患病草鱼肠道病原菌的分离鉴定及毒力研究
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摘要
自环洞庭湖区精养鱼池患病草鱼肠道中分离获得4种病原菌,进行16S rRNA基因和gyrB基因序列测定和在线比对,并对草鱼进行回归感染。随后进行4种病原菌的药敏特征、溶血活性试验,并利用PCR方法检测了6种毒力因子的携带情况。试验结果表明,病原菌分别为温和气单胞菌、异常嗜糖气单胞菌、类志贺邻单胞菌和格氏乳球菌;4种病原菌回归感染草鱼,均能使草鱼患病致死,自死亡的草鱼体内均能分离得到相应感染的病原菌;4种病原菌对多种抗生素具有耐药性,且在脱纤维绵羊血血平板上检测到了温和气单胞菌、异常嗜糖气单胞菌AaB007的溶血活性;PCR结果表明,4种病原菌携带多种毒力因子。研究结果可为环洞庭湖区草鱼精养鱼池健康养殖及病害防治提供科学依据。
Four pathogenic strains were isolated from intestine of diseased grass carp(Ctenopharyngodon idellus) collected from an intensive fish farming ponds in Dongting Lake area. The 16 S rRNA gene sequencing, gyrB gene sequencing and BLAST analysis revealed that these four pathogenic bacterial strains were Aeromonas sobria, A. allosaccharophila, Plesiomonas shigelloide and Lactococcus garvieae. The infection tests indicated that all these four pathogenic strains were lethal to grass carp, and the corresponding pathogenic bacteria were isolated from the dead grass carp. Subsequently, we studied the drug sensitivity and hemolytic activity of these four strains, and it was approved that they carried the six toxicity genes by PCR. The results showed that the four pathogenic strains had resistance to a variety of antibiotics and many toxicity genes. The findings provide important scientific basis for healthy aquaculture of grass carp in intensive fish culture ponds in Dongting Lake area and that more attention should be paid to prevention and control of freshwater fish diseases.
Four pathogenic strains were isolated from intestine of diseased grass carp(Ctenopharyngodon idellus) collected from an intensive fish farming ponds in Dongting Lake area. The 16 S rRNA gene sequencing, gyrB gene sequencing and BLAST analysis revealed that these four pathogenic bacterial strains were Aeromonas sobria, A. allosaccharophila, Plesiomonas shigelloide and Lactococcus garvieae. The infection tests indicated that all these four pathogenic strains were lethal to grass carp, and the corresponding pathogenic bacteria were isolated from the dead grass carp. Subsequently, we studied the drug sensitivity and hemolytic activity of these four strains, and it was approved that they carried the six toxicity genes by PCR. The results showed that the four pathogenic strains had resistance to a variety of antibiotics and many toxicity genes. The findings provide important scientific basis for healthy aquaculture of grass carp in intensive fish culture ponds in Dongting Lake area and that more attention should be paid to prevention and control of freshwater fish diseases.
引文
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[19] 王小亮,徐立蒲,曹欢,等.鲟致病性类志贺邻单胞菌的鉴定及药物敏感性[J].微生物学报,2013,53(7):723-729.
[20] 胡钱东,林强,石存斌,等.草鱼致病性类志贺邻单胞菌的分离与鉴定[J].微生物学报,2014,54(2):229-235.
[21] 陆文浩,杨家新,陈辉,等.异育银鲫类志贺邻单胞菌的鉴定[J].淡水渔业,2009,39(2):48-53.
[22] 刘志刚,可小丽,卢迈新,等.尼罗罗非鱼致病性类志贺邻单胞菌(Plesiomonas shigelloides)的分离鉴定及其病理学观察[J].微生物学报,2015,55(1):96-106.
[23] 叶键,刘晓宁,王晓,等.青鱼(Mylopharyngodon piceus)新发病病原类志贺邻单胞菌(Plesiomonas shigelloides)的分离鉴定[J].海洋与湖沼,2016,47(3):633-639.
