广西北部湾大宗海水养殖鱼类卵形鲳鲹感染溶藻弧菌及其致病性研究
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摘要
【目的】2016年广西钦州湾近海网箱养殖的卵形鲳鲹(Trachinotus ovatus)发生以体表皮肤溃烂、皮下出血、内脏器官病变为典型症状的细菌性疾病,通过对患病卵形鲳鲹中病原菌进行分离、鉴定与保存,为后续开展病原菌快速检测技术和防控技术等研究,以及控制相关病原菌在海水养殖中的暴发和流行奠定基础。【方法】利用LB平板和TCBS平板从患病卵形鲳鲹体表溃烂病灶、肾脏、肝脏组织中分离纯化疑似病原菌,并对其进行常规形态特征和生理生化反应的生物学检验,通过16SrDNA基因测序对疑似病原菌株进行分子生物学鉴定,在细胞和鱼体水平开展病原菌的致病性研究,同时进行人工感染的回接试验。【结果】分离得到4株疑似病原菌(TOQZ01,TOQZ02,TOQZ03,TOQZ04),基于其表型、分子生物学特征和进化关系,判定4株菌株均为弧菌属的溶藻弧菌(Vibrio alginolyticus),且细胞毒性试验和回归试验证实,分离得到的溶藻弧菌为此次卵形鲳鲹发病的病原菌。【结论】溶藻弧菌是引起广西沿海地区海水养殖鱼类发生细菌性鱼病的主要致病菌之一,未来将基于本研究中分离得到的溶藻弧菌开展病害快速检测技术和防控技术等现代海水生态健康养殖关键技术研究,以实现对广西海水养殖溶藻弧菌病的快速诊断、实时监控和有效预防。
【Objective】The Trachinotus ovatus cultured in offshore cages in Qinzhou Bay of Guangxi Province showed typical symptoms of bacterial diseases in 2016,including skin ulceration,subcutaneous bleeding and visceral lesions.By isolating,identifying and preserving the pathogenic bacteria in the diseased T.ovatus,it established the foundation for the follow-up research on the rapid detection technology and prevention and control technology of pathogenic bacteria,as well as controlling the outbreak and epidemic of related pathogenic bacteria in the marine culture.【Methods】Four suspected pathogen strains were isolated from the ulcer lesions,kidney and liver of diseased T.ovatus by using LB and TCBS plates.Biological tests were conducted to check the conventional morphological characteristics and physiological and biochemical reactions.Molecular biology identification of the suspected pathogenic strains was performed by 16 SrDNA gene sequencing.The pathogenicity of pathogenic bacteria was carried out at the level of cells and fish,and the re-inoculation of artificial infection was carried out simultaneously.【Results】Four strains of suspected pathogens(TOQZ01,TOQZ02,TOQZ03,TOQZ04)were isolated and these four strains were identified as Vibrio alginolyticus based on their phenotypes,molecular biological characteristics and evolutionary relationships.The cytotoxicity and regression tests confirmed that the isolated V.alginolyticus were the pathogen,which caused the bacterial diseases in T.ovatus.【Conclusion】Vibrio alginolyticus is one of the main pathogenic bacteria that cause bacterial fish disease in marine culture fish in the coastal areas of Guangxi.In the future,Vibrio alginolyticusisolated from this study will be used to carry out rapid disease detection technology and prevention and control technology and other key technologies of modern seawater eco-health farming in order to realize the rapid diagnosis,real-time monitoring and effective prevention of Vibrio alginolyticusin marine culture of Guangxi.
【Objective】The Trachinotus ovatus cultured in offshore cages in Qinzhou Bay of Guangxi Province showed typical symptoms of bacterial diseases in 2016,including skin ulceration,subcutaneous bleeding and visceral lesions.By isolating,identifying and preserving the pathogenic bacteria in the diseased T.ovatus,it established the foundation for the follow-up research on the rapid detection technology and prevention and control technology of pathogenic bacteria,as well as controlling the outbreak and epidemic of related pathogenic bacteria in the marine culture.【Methods】Four suspected pathogen strains were isolated from the ulcer lesions,kidney and liver of diseased T.ovatus by using LB and TCBS plates.Biological tests were conducted to check the conventional morphological characteristics and physiological and biochemical reactions.Molecular biology identification of the suspected pathogenic strains was performed by 16 SrDNA gene sequencing.The pathogenicity of pathogenic bacteria was carried out at the level of cells and fish,and the re-inoculation of artificial infection was carried out simultaneously.【Results】Four strains of suspected pathogens(TOQZ01,TOQZ02,TOQZ03,TOQZ04)were isolated and these four strains were identified as Vibrio alginolyticus based on their phenotypes,molecular biological characteristics and evolutionary relationships.The cytotoxicity and regression tests confirmed that the isolated V.alginolyticus were the pathogen,which caused the bacterial diseases in T.ovatus.【Conclusion】Vibrio alginolyticus is one of the main pathogenic bacteria that cause bacterial fish disease in marine culture fish in the coastal areas of Guangxi.In the future,Vibrio alginolyticusisolated from this study will be used to carry out rapid disease detection technology and prevention and control technology and other key technologies of modern seawater eco-health farming in order to realize the rapid diagnosis,real-time monitoring and effective prevention of Vibrio alginolyticusin marine culture of Guangxi.
