引起凡纳滨对虾急性肝胰腺坏死病的副溶血弧菌MLST新序列型
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摘要
针对12株引起凡纳滨对虾急性肝胰腺坏死病(AHPND)的致病性副溶血弧菌,运用多位点测序技术,分析致病菌株的遗传特征。实验选择副溶血弧菌的7个管家基因dnaE、gyrB、recA、dtdS、pntA、pyrC及tnaA,对12株AHPND致病菌扩增测序。将核酸序列上传至PubMLST数据库进行比对后获得每株菌的序列型。收集其他地区AHPND致病性副溶血弧菌的多位点序列分型(MLST)数据,采用eBURSTV3及MEGA5.0软件进行遗传进化分析。结果显示,2014年中国广东分离的AHPND致病株属于一个新序列型ST1710(42、134、99、79、141、41、51),仅与库中ST415及ST975同源性较高。目前,急性肝胰腺坏死致病株的9种ST型可归为2个克隆组和5个单体。经系统发育树进一步分析可知新序列型ST1710与单体ST975遗传关系相近。本研究首次报道中国AHPND分离株的序列型,丰富了PubMLST数据库,并为其遗传进化研究奠定了理论基础。
Twelve strains of pathogenic Vibrio parahaemolyticus isolated from Litopenaeus vannamei with acute hepatopancreatic necrosis disease(AHPND) were chosen, and their genetic characteristics were analyzed by the multilocus sequence typing method. Seven house-keeping genes(dna E, gyr B, rec A, dtd S, pnt A, pyr C and tna A) of V. parahaemolyticus were selected as target genes for PCR amplification and nucleic acid sequencing. The sequences of AHPND-causing V. parahaemolyticus in this study were uploaded to the Pub MLST database for multialignment to determine the sequence type of each strain. MLST data of AHPND-causing V. parahaemolyticus from other areas were collected and the genetic evolution was analyzed by MEGA 5.0 and e BURST V3 software.AHPND-causing V. parahaemolyticus isolated from Guangdong province in 2014 belonged to the new sequence type ST1710(42, 134, 99, 79, 141, 41, 51). Compared with other sequence types in Pub MLST database, it has the high homology with ST415 and ST975. The 9 STs of AHPND-causing V. parahaemolyticus were separated by e BURST into two doublets and five singletons. Phylogenetic tree analysis showed that the new sequence type ST1710 was similar to the singletons ST975. Our study is the first report of molecular epidemiology of AHPNDcausing V. parahaemolyticus strains in China using multilocus sequence typing, and enriches the Pub MLST database and provides an insight into their evolutionary mechanisms.
Twelve strains of pathogenic Vibrio parahaemolyticus isolated from Litopenaeus vannamei with acute hepatopancreatic necrosis disease(AHPND) were chosen, and their genetic characteristics were analyzed by the multilocus sequence typing method. Seven house-keeping genes(dna E, gyr B, rec A, dtd S, pnt A, pyr C and tna A) of V. parahaemolyticus were selected as target genes for PCR amplification and nucleic acid sequencing. The sequences of AHPND-causing V. parahaemolyticus in this study were uploaded to the Pub MLST database for multialignment to determine the sequence type of each strain. MLST data of AHPND-causing V. parahaemolyticus from other areas were collected and the genetic evolution was analyzed by MEGA 5.0 and e BURST V3 software.AHPND-causing V. parahaemolyticus isolated from Guangdong province in 2014 belonged to the new sequence type ST1710(42, 134, 99, 79, 141, 41, 51). Compared with other sequence types in Pub MLST database, it has the high homology with ST415 and ST975. The 9 STs of AHPND-causing V. parahaemolyticus were separated by e BURST into two doublets and five singletons. Phylogenetic tree analysis showed that the new sequence type ST1710 was similar to the singletons ST975. Our study is the first report of molecular epidemiology of AHPNDcausing V. parahaemolyticus strains in China using multilocus sequence typing, and enriches the Pub MLST database and provides an insight into their evolutionary mechanisms.
引文
[1]Han J E,Tang K F J,Pantoja C R,et al.q PCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease(AHPND)due to pathogenic Vibrio parahaemolyticus[J].Aquaculture,2015,442:12-15.
[2]Lightner D V,Redman R M,Pantoja C R,et al.Early mortality syndrome affects shrimp in Asia[J].Global Aquaculture Advocate,2012,15(1):40.
[3]Nunan L,Lightner D,Pantoja C,et al.Detection of acute hepatopancreatic necrosis disease(AHPND)in Mexico[J].Diseases of Aquatic Organisms,2014,111(1):81-86.
[4]Soto-Rodriguez S A,Gomez-Gil B,Lozano-Olvera R,et al.Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp(Litopenaeus vannamei)in Northwestern Mexico[J].Applied and Environmental Microbiology,2015,81(5):1689-1699.
[5]Joshi J,Srisala J,Truong V H,et al.Variation in Vibrio parahaemolyticus isolates from a single Thai shrimp farm experiencing an outbreak of acute hepatopancreatic necrosis disease(AHPND)[J].Aquaculture,2014,428-429:297-302.