[24] Samannodi M, Zhao A, Nemshah Y, et al. Plesiomonas shigelloides septic shock leading to death of postsplenectomy patient with pyruvate kinase deficiency and hemochromatosis[J]. Case Rep Infect Dis,2016(2016):1538501.
[25] Meyburgh C M, Bragg R R, Boucher C E. Lactococcus garvieae: an emerging bacterial pathogen of fish[J]. Dis Aquat Organ, 2017,123(1):67-79.
[26] 吴晓丰,杨鹭花.氯霉素残留的危害及其检测方法[J].动物医学进展,2004,25(3):41-43.
[27] Takino M, Daishima S, Nakahara T. Determination of chloramphenicol residues in fish meats by liquid chromatography-atmospheric pressure photoionization mass spectrometry[J]. J Chromatogr A,2003,1011(1/2):67-75.
[28] 李晓川,孔轶群.水产品中氯霉素的问题与检测[J].农业质量标准,2003(3):18-19.
[29] 程方俊,徐兴然,宋振辉,等.温和气单胞菌RC-07-KA株溶血素基因克隆、表达及活性检测[J].中国预防兽医学报,2012,34(3):201-205.
[30] 李烨,李楠,张晓林,等.鱼类烂鳃病病原柱状黄杆菌溶血素基因的初步研究[J].水生生物学报,2015,39(3):604-607.
[31] Kothary M H, Lowman H, McCardell B A, et al. Purification and characterization of enterotoxigenic El Tor-like hemolysin produced by Vibrio fluvialis[J]. Infect Immun,2003,71(6):3213-3220.
[32] Ji Y, Li J, Qin Z, et al. Contribution of nuclease to the pathogenesis of Aeromonas hydrophila[J]. Virulence,2015,6(5):515-522.
[2] Li F Y, Ma S J, Li Y Y, et al. Impact of the Three Gorges project on ecological environment changes and snail distribution in Dongting Lake area[J]. PloS Neglected Tropical Diseases,2017,11(7):5661-5675.
[3] 李成.洞庭湖主要经济鱼类资源调查及其变化规律研究[D].长沙:湖南农业大学,2006:1-38.
[4] 张德锋,刘礼辉,李宁求,等.我国南方地区鱼源气单胞菌不同种类的流行特征[J].水产科学,2015,34(11):673-682.
[5] 周文,江为民,徐永福.2012年湖南省水产养殖病害监测报告[J].当代水产,2013(2):80-82.
[6] 张晓君,阎斌伦,邴旭文,等.异育银鲫病原温和气单胞菌表型及分子鉴定与溶血素基因检测[J].水生态学杂志,2010,3(4):102-107.
[7] 王海娟,王利.温和气单胞菌毒力基因的检测及其对鲫鱼致病性试验[J].动物医学进展,2015,36(3):17-20.
[8] 李光超, 武小虎, 马友记, 等. 奶牛乳腺炎金黄色葡萄球菌溶血素基因型和表现型分析[J]. 中国兽医学报,2016,36(10):1714-1717.
[9] Nawaz M, Khan S A, Khan A A, et al. Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish[J]. Food Microbiol,2010,27(3):327-331.
[10] 隗黎丽,王自蕊,杨竹青.草鱼2株气单胞菌的分离与鉴定[J].水产科学,2013,32(6):348-352.
[11] Coscelli G A, Casabonne C, Moron-Alcain E, et al. Aeromonas sobria, an outbreak of natural infection in cultured silver catfish Rhamdia quelen (Quoy & Gaimard, 1824) in Argentina[J]. J Fish Dis,2017,40(12):1929-1933.
[12] Janda J M, Abbott S L. Evolving concepts regarding the genus Aeromonas: an expanding Panorama of species, disease presentations, and unanswered questions[J]. Clin Infect Dis,1998,27(2):332-344.
