海南地区养殖石斑鱼皮肤溃疡病病原的分离、鉴定、药敏分析及病理学研究
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摘要
2016年,针对海南岛沿岸9个石斑鱼养殖场的石斑鱼皮肤溃疡病的调查发现,该疾病的发病面积广、死亡率高,对养殖产业的危害严重.从9个采样点3种患病石斑鱼样品的组织中分离得到了9株优势菌株,人工回感试验表明,其感染特征与自然病症一致,证明这9株菌均为皮肤溃疡病的致病原,在经过生理生化和16S r DNA序列分析后,这9株菌均被鉴定为哈维氏弧菌(Vibrio harveyi).人工回感试验证实,菌株SY-1对珍珠龙胆石斑鱼的毒力最强,其14天每克鱼体重的半致死剂量(LD50值)为7.18×10~4cfu;药敏实验的分析表明,这9株病原菌对34种抗菌药物的药敏谱基本一致,但对氟苯尼考、强力霉素、四环素等药物高度敏感;病理学的研究表明,患病鱼的鳃丝、皮肤、肌肉、肝脏、脾脏、肾脏、肠道和脑等组织器官都发生了不同程度的病理变化,因此认为该病为全身性感染.本研究表明,目前海南地区养殖石斑鱼皮肤溃疡病的致病原有较高的病原同一性,主要为哈维氏弧菌;而较早报道的溶藻弧菌、副溶血弧菌等致病菌的优势度大大降低了,其致病力有所下降,并且与海南地区养殖石斑鱼皮肤溃疡病的相关度不大.
In 2016,the disease survey in nine fish farms along the coast of Hainan island were performed. The results indicated that skin-ulcer disease(SUD) is a serious bacteria disease to cultured groupers. Nine strains were isolated from the diseased groupers,and the results suggested that all the 9 strains were shown to be the causative pathogens by artificial infection trails. Based on the biochemical tests and 16 S r DNA sequence analysis,the 9 strains were all identified as Vibrio harveyi. Among them,SY~(-1) was shown as the most virulent strain and its LD50 for the cultured grouper(E. fuscoguttatus♀ × E. lanceolatus♂) was 7. 18 × 104 CFU·g~(-1) body weight. The sensitivity test results suggested that the 9 strains were basically consistent with the drug sensitivity spectrum for 34 antimicrobial agents,which were highly sensitive to drugs such as fluphenicol,doxycycline and tetracycline. The pathological analysis results showed that the pathological changes occurred in multiple tissues and organs,including gill filament,skin,muscle,liver,spleen,kidney,intestine and brain. Based on these findings,the conclusions can be obtained that the causative pathogen of SUD in Hainan Island was mainly V.harveyi.
In 2016,the disease survey in nine fish farms along the coast of Hainan island were performed. The results indicated that skin-ulcer disease(SUD) is a serious bacteria disease to cultured groupers. Nine strains were isolated from the diseased groupers,and the results suggested that all the 9 strains were shown to be the causative pathogens by artificial infection trails. Based on the biochemical tests and 16 S r DNA sequence analysis,the 9 strains were all identified as Vibrio harveyi. Among them,SY~(-1) was shown as the most virulent strain and its LD50 for the cultured grouper(E. fuscoguttatus♀ × E. lanceolatus♂) was 7. 18 × 104 CFU·g~(-1) body weight. The sensitivity test results suggested that the 9 strains were basically consistent with the drug sensitivity spectrum for 34 antimicrobial agents,which were highly sensitive to drugs such as fluphenicol,doxycycline and tetracycline. The pathological analysis results showed that the pathological changes occurred in multiple tissues and organs,including gill filament,skin,muscle,liver,spleen,kidney,intestine and brain. Based on these findings,the conclusions can be obtained that the causative pathogen of SUD in Hainan Island was mainly V.harveyi.
引文
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[24]王印庚,杨洋,张正,等.津冀地区养殖三疣梭子蟹大量死亡的病原和病理分析[J].中国水产科学,2017(3):596-605.
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[27]姚欢,黄郁葱,蔡双虎,等.眼斑拟石首鱼烂尾病病原菌的分离鉴定及灭活疫苗的免疫效果[J].广东海洋大学学报,2016(4):37-44.
[28]徐晓丽,邵蓬,李贺密,等.半滑舌鳎体表溃疡病病原的分离鉴定[J].中国海洋大学学报(自然科学版),2015(11):29-35.
[29]高桂生,张艳英,张召兴,等.半滑舌鳎皮肤溃疡病的病原分离鉴定与药物敏感性分析[J].中国兽医学报,2016(4):595-599.
[30]刘巧红,王世锋,蔡岩,等.海南养殖方斑东风螺暴发性疾病病原分离鉴定及药敏分析[J].渔业科学进展,2014(1):74-81.
[31]崔婧.华南地区鱼类病原哈维氏弧菌分型及其防治策略研究[D].海口:海南大学,2014.
[32]杨宁,谢珍玉,黄纪徽,等.海南养殖的奥尼罗非鱼暴发性出血症病原的分离与鉴定[J].海南大学学报(自然科学版),2009(1):43-47.
[33]徐先栋.华南海水养殖地区哈氏弧菌分子流行病学特征及其毒力菌株基因组分析[D].海口:海南大学,2017.
[34]刘开放,步君,章翔,等.华南地区海水养殖水域中哈维氏弧菌的抗药性分析[J].海南大学学报(自然科学版),2017,35(2):131-139.
[2]农业部渔业渔政管理局.2017年中国渔业统计年鉴[R].北京:中国农业出版社,2017.
[3]沈铭辉,王永波,周永灿,等.海南省热带水产苗种产业发展行动计划[Z].海南省海洋与渔业厅,2015.
