文章摘要
刘小芳,任燕,张德锋,巩华,石存斌,常藕琴,潘厚军.鱼源气单胞菌的毒力基因检测、分型及致病力[J].水产学报,2021,45(3):462~471
鱼源气单胞菌的毒力基因检测、分型及致病力
Detection, genotyping and pathogenicity of virulence genes in Aeromonas species isolated from diseased freshwater fish
投稿时间:2020-03-05  修订日期:2020-06-20
DOI:10.11964/jfc.20200312180
中文关键词: 气单胞菌  毒力基因  致病性  患病鱼
英文关键词: Aeromonas spp.  virulence gene  pathogenicity  diseased fish
基金项目:国家重点研发计划(2019YFD0900102);国家大宗淡水鱼产业技术体系(CARS-45);广州市科技计划项目(201904020004);中国水产科学研究院基本科研业务费专项(2020XT0405)
作者单位E-mail
刘小芳 中国水产科学研究院珠江水产研究所广东省水产动物免疫技术重点实验室农业农村部渔用药物创制重点实验室广东 广州 510380
上海海洋大学水产与生命学院上海 201306 
 
任燕 中国水产科学研究院珠江水产研究所广东省水产动物免疫技术重点实验室农业农村部渔用药物创制重点实验室广东 广州 510380 renyannj@126.com 
张德锋 中国水产科学研究院珠江水产研究所广东省水产动物免疫技术重点实验室农业农村部渔用药物创制重点实验室广东 广州 510380  
巩华 中国水产科学研究院珠江水产研究所广东省水产动物免疫技术重点实验室农业农村部渔用药物创制重点实验室广东 广州 510380  
石存斌 中国水产科学研究院珠江水产研究所广东省水产动物免疫技术重点实验室农业农村部渔用药物创制重点实验室广东 广州 510380  
常藕琴 中国水产科学研究院珠江水产研究所广东省水产动物免疫技术重点实验室农业农村部渔用药物创制重点实验室广东 广州 510380  
潘厚军 中国水产科学研究院珠江水产研究所广东省水产动物免疫技术重点实验室农业农村部渔用药物创制重点实验室广东 广州 510380  
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中文摘要:
      为调查鱼源气单胞菌毒力基因与其致病力的相关性,以2009—2018年从不同患病鱼分离的173株气单胞菌为研究对象,通过检测毒力相关基因、测定溶血活性、腹腔注射感染异育银鲫等方法开展评价。通过管家基因gyrB分子鉴定结果显示,173株气单胞菌中维氏气单胞菌(119/173,68.9%)和嗜水气单胞菌(50/173,28.9%)是主要流行的菌株。10个毒力基因aer(162/173,93.64%)、act(131/173,75.72%)、ast(55/173,31.79%)、alt(58/173,33.53%)、lip(152/173,87.86%)、exu(154/173,89.02%)、fla(143/173,82.66%)、gcaT(148/173,85.55%)、eprCAI(41/173,23.70%)和ahyB(51/173,29.48%)普遍存在于173株气单胞菌中。依据检测到的毒力基因数量从多到少分布情况,这些菌株可分为7大类(Ⅰ~Ⅶ)53个毒力基因型。大部分嗜水气单胞菌检测到8~10个毒力基因,主要分布于Ⅰ、Ⅱ和Ⅲ类基因型;维氏气单胞菌的eprCAIahyBastalt等4个毒力基因检测率低,主要分布于Ⅳ、Ⅴ和Ⅵ类基因型。大部分气单胞菌(94.22%,163/173)具有溶血活性。代表性毒力基因型的38株维氏气单胞菌和20株嗜水气单胞菌腹腔注射异育银鲫攻毒结果显示,3.0×106 CFU/尾的剂量下,3株维氏气单胞菌使鲫死亡率达80%~100%,16株嗜水气单胞菌使鲫死亡率达90%~100%。研究表明,维氏气单胞菌是目前最主要流行的气单胞菌,但其检测到的毒力基因普遍少于嗜水气单胞菌,且对异育银鲫的致病力普遍弱于嗜水气单胞菌。本研究能够为气单胞菌败血症的流行病学调查和疫苗研究提供理论依据。
英文摘要:
      Aeromonas spp. are gram-negative bacteria that can infect a variety of aquatic and terrestrial animals, including humans and cause motile aeromonad septicemia. The prevalence of Aeromonas spp. in various water and food sources poses a significant public health threat. Aeromonas spp. encompass a diversity of strains or genotypes with varying pathogenic potentials. Many strains are pathogenic, but others are avirulent and produce little harm to the host. The availability of methods to assess the pathogenic potential of Aeromonas spp. would contribute to the improved control and prevention strategies for this bacterial infection. An approach to the direct detection of potentially pathogenic Aeromonas spp. is the use of virulence determinants as genetic markers. To investigate the correlation between virulence genes and pathogenicity of Aeromonas spp., 173 strains of Aeromonas were isolated from different diseased freshwater fish from 2009 to 2018 as the research object. Virulence genes including aer, act, fla, lip, gcaT, exu, ast, alt, eprCAI and ahyB were detected and the bacterial pathogenicity with different virulence genotypes was assessed through intraperitoneal (IP) injection challenged Carassius auratus gibelio (gibel carp). Molecular identification revealed that A.veronii (119/173, 68.8%) and A.hydrophila (50/173, 28.9%) were the prevalent species based on gyrB gene sequencing. The distribution of 10 virulence genes including aer (162/173, 93.64%), act (131/173, 75.72%), ast (55/173, 31.79%), alt (58/173, 33.53%), lip (152/173, 87.86%), exu (154/173, 89.02%), fla (143/173, 82.66%), gcaT (148/173, 85.55%), eprCAI(41/173, 23.70%) and ahyB (51/173, 29.48%) were determined and these strains were sorted into 7 clusters (Ⅰ-Ⅶ) and formed 53 genotypes according to the virulence distribution profile. 8-10 virulence genes were present in 24.86% (43/173) Aeromonas strains, among which 38 strains were identified as A. hydrophila and 4 strains A.veronii. The eprCAI (0/119, 0%), ahyB (19/119, 15.13%), ast (7/119, 5.88%) and alt (24/119, 20.17%) genes were detected from the A.veronii isolates. Most of the Aeromonas strains (94.22%, 163/173) had hemolytic activity. At the challenge dose of 3.0×106CFU per fish, 3 strains of A.veronii (Ⅳ-5, Ⅴ-1 and Ⅵ genotype) and 16 strains of A.hydrophila (Ⅰ, Ⅱ-1, Ⅱ-3, Ⅱ-5, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅲ-5, Ⅳ-1 and Ⅳ-2 genotype) were highly pathogenic and the mortality reached 80%-100%.The study suggested that A.veronii was the most prevalent species in the diseased fish. A.veronii isolates had fewer virulence genes and lower pathogenicity than A.hydrophila ones. The result may provide reference for epidemiological investigation of motile aeromonad septicemia and vaccine research.
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