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美洲黑石斑鱼(Centropristis striata)“突眼症”的病原菌分离鉴定
陈建国,陈 超,李炎璐,孙曙光,邵彦翔,张廷廷,刘 莉,孙 涛
作者单位
陈建国 上海海洋大学水产与生命学院 上海 201306农业部海洋渔业可持续发展重点实验室 青岛市海水鱼类种子工程与生物技术重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
陈 超 上海海洋大学水产与生命学院 上海 201306农业部海洋渔业可持续发展重点实验室 青岛市海水鱼类种子工程与生物技术重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
李炎璐 农业部海洋渔业可持续发展重点实验室 青岛市海水鱼类种子工程与生物技术重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
孙曙光 上海海洋大学水产与生命学院 上海 201306农业部海洋渔业可持续发展重点实验室 青岛市海水鱼类种子工程与生物技术重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
邵彦翔 大连海洋大学 大连 116023 
张廷廷 上海海洋大学水产与生命学院 上海 201306农业部海洋渔业可持续发展重点实验室 青岛市海水鱼类种子工程与生物技术重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
刘 莉 上海海洋大学水产与生命学院 上海 201306农业部海洋渔业可持续发展重点实验室 青岛市海水鱼类种子工程与生物技术重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
孙 涛 烟台开发区天源水产有限公司 烟台 261418 
摘要:
养殖中患有“突眼症”的美洲黑石斑鱼(Centropristis striata)的症状表现为眼球白浊、充血、异常增生。从眼球病灶部位分离纯化得到1株优势菌,在TCBS培养基上生长迅速,菌落中部隆起,黄色,有黏性,杆状菌,端生单鞭毛,属于革兰氏阴性菌,定名为CJG01。人工感染实验证实,其对美洲黑石斑鱼有较强的致病性,可引起幼鱼眼球突出、脱落,肌肉溃烂,骨胳外露。解剖感染组的幼鱼发现,患病幼鱼的肝脏、肾脏红肿,脾脏肿大,肠道内有淡黄色液体。其半致死浓度LD50为2.67×105 CFU/ml。API 20NE快速鉴定及相关生理生化实验结果显示,菌株CJG01的生长温度为28–37℃,最适温度为28℃,在含盐量为0–5%之间的TSB培养基可生长,对弧菌抑制剂O/129敏感,氧化酶反应阳性,鸟氨酸脱羧酶反应阳性,V-P反应阴性,可同化甘露醇、麦芽糖、苹果酸,不能同化葡萄糖、阿拉伯糖、甘露糖、癸酸、已二酸、柠檬酸、苯乙酸等,菌株CJG01的生理生化特性与哈维氏弧菌(Vibrio harveyi)一致。对病原菌的16S rDNA序列对比分析及系统进化树分析显示,菌株CJG01与哈维氏弧菌序列同源性最高,达99%。药敏实验证实,该菌株对氨苄西林、头孢氨苄、头孢拉定、诺氟沙星、青霉素、多粘菌素B、阿奇霉素等药物不敏感,对头孢唑林、恩诺沙星、链霉素、红霉素、克拉霉素等药物中度敏感,对抗生素头孢哌酮、头孢曲松、头孢他啶、氧氟沙星、洛美沙星、氟罗沙星、环丙沙星、氯霉素、新生霉素、新霉素、庆大霉素、卡那霉素、呋喃唑酮、利福平、四环素、米诺环素等种药物敏感。
关键词:  美洲黑石斑鱼  “突眼症”  分离鉴定  哈维氏弧菌
DOI:10.11758/yykxjz.20160504001
分类号:
基金项目:
Isolation and Classification of Exophthalmic Disease-Inducing Pathogenic Bacterium from Centropristis striata
CHEN Jianguo,CHEN Chao,LI Yanlu,SUN Shuguang,SHAO Yanxiang,ZHANG Tingting,LIU Li,SUN Tao
Abstract:
A strain of bacterial pathogen was isolated from eyeballs of cultured Centropristis striata with the symptoms such as white chaotic, hyperaemia and paraplasm in the eyeballs. The strain grew quickly in the TCBS culture medium, and the colonies were protruding, yellow, and tackiness. The strain named CJG01 was Gram-negative, bacillus and monotricha. The intramuscular injection experiment revealed that CJG01 was virulent to C. striata with LD50 value of 2.67×105 CFU/ml. CJG01 could cause symptoms including larval exophthalmos, eyeball fall off, muscle ulceration, and fishbone exposure. Anatomical observation showed liver inflammation, splenic organ enlargement, kidney inflammation and yellow liquid in the intestine. The results of API 20NE rapid identification and related physiological and biochemical experiments showed that the growth temperature for CJG01 was 28℃–37℃, and the optimum temperature was 28℃. CJG01 could grow in TSB culture medium with 0–5% salinity. It was sensitive to vibrio inhibitors O/129, was oxidase and ornithine decarboxylase positive, and was V-P negative. CJG01 could assimilate mannitol, maltose and malic acid, but not glucose, arabinose, mannose, decanoic acid, acetic acid, citric acid, benzene, etc. The strain shared similarities with Vibrio harveyi in biochemical and biophysical properties. Analysis of the 16S rDNA sequence and the phylogenetic tree suggested that CJG01 was highly similar to V. harveyi. Based on the results above, CJG01 was identified as V. harveyi. The chemotherapyeutant sensitivity test showed that CJG01 was insensitive to ampicillin, cephalosporins ammonia benzyl, cephalosporins, norfloxacin, penicillin, azithromycin, polymyxin B, etc. It was moderately sensitive to cefazolin, streptomycin, erythromycin, clarithromycin, etc. Novobiocin, neomycin, cefoperazone, ceftriaxone, gentamicin, cefoperazone, kanamycin, ofloxacin, ciprofloxacin, fleroxacin, furazolidone, rifampicin, ciprofloxacin, tetracycline, chloroamphenicol and minocycline could effectively inhibit the growth of CJG01.
Key words:  Centropristis striata  Exophthalmos  Separation and identification  Vibrio harveyi