引文
[1]刘锡强,马学坤,刘康,等.华南地区金鲳鱼养殖报告[J].当代水产,2014(2):26-29.LIU X Q,MA X K,LIU K,et al.Reports on the cultured Trachinotus ovatus in South China[J].Current Fisheries,2014(2):26-29.
[2]黄和,王娜,陈良,等.金鲳鱼鱼糜制品加工工艺优化[J].食品与机械,2015,31(6):193-198.HUANG H,WANG N,CHEN L,et al.Optimization on processing of Trachinotus ovatus surimi gel by response surface methodology[J].Food&Machinery,2015,31(6):193-198.
[3]张少峰,张春华,邢素坤.广西海洋经济发展现状与对策分析[J].海洋开发与管理,2015(4):103-106.ZHANG S F,ZHANG C H,XING S K.Analysis on the current situation and countermeasures of marine economic development in Guangxi[J].Ocean Development and Management,2015(4):103-106.
[4]李坚明.“十三五”广西水产养殖业发展战略研究[J].中国渔业经济,2016,34(4):25-31.LI J M.Research on the development strategy of Guangxi aquaculture in 13th Five-Year[J].Chinese Fisheries Economics,2016,34(4):25-31.
[5]马振国,洪泉.广西推动石斑鱼产业快速发展[N].中国食品安全报,2013-11-30(B1).MA Z G,HONG Q.Guangxi promotes the rapid development of grouper fish industry[N].China Food Safety News,2013-11-30(B1).
[6]罗春业,廖愚.广西大力发展“九化渔业”[J].中国水产,2015(1):20-22.LUO C Y,LIAO Y.Guangxi vigorously develops“nine oriented fishery”[J].China Fisheries,2015(1):20-22.
[7]夏立群,黄郁葱,鲁义善.卵形鲳鲹主要病害及其研究进展[J].安徽农学通报,2012,18(23):140-143,150.XIA L Q,HUANG Y C,LU Y S.The diseases in Trachinotus ovatus culture and their research progress[J].Anhui Agricultural Science Bulletin,2012,18(23):140-143,150.
[8]LIU C H,CHEN J C.Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus[J].Fish Shellfish Immunol,2004,16(3):321-334.
[9]张彬,黄婷,熊建华,等.文蛤主要弧菌性病害研究进展[J].广东农业科学,2012(17):128-130,134.ZHANG B,HUANG T,XIONG J H,et al.Research progress of Vibriosis in hard calm,Meretrix meretrix Linnaeus[J].Guangdong Agricultural Sciences,2012(17):128-130,134.
[10]陈雨生,房瑞景,乔娟.中国海水养殖业发展研究[J].农业经济问题,2012(6):72-77.CHEN Y S,FANG R J,QIAO J.Study on the development of mariculture in China[J].Issues in Agricultural Economy,2012(6):72-77.
[11]PARVATHI A,MENDEZ D,ANTO C.Distribution of putative virulence genes and antimicrobial drug resistance in Vibrio harveyi[J].Indian J Microbiol,2011,51(3):332-337.
[12]LI P,ZHOU L,WEI S,et al.Establishment and characterization of a cell line from the head kidney of Golden pompano Trachinotus ovatus and its application in toxicology and virus susceptibility[J].J Fish Biol,2017,90(5):1944-1959.
[13]HRMANSDORFER S,WENTGES H,NEUGEBAUR-BCHLER K,et al.Isolation of Vibrio alginolyticus from seawater aquaria[J].Int J Hyg Environ Health,2000,203(2):169-175.
[14]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.DONG X Z,CAI M Y.Handbook of systematic identification of common bacteria[M].Beijing:Science Press,2001.
[15]赖迎迢,陶家发,孙承文,等.鱼源溶藻弧菌生物学特性和病理组织学观察[J].微生物学报,2014,54(11):1378-1384.LAI Y T,TAO J F,SUN C W,et al.Biological characteristics and histopathological observation of Vibrio alginolyticus from diseased fish[J].Acta Microbiologica Sinica,2014,54(11):1378-1384.
[16]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,2014,32(3):244-251.CUI J,FAN X T,LIU W Z,et al.Isolation and identification of Vibriosis pathogens of marine cultured fishes in Southern China[J].Natural Science Journal of Hainan University,2014,32(3):244-251.
[17]ABUBAKAR I,IRVINE L,ALDUS C F,et al.A systematic review of the clinical,public health and cost-effectiveness of rapid diagnostic tests for the detectionand identification of bacterial intestinal pathogens in faeces and food[J].Health Technol Assess,2007,11(36):1-216.
