一株大黄鱼致病性溶藻弧菌的分离鉴定与毒力相关基因分析

Isolation,identification and virulence-related gene analysis of a pathogenic Vibrio alginolyticus from the Large yellow croaker

2018年,宁德蕉城大湾渔排大黄鱼出现体表和鳍条溃疡、出血,鳃缺损等症状。为鉴定该菌株并评估其致病性,本研究从患病大黄鱼鳃部分离得到一株优势菌株,生理生化特征显示,该菌株可在TCBS培养基上生长,菌落颜色为黄色,为革兰阴性短杆菌,具有嗜盐性,生理生化指标与标准株17700一致。结合生理生化指标与16S rDNA分析,确定该菌为溶藻弧菌,命名为Val180620。攻毒结果显示,Val180620菌株对大黄鱼具有较强的致病力,在水温25℃腹腔注射感染时,对规格(20±2)g大黄鱼的LD 50为2.38×10^6 CFU。毒力相关基因分析结果表明,Val180620菌株携带有CollagenaseVA、ToxR、FlaA和TRH等毒力相关基因。此外,本研究未从菌株Val180620中检测出UreR基因,但菌株Val180620仍表现出较强的致病性,说明UreR不是决定溶藻弧菌毒力的主要因子。

Vibriosis is the most prevalent and the most harmful marine disease of marine aquaculture animals.Large yellow croaker(Pseudosciaena crocea),as the largest aquaculture fish,is the main infection host of Vibrio.Body surface ulcers,hemorrhage,and silk rot are the main symptoms of Vibriosis.In this study,a dominant strain was isolated from the diseased large yellow croaker of the Ningde Jiaocheng Dawan fish raft of China,which had surface and fin ulcer,hemorrhage and gill defect.In order to identify the strain and evaluate its pathogenicity,the culture was purified and the physiological and biochemical characteristics of the strain were analyzed.The 16S rDNA of the strain was identified,the pathogenicity of the strain to the Large yellow croaker was determined,and the virulence factor of the strain was also tested.The culture results showed that the strain could grow on TCBS medium,and the colony color was yellow.The physiological and biochemical identification results indicated that the strain was gram-negative Brevibacterium,which was halophilic,and the physiological and biochemical indexes were consistent with the standard strain 17700;16S rDNA analysis results showed that the homology rate of the strain with Vibrio alginolyticus was 99%,and it was confirmed that the strain was Vibrio alginolyticus and named as Val180620.The results of the challenge showed that the Val180620 strain had strong pathogenicity to the Large yellow croaker.When injected at a water temperature of 25℃,the LD 50 of the Large yellow croaker of the average body weight(20±2)g was 2.38×10^6 CFU.The results of virulence-related gene analysis indicated that the strain Val180620 carried virulence-related genes such as CollagenaseVA,ToxR,FlaA and TRH,which explained why the strain Val180620 has high pathogenicity and caused ulceration and bleeding of the infected fish.In addition,the UreR gene was not detected in the strain Val180620,but the strain Val180620 showed high pathogenicity,which indicated that the UreR gene maybe not the determinant of the virulence of Vibrio alginolyticus.