养殖大菱鲆感染嗜水气单胞菌的分离、毒力分析及ERIC-PCR分型
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摘要
为对辽宁省葫芦岛市养殖患病大菱鲆Scophthalmus maximus(L.)全年感染嗜水气单胞菌Aeromonas hydrophila情况进行调查,通过细菌16S rRNA基因序列分析,以及嗜水气单胞菌特异性引物和气溶素基因的双重PCR扩增对分离株进行鉴定。结果表明:调查中共分离到7株嗜水气单胞菌(AP40301、AP40401、AP40402、AP40403、AP40501、AP40502和AP40503),且均含有气溶素基因(aer A);ERIC-PCR结果进一步显示,7株嗜水气单胞菌存在独立基因型,分别标记为Ⅰ和Ⅱ型,其中Ⅰ型分离株中有3株来自绥中,1株来自兴城,Ⅱ型分离株3株均来自兴城;人工感染试验显示,7株嗜水气单胞菌均具有强致病性,致病率为100%。本研究结果可为养殖大菱鲆的临床诊断以及疾病防控提供理论依据。
The isolates from naturally diseased turbot Scophthalmus maximus(L.) in Huludao City,Liaoning Province from 2014 to 2015 were indentified and characterized by 16 S rRNA gene sequence analysis and double PCR using Aeromonas hydrophila-specific and aerosol gene primers. The seven isolates were shown to be A. hydrophila,with aerosol genes. The isolates genotyping by ERIC-PCR demonstrated that seven A.hydrophila strains belonged to genotype I and genotype II,in which three strains of type I were isolated from Suizhong farms,one strain of type I from Xingcheng farms and another three strains of type II were from Xingcheng farm. Moreover,the challenge tests by these seven A.hydrophila strains showed that all the strains were highly pathogenic to turbot,with the 7-days cumulative mortality(1.0×10~8 cells/m L) of 100%. The finding provides a theoretical basis for future clinical diagnosis and disease prevention and control of cultured turbot.
The isolates from naturally diseased turbot Scophthalmus maximus(L.) in Huludao City,Liaoning Province from 2014 to 2015 were indentified and characterized by 16 S rRNA gene sequence analysis and double PCR using Aeromonas hydrophila-specific and aerosol gene primers. The seven isolates were shown to be A. hydrophila,with aerosol genes. The isolates genotyping by ERIC-PCR demonstrated that seven A.hydrophila strains belonged to genotype I and genotype II,in which three strains of type I were isolated from Suizhong farms,one strain of type I from Xingcheng farms and another three strains of type II were from Xingcheng farm. Moreover,the challenge tests by these seven A.hydrophila strains showed that all the strains were highly pathogenic to turbot,with the 7-days cumulative mortality(1.0×10~8 cells/m L) of 100%. The finding provides a theoretical basis for future clinical diagnosis and disease prevention and control of cultured turbot.
引文
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[2]夏飞,梁利国,谢骏.团头鲂病原嗜水气单胞菌的分离鉴定及药敏试验[J].水产科学,2012,31(10):606-610.
[3]冷闯,邓舜洲,张文波,等.中华鳖致病性嗜水气单胞菌的分离鉴定及药敏试验[J].动物医学进展,2012,33(2):124-129.
[4]田甜.鱼源嗜水气单胞菌的分离鉴定及耐药性研究[D].武汉:华中农业大学,2010.
[5]邓国成,江小燕,叶星,等.草鱼出血病混合感染的嗜水气单胞菌的分离、鉴定与理化特性[J].微生物学通报,2009,36(8):1170-1177.
[6]王印庚,张正,秦蕾,等.养殖大菱鲆主要疾病及防治技术[J].海洋水产研究,2004,25(6):61-68.
[7]张晓君.三种海水养殖鱼类的主要细菌性疾病研究[D].青岛:中国海洋大学,2006.
[8]张正.养殖大菱鲆流行病调查及主要细菌性疾病的病原学研究[D].青岛:中国海洋大学,2004.
[9]乔毅,沈辉,万夕和,等.一株黑鲷致病性假交替单胞菌的鉴定及毒力基因分析[J].江苏农业科学,2015,43(4):229-233.
[10]王友娟,李荣峰,李华,等.辽宁地区养殖淡水鱼感染嗜水气单胞菌的流行病学调查[J].中国农业科技导报,2012,14(4):128-134.
[11]刘亚楠,梁利国,顾伟,等.异育银鲫致病性嗜水气单胞菌的分离鉴定与药敏特性研究[J].水生态学杂志,2012,33(5):108-113.
[12]梁利国,谢骏.青鱼病原嗜水气单胞菌分离鉴定、毒力因子检测及药敏试验[J].生态学杂志,2013,32(12):3236-3242.
[13]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.
[14]Hunter R P,Gaston A M.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.
[15]Pemberton J M,Kidd S P,Schmidt R.Secreted enzymes of Aeromonas[J].FEMS Microbiology Letters,1997,152(1):l-10.
[16]朱建萍,叶展翔,朱烨,等.嗜水气单胞菌气溶素毒力基因检测研究[J].中国热带医学,2009,9(5):810-811.
[17]卢强,任瑞文,胡岩,等.嗜水气单胞菌的随机扩增多态DNA分型[J].中国兽医学报,2002,22(1):34-36.
[18]Kato H,Kato N,Watanabe K,et al.Application of typing by pulsed-field gel electrophoresis to the study of Clostridium difficile in a neonatal intensive care unit[J].Journal of Clinical Microbiology,1994,32(9):2067-2070.
[19]Coscelli G A,Bermúdez R,Silva A R S,et al.Granulomatous dermatitis in turbot(Scophthalmus maximus L.)associated with natural Aeromonas salmonicida subsp.salmonicida infection[J].Aquaculture,2014,428-429:111-116.
[20]Liu P Y F,Shi Z Y,Lau Y J,et al.Comparison of different PCR approaches for characterization of Burkholderia(Pseudomonas)cepacia isolates[J].Journal of Clinical Microbiology,1995,33(12):3304-3307.
[21]罗志飞,胡萌,陆承平,等.嗜水气单胞菌分离株ERIC-PCR及RAPD分型比较[J].中国兽医学报,2011,31(4):504-508.
[22]Aguilera-Arreola M G,Hernández-Rodríguez C,Zú1iga G,et al.Aeromonas hydrophila clinical and environmental ecotypes as revealed by genetic diversity and virulence genes[J].FEMS Microbiology Letters,2005,242(2):231-240.
[23]肖丹,曹海鹏,胡鲲,等.淡水养殖动物致病性嗜水气单胞菌ERIC-PCR分型与耐药性[J].中国水产科学,2011,18(5):1092-1099.
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