[18]王一娴,叶尊忠,斯城燕,等.适配体生物传感器在病原微生物检测中的应用[J].分析化学评述与进展,2012,40(4):634-642.WANG Y X,YE Z Z,SI C Y,et al.Application of aptamer based biosenor for detection of pathogenic microorganism[J].Chinese Journal of Analytical Chemistry,2012,40(4):634-642.
[2]黄和,王娜,陈良,等.金鲳鱼鱼糜制品加工工艺优化[J].食品与机械,2015,31(6):193-198.HUANG H,WANG N,CHEN L,et al.Optimization on processing of Trachinotus ovatus surimi gel by response surface methodology[J].Food&Machinery,2015,31(6):193-198.
[3]张少峰,张春华,邢素坤.广西海洋经济发展现状与对策分析[J].海洋开发与管理,2015(4):103-106.ZHANG S F,ZHANG C H,XING S K.Analysis on the current situation and countermeasures of marine economic development in Guangxi[J].Ocean Development and Management,2015(4):103-106.
[4]李坚明.“十三五”广西水产养殖业发展战略研究[J].中国渔业经济,2016,34(4):25-31.LI J M.Research on the development strategy of Guangxi aquaculture in 13th Five-Year[J].Chinese Fisheries Economics,2016,34(4):25-31.
[5]马振国,洪泉.广西推动石斑鱼产业快速发展[N].中国食品安全报,2013-11-30(B1).MA Z G,HONG Q.Guangxi promotes the rapid development of grouper fish industry[N].China Food Safety News,2013-11-30(B1).
[6]罗春业,廖愚.广西大力发展“九化渔业”[J].中国水产,2015(1):20-22.LUO C Y,LIAO Y.Guangxi vigorously develops“nine oriented fishery”[J].China Fisheries,2015(1):20-22.
[7]夏立群,黄郁葱,鲁义善.卵形鲳鲹主要病害及其研究进展[J].安徽农学通报,2012,18(23):140-143,150.XIA L Q,HUANG Y C,LU Y S.The diseases in Trachinotus ovatus culture and their research progress[J].Anhui Agricultural Science Bulletin,2012,18(23):140-143,150.
[8]LIU C H,CHEN J C.Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus[J].Fish Shellfish Immunol,2004,16(3):321-334.
[9]张彬,黄婷,熊建华,等.文蛤主要弧菌性病害研究进展[J].广东农业科学,2012(17):128-130,134.ZHANG B,HUANG T,XIONG J H,et al.Research progress of Vibriosis in hard calm,Meretrix meretrix Linnaeus[J].Guangdong Agricultural Sciences,2012(17):128-130,134.
[10]陈雨生,房瑞景,乔娟.中国海水养殖业发展研究[J].农业经济问题,2012(6):72-77.CHEN Y S,FANG R J,QIAO J.Study on the development of mariculture in China[J].Issues in Agricultural Economy,2012(6):72-77.
[11]PARVATHI A,MENDEZ D,ANTO C.Distribution of putative virulence genes and antimicrobial drug resistance in Vibrio harveyi[J].Indian J Microbiol,2011,51(3):332-337.
[12]LI P,ZHOU L,WEI S,et al.Establishment and characterization of a cell line from the head kidney of Golden pompano Trachinotus ovatus and its application in toxicology and virus susceptibility[J].J Fish Biol,2017,90(5):1944-1959.
[13]HRMANSDORFER S,WENTGES H,NEUGEBAUR-BCHLER K,et al.Isolation of Vibrio alginolyticus from seawater aquaria[J].Int J Hyg Environ Health,2000,203(2):169-175.
[14]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.DONG X Z,CAI M Y.Handbook of systematic identification of common bacteria[M].Beijing:Science Press,2001.
[15]赖迎迢,陶家发,孙承文,等.鱼源溶藻弧菌生物学特性和病理组织学观察[J].微生物学报,2014,54(11):1378-1384.LAI Y T,TAO J F,SUN C W,et al.Biological characteristics and histopathological observation of Vibrio alginolyticus from diseased fish[J].Acta Microbiologica Sinica,2014,54(11):1378-1384.
[16]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,2014,32(3):244-251.CUI J,FAN X T,LIU W Z,et al.Isolation and identification of Vibriosis pathogens of marine cultured fishes in Southern China[J].Natural Science Journal of Hainan University,2014,32(3):244-251.
[17]ABUBAKAR I,IRVINE L,ALDUS C F,et al.A systematic review of the clinical,public health and cost-effectiveness of rapid diagnostic tests for the detectionand identification of bacterial intestinal pathogens in faeces and food[J].Health Technol Assess,2007,11(36):1-216.
[18]王一娴,叶尊忠,斯城燕,等.适配体生物传感器在病原微生物检测中的应用[J].分析化学评述与进展,2012,40(4):634-642.WANG Y X,YE Z Z,SI C Y,et al.Application of aptamer based biosenor for detection of pathogenic microorganism[J].Chinese Journal of Analytical Chemistry,2012,40(4):634-642.