湖北地区暴发病池塘中嗜水气单胞菌的遗传多样性和毒力特征研究
THE GENETIC DIVERSITY AND VIRULENCE CHARACTERISTICS OF AEROMONAS HYDROPHILA ISOLATED FROM FISHPONDS WITH DISEASE OUTBREAKS IN HUBEI PROVINCE
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摘要: 为了调查引起鱼类运动型气单胞菌败血症(俗称暴发病)的嗜水气单胞菌的遗传多样性和毒力特征, 阐明其流行规律, 研究于2006-2009年度从湖北省内3个不同地区的6个发病鱼塘中分离了30株嗜水气单胞菌, 其中20株为临床株(分离自血液、肝脏、肾脏或腹水), 6株为肠道株, 4株为池水株。基于所有菌株gyrB基因序列, 构建了系统发育树; 通过ERIC (Enterobacterial repetitive intergenic consensus, 肠道细菌基因间重复序列)指纹图谱进行菌株的遗传分型; 用PCR方法检测了7个毒力基因在菌株中的分布模式。这7个基因包括气溶素(aerA)、溶血素(hlyA)、热不稳定性细胞兴奋性肠毒素(alt)、热稳定性细胞兴奋性肠毒素(ast)、弹性蛋白酶(ahpB)、脂酶(lip)和鞭毛基因(fla)。此外, 以斑马鱼为感染对象, 通过腹腔注射测定了15株代表菌株的毒力。结果表明: 不同来源的20株临床株、1株肠道株和3株池水株具有相同的遗传特性, 体现为在系统树上聚为一枝, 序列相似性为100%, 具有相同的ERIC指纹图谱, 毒力基因分布模式为: aerA+hlyA+alt+ast+ahpB+lip+fla+, 且均为强毒株(LD50 9.74104cfu/尾)。与临床株相比, 其余5株肠道株和1株池水株或具有不同的ERIC指纹图谱或具有不同的毒力基因分布模式, 显示出了遗传多样性, 且毒力均弱于临床株(LD501.01106cfu/尾)。这说明在一定时间、一定区域内, 作为暴发病病原的嗜水气单胞菌为同一克隆系在流行, 不存在明显的变异或遗传多样性。此结果有助于阐明嗜水气单胞菌引起的暴发病的流行规律, 制定相应的防御措施。多种毒力基因在致病性菌株中的联合流行为发病机理的解析奠定了基础。此外, 鉴于毒力基因谱与致病性之间的相关性, 表明毒力基因可作为标记基因, 用于致病性菌株的检测。Abstract: to investigate the genetic diversity and virulence characteristics, and to clarify the epidemic regularity of Aeromonas hydrophila which caused motile Aeromonas septicaemia outbreak (commonly known as disease outbreaks) in fish, 30 strains were isolated from 6 diseased fishponds which located in three different regions of Hubei province from 2006 to 2009. Among these strains, 20 were clinical strains (isolated from blood, liver, kidney or ascites fluid), 6 were intestinal strains and 4 were pond-water strains. A phylogenetic tree was constructed on the basis of the gyrB gene sequence of all 30 strains; the genetic typing of these strains were done through ERIC (enterobacterial repetitive intergenic consensus)-fingerprinting; the distribution patterns of seven virulence genes in these strains were tested by PCR method, including aerolysin (aerA), haemolysin (hlyA), heat-labile cytotonic enterotoxin (alt), heat-stable cytotonic enterotoxin (ast), elastase (ahpB), lipase (lip), and flagella (fla). In addition, the virulence of 15 representative strains was assayed in zebrafish by intraperitoneal injection. The results showed that regardless of the origin, all the 20 clinical strains, 1 intestinal strain and 3 pond-water strains had the same genetic characteristics. These included that clustered together in the phylogenetic tree, with 100% sequence similarities among these strains; have the same ERIC-finger-printing and the same virulence-gene pattern: aerA+hlyA+alt+ast+ahpB+lip+fla+; all of them were high virulent strains (LD50 9.74104 cfu/fish). Compared with clinical strains, the rest 5 intestinal strains and 1 pond-water strain either had different ERIC-fingerprintings or have different virulence-gene patterns, showing higher genetic diversity and lower virulence (LD501.01106 cfu/fish). These results indicated that as the pathogen of disease outbreaks, a clonal lineage of A. hydrophila was in popular within a certain time and areas, and no obvious variation or genetic diversity was found among these strains. This result would help in the clarification of the epidemic regularity of A. hydrophila underlying outbreaks, and help to make the right defense measures to prevent this infection. Meanwhile, the combined prevalence of multiple virulence genes in pathogenic strains of A. hydrophila laid the foundation for clarify the pathogenic mechanism underlie this infection. Moreover, the correlation of virulence and virulence-gene patterns of A. hydrophila showed that virulence genes can be chosen as gene-marker to detect pathogenic strains of this bacterium.
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Keywords:
- Diseased fishponds /
- Aeromonas hydrophila /
- Genetic diversity /
- Virulence gene /
- Virulence
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