[4]罗鸣,陈傅晓,刘龙龙,等.我国石斑鱼养殖疾病的研究进展[J].水产科学,2013(9):549-554.
[5]杨亚惟,冯乾生.石斑鱼的常见疾病及诊治[J].现代畜牧科技,2017(1):51.
[6]王永波,王秀英,刘金叶,等.海南岛石斑鱼刺激隐核虫病发病规律调查[J].广东农业科学,2016(5):152-156.
[7]崔婧,范雪亭,刘文竹,等.华南地区海水养殖鱼类主要弧菌病原的分离与鉴定[J].海南大学学报(自然科学版),2014(3):244-251.
[8]朱志煌,林克冰,宸周,等.闽南地区斜带石斑鱼(Epinephelus coioides)神经坏死病毒的基因型与分子进化研究[J].渔业研究,2017,6(39):429-436.
[9]朱传华,何建国,黄志坚.网箱养殖石斑鱼暴发性溃疡病病原菌分离、鉴定及致病性研究[J].中山大学学报(自然科学版),2000(S1):278-282.
[10]陈晓燕,胡超群,陈偿,等.人工养殖点带石斑鱼弧菌病病原菌的分离及鉴定[J].海洋科学,2003,6(27):68-72.
[11]胡学峰,石存斌,吴淑勤,等.海水养殖斜带石斑鱼溃疡病病原菌(溶藻弧菌)的初步研究[J].中国海洋大学学报(自然科学版),2005,2(35):232-236.
[12]梅冰,周永灿,徐先栋,等.斜带石斑鱼烂尾病病原菌的分离与鉴定[J].热带海洋学报,2010,6(29):118-124.
[13]徐先栋,谢珍玉,欧阳吉隆,等.褐点石斑鱼脱鳞病病原菌的分离与鉴定[J].海洋科学,2012,2(36):67-74.
[14]林克冰,吴建绍,黄兆斌,等.一株斜带石斑鱼(Epinephelus coioides)病原菌的分离与鉴定[J].渔业研究,2014(6):419-427.
[15]沈桂明.珍珠龙胆石斑鱼幼鱼皮肤溃疡病病原的研究[D].上海:上海海洋大学,2016.
[16]饶颖竹,梁静真,陈蓉,等.海水养殖斜带石斑鱼致病性鳗弧菌的分离鉴定及药敏试验[J].南方农业学报,2016(08):1416-1422.
[17]王雪惠,耿绪云,李翔,等.点带石斑鱼溃烂病病原菌的分离与鉴定[J].天津农学院学报,2012(3):19-25.
[18]龚艳清,陈信忠,王军,等.福建南部养殖石斑鱼暴发性疾病流行调查[J].福建农林大学学报(自然科学版),2006,35(5):532-537.
[19]蒋魁,徐力文,冯娟,等.两株珍珠龙趸病原性哈维弧菌(Vibrio harveyi)的分离与鉴定[J].生态科学,2017,6(36):16-24.
[20]蔡妙英,东秀珠.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[21]Holt J G,Krieg N R,Sneath P H A,et al.Bergey's Manual of Determinative Bacteriology[M].The ninth edition.Maryland:Lippincott,Williams&Wilkins,1994:565-786.
[22]萨姆布鲁克·J,拉塞尔·D W,萨姆布鲁克,等.分子克隆实验指南[M].第三版.北京:科学出版社,2002.
[23]沈建忠.动物毒理学[M].北京:中国农业出版社,2012.
[24]王印庚,杨洋,张正,等.津冀地区养殖三疣梭子蟹大量死亡的病原和病理分析[J].中国水产科学,2017(3):596-605.
[25]Benediktsdóttir E,Verdonck L,Spr9er C,et al.Characterization of Vibrio viscosus and Vibrio wodanis isolated at different geographical locations:a proposal for reclassification of Vibrio viscosus as Moritella viscosa comb.nov.[J].International Journal of Systematic&Evolutionary Microbiology,2000,50 2(2):479-488.
[26]纪荣兴,邹文政,李印龙.大黄鱼皮肤溃烂病病原的研究[J].海洋科学,2004(11):57-60.
[27]姚欢,黄郁葱,蔡双虎,等.眼斑拟石首鱼烂尾病病原菌的分离鉴定及灭活疫苗的免疫效果[J].广东海洋大学学报,2016(4):37-44.
[28]徐晓丽,邵蓬,李贺密,等.半滑舌鳎体表溃疡病病原的分离鉴定[J].中国海洋大学学报(自然科学版),2015(11):29-35.
[29]高桂生,张艳英,张召兴,等.半滑舌鳎皮肤溃疡病的病原分离鉴定与药物敏感性分析[J].中国兽医学报,2016(4):595-599.
[30]刘巧红,王世锋,蔡岩,等.海南养殖方斑东风螺暴发性疾病病原分离鉴定及药敏分析[J].渔业科学进展,2014(1):74-81.
[31]崔婧.华南地区鱼类病原哈维氏弧菌分型及其防治策略研究[D].海口:海南大学,2014.
[32]杨宁,谢珍玉,黄纪徽,等.海南养殖的奥尼罗非鱼暴发性出血症病原的分离与鉴定[J].海南大学学报(自然科学版),2009(1):43-47.
[33]徐先栋.华南海水养殖地区哈氏弧菌分子流行病学特征及其毒力菌株基因组分析[D].海口:海南大学,2017.
[34]刘开放,步君,章翔,等.华南地区海水养殖水域中哈维氏弧菌的抗药性分析[J].海南大学学报(自然科学版),2017,35(2):131-139.