鱼源溶藻弧菌生物学特性和病理组织学观察
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摘要
【目的】溃烂病是我国海水养殖及南方养殖斜带石斑鱼的常见严重病害之一。通过对患病的斜带石斑鱼病原菌的分离与鉴定,为检测其耐药性和控制石斑鱼在海水养殖中溃烂病的爆发和流行奠定基础。本文目的是研究斜带石斑鱼相关的病原菌的特性。【方法】从患病斜带石斑鱼肾脏、肝脏病灶组织,分离纯化得到病原菌,进行纯化培养、人工感染和回归感染、ATB细菌自动鉴定及药敏试验,并进行菌体常规形态特征、培养特性和生理生化反应指标测定。【结果】确定为溶藻弧菌(Vibrio alginolyticus)。两株典型菌株经人工感染和回归感染均显示自然发病症状。药敏试验表明,该菌具有较强的耐药性,在所检测的抗菌药物中,对青霉素等3类抗菌药物高度耐药,而对氯霉素等5类抗菌药物敏感。器官主要组织病理学变化为:肝细胞、肾小管细胞等变性、坏死,病变组织有炎性细胞浸润且呈变质性炎症。溶藻弧菌对机体的主要器官造成严重病理损伤。【结论】生理生化等结果均证实分离菌株是溶藻弧菌,而且产生多重耐药情况较为普遍,未来应加强对养殖海水中病原耐药性和斜带石斑鱼病原感染的监测。
[Objective]Ulcer disease is one of the most serious diseases and a common problem in various stages marine culture including Epinephelus coioides culture of southern China. The isolation and identification of pathogenic bacteria from E. coioides will be useful for monitoring of drug resistance and controlling the outbreak and spread of ulcer disease in E. coioides. The purpose of this study was to characterize the pathogen of E. coioides. [Methods] The pathogenic bacteria separated from the liver and kidney of diseased fish were identified through pure culture,artificial infection,automatic tests in bacteriology automatic identification, drug sensitive tests, morphometry, and physiological and biochemical determination. [Results] The strains were characterized and identified as Vibrio alginolyticus. Two strain were selected for virulence tests and all the moribund /dead fish exhibited ulcer disease as that observed in natural outbreak. Drug sensitive tests show that V. alginolyticus was highly resistant to 3 agents including penicillin,whereas sensitive to 5 agents including chloromycetin. Histopathological changes were mainly shown as cell degeneration and necrosis of gill,liver and kidney,and alterative inflammation as a result of inflammatory cell infiltration in the diseased tissue. [Conclusion] The biochemical,physiological tests confirm that V. alginolyticus is the pathogen causing E.coioides vibriosis. The multi-drug resistance among V. alginolyticus suggests strengthened monitoring of outbreaks of V.alginolyticus caused disease in E. coioides culture.
[Objective]Ulcer disease is one of the most serious diseases and a common problem in various stages marine culture including Epinephelus coioides culture of southern China. The isolation and identification of pathogenic bacteria from E. coioides will be useful for monitoring of drug resistance and controlling the outbreak and spread of ulcer disease in E. coioides. The purpose of this study was to characterize the pathogen of E. coioides. [Methods] The pathogenic bacteria separated from the liver and kidney of diseased fish were identified through pure culture,artificial infection,automatic tests in bacteriology automatic identification, drug sensitive tests, morphometry, and physiological and biochemical determination. [Results] The strains were characterized and identified as Vibrio alginolyticus. Two strain were selected for virulence tests and all the moribund /dead fish exhibited ulcer disease as that observed in natural outbreak. Drug sensitive tests show that V. alginolyticus was highly resistant to 3 agents including penicillin,whereas sensitive to 5 agents including chloromycetin. Histopathological changes were mainly shown as cell degeneration and necrosis of gill,liver and kidney,and alterative inflammation as a result of inflammatory cell infiltration in the diseased tissue. [Conclusion] The biochemical,physiological tests confirm that V. alginolyticus is the pathogen causing E.coioides vibriosis. The multi-drug resistance among V. alginolyticus suggests strengthened monitoring of outbreaks of V.alginolyticus caused disease in E. coioides culture.
引文
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[2]Liu X,Zou X,Mo X,Li J,Ye Z,Weng R,Wu T,Zhu S.A Study on the pathogen of vibrisis isolated from diseased Epinephelus sp.in cage mariculture.Tropic Oceanology,1994,13(1):81-86.(in Chinese)刘秀珍,邹晓理,莫小燕,李家炳,叶振腾,翁容生,巫特坚,朱世雄.海水网箱养殖石斑鱼病原菌研究.热带海洋,1994,13(1):81-86.