[6]FAO.FAO Fisheries and Aquaculture Report[C]//FAOSecond International Technical Seminar/Workshop on acute Hepatopancreatic Necrosis Disease(AHPND):There is A Way Forward(under TCP/INT/3502).Panama:FAO,2016:9.
[7]徐含颖,张婉蓉,张学舒,等.对虾早期死亡综合症的研究进展[J].安徽农业科学,2015(6):166-167.Xu H Y,Zhang W R,Zhang X S,et al.Research advances in early mortality syndrome of shrimp[J].Journal of Anhui Agricultural Sciences,2015(6):166-167(in Chinese).
[8]唐小千,徐洪森,战文斌.对虾急性肝胰腺坏死综合症研究进展[J].海洋湖沼通报,2016(2):90-93.Tang X Q,Xu H S,Zhan W B.Advances in study of shrimp acute hepatopancreas necrosis syndrome[J].Transactions of Oceanology and Limnology,2016(2):90-93(in Chinese).
[9]Tran L,Nunan L,Redman R M,et al.Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp[J].Diseases of Aquatic Organisms,2013,105(1):45-55.
[10]Lee C T,Chen I T,Yang Y T,et al.The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(34):10798-10803.
[11]Sirikharin R,Taengchaiyaphum S,Sanguanrut P,et al.Characterization and PCR detection of binary,Pir-like toxins from Vibrio parahaemolyticus isolates that cause acute hepatopancreatic necrosis disease(AHPND)in shrimp[J].PLo S One,2015,10(5):e0126987.
[12]Marri P R,Harris L K,Houmiel K,et al.The effect of chromosome geometry on genetic diversity[J].Genetics,2008,179(1):511-516.
[13]Vázquez J A,Berrón S.Multilocus sequence typing:the molecular marker of the internet era[J].Enfermedades Infecciosas y Microbiología Clínica,2004,22(2):113-120.
[14]黄东红,范春梅,朱炎,等.耐甲氧西林金黄色葡萄球菌的MLST分型及耐药性分析[J].中华医院感染学杂志,2016,26(23):5312-5314.Huang D H,Fan C M,Zhu Y,et al.MLST typing anddrug resistance of methicillin-resistant Staphylococcus aureus[J].Chinese Journal of Nosocomiology,2016,26 (23):5312-5314(in Chinese).
[15]Maiden M C J.Multilocus sequence typing of bacteria[J].Annual Review of Microbiology,2006,60:561-588.
[16]Feil E J,Li B C,Aanensen D M,et al.e BURST:inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data[J].Journal of Bacteriology,2004,186(5):1518-1530.
[17]Chonsin K,Matsuda S,Theethakaew C,et al.Genetic diversity of Vibrio parahaemolyticus strains isolated from farmed Pacific white shrimp and ambient pond water affected by acute hepatopancreatic necrosis disease outbreak in Thailand[J].FEMS Microbiology Letters,2016,363(2):fnv222.
[18]Chao G X,Wang F,Zhou X H,et al.Origin of Vibrio parahaemolyticus O3:K6 pandemic clone[J].International Journal of Food Microbiology,2011,145(2-3):459-463.
[19]陈健舜,朱凝瑜,郑天伦,等.10株副溶血弧菌多位点序列分型新序列型[J].水产科学,2012,31(9):540-543.Chen J S,Zhu N Y,Zheng T L,et al.Ten new sequencetype isolates of Vibrio parahaemolyticus by multilocus sequence typing[J].Fisheries Science,2012,31(9):540-543 (in Chinese).
[20]Hong X P,Lu L Q,Xu D.Progress in research on acute hepatopancreatic necrosis disease(AHPND)[J].Aquaculture International,2016,24(2):577-593.
[21]文国樑,曹煜成,徐煜,等.养殖对虾肝胰腺坏死综合症研究进展[J].广东农业科学,2015,42(11):118-123.Wen G L,Cao Y C,Xu Y,et al.Review on hepatopancreas necrosis syndrome of shrimp[J].Guangdong Agricultural Sciences,2015,42(11):118-123(in Chinese).
[22]Hanage W P,Fraser C,Spratt B G.Sequences,sequence clusters and bacterial species[J].Philosophical Transactions of the Royal Society B:Biological Sciences,2006,361(1475):1917-1927.
[23]Maiden M C J,Bygraves J A,Feil E,et al.Multilocus sequence typing:a portable approach to the identification of clones within populations of pathogenic microorganisms[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(6):3140-3145.
[24]Jiang S C,Paul J H.Gene transfer by transduction in the marine environment[J].Applied and Environmental Microbiology,1998,64(8):2780-2787.
[2]Lightner D V,Redman R M,Pantoja C R,et al.Early mortality syndrome affects shrimp in Asia[J].Global Aquaculture Advocate,2012,15(1):40.
[3]Nunan L,Lightner D,Pantoja C,et al.Detection of acute hepatopancreatic necrosis disease(AHPND)in Mexico[J].Diseases of Aquatic Organisms,2014,111(1):81-86.
