虹鳟致病性杀鲑气单胞菌的分离鉴定及其毒力因子的检测
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摘要
为研究虹鳟Oncorhynchus mykiss致病性杀鲑气单胞菌Aeromonas salmonicida的致病机理,利用微生物学及分子生物学方法,从患皮肤溃疡病的虹鳟病灶处分离纯化获得1株优势菌株RBWF-1,通过人工感染试验证实其具有较强的致病性。结果表明:菌株RBWF-1对虹鳟的半致死浓度(LD50)为5.5×10~6CFU/m L;通过对菌株RBWF-1 16S r DNA基因序列扩增、测序及Blast对比,发现其与杀鲑气单胞菌杀日本鲑亚种Asalmonicida subsp.masoucida菌株NBRC 13748及无色亚种A.salmonicida subsp.achromogens菌株NCIMB 1110的16S rDNA基因序列同源性最高,一致性均为99.6%;构建系统发育树显示,菌株RBWF-1与杀鲑气单胞菌杀鲑亚种A.salmonicida subsp.salmonicida、杀日本鲑亚种、无色亚种和史氏亚种A.salmonicida subsp.smithia聚为一支;为进一步明确菌株的分类地位,利用Biolog微生物鉴定系统结合传统生理生化试验对菌株RBWF-1各项理化指标进行检测,经对比,与杀鲑气单胞菌杀日本鲑亚种吻合度最高,因而判断该分离株为杀鲑气单胞菌杀日本鲑亚种;同时,对分离菌株的气溶素、溶血素、封闭带毒素、DNA酶、丝氨酸蛋白酶、磷脂酶、S层蛋白7个毒力相关基因进行了PCR检测,检测结果均呈阳性;酶活性检测结果显示,菌株RBWF-1胞外产物具有蛋白酶、淀粉酶、脂酶、DNA酶和溶血活性。本研究结果可为中国虹鳟养殖过程中杀鲑气单胞菌的感染特征、分类鉴定和疫苗研制提供必要的数据和参考。
One dominant bacterial strain RBWF-1 was isolated from the skin ulcer lesion of diseased rainbow trout Oncorhynchus mykiss,and its virulence was proved to be high to healthy rainbow trout by artificial LD50 value of 5.5×10~6 CFU/m L infection test to elucidate the mechanism of Aeromonas salmonicida in rainbow thout. 16S rDNA gene of strain RBWF-1 was amplified and sequenced,and was highly homologous with Aeromonas salmonicida subsp. masoucida strain NBRC 13748 and A. salmonicida subsp. achromogenes strain NCIMB 1110,with similarity of99.6%. Phylogenetic tree based on 16S rDNA gene revealed that strain RBWF-1 clustered together with A.salmonicida subsp. salmonicida,A. salmonicida subsp. masoucida,A. salmonicida subsp. achromogenes and A. salmonicida subsp. smithia. Biolog's identification system combined with physiological and biochemical test indicated that strain RBWF-1 had the highest similarity with those of A.salmonicida subsp. masoucida,and was identified to be A. salmonicida subsp. salmonicida. Meanwhile,7 virulence-related genes including haemolysin,serine protease,lipase,DNases,aerolysin,zonula occludens toxin and S-layer protein,were all detected in the isolated strain. The enzymatic activity test showed that the extracellular products of strain RBWF-1 had the activities of protease,lipase,amylase,DNase and hemolysin. The findings provide the necessary data and information for the infectious characteristics,taxonomic identification and vaccine development of A.salmonicida in rainbow trout cultured in China.
