养殖大菱鲆感染迟缓爱德华氏菌的分离、毒力基因及ERIC-PCR分析
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摘要
为了调查辽宁省葫芦岛市养殖患病大菱鲆Scophthalmus maximus病原菌感染情况,选用迟缓爱德华氏菌Edwardsiella tarda特异性引物对分离菌株进行PCR鉴定,共分离得到22株迟缓爱德华氏菌。结果表明:迟缓爱德华氏菌毒力基因的PCR扩增显示,22株菌均含有kat B、fim A、gad B、esa V等基因;人工感染试验表明,22株迟缓爱德华氏菌感染并致大菱鲆累积死亡率均为100%;ERIC-PCR结果显示,迟缓爱德华氏菌模式菌株(ATCC15947)与分离株可分为2个基因型,分别标记为Ⅰ和Ⅱ型,其中22株迟缓爱德华氏菌分离株均为Ⅱ型,与ATCC15947菌株为Ⅰ型明显不同。本研究结果为养殖大菱鲆迟缓爱德华氏菌感染的防控提供了依据。
Pathogen Edwardsiella tarda was isolated from diseased farmed turbot Scophthalmus maximus( L.),identified by PCR with a specific primer and further genotyping by ERIC-PCR analysis to explore the pathogenic bacteria infection of diseased farmed turbot. The PCR amplification of virulence gene of the pathogen E. tarda revealed that all the 22 isolates contained the four tested virulence genes( kat B,fim A,gad B and esa V). Challenge tests showed that the cumulative mortality of 100% was observed in the turbot challenged with the 22 strains of pathogen. In addition,ERIC-PCR showed that the standard E.tarda( ATCC15947) and all 22 isolates were divided into two genotypes including E.tarda Ⅰ and E.tarda Ⅱ. The reference strains belonged to E.tarda Ⅰ,and the isolates belonged to E.tarda Ⅱ. The findings will provide very important references for disease prevention in turbot.
Pathogen Edwardsiella tarda was isolated from diseased farmed turbot Scophthalmus maximus( L.),identified by PCR with a specific primer and further genotyping by ERIC-PCR analysis to explore the pathogenic bacteria infection of diseased farmed turbot. The PCR amplification of virulence gene of the pathogen E. tarda revealed that all the 22 isolates contained the four tested virulence genes( kat B,fim A,gad B and esa V). Challenge tests showed that the cumulative mortality of 100% was observed in the turbot challenged with the 22 strains of pathogen. In addition,ERIC-PCR showed that the standard E.tarda( ATCC15947) and all 22 isolates were divided into two genotypes including E.tarda Ⅰ and E.tarda Ⅱ. The reference strains belonged to E.tarda Ⅰ,and the isolates belonged to E.tarda Ⅱ. The findings will provide very important references for disease prevention in turbot.
引文
[1]秦蕾,王印庚,张晓君.迟钝爱德华氏菌感染大菱鲆的病理学研究[J].中国水产科学,2009,16(3):411-419.
[2]Alcaide E,Herraiz S,Esteve C.Occurrence of Edwardsiella tarda in wild European eels Anguilla anguilla from Mediterranean Spain[J].Diseases of Aquatic Organisms,2006,73(1):77-81.
[3]王波,莫照兰.迟缓爱德华氏菌及其致病机理[J].海洋科学集刊,2007(48):133-139.
[4]荣小军,廖梅杰,张正,等.迟缓爱德华氏菌SYBR Green I实时荧光定量PCR检测方法的建立及其应用[J].水产学报,2013,37(12):1829-1838.
[5]张晓君,白雪松,毕可然,等.病原迟钝爱德华菌毒力基因及双重PCR与LAMP检测方法的建立[J].水产学报,2013,37(7):1087-1094.
[6]李杰.致病性迟缓爱德华氏菌的鉴定、PCR检测及esa C基因的功能鉴定[D].青岛:中国海洋大学,2008:1-55.
[7]Maiti B,Raghunath P,Karunasagar I,et al.Typing of clinical and environmental strains of Aeromonas spp.using two PCR based methods and whole cell protein analysis[J].Journal of Microbiological Methods,2009,78(3):312-318.
