摘要
为研究2023年广西壮族自治区钦州湾网箱养殖多纹钱蝶鱼(Selenotoca multifasciata)发生溃疡病的原因,从患病多纹钱蝶鱼的肝、脾和肾组织中分离纯化出优势菌株,并对其进行形态学观察、生理生化特性鉴定、16SrRNA与dnaJ基因序列测序分析、毒力基因检测、人工感染试验及药敏试验.结果表明:分离获得的优势菌在TCBS平板上形成黄色、圆形的光滑菌落,在TSA平板上形成乳白色微凸圆润菌落;生理生化特征分析显示,菌株为β溶血的革兰氏阴性菌,可发酵葡萄糖、蔗糖、甘露糖和阿拉伯糖,V-P试验呈阳性;基于16S rRNA和dnaJ基因序列构建的系统发育树显示,3株优势菌均与鳗弧菌(Vibrio anguil-larum)聚为一支,综合鉴定表明菌株QZ991、QZ992和QZ993均为鳗弧菌;毒力基因检测显示,3株优势菌均携带virA、vah1、virC、empA、angM、flaA和ompU 7种毒力基因;人工感染试验显示,QZ991、QZ992和QZ993对多纹钱蝶鱼的半致死浓度(LD50)分别为5.10×105、4.33×105、2.45×106 CFU/mL;药敏试验显示,3株优势菌均对多粘菌素B、庆大霉素、四环素类、喹诺酮类和多数头孢烯类等多种抗菌药物敏感;组织病理切片观察发现,患病多纹钱蝶鱼多脏器发生了不同程度的损伤,主要表现为充血、出血、坏死和炎性细胞浸润等.研究表明,鳗弧菌为广西钦州湾地区多纹钱蝶鱼溃疡病的病原,对多纹钱蝶鱼具有较强的致病性,并具有一定的耐药性,建议交替使用水产用多西环素和恩诺沙星进行治疗.
Abstract
To investigate the cause of ulcer disease of butterflyfish Selenotoca multifasciata in cage culture systems in Qinzhou Bay,Guangxi Zhuang Autonomous Region,in 2023,the dominant strains were isolated from the liver,spleen and kidney tissues of the diseased S.multifasciata.They were subjected to morphological observation,identi-fication of physicochemical and biochemical characteristics,16S rRNA and dnaJ gene sequence analysis,detection of virulence genes,artificial infection tests and antimicrobial drug susceptibility test.The results showed that the i-solated dominant bacteria formed yellow,round and smooth colonies on TCBS plates,and creamy-white in color,slightly convex,rounded colonies on TSA plates.The physiological and biochemical characteristics showed that the three dominant bacteria were β-haemolytic Gram-negative bacteria,which fermented glucose,sucrose,mannose and arabinose,and were positive in the V-P test.The phylogenetic analysis,encompassing 16S rRNA and dnaJ gene sequences,revealed that the three dominant strains were clustered with Vibrio anguillarum,and the isolates QZ991,QZ992 and QZ993 were finally identified as V.anguillarum,and carried the seven virulence genes(virA,vah1,virC,empA,angM,flaA,and ompU)with the LD50 of 5.10×105,4.33×105 and 2.45×106 CFU/mL,respec-tively.The antibiotic susceptibility test revealed that the three isolated strains were susceptible to different antimi-crobial drugs,including polymyxin B,gentamicin,tetracyclines,quinolones and most of the cephalosporins.His-topathological examination of infected fish tissue delineated various degrees of organ impairment,predominantly characterized by congestion,hemorrhage,necrosis and inflammatory cell in filtration.It is found that V.anguillarum is the pathogen of ulcerative disease of S.multifasciata in the Qinzhou Bay,which is highly pathogenic and some ex-tent to drug resistance,and can be treated with doxycycline and enrofloxacin for aquaculture alternatively.These findings offer scientific reference for the prevention and control of Vibrio infections in the S.multifasciata.
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