石斑鱼腐皮病病原的分离鉴定及药物敏感性分析  

Isolation, identification and drug sensitivity analysis of the pathogen of grouper skin ulcer

作　　者：赵奇 赵捷雄 鲁东东 韩雅静 赵欣雅 唐嘉宝 王娜[1] 高桂生[1] ZHAO Qi;ZHAO Jiexiong;LU Dongdong;HAN Yajing;ZHAO Xinya;TANG Jiabao;WANG Na;GAO Guisheng(Ocean College of Hebei Normal University of Science&Technology,Qinhuangdao 066004,China;Changli County Bureau of Agriculture and Rural Affairs,Changli 066004,China)

机构地区：[1]河北科技师范学院海洋学院,河北秦皇岛066004 [2]昌黎县农业农村局,河北昌黎066004

出　　处：《黑龙江畜牧兽医》2023年第6期133-138,144,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：河北省重大科技成果转化项目(20286701Z)。

摘　　要：为了查明河北省秦皇岛市某大型海水鱼类养殖场石斑鱼腐皮病的病原,进而精准有效地对患病石斑鱼进行治疗,试验首先采用形态学观察、生化鉴定法对从患病石斑鱼腐皮病病灶中分离出的一株优势菌进行初步鉴定,通过16S rRNA、hsp 60基因序列分析对分离菌进行进一步鉴定,然后采用PCR方法检测分离菌的毒力基因和耐药基因,最后检测该分离菌对16种抗生素和19味中草药的药物敏感性。结果表明:分离菌为革兰氏阴性短杆菌,呈弧状、杆状等多种形态;其生化特性与副溶血弧菌生化特性符合率高达99%;BLAST分析发现,分离菌16S rRNA基因序列与多种弧菌的基因相似性高达99.7%;基于分离菌hsp 60基因序列构建的系统进化树显示,该菌与副溶血弧菌自动聚为一支;分离菌含有不耐热溶血素基因tlh和相对耐热直接溶血毒素基因trh,并且含有喹诺酮类耐药基因qnr、大环内酯类耐药基因ermB和四环素类耐药基因tetA,分离菌对泰乐菌素、环丙沙星、头孢拉定、恩诺沙星、替米考星、氧氟沙星、土霉素、卡那霉素、强力霉素、新霉素、头孢曲松共11种抗生素表现为高度敏感,对氨苄西林表现为中度敏感,对青霉素、大观霉素、林可霉素、阿莫西林共4种杭生素药物具有耐药性;分离菌对连翘、黄芩、竹叶紫胡、紫花地丁、甘草、黄柏共6味中草药表现为高度敏感,对大青叶、苦地丁、金银花、干姜、千里光、薄荷、蒲公英、苦参、马鞭草共9味中草药表现为中度敏感,对使君子、路边青、穿心莲和丁香叶共4味中草药具有耐药性。说明该养殖场石斑鱼腐皮病的病原为副溶血弧菌,该副溶血弧菌耐药情况复杂,对4种抗生素和4味中草药耐药。In order to identify the pathogen of skin ulcer in groupers from a large marine fish farm in Qinhuangdao City, Hebei Province, and to accurately and effectively treat the infected groupers, a dominant strain isolated from the lesion of putrefactive disease in diseased groupers was initially identified by morphological observation and biochemical identification. The isolated strains were further identified by 16S rRNA and hsp 60 gene sequence analysis, and then the virulence genes and drug resistance genes of the isolated strains were detected by PCR method. Finally, the drug sensitivity of the isolated strains to 16 antibiotics and 19 Chinese herbs was detected. The results showed that the isolated bacteria were gram-negative brachybacterium, with various forms such as arc-shaped and rod-shaped. The coincidence rate of biochemical characteristics with those of Vibrio parahaemolyticus was up to 99%. BLAST analysis showed that the 16S rRNA sequence of the isolate shared 99.7% gene similarity with a variety of Vibrios. The phylogenetic tree based on the hsp 60 gene sequence of the isolate showed that the isolate and Vibrio parahaemolyticus were automatically clustered into one branch. The isolates contained the heat-labile hemolysin gene tlh and the relatively heat-labile direct hemolysin gene trh. It also contained quinolone resistance gene qnr, macrolide resistance gene ermB and tetA. The isolates were highly sensitive to 11 antibiotics(tylosin, ciprofloxacin, cephradine, enrofloxacin, tilmicocin, ofloxacin, terramycin, kanamycin, doxycycline, neomycin and ceftriaxone), moderately sensitive to ampicillin, and resistant to four antibiotics(sinomycin, spectinomycin, lincomycin and amoxicillin). The isolates were highly sensitive to Forsythia suspensa, Scutellaria baicalensis Georgi, Bupleurum marginatum, Philippica Herba, Glycyrrhiza uralensis and Phellodendri Chinensis Cortex.They were moderately sensitive to Isatidis Folium, Herba corydalis Bungeanae, Lonicerae Japonicae Flos, Rhizoma Zingiberis, Senecionid Scande

关 键 词：副溶血弧菌 石斑鱼 腐皮病 药敏试验 毒力基因 耐药基因 

分 类 号：S943[农业科学—水产养殖]