One dominant bacterial strain RBWF-1 was isolated from the skin ulcer lesion of diseased rainbow trout Oncorhynchus mykiss,and its virulence was proved to be high to healthy rainbow trout by artificial LD50 value of 5.5×10~6 CFU/m L infection test to elucidate the mechanism of Aeromonas salmonicida in rainbow thout. 16S rDNA gene of strain RBWF-1 was amplified and sequenced,and was highly homologous with Aeromonas salmonicida subsp. masoucida strain NBRC 13748 and A. salmonicida subsp. achromogenes strain NCIMB 1110,with similarity of99.6%. Phylogenetic tree based on 16S rDNA gene revealed that strain RBWF-1 clustered together with A.salmonicida subsp. salmonicida,A. salmonicida subsp. masoucida,A. salmonicida subsp. achromogenes and A. salmonicida subsp. smithia. Biolog's identification system combined with physiological and biochemical test indicated that strain RBWF-1 had the highest similarity with those of A.salmonicida subsp. masoucida,and was identified to be A. salmonicida subsp. salmonicida. Meanwhile,7 virulence-related genes including haemolysin,serine protease,lipase,DNases,aerolysin,zonula occludens toxin and S-layer protein,were all detected in the isolated strain. The enzymatic activity test showed that the extracellular products of strain RBWF-1 had the activities of protease,lipase,amylase,DNase and hemolysin. The findings provide the necessary data and information for the infectious characteristics,taxonomic identification and vaccine development of A.salmonicida in rainbow trout cultured in China.
引文
[1]赵鹏.虹鳟鱼常见细菌性疾病的防治技术[J].黑龙江水产,2015(6):24-26.
[2]Garrity G M.Bergey's Manual of Systematic Bacteriology[M].9 th ed.New York:Springer,1994.
[3]汪建国.鱼病学[M].北京:中国农业出版社,2013:314-315.
[4]李绍戊,王荻,连浩淼,等.大西洋鲑杀鲑气单胞菌无色亚种的分离鉴定和致病性研究[J].水生生物学报,2015,39(1):234-240.
[5]田会芹,李贤,傅松哲,等.温度及p H对杀鲑气单胞菌生长的影响研究[J].海洋科学,2015,39(7):7-12.
[6]王家祯,耿昕颖,董文龙,等.3株杀鲑气单胞菌的分离鉴定及药敏试验[J].中国兽医杂志,2017,53(2):78-80.
[7]Ji Liqin,Sun Guoxiangun,Li J,et al.Effect of dietaryβ-glucan on growth,survival and regulation of immune processes in rainbow trout(Oncorhynchus mykiss)infected by Aeromonas salmonicida[J].Fish&Shellfish Immunology,2017,64:56-67.
[8]Liu Pengfei,Du Yishuai,Meng Lingjie,et al.ALDH7A1 is a protein that protects Atlantic salmon against Aeromonas salmonicida at the early stages of infection[J].Fish&Shellfish Immunology,2017,70:30-39.
[9]陆梦莹,胡秀彩,吕爱军,等.鲶源维氏气单胞菌的分离鉴定及药敏特性[J].大连海洋大学学报,2017,32(5):563-567.
[10]Reith M E,Singh R K,Curtis B,et al.The genome of Aeromonas salmonicida subsp.salmonicida A449:insights into the evolution of a fish pathogen[J].BMC Genomics,2008,9:427.
[11]贺扬,华丽,汪开毓,等.高致病性维氏气单胞菌胞外产物对斑点鮰的致病性[J].水产学报,2016,40(3):457-467.
[12]Buller N B.鱼类及其他水生动物细菌实用鉴定指南[M].徐高蓉,常亚青,王诗欢,译.北京:海洋出版社,2013:151-153.
[13]Wiklund T,Dalsgaard I.Occurrence and significance of atypical Aeromonas salmonicida in non-salmonid and salmonid fish species:a review[J].Diseases of Aquatic Organisms,1998,32(1):49-69.
[14]Wiklund T,Dalsgaard I,Eerola E,et al.Characteristics of‘atypical',cytochrome oxidase-negative Aeromonas salmonicida isolated from ulcerated flounders(Platichthys flesus(L.))[J].Journal of Applied Bacteriology,1994,76(5):511-520.
[15]张晓君,房海,陈翠珍,等.杀鲑气单胞菌一新亚种的生物学特性及系统发育学分析[J].中国水产科学,2006,13(6):917-923.
[16]Bernheimer A W,Avigad L S.Partial characterization of aerolysin,a lytic exotoxin from Aeromonas hydrophila[J].Infection and Immunity,1974,9(6):1016-1021.
[17]Hirono I,Aoki T.Cloning and characterization of three hemolysin genes from Aeromonas salmonicida[J].Microbial Pathogenesis,1993,15(4):269-282.
[18]Vipond R,Bricknell I R,Durant E,et al.Defined deletion mutants demonstrate that the major secreted toxins are not essential for the virulence of Aeromonas salmonicida[J].Infection and Immunity,1998,66(5):1990-1998.
