凡纳滨对虾(Litopenaeus vannamei)病原副溶血弧菌(Vibrio parahaemolyticus)的表型及分子特征

PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF PATHOGENIC VIBRIO PARAHAEMOLYTICUS ISOLATED FROM PENAEUS VANNAMEI

采用常规的形态及生理生化特性检验及分子生物学等方法,对引起凡纳滨对虾幼虾发生毁灭性死亡的病原菌进行了致病性、表型生物学及分子生物学的系统研究。结果表明,分离菌为弧菌属(Vibrio Pacini 1954)的副溶血弧菌(Vibrio parahaemolyticus),对凡纳滨对虾、中国对虾及日本对虾仔虾均有强致病性。分离鉴定的4株副溶血弧菌具有淀粉酶、明胶酶、卵磷脂酶活性,但不具有蛋白酶、脂肪酶活性,KP溶血均呈阴性,且均具有K抗原;代表菌株(JGB080708-1株)的16S rRNA基因序列(长度为1456bp,GenBank登录号为GQ205448)和gyrB基因序列(长度为1195bp,GenBank登录号为GQ205453)与副溶血弧菌的两种基因序列相似性均可达98%以上。病原菌的耐药性检测结果显示,对供试49种抗菌药物中的林可霉素等6种药物耐药。

A mass mortality of cultured Litopenaeus vannamei broke out during summer in a local farm in northern Jiangsu Province. Syndromes include swimming near water surface and the pond walls, with black gill and opaque muscle, Tissue samples were taken in the liver, and strong pathogenicity to L. vannamei, P. japonicus and R chinensis were determined by immersion. The phenotypic characteristics of isolates （4 strains） were examined in morphology, physiology and biochemistry; the 16S rRNA and gyrB were amplified by PCR and compared with sequences in databases, from which molecular phylogenetic trees were constructed. The sequenced 16S rRNA gene of strain JGB080708-1 （GenBank accession No.GQ205448） is 1456bp in length, the sequenced gyrB gene of strain JGB080708-1 （GenBank accession No.GQ205453） is 1195bp in length. Results of phylogenetic analysis on 16S rRNA and gyrB genes revealed a high homogeneity between the isolates and V. parahaemolyticus from GenBank database. Comparison in method between 16S rRNA and gyrB genes confirms that gyrB gene analysis is more useful. The isolates were identified as V. parahaemolyticus [（Fujino et al 1951） Sakazaki Nakamura and Takizawa 1963] of Vibrio （Pacini 1854） in phenotypic and molecular characteristics. Test on the activity of extracellulase and hemolysin shows that the isolates could produce diastase, gelatinase, and lecithinase, but no proteinase and lipase, and KP haemolysis was negative. Examination of K-antigen and agglutination titers show that the isolates have K-antigen, and the titer of OK-agglutinogen with OK-antiserum and O-agglutinogen with O-antiserum was the same （7log2）; the titer of OK-antiserum with O-agglutinogen and O-antiserum with OK-agglutinogen was 2log2 and 3log2, respectively. The drug resistance of isolates to 49 antimicrobial agents was detected, the results show that the isolates are resistant to penicillin G, ampicillin, oxacillin, lincomycin, bacitracin and amoxicillin.