诸氏鲻虾虎鱼出血病病原鉴定及传播途径分析

Identification of the pathogen responsible for causing hemorrhage in Mugilogobius chulae and its transmission routes

目的分离鉴定诸氏鲻虾虎鱼(Mugilogobius chulae)出血病病原,并探讨其传播途径,为虾虎鱼微生物质量控制提供技术依据。方法从患出血病虾虎鱼肝脏取样分离病原菌,经菌落形态、生化特性和分子特征鉴定细菌,回归感染确定其致病性,并采用双重PCR方法对虾虎鱼、轮虫、卤虫、水进行嗜水气单胞菌(Aeromonas hydrophila)检测。结果分离到1株优势菌PYMc15-1,该菌株API生化反应结果与嗜水气单胞菌一致,gry B序列与Gen Bank上的嗜水气单胞菌相应基因序列同源性最高;根据已知序列构建gry B分子系统进化树,分离株PYMc15-1与嗜水气单胞菌聚类;分离株PYMc15-1溶血素基因(hly A)和气溶素基因(aer A)检测结果阳性,对虾虎鱼半致死剂量(median lethal dose,LD50)为每尾1.2×104cfu。野生虾虎鱼、卤虫和野生轮虫致病性嗜水气单胞菌检测出现阳性结果,虾虎鱼封闭群、室内培育的轮虫以及海水样品致病性嗜水气单胞菌检测均为阴性。结论嗜水气单胞菌可自然感染虾虎鱼;野生虾虎鱼和生物饵料是其潜在传播媒介,引进虾虎鱼和生物饵料时应检测该病原。

Objective To isolate and identify the pathogen responsible for causing hemorrhage in Mugilogobius chulae,and to investigate its route of transmission,providing a technical basis for microbial quality control in M.chulae.Methods The pathogen was isolated from the liver of diseased M.chulae.The morphological,biochemical,and molecular characteristics were used to identify the isolated bacteria.An artificial infection experiment was carried out to assess the pathogenicity of isolated strains.Double PCR of Aeromonas hydrophila virulence genes was used to screen samples of M.chulae,Brachionus plicatilis,Artemia nauplii,and water.Results The dominant strain PYMc15-1 was isolated from diseased M.chulae.The characteristics(API ID32 E)of PYMc15-1 corresponded to A.hydrophila.The gyrase subunit B(gry B)gene sequence of the strain possessed high similarity with those of A.hydrophila registered in Gen Bank.Phylogenetic tree analysis of Aeromonas based on the gry B gene showed that strain PYMc15-1 clustered together with A.hydrophila.The hemolysin(hly A)and gas lysin(aer A)genes could be amplified from the strain PYMc15-1,and the median lethal dose(LD50)of PYMc15-1 to healthy M.chulae was 1.2×104 cfu/fish.Wild M.chulae,Artemia nauplii,and wild B.plicatilis were positive for pathogenic A.hydrophila,while the closed colony of M.chulae,indoor cultured B.plicatilis,and seawater samples were negative for pathogenic A.hydrophila.Conclusions A.hydrophila can naturally infect M.chulae.For wild M.chulae,living feeds are potential vectors of A.hydrophila.A.hydrophila should be tested when introducing M.chula and living feeds.