[4]Soto-Rodriguez S A,Gomez-Gil B,Lozano-Olvera R,et al.Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp(Litopenaeus vannamei)in Northwestern Mexico[J].Applied and Environmental Microbiology,2015,81(5):1689-1699.
[5]Joshi J,Srisala J,Truong V H,et al.Variation in Vibrio parahaemolyticus isolates from a single Thai shrimp farm experiencing an outbreak of acute hepatopancreatic necrosis disease(AHPND)[J].Aquaculture,2014,428-429:297-302.
[6]FAO.FAO Fisheries and Aquaculture Report[C]//FAOSecond International Technical Seminar/Workshop on acute Hepatopancreatic Necrosis Disease(AHPND):There is A Way Forward(under TCP/INT/3502).Panama:FAO,2016:9.
[7]徐含颖,张婉蓉,张学舒,等.对虾早期死亡综合症的研究进展[J].安徽农业科学,2015(6):166-167.Xu H Y,Zhang W R,Zhang X S,et al.Research advances in early mortality syndrome of shrimp[J].Journal of Anhui Agricultural Sciences,2015(6):166-167(in Chinese).
[8]唐小千,徐洪森,战文斌.对虾急性肝胰腺坏死综合症研究进展[J].海洋湖沼通报,2016(2):90-93.Tang X Q,Xu H S,Zhan W B.Advances in study of shrimp acute hepatopancreas necrosis syndrome[J].Transactions of Oceanology and Limnology,2016(2):90-93(in Chinese).
[9]Tran L,Nunan L,Redman R M,et al.Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp[J].Diseases of Aquatic Organisms,2013,105(1):45-55.
[10]Lee C T,Chen I T,Yang Y T,et al.The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(34):10798-10803.
[11]Sirikharin R,Taengchaiyaphum S,Sanguanrut P,et al.Characterization and PCR detection of binary,Pir-like toxins from Vibrio parahaemolyticus isolates that cause acute hepatopancreatic necrosis disease(AHPND)in shrimp[J].PLo S One,2015,10(5):e0126987.
[12]Marri P R,Harris L K,Houmiel K,et al.The effect of chromosome geometry on genetic diversity[J].Genetics,2008,179(1):511-516.
[13]Vázquez J A,Berrón S.Multilocus sequence typing:the molecular marker of the internet era[J].Enfermedades Infecciosas y Microbiología Clínica,2004,22(2):113-120.
[14]黄东红,范春梅,朱炎,等.耐甲氧西林金黄色葡萄球菌的MLST分型及耐药性分析[J].中华医院感染学杂志,2016,26(23):5312-5314.Huang D H,Fan C M,Zhu Y,et al.MLST typing anddrug resistance of methicillin-resistant Staphylococcus aureus[J].Chinese Journal of Nosocomiology,2016,26 (23):5312-5314(in Chinese).
[15]Maiden M C J.Multilocus sequence typing of bacteria[J].Annual Review of Microbiology,2006,60:561-588.
[16]Feil E J,Li B C,Aanensen D M,et al.e BURST:inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data[J].Journal of Bacteriology,2004,186(5):1518-1530.
[17]Chonsin K,Matsuda S,Theethakaew C,et al.Genetic diversity of Vibrio parahaemolyticus strains isolated from farmed Pacific white shrimp and ambient pond water affected by acute hepatopancreatic necrosis disease outbreak in Thailand[J].FEMS Microbiology Letters,2016,363(2):fnv222.
[18]Chao G X,Wang F,Zhou X H,et al.Origin of Vibrio parahaemolyticus O3:K6 pandemic clone[J].International Journal of Food Microbiology,2011,145(2-3):459-463.
[19]陈健舜,朱凝瑜,郑天伦,等.10株副溶血弧菌多位点序列分型新序列型[J].水产科学,2012,31(9):540-543.Chen J S,Zhu N Y,Zheng T L,et al.Ten new sequencetype isolates of Vibrio parahaemolyticus by multilocus sequence typing[J].Fisheries Science,2012,31(9):540-543 (in Chinese).
[20]Hong X P,Lu L Q,Xu D.Progress in research on acute hepatopancreatic necrosis disease(AHPND)[J].Aquaculture International,2016,24(2):577-593.
[21]文国樑,曹煜成,徐煜,等.养殖对虾肝胰腺坏死综合症研究进展[J].广东农业科学,2015,42(11):118-123.Wen G L,Cao Y C,Xu Y,et al.Review on hepatopancreas necrosis syndrome of shrimp[J].Guangdong Agricultural Sciences,2015,42(11):118-123(in Chinese).
[22]Hanage W P,Fraser C,Spratt B G.Sequences,sequence clusters and bacterial species[J].Philosophical Transactions of the Royal Society B:Biological Sciences,2006,361(1475):1917-1927.
[23]Maiden M C J,Bygraves J A,Feil E,et al.Multilocus sequence typing:a portable approach to the identification of clones within populations of pathogenic microorganisms[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(6):3140-3145.
[24]Jiang S C,Paul J H.Gene transfer by transduction in the marine environment[J].Applied and Environmental Microbiology,1998,64(8):2780-2787.