[3]Zhu Z,He J,huang Z.Identificatio and pathogenicity of pathogen of Epinephalus fario and E.awoara ulceration disease.Acta Cientiarum Naturalium Universitatis Sunyatseni,1994,13(1):81-86.(in Chinese)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.中山大学学报(自然科学版).2000,39(增刊):78-281.
[4]吴后波,吴灶和,潘金培.海水网箱养殖赤点石斑鱼溃疡病致病菌研究.热带海洋研究(五).北京:科学出版社,1997:91-95.
[5]Anderson DP.鱼类免疫学.张寿山,鼎可译,等译.北京:中国农业出版社,1984:51-52.
[6]东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001:83-90.
[7]Aznar R,Ludwig W,Amarm RI,Schleifer KH.Sequence determination of rRNA genes of pathogenic Vibrio species and whole-cell identification of Vibrio vulnificus with rRNA-targeted oLigonucleotide probes.SystBacteriol,1994,44(2):330-337.
[8]Gauger EJ,Gomez-Chiarri M.16S ribosomal DNA sequencing confirms the synonymy of Vibrio and V.carchariae.Diseases of Aquatic Organisms,2002,52(1):39-46.
[9]Yamada Y,Kaku Y,Wakabayashi H.Phylogenetic intrarelationships of atypical Aeromonas salmonicida isolated in Japan as determined by 16S rDNA sequencing.Fish Patholgy,2000,35(I):35-40.
[10]Chen Y,Zhong S,Zhou C.Prevention and treatment of luminous disease of adult prawns.Acta Cientiarum Naturalium Universitatis Sunyatseni,2000,39:218-223.(in Chinese)陈月忠,钟硕良,周宸.成虾发光病病原体的分离鉴定及防治技术研究.中山大学学报(自然科学版),2000,39(增刊):218-223.
[11]Wu X,Zhou Y,Ouyang J,Dai X,Wang S,Xie Z.Resistance to drugs and plasmid diversity in bacterium Vibrio Alginolyticus in Hainan Province.Fisheries Science,2012,31(1):50-54.(in Chinese)吴学贵,周永灿,欧阳吉隆,戴小连,王世锋,谢珍玉.海南地区溶藻弧菌耐药性及其质粒多样性的研究.水产科学,2012,31(1):50-54.
[12]Eun-Gyoung1,Kwang-Tae,Hongsik.Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from farmed fish in Korea from 2005 through 2007.Journal of Food Protection,2011,74(3):380-386.
[13]Wang R,Wang J,Xu L,Feng J.Antibiotic resistance of vibrio strains isolated from cobia(Rachycentron canadum)farming water and their digestion guts.South China Fisheries Science,2007,3(5):1-6.(in Chinese)王瑞旋,王江勇,徐力文,冯娟.军曹鱼养殖水体及其肠道弧菌的耐药性研究.南方水产,2007,3(5):1-6.
[14]Zhang X,Cai J.Determination of the Antibiotic Susceptibilities of Four Marine Pathogenic Vibrios.Journal of South China University of Technology(Natural Science),2003,31(9):66-69.(in Chinese)张昕,蔡俊鹏.四种海洋致病弧菌对抗生素敏感性的测定.华南理工大学学报,2003,31(9):66-69.
[15]Han S,Yu J,Jiang T,Wang D.An analysis of antimicrobial susceptibility and isolation of pathogenic Vibrio in brine.Chinese Journal of Antibiotics,2008,34(16):195-199.(in Chinese)韩善桥,虞积耀,姜涛.海水中致病性弧菌分离及抗菌药物敏感性分析,中国抗生素杂志,2008,34(16):195-199.
[16]Liu H,Wang Q,Liu Q.Roles of Hfq in the stress adaptation and virulence in fish pathogen Vibrio alginolyticus and its potential application as a target for live attenuated vaccine.Applied Microbiology and Biotechnology,2011,91(2):353-364.
[17]Zhou Z,Pan H,Ding Y,Cai J,Huang Y,Jian J,Wu Z.VscO,a putative T3SS chaperone escort of Vibrio alginolyticus,contributes to virulence in fish and is a target for vaccine development.Fish&Shellfish Immunology,2013,35(5):1523-1531.
[18]徐建国.分子医学细菌学.北京:科学出版社,2000:131-152.