[13] 邢根娣,胡秀彩,李雪,等.鲫鱼肠道温和气单胞菌的分离鉴定及药敏试验[J].江苏师范大学学报:自然科学版,2013,31(4):52-55.
[14] 刘敏,韩英.鲤鱼温和气单胞菌的分离鉴定和药敏试验[J].东北农业大学学报,2005,36(4):486-489.
[15] 巴翠玉,张雅斌,张培军,等.大麻哈鱼温和气单胞菌的分离鉴定及药敏试验[J].中国兽医科学,2016(6):775-780.
[16] 胡琳琳,房文红,梁思成,等.金鱼温和气单胞菌的分离鉴定及药敏试验[J].上海水产大学学报,2008,17(3):285-290.
[17] Martinez-Murcia A J, Esteve C, Garay E, et al. Aeromonas allosaccharophila sp. nov., a new mesophilic member of the genus Aeromonas[J]. FEMS Microbiol Lett,1992,70(3):199-205.
[18] 刘双凤,邹作宇,袁美云,等.施氏鲟肿嘴病快速检测方法的研究[J].江西水产科技,2015(1):3-8.
[19] 王小亮,徐立蒲,曹欢,等.鲟致病性类志贺邻单胞菌的鉴定及药物敏感性[J].微生物学报,2013,53(7):723-729.
[20] 胡钱东,林强,石存斌,等.草鱼致病性类志贺邻单胞菌的分离与鉴定[J].微生物学报,2014,54(2):229-235.
[21] 陆文浩,杨家新,陈辉,等.异育银鲫类志贺邻单胞菌的鉴定[J].淡水渔业,2009,39(2):48-53.
[22] 刘志刚,可小丽,卢迈新,等.尼罗罗非鱼致病性类志贺邻单胞菌(Plesiomonas shigelloides)的分离鉴定及其病理学观察[J].微生物学报,2015,55(1):96-106.
[23] 叶键,刘晓宁,王晓,等.青鱼(Mylopharyngodon piceus)新发病病原类志贺邻单胞菌(Plesiomonas shigelloides)的分离鉴定[J].海洋与湖沼,2016,47(3):633-639.
[24] Samannodi M, Zhao A, Nemshah Y, et al. Plesiomonas shigelloides septic shock leading to death of postsplenectomy patient with pyruvate kinase deficiency and hemochromatosis[J]. Case Rep Infect Dis,2016(2016):1538501.
[25] Meyburgh C M, Bragg R R, Boucher C E. Lactococcus garvieae: an emerging bacterial pathogen of fish[J]. Dis Aquat Organ, 2017,123(1):67-79.
[26] 吴晓丰,杨鹭花.氯霉素残留的危害及其检测方法[J].动物医学进展,2004,25(3):41-43.
[27] Takino M, Daishima S, Nakahara T. Determination of chloramphenicol residues in fish meats by liquid chromatography-atmospheric pressure photoionization mass spectrometry[J]. J Chromatogr A,2003,1011(1/2):67-75.
[28] 李晓川,孔轶群.水产品中氯霉素的问题与检测[J].农业质量标准,2003(3):18-19.
[29] 程方俊,徐兴然,宋振辉,等.温和气单胞菌RC-07-KA株溶血素基因克隆、表达及活性检测[J].中国预防兽医学报,2012,34(3):201-205.
[30] 李烨,李楠,张晓林,等.鱼类烂鳃病病原柱状黄杆菌溶血素基因的初步研究[J].水生生物学报,2015,39(3):604-607.
[31] Kothary M H, Lowman H, McCardell B A, et al. Purification and characterization of enterotoxigenic El Tor-like hemolysin produced by Vibrio fluvialis[J]. Infect Immun,2003,71(6):3213-3220.
[32] Ji Y, Li J, Qin Z, et al. Contribution of nuclease to the pathogenesis of Aeromonas hydrophila[J]. Virulence,2015,6(5):515-522.