[8]Hunter P R,Gaston M A.Numerical index of the discriminatory ability of typing systems:an application of Simpson's index of diversity[J].Journal of Clinical Microbiology,1988,26(11):2465-2466.
[9]王斌,孙岑,范薇,等.养殖大菱鲆出血性败血症病原菌致病性的研究及鉴定[J].大连水产学院学报,2006,21(2):100-104.
[10]王亚婷,李晓玥,赵宝华.鱼类迟钝爱德华菌病诊断与防治研究进展[J].动物医学进展,2009,30(3):77-81.
[11]李筠,颜显辉,陈吉祥,等.养殖大菱鲆腹水病病原的研究[J].中国海洋大学学报,2006,36(4):649-654.
[12]Choi H S.Study on Edwardsiella tarda isolated from cultured bastard halibut(Paralichthys olivaceus)[J].Bulletin of National Fisheries Research and Development Agency(Korea).Yangsan,1991(45):197-205.
[13]Mohanty B R,Sahoo P K.Edwardsiellosis in fish:a brief review[J].Journal of Biosciences,2007,32(S3):1331-1344.
[14]朱文进,陈翠珍,苏咏梅,等.牙鲆致病性迟钝爱德华菌毒力基因的检测及序列分析[J].动物医学进展,2014,35(11):20-24.
[15]Rao P S S,Lim T M,Leung K Y.Functional genomics approach to the identification of virulence genes involved in Edwardsiella tarda pathogenesis[J].Infection and Immunity,2003,71(3):1343-1351.
[16]Wang Xin,Wang Qiyao,Xiao Jingfan,et al.Edwardsiella tarda T6SS component evp P is regulated by esr B and iron,and plays essential roles in the invasion of fish[J].Fish&Shellfish Immunology,2009,27(3):469-477.
[17]Rao P S S,Lim T M,Leung K Y.Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates[J].Infection and Immunity,2001,69(9):5689-5697.
[18]Ishibe K,Osatomi K,Hara K,et al.Comparison of the responses of peritoneal macrophages from Japanese flounder(Paralichthys olivaceus)against high virulent and low virulent strains of Edwardsiella tarda[J].Fish&Shellfish Immunology,2008,24(2):243-251.
[19]江云,李寿崧,王寿昆,等.致病性迟钝爱德华氏菌毒力基因的PCR检测[J].中国食品学报,2008,8(4):123-129.
[20]Sakai T,Kanai K,Osatomi K,et al.Identification of a 19.3-k Da protein in MRHA-positive Edwardsiella tarda:putative fimbrial major subunit[J].FEMS Microbiology Letters,2003,226(1):127-133.
[21]Liu Ying,Oshima S I,Kurohara K,et al.Vaccine efficacy of recombinant GAPDH of Edwardsiella tarda against Edwardsiellosis[J].Microbiology and Immunology,2005,49(7):605-612.
[22]李墨非.海水鱼类病原菌迟缓爱德华氏菌和海豚链球菌的致病机制[D].青岛:中国科学院研究生院(海洋研究所),2015.
[23]Acharya M,Maiti N K,Mohanty S,et al.Genotyping of Edwardsiella tarda isolated from freshwater fish culture system[J].Comparative Immunology,Microbiology and Infectious Diseases,2007,30(1):33-40.
[24]Abayneh T,Colquhoun D J,Srum H.Multi-locus sequence analysis(MLSA)of Edwardsiella tarda isolates from fish[J].Veterinary Microbiology,2012,158(3-4):367-375.
[2]Alcaide E,Herraiz S,Esteve C.Occurrence of Edwardsiella tarda in wild European eels Anguilla anguilla from Mediterranean Spain[J].Diseases of Aquatic Organisms,2006,73(1):77-81.
[3]王波,莫照兰.迟缓爱德华氏菌及其致病机理[J].海洋科学集刊,2007(48):133-139.
[4]荣小军,廖梅杰,张正,等.迟缓爱德华氏菌SYBR Green I实时荧光定量PCR检测方法的建立及其应用[J].水产学报,2013,37(12):1829-1838.
