美洲黑石斑鱼(Centropristis striata)“突眼症”的病原菌分离鉴定
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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2.67×10~5 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 r DNA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。
A strain of bacterial pathogen was isolated from eyeballs of cultured Centropristis striatawith 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 LD_(50) value of 2.67×10~5 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 20 NE 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 16 SrDNA 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.
A strain of bacterial pathogen was isolated from eyeballs of cultured Centropristis striatawith 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 LD_(50) value of 2.67×10~5 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 20 NE 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 16 SrDNA 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.
引文
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Chen XG,Wu SQ,Shi CB,et al.Isolation and identification of pathogenetic Vibrio harveyi from estuary cod Epinephelus coioides.Journal of Fishery Sciences of China,2004,11(4):313 –317[陈献稿,吴淑勤,石存斌,等.斜带石斑鱼病原菌(哈维氏弧菌)的分离与鉴定.中国水产科学,2004,11 (4):313–317]
Chen XY,Hu CQ,Chen C.Characteristics of pathogenic bacteria of vibriosis in caged Malabar grouper,Epinephelus malabaricus.Marine Sciences,2003,27(6):68–71[陈晓燕,胡超群,陈偿.人工养殖点带石斑鱼弧菌病病原菌的分离及鉴定.海洋科学,2003,27(6):68–71]
Fan WH,Huang J,Wang XH,et al.Identification and phylogenetic study of pathogenic bacteria causing ulcer disease of cultured Turbot(Scophthalmus maximus).Acta Microbiologica Sinica,2005,45(5):665–670[范文辉,黄倢,王秀华,等.养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析.微生物学报,2005,45(5):665–670]
Harikrishnan R,Balasundaram C,Heo MS.Molecular studies,disease status and prophylactic measures in grouper aquaculture:Economic importance,diseases and immunology.Aquaculture,2010,30(9):1–14
He PP,Zhao YZ,Chen XL,et al.Isolation and identification of Vibrio in cultured Litopenaeus vannamei.Jiangsu Agricultural Sciences,2013,41(6):199–202[何苹萍,赵永贞,陈秀荔,等.凡纳滨对虾及养殖水体中弧菌的分离鉴定.江苏农业科学,2013,41(6):199–202]
Hu CQ,Tao BH.Penaeid shrimp vibriosis and immune prevention:A review.Tropic Oceanology,2010,19(3):84–92[胡超群,陶保华.综述:对虾弧菌病及其免疫预防的研究进展.热带海洋,2010,19(3):84–92]
Huang ZJ,He JG.Isolation,identification and pathogenicity of the bacterial pathogen in Epinephlus fario.Acta Scientiarum Naturalium Universitatis Sunyatseni,2002,41(5):64–67[黄志坚,何建国.鲑点石斑鱼细菌病原的分离鉴定和致病性.中山大学学报(自然版),2002,41(5):64–67]
Jin S,Wang GL,Zhao QS,et al.Epidemiology of vibriosis in large yellow croaker Pseudosciaena crocea(Richardson)in marine cage culture.Fisheries Science,2005,24(1):17–19[金珊,王国良,赵青松,等.海水网箱养殖大黄鱼弧菌病的流行病学研究.水产科学,2005,24(1):17–19]
Lin KB,Wu JS,Huang ZB,et al.Isolation and identification of a bacterial pathogen from Epinephelus coioides.Journal of Fujian Fisheries,2014,36(6):419–427[林克冰,吴建绍,黄兆斌.一株斜带石斑鱼(Epinephelus coioides)病原菌的分离与鉴定.福建水产,2014,36(6):419–427]
Lin KB,Zhou C,Liu JF,et al.Studies on the pathogenic bacteria of Pseudosciaena crocea in marine cage culture.Marine Sciences,1999(4):58–62[林克冰,周宸,刘家富,等.海水网箱养殖大黄鱼病原菌研究,海洋科学,1999(4)58 –62]
Liu XZ,Zou XL,Mo XY,et al.A study on the pathogen of vibriosis isolated from diseased Epinephelus sp.in cage mariculture.Tropic Oceanology,1994,13(1):81–86[刘秀珍,邹晓理,莫小燕,等.海水网箱养殖石斑鱼病原菌研究.热带海洋,1994,13(1):81–86]
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Mao ZJ,Liu GY,Chen CF.Isolation and identification of pathogenic bacteria causing ulcerosis in large yellow croaker(Pseudosciaena crocea).Journal of Anhui Agricultural University,2002,29(2):178–181[毛芝娟,刘国勇,陈昌福.大黄鱼溃疡病致病菌的初步分离与鉴定.安徽农业大学学报,2002,29(2):178–181]
Qin YX,Chi XC,Su YQ,et al.The pathogeny of ulcer disease in Epinephlus awoara.Journal of Fisheries of China,2004,28 (3):297–302[覃映雪,池信才,苏永全,等.网箱养殖青石斑鱼的溃疡病病原.水产学报,2004,28(3):297–302]
Ren HM,He Z,Yang DY,et al.Pathological study on rice field eel artificial infected by Aeromonas sobria.Journal of Hydroecology,2011,32(1):118–120[任红梅,何智,杨德英,等.黄鳝人工感染温和气单胞菌的组织病理学研究.水生态学杂志,2011,32(1):118–120]
Tamura K,Peterson D,Peterson N,et al.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Molecular Biology and Evolution,2011,28(10):2731–2739
Wang B,Zhu MY,Mao XH.The new variety-Centropristis striata for artificial culture.Hebei Fisheries,2003(5):26–27[王波,朱明远,毛兴华.养殖新品种–美洲黑石斑鱼.河北渔业,2003(5):26–27]
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