Bacterial pathogens of freshwater fish: environmental survival and evolution of virulence

淡水鱼的细菌病原体：环境生存和毒力进化

• 批准号: 6559-2012
• 负责人: Stevenson, Roselynn
• 金额: $ 1.89万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=592848
• 关键词: Bacterial; pathogens; freshwater; fish; environmental

The bacterium Yersinia ruckeri infects rainbow trout and other fish, moving from the aquatic environment into the host animal through gills, skin and digestive tract and persisting in organs of the fish. Enteric redmouth disease appears when carrier fish are stressed by water quality, crowding, or handling. Not all isolates are identical, and the diversity affects their ability to infect different fish species and to persist and spread in the aquatic environment. We will identify genetic markers that can be used to clearly differentiate strains of Yersinia ruckeri, based on the genes involved in synthesizing their major surface antigen (lipopolysaccharide) and virulence-related genes. Sets of PCR primers will allow laboratories to quickly characterize isolates and help us understand distribution of strain types in fish populations. Our approach to finding the markers is genomic subtractive hybridization, in which single-stranded DNA isolated from two strains is mixed and allowed to reanneal to double-strands. DNA fragments from a tester strain that do not find a match, even in an excess of DNA from the second (driver) strain will be cloned and used to screen panels of bacterial strains collected from many sources. Fish responses to pathogen strains would ideally be studied in fish challenge experiments, but high individual variability in fish responses and the effect of stress mean that experiments require large numbers of fish. Single-celled protozoans can be useful as a model to indicate bacterial genes that affect virulence. Protozoa, such as amoeba, are voracious feeders on bacteria but some resist digestion. Resistant bacteria are often pathogens, perhaps because the characteristics that allow them to resist amoeba are also effective against host macrophages. We will use cultured amoeba as a way to compare bacterial strains (and mutants constructed in the studied genes) for survival. Research on amoeba-Yersinia ruckeri interactions can also indicate how amoeba in an aquatic environment serve as a reservoir for pathogen, allowing survival and transmission to new fish hosts. This can provide new ways of testing for significant fish pathogens in the environment and in aquaculture recirculation systems.

里氏耶尔森氏菌感染虹鳟鱼和其他鱼类，通过鳃、皮肤和消化道从水环境进入宿主动物，并持续存在于鱼类的器官中。当携带鱼受到水质、拥挤或搬运的压力时，就会出现肠源性赤口病。并不是所有的分离株都是相同的，多样性影响它们感染不同鱼类物种以及在水环境中持续和传播的能力。我们将根据参与合成主要表面抗原(脂多糖)的基因和毒力相关基因，寻找可用于明确区分ruckeri耶尔森菌菌株的遗传标记。一套聚合酶链式反应引物将使实验室能够快速确定分离株的特征，并帮助我们了解鱼类种群中菌株类型的分布。我们寻找标记的方法是基因组消减杂交，即从两个菌株中分离出的单链DNA混合，并允许重新退火为双链。来自测试菌株的DNA片段如果找不到匹配，即使是来自第二个(驱动)菌株的过量DNA，也将被克隆并用于筛选从许多来源收集的细菌菌株。理想的做法是在鱼类挑战实验中研究鱼类对病原体菌株的反应，但鱼类反应的高度个体变异性和应激的影响意味着实验需要大量的鱼。单细胞原生动物可以作为指示影响毒力的细菌基因的模型。原生动物，如阿米巴，是贪婪的细菌食客，但有些抵抗消化。耐药细菌通常是病原体，可能是因为它们抵抗阿米巴的特性也对宿主巨噬细胞有效。我们将使用培养的阿米巴作为比较细菌菌株(和在研究的基因中构建的突变株)存活的一种方式。对阿米巴和鲁氏耶尔森菌相互作用的研究还可以表明，水环境中的阿米巴是如何作为病原体的储存库，使其存活并传播给新的鱼类宿主的。这可以为在环境和水产养殖再循环系统中检测重要的鱼类病原体提供新的方法。