[2]Liu X,Zou X,Mo X,Li J,Ye Z,Weng R,Wu T,Zhu S.A Study on the pathogen of vibrisis isolated from diseased Epinephelus sp.in cage mariculture.Tropic Oceanology,1994,13(1):81-86.(in Chinese)刘秀珍,邹晓理,莫小燕,李家炳,叶振腾,翁容生,巫特坚,朱世雄.海水网箱养殖石斑鱼病原菌研究.热带海洋,1994,13(1):81-86.
[3]Zhu Z,He J,huang Z.Identificatio and pathogenicity of pathogen of Epinephalus fario and E.awoara ulceration disease.Acta Cientiarum Naturalium Universitatis Sunyatseni,1994,13(1):81-86.(in Chinese)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.中山大学学报(自然科学版).2000,39(增刊):78-281.
[4]吴后波,吴灶和,潘金培.海水网箱养殖赤点石斑鱼溃疡病致病菌研究.热带海洋研究(五).北京:科学出版社,1997:91-95.
[5]Anderson DP.鱼类免疫学.张寿山,鼎可译,等译.北京:中国农业出版社,1984:51-52.
[6]东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001:83-90.
[7]Aznar R,Ludwig W,Amarm RI,Schleifer KH.Sequence determination of rRNA genes of pathogenic Vibrio species and whole-cell identification of Vibrio vulnificus with rRNA-targeted oLigonucleotide probes.SystBacteriol,1994,44(2):330-337.
[8]Gauger EJ,Gomez-Chiarri M.16S ribosomal DNA sequencing confirms the synonymy of Vibrio and V.carchariae.Diseases of Aquatic Organisms,2002,52(1):39-46.
[9]Yamada Y,Kaku Y,Wakabayashi H.Phylogenetic intrarelationships of atypical Aeromonas salmonicida isolated in Japan as determined by 16S rDNA sequencing.Fish Patholgy,2000,35(I):35-40.
[10]Chen Y,Zhong S,Zhou C.Prevention and treatment of luminous disease of adult prawns.Acta Cientiarum Naturalium Universitatis Sunyatseni,2000,39:218-223.(in Chinese)陈月忠,钟硕良,周宸.成虾发光病病原体的分离鉴定及防治技术研究.中山大学学报(自然科学版),2000,39(增刊):218-223.
[11]Wu X,Zhou Y,Ouyang J,Dai X,Wang S,Xie Z.Resistance to drugs and plasmid diversity in bacterium Vibrio Alginolyticus in Hainan Province.Fisheries Science,2012,31(1):50-54.(in Chinese)吴学贵,周永灿,欧阳吉隆,戴小连,王世锋,谢珍玉.海南地区溶藻弧菌耐药性及其质粒多样性的研究.水产科学,2012,31(1):50-54.
[12]Eun-Gyoung1,Kwang-Tae,Hongsik.Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from farmed fish in Korea from 2005 through 2007.Journal of Food Protection,2011,74(3):380-386.
[13]Wang R,Wang J,Xu L,Feng J.Antibiotic resistance of vibrio strains isolated from cobia(Rachycentron canadum)farming water and their digestion guts.South China Fisheries Science,2007,3(5):1-6.(in Chinese)王瑞旋,王江勇,徐力文,冯娟.军曹鱼养殖水体及其肠道弧菌的耐药性研究.南方水产,2007,3(5):1-6.
[14]Zhang X,Cai J.Determination of the Antibiotic Susceptibilities of Four Marine Pathogenic Vibrios.Journal of South China University of Technology(Natural Science),2003,31(9):66-69.(in Chinese)张昕,蔡俊鹏.四种海洋致病弧菌对抗生素敏感性的测定.华南理工大学学报,2003,31(9):66-69.
[15]Han S,Yu J,Jiang T,Wang D.An analysis of antimicrobial susceptibility and isolation of pathogenic Vibrio in brine.Chinese Journal of Antibiotics,2008,34(16):195-199.(in Chinese)韩善桥,虞积耀,姜涛.海水中致病性弧菌分离及抗菌药物敏感性分析,中国抗生素杂志,2008,34(16):195-199.
[16]Liu H,Wang Q,Liu Q.Roles of Hfq in the stress adaptation and virulence in fish pathogen Vibrio alginolyticus and its potential application as a target for live attenuated vaccine.Applied Microbiology and Biotechnology,2011,91(2):353-364.
[17]Zhou Z,Pan H,Ding Y,Cai J,Huang Y,Jian J,Wu Z.VscO,a putative T3SS chaperone escort of Vibrio alginolyticus,contributes to virulence in fish and is a target for vaccine development.Fish&Shellfish Immunology,2013,35(5):1523-1531.
[18]徐建国.分子医学细菌学.北京:科学出版社,2000:131-152.