[19]Kay W W,Buckley J T,Ishiguro E E,et al.Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida[J].Journal of Bacteriology,1981,147(3):1077-1084.
[20]Boyd J M,Dacanay A,Knickle L C,et al.Contribution of type IV pili to the virulence of Aeromonas salmonicida subsp.salmonicida in Atlantic salmon(Salmo salar L.)[J].Infection and Immunity,2008,76(4):1445-1455.
[21]Dacanay A,Knickle L,Solanky K S,et al.Contribution of the type III secretion system(TTSS)to virulence of Aeromonas salmonicida subsp.salmonicida[J].Microbiology,2006,152(6):1847-1856.
[22]Najimi M,Lemos M L,Osorio C R.Identification of siderophore biosynthesis genes essential for growth of Aeromonas salmonicida under iron limitation conditions[J].Applied and EnvironmentalMicrobiology,2008,74(8):2341-2348.
[23]Swift S,Karlyshev A V,Fish L,et al.Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida:identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules[J].Journal of Bacteriology,1997,179(17):5271-5281.
[24]Li J,Ni X D,Liu Y J,et al.Detection of three virulence genes alt,ahp and aer A in Aeromonas hydrophila and their relationship with actual virulence to zebrafish[J].Journal of Applied Microbiology,2011,110(3):823-830.
[25]刘杰,黄艳华,黄钧,等.黄沙鳖源嗜水气单胞菌的致病力与毒力基因型相关性[J].中国水产科学,2015,22(4):698-706.
[26]Zhou Q L,Wang Y J,Xie J,et al.Distribution and virulence gene comparison of Aeromonas strains isolated from diseased fish and water environment[J].Polish Journal of Microbiology,2013,62(3):299-302.
[27]王一娟.江苏地区大宗淡水鱼养殖池塘气单胞菌分离、鉴定和遗传多样性研究[D].南京:南京农业大学,2010.
[28]Fivaz M,Abrami L,Tsitrin Y,et al.Aerolysin from Aeromonas hydrophila and related toxins[J].Current Topics in Microbiology and Immunology,2001,257:35-52.
[29]Ellis A E,Hastings T S,Munro A L S.The role of Aeromonas salmonicida extracellular products in the pathology of furunculosis[J].Journal of Fish Diseases,1981,4(1):41-51.
[30]Kong P Y C,Lee S K Y,Law T W F,et al,Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR[J].Water Research,2002,36(11):2802-2812.
[31]Gustafson C E,Thomas C J,Trust T J.Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene[J].Applied and Environmental Microbiology,1992,58(12):3816-3825.
[32]刘帅,卢彤岩,王荻,等.杀鲑气单胞菌SYBRGreenⅠ实时荧光定量PCR检测方法的建立与应用[J].基因组学与应用生物学,2017(7):2884-2891.
[33]Zhang D H,Xu D H,Shoemaker C A.Immunization with recombinant aerolysin and haemolysin protected channel catfish against virulent Aeromonas hydrophila[J].Aquaculture Research,2017,48(3):875-882.
[34]Ni X D,Wang N,Liu Y J,et al.Immunoproteomics of extracellular proteins of the Aeromonas hydrophila China vaccine strain J-1 reveal a highly immunoreactive outer membrane protein[J].FEMS Immunology&Medical Microbiology,2010,58(3):363-373.
[35]Poobalane S,Thompson K D,ArdóL,et al.Production and efficacy of an Aeromonas hydrophila recombinant S-layer protein vaccine for fish[J].Vaccine,2010,28(20):3540-3547.
[36]Marana M H,Von Gersdorff Jrgensen L,Skov J,et al.Subunit vaccine candidates against Aeromonas salmonicida in rainbow trout Oncorhynchus mykiss[J].PLo S One,2017,12(2):e0171944.
[37]Thornton J C,Gardu1o R A,Kay W W.The development of live vaccines for furunculosis lacking the A-layer and O-antigen of Aeromonas salmonicida[J].Journal of Fish Diseases,1994,17(3):195-204.
[38]Locke J B,Aziz R K,Vicknair M R,et al.Streptococcus iniae Mlike protein contributes to virulence in fish and is a target for live attenuated vaccine development[J].PLo S One,2008,3(7):e2824.
