四株鳖源致病性嗜水气单胞菌(Aeromonas hydrophila)的表型、分子鉴定及其毒力基因检测

PHENOTYPE AND MOLECULAR INDENTIFICATION OF VIRULENCE GENES FOR FOUR PATHOGENIC AEROMONAS HYDROPHILA ISOLATED FROM A DYING TRIONYX SINENSIS

采用生态毒理学、表观分类学及分子生物学方法,对具白底板病典型症状濒死中华鳖内脏中分离获得的4株病原菌开展了以致病性、表型分析、分子鉴定及毒力基因检测为内容的实验研究,结果表明:(1)4株病原菌均具致病性,致死力由大到小依次为ZHYYZ-2、ZHYYZ-4、ZHYYZ-1、ZHYYZ-3;(2)4株病原菌均为呈短杆状、具溶血活性的革兰氏阴性细菌,VITEK2型全自动细菌鉴定与药敏系统和ATBExpression型细菌鉴定与药敏智能系统均显示为嗜水气单胞菌;(3)ZHYYZ-1、ZHYYZ-2、ZHYYZ-3、ZHYYZ-4的16SrDNA序列长度分别为1460、1464、1466、1461,经Blast同源性检索表明它们所扩增的16SrDNA序列与GenBank数据库中登记的71株嗜水气单胞菌的相似性均为99%;(4)经PCR特异性检测,各实验菌均含有Aha、AHH、AerA和OMP。根据4株实验菌的表型和分子生物学特征,判定它们均为气单胞菌属的致病性嗜水气单胞菌。

Based on exotoxicology, phenotypic information and molecular biological methods, the identification of pathogenicity, phenotypic characteristics and virulence genes were carried out for four pathogenic bacteria, which were isolated from a dying Trionyx sinensis with typical white abdominal shell disease. We found all of four bacterial strains were pathogenic. The order of killing power of these strains was ZHYYZ-2 ZHYYZ-4 ZHYYZ-1 ZHYYZ-3. After an injection of 2.2×107 CFU/g, the occurrence of clinical infection symptoms or death in experimental turtles was 1 or 19h, respectively. These strains were gram-negative bacteria with short pole-like and hemolytic activity. Both VITEK2 system and ATB expression system showed that the bacteria were Aeromonas hydrophila. ZHYYZ-1, ZHYYZ-2, ZHYYZ-3, and ZHYYZ-4, whose 16S rDNA sequence were of 1460, 1464, 1466, and 1461, respectively, were registered in GenBank, with the accession numbers being GU563992, GU563993, GU563994, and GU563995 respectively. Blast analysis indicated that the similarity of their 16S rDNA sequences and 71 strains of A. hydrophila in the GenBank database was 99%. PCR test showed that all the experimental bacteria contained Aha, AHH, AerA and OMP gene. Based on these phenotype and mo- lecular biology characteristics, the isolated bacteria were identified as A. hydrophila, and they were the strains of patho- genicity.