[5]张晓君,白雪松,毕可然,等.病原迟钝爱德华菌毒力基因及双重PCR与LAMP检测方法的建立[J].水产学报,2013,37(7):1087-1094.
[6]李杰.致病性迟缓爱德华氏菌的鉴定、PCR检测及esa C基因的功能鉴定[D].青岛:中国海洋大学,2008:1-55.
[7]Maiti B,Raghunath P,Karunasagar I,et al.Typing of clinical and environmental strains of Aeromonas spp.using two PCR based methods and whole cell protein analysis[J].Journal of Microbiological Methods,2009,78(3):312-318.
[8]Hunter P R,Gaston M A.Numerical index of the discriminatory ability of typing systems:an application of Simpson's index of diversity[J].Journal of Clinical Microbiology,1988,26(11):2465-2466.
[9]王斌,孙岑,范薇,等.养殖大菱鲆出血性败血症病原菌致病性的研究及鉴定[J].大连水产学院学报,2006,21(2):100-104.
[10]王亚婷,李晓玥,赵宝华.鱼类迟钝爱德华菌病诊断与防治研究进展[J].动物医学进展,2009,30(3):77-81.
[11]李筠,颜显辉,陈吉祥,等.养殖大菱鲆腹水病病原的研究[J].中国海洋大学学报,2006,36(4):649-654.
[12]Choi H S.Study on Edwardsiella tarda isolated from cultured bastard halibut(Paralichthys olivaceus)[J].Bulletin of National Fisheries Research and Development Agency(Korea).Yangsan,1991(45):197-205.
[13]Mohanty B R,Sahoo P K.Edwardsiellosis in fish:a brief review[J].Journal of Biosciences,2007,32(S3):1331-1344.
[14]朱文进,陈翠珍,苏咏梅,等.牙鲆致病性迟钝爱德华菌毒力基因的检测及序列分析[J].动物医学进展,2014,35(11):20-24.
[15]Rao P S S,Lim T M,Leung K Y.Functional genomics approach to the identification of virulence genes involved in Edwardsiella tarda pathogenesis[J].Infection and Immunity,2003,71(3):1343-1351.
[16]Wang Xin,Wang Qiyao,Xiao Jingfan,et al.Edwardsiella tarda T6SS component evp P is regulated by esr B and iron,and plays essential roles in the invasion of fish[J].Fish&Shellfish Immunology,2009,27(3):469-477.
[17]Rao P S S,Lim T M,Leung K Y.Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates[J].Infection and Immunity,2001,69(9):5689-5697.
[18]Ishibe K,Osatomi K,Hara K,et al.Comparison of the responses of peritoneal macrophages from Japanese flounder(Paralichthys olivaceus)against high virulent and low virulent strains of Edwardsiella tarda[J].Fish&Shellfish Immunology,2008,24(2):243-251.
[19]江云,李寿崧,王寿昆,等.致病性迟钝爱德华氏菌毒力基因的PCR检测[J].中国食品学报,2008,8(4):123-129.
[20]Sakai T,Kanai K,Osatomi K,et al.Identification of a 19.3-k Da protein in MRHA-positive Edwardsiella tarda:putative fimbrial major subunit[J].FEMS Microbiology Letters,2003,226(1):127-133.
[21]Liu Ying,Oshima S I,Kurohara K,et al.Vaccine efficacy of recombinant GAPDH of Edwardsiella tarda against Edwardsiellosis[J].Microbiology and Immunology,2005,49(7):605-612.
[22]李墨非.海水鱼类病原菌迟缓爱德华氏菌和海豚链球菌的致病机制[D].青岛:中国科学院研究生院(海洋研究所),2015.
[23]Acharya M,Maiti N K,Mohanty S,et al.Genotyping of Edwardsiella tarda isolated from freshwater fish culture system[J].Comparative Immunology,Microbiology and Infectious Diseases,2007,30(1):33-40.
[24]Abayneh T,Colquhoun D J,Srum H.Multi-locus sequence analysis(MLSA)of Edwardsiella tarda isolates from fish[J].Veterinary Microbiology,2012,158(3-4):367-375.
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