[39]Xiao J,Chen T,Wang Q,et al.Search for live attenuated vaccine candidate against edwardsiellosis by mutating virulence-related genes of fish pathogen Edwardsiella tarda[J].Letters in Applied Microbiology,2011,53(4):430-437.
[40]Sun D W.Mutagenesis of virulence genes in an epidemic strain of Aeromonas hydrophila ML09-119 for development of anattenuated vaccine strain[D].Alabama:Auburn University,2014.
[2]Garrity G M.Bergey's Manual of Systematic Bacteriology[M].9 th ed.New York:Springer,1994.
[3]汪建国.鱼病学[M].北京:中国农业出版社,2013:314-315.
[4]李绍戊,王荻,连浩淼,等.大西洋鲑杀鲑气单胞菌无色亚种的分离鉴定和致病性研究[J].水生生物学报,2015,39(1):234-240.
[5]田会芹,李贤,傅松哲,等.温度及p H对杀鲑气单胞菌生长的影响研究[J].海洋科学,2015,39(7):7-12.
[6]王家祯,耿昕颖,董文龙,等.3株杀鲑气单胞菌的分离鉴定及药敏试验[J].中国兽医杂志,2017,53(2):78-80.
[7]Ji Liqin,Sun Guoxiangun,Li J,et al.Effect of dietaryβ-glucan on growth,survival and regulation of immune processes in rainbow trout(Oncorhynchus mykiss)infected by Aeromonas salmonicida[J].Fish&Shellfish Immunology,2017,64:56-67.
[8]Liu Pengfei,Du Yishuai,Meng Lingjie,et al.ALDH7A1 is a protein that protects Atlantic salmon against Aeromonas salmonicida at the early stages of infection[J].Fish&Shellfish Immunology,2017,70:30-39.
[9]陆梦莹,胡秀彩,吕爱军,等.鲶源维氏气单胞菌的分离鉴定及药敏特性[J].大连海洋大学学报,2017,32(5):563-567.
[10]Reith M E,Singh R K,Curtis B,et al.The genome of Aeromonas salmonicida subsp.salmonicida A449:insights into the evolution of a fish pathogen[J].BMC Genomics,2008,9:427.
[11]贺扬,华丽,汪开毓,等.高致病性维氏气单胞菌胞外产物对斑点鮰的致病性[J].水产学报,2016,40(3):457-467.
[12]Buller N B.鱼类及其他水生动物细菌实用鉴定指南[M].徐高蓉,常亚青,王诗欢,译.北京:海洋出版社,2013:151-153.
[13]Wiklund T,Dalsgaard I.Occurrence and significance of atypical Aeromonas salmonicida in non-salmonid and salmonid fish species:a review[J].Diseases of Aquatic Organisms,1998,32(1):49-69.
[14]Wiklund T,Dalsgaard I,Eerola E,et al.Characteristics of‘atypical',cytochrome oxidase-negative Aeromonas salmonicida isolated from ulcerated flounders(Platichthys flesus(L.))[J].Journal of Applied Bacteriology,1994,76(5):511-520.
[15]张晓君,房海,陈翠珍,等.杀鲑气单胞菌一新亚种的生物学特性及系统发育学分析[J].中国水产科学,2006,13(6):917-923.
[16]Bernheimer A W,Avigad L S.Partial characterization of aerolysin,a lytic exotoxin from Aeromonas hydrophila[J].Infection and Immunity,1974,9(6):1016-1021.
[17]Hirono I,Aoki T.Cloning and characterization of three hemolysin genes from Aeromonas salmonicida[J].Microbial Pathogenesis,1993,15(4):269-282.
[18]Vipond R,Bricknell I R,Durant E,et al.Defined deletion mutants demonstrate that the major secreted toxins are not essential for the virulence of Aeromonas salmonicida[J].Infection and Immunity,1998,66(5):1990-1998.
[19]Kay W W,Buckley J T,Ishiguro E E,et al.Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida[J].Journal of Bacteriology,1981,147(3):1077-1084.
[20]Boyd J M,Dacanay A,Knickle L C,et al.Contribution of type IV pili to the virulence of Aeromonas salmonicida subsp.salmonicida in Atlantic salmon(Salmo salar L.)[J].Infection and Immunity,2008,76(4):1445-1455.
