.The phenotypic characteristics of isolate strain were examined,including morphological characteristics,physiological,and biochemical characteristics,the 16S rRNA and gyrB genes were amplified and compared with those sequences deposited in databases.Phylogenetic analysis of 16S rRNA and gyrB genes indicated high homogeneity between the isolate strain and Citrobacter freundii from GenBank database.The 16S rRNA gene fragment length is 1 451 bp(GenBank accession number HQ170626)and gyrB gene fragment length is 1 186 bp(GenBank accession HQ170627)The results showed that the identified isolate strain belonged to C.freundii.Colonization factor antigen was a pathogenic factor of enterobacteriaceae which could produce enterotoxin that was capable of causing death.And the cfa gene(100 bp)could be amplified by PCR.It was concluded that the isolated strain C.freundii carried cfa and provided an effective method for detecting the C.freundii.C.freundii was a conditional pathogen that is gram-negative,bacillus,aerobic or amphimicrobian.And many diseases were attributed to the C.freundii for man,reptiles,amphibians.In recent years,a lot of aquatic animals such as Cherax quadricianalus,Ophicephalus argus,Oreochromis niloticus,Eriocheir sinesis etc.,were also infected by the C.freundii.In order to choose the appropriate antibiotic the drug sensitive was tested,and testing results by paper extension methods for drug sensitive showed that among 48 antibiotics,ciprofloxacin,fleroxacin,ceftriaxone,enoxacin,enrofloxacin,floxacin and levofloxacin had the most inhibitive effects on the strain.The paper reported for the first time that C.freundii could lead to the mass mortality of P.trituberculatus in Jiangsu coastal waters and a detailed study of the pathogen was made,which would be helpful in the disease control and health management in P.trituberculatus cultivation.