溶藻弧菌的毒力相关基因及其对小鼠的致病力

Virulence-related genes of Vibrio alginolyticus and its virulence in mice

【目的】通过多重PCR检测和小鼠动物实验,对溶藻弧菌环境分离株的毒力因子进行评估,以期获得较强致病菌株和弱致病菌株之间的差别,并初步探讨该菌毒力因子对小鼠的致病机理。【方法】采用多重PCR体系检测毒力相关基因,我妻氏血平板溶血实验和平板酶活实验检测溶藻弧菌株的溶血素和胞外酶;以昆明小白鼠为实验动物,攻毒方式为灌胃和腹腔注射,根据小鼠的致病症状和死亡情况来分析和对比溶藻弧菌的胞外分泌物以及菌体本身的毒性。【结果】10株溶藻弧菌产淀粉酶、卵磷脂酶的比例为100%,脂肪酶、明胶酶次之(为70%),脲酶均未被检出;神奈川现象阳性菌株率为60%。毒力基因检测的结果显示10株溶藻弧菌中toxR、Collagenase、tlh、FlaA、ompW、AspA、fur这些与毒力有关的基因均有分布,而toxS、trh、tdh、UreR并未检出。10株溶藻弧菌中的VA009对小鼠显示了较强的致病性,能造成腹腔积液,经腹腔注射感染此菌后7 d内死亡率高达80%。【结论】不同的溶藻弧菌对小鼠的致病性存在较大差异,溶藻弧菌菌体本身比胞外分泌物对其毒性的贡献要大,而副溶血弧菌的毒性则由其胞外分泌物起主要作用;比较我们筛选出的强致病菌株与弱致病菌株,其上述毒力基因的分布并没有差别,说明溶藻弧菌可能存在一套与副溶血弧菌不同的独立的毒力基因系统。

[Objective]We determinated the virulence factors of Vibrio alginolyticus strains isolated from the environment by multiplex PCRs and animal experiments,in order to compare the differences between the highly virulent strain and attenuated virulent strain,and to explore the virulent mechanism of V. alginolyticus in mammals.[Methods] The virulence-related genes of V. alginolyticus were investigated by multiplex PCRs.Hemolysin and pathogenic proteins were detected using Kanagawa phenomenon tests and enzyme activity tests.In vivo pathogenetic tests of V.alginolyticus were done through orogastric and intraperitoneal Kunming mousel.[Results]Amylase and lecithinase activities were observed in 100% of the strains,whereas lipase and gelatinase activities were found in only 70% and urease activity was not detected. In Kanagawa phenomenon tests 60% of the strains gave positive results. The related virulence genes such as toxR,Collagenase,tlh,FlaA,ompW,AspA and fur were distributed among 10 strains of V.alginolyticus collected,with the exception of toxS,trh,tdh and UreR.Among those 10 strains,VA009 has shown a strong pathogenesis to the mouse,which caused fluid accumulation and led the mortality rate as high as 80% within 7 days by intraperitoneal infection.[Conclusion]This study indicates that there is a great difference in pathogenicity among V. alginolyticus strains to mouse.The cell toxicity of V.alginolyticus made more contribution than extracellular secretion,while the extracellular secretion of V. parahaemolyticus played a major role in its toxicity. The virulence gene profiles were consistent between the highly virulent and attenuated virulent strains,indicating that V. alginolyticus might have a different virulence system and different pathogenic mechanism compared with V. parahaemolyticus.