鲢病原嗜水气单胞菌分离鉴定及检测方法的建立

Identification and detection of the pathogenic Aeromonas hydrophila isolated from Hypophthalmichthys molitrix

为鉴定江苏高邮市某养殖场混养鲢死亡原因,本研究对濒死鱼进行细菌分离纯化、常规理化特性、分子生物学和致病性试验鉴定以及药敏性检测。鉴定结果表明,分离菌对健康鲢具有较强的致病性,其LD50为5×106.75cfu/mL,根据其表型特征、理化特性及分子生物学鉴定,分离菌为气单胞菌属(Aeromonas)的嗜水气单胞菌(A.hydrophila),其16S rRNA和gyrB基因序列分别为1 460 bp和1 171 bp;药敏试验结果显示,分离菌对37种抗生素中氨曲南、氟苯尼考、庆大霉素等12种抗生素敏感。另外,根据A.hydrophila的气溶素aerA基因设计引物,建立了PCR检测方法,该方法可以扩增出280 bp的aerA基因片段,其最低检出量为3×104cfu/mL。本研究对A.hydrophila导致鲢出血死亡病原菌的分离鉴定以及建立的该菌检测方法,对鲢健康养殖和病害防治具有重要的意义。

In October 2011, a continuous high mortality of Hypophthalmichthys molitn＇x occurred in a farm of Gaoyou area in Jiangsu province, and the diseased fish characterized by haemorrhages at gill, base of fin, and swelling of the abdomen with yellow fliud of some diseased fish. To identify the pathogen, the bacteria was isolated from the diseased H.molitrix, which shown the median lethal dose was 5x1067s cftffmL to H.molitrix. The pathogenic isolate was identified to be Aeromonas hydrophila based on physiological and biochemical tests and amplifications of 16S rRNA （1,460 bp） and gyrB （1,171 bp） gene. The drug sensitivity tests displayed that among 37 antibiotics, the isolate was only sensitive to 12 drugs including Ammonia-South, Florfenicol, Gentamicin and so on. In addition, Aerolysin （aer） was main virulence genes of A.hydrophila and the 280 bp DNA fragment of aerA gene could be amplified by PCR. It was confirmed that the isolate carried the aerA gene. Furthermore, it provided the PCR method was applicable for specifically detecting of the A.hydrophila. These results demonstrated that A.hydrophila was the main causal agent responsible for the mass mortality of H.molitrix in this case.