[21]Dacanay A,Knickle L,Solanky K S,et al.Contribution of the type III secretion system(TTSS)to virulence of Aeromonas salmonicida subsp.salmonicida[J].Microbiology,2006,152(6):1847-1856.
[22]Najimi M,Lemos M L,Osorio C R.Identification of siderophore biosynthesis genes essential for growth of Aeromonas salmonicida under iron limitation conditions[J].Applied and EnvironmentalMicrobiology,2008,74(8):2341-2348.
[23]Swift S,Karlyshev A V,Fish L,et al.Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida:identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules[J].Journal of Bacteriology,1997,179(17):5271-5281.
[24]Li J,Ni X D,Liu Y J,et al.Detection of three virulence genes alt,ahp and aer A in Aeromonas hydrophila and their relationship with actual virulence to zebrafish[J].Journal of Applied Microbiology,2011,110(3):823-830.
[25]刘杰,黄艳华,黄钧,等.黄沙鳖源嗜水气单胞菌的致病力与毒力基因型相关性[J].中国水产科学,2015,22(4):698-706.
[26]Zhou Q L,Wang Y J,Xie J,et al.Distribution and virulence gene comparison of Aeromonas strains isolated from diseased fish and water environment[J].Polish Journal of Microbiology,2013,62(3):299-302.
[27]王一娟.江苏地区大宗淡水鱼养殖池塘气单胞菌分离、鉴定和遗传多样性研究[D].南京:南京农业大学,2010.
[28]Fivaz M,Abrami L,Tsitrin Y,et al.Aerolysin from Aeromonas hydrophila and related toxins[J].Current Topics in Microbiology and Immunology,2001,257:35-52.
[29]Ellis A E,Hastings T S,Munro A L S.The role of Aeromonas salmonicida extracellular products in the pathology of furunculosis[J].Journal of Fish Diseases,1981,4(1):41-51.
[30]Kong P Y C,Lee S K Y,Law T W F,et al,Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR[J].Water Research,2002,36(11):2802-2812.
[31]Gustafson C E,Thomas C J,Trust T J.Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene[J].Applied and Environmental Microbiology,1992,58(12):3816-3825.
[32]刘帅,卢彤岩,王荻,等.杀鲑气单胞菌SYBRGreenⅠ实时荧光定量PCR检测方法的建立与应用[J].基因组学与应用生物学,2017(7):2884-2891.
[33]Zhang D H,Xu D H,Shoemaker C A.Immunization with recombinant aerolysin and haemolysin protected channel catfish against virulent Aeromonas hydrophila[J].Aquaculture Research,2017,48(3):875-882.
[34]Ni X D,Wang N,Liu Y J,et al.Immunoproteomics of extracellular proteins of the Aeromonas hydrophila China vaccine strain J-1 reveal a highly immunoreactive outer membrane protein[J].FEMS Immunology&Medical Microbiology,2010,58(3):363-373.
[35]Poobalane S,Thompson K D,ArdóL,et al.Production and efficacy of an Aeromonas hydrophila recombinant S-layer protein vaccine for fish[J].Vaccine,2010,28(20):3540-3547.
[36]Marana M H,Von Gersdorff Jrgensen L,Skov J,et al.Subunit vaccine candidates against Aeromonas salmonicida in rainbow trout Oncorhynchus mykiss[J].PLo S One,2017,12(2):e0171944.
[37]Thornton J C,Gardu1o R A,Kay W W.The development of live vaccines for furunculosis lacking the A-layer and O-antigen of Aeromonas salmonicida[J].Journal of Fish Diseases,1994,17(3):195-204.
[38]Locke J B,Aziz R K,Vicknair M R,et al.Streptococcus iniae Mlike protein contributes to virulence in fish and is a target for live attenuated vaccine development[J].PLo S One,2008,3(7):e2824.
[39]Xiao J,Chen T,Wang Q,et al.Search for live attenuated vaccine candidate against edwardsiellosis by mutating virulence-related genes of fish pathogen Edwardsiella tarda[J].Letters in Applied Microbiology,2011,53(4):430-437.
[40]Sun D W.Mutagenesis of virulence genes in an epidemic strain of Aeromonas hydrophila ML09-119 for development of anattenuated vaccine strain[D].Alabama:Auburn University,2014.