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

拉克氏耶尔森氏菌感染虹鳟鱼和其他鱼类，从水生环境通过鳃、皮肤和消化道进入宿主动物，并在鱼的器官中存留。当带鱼受到水质、拥挤或处理的压力时，就会出现肠性红口病。并不是所有的分离株都是相同的，这种多样性影响了它们感染不同鱼类的能力，以及在水生环境中持续存在和传播的能力。我们将根据其主要表面抗原（脂多糖）的合成基因和毒力相关基因，确定可用于明确区分拉克氏耶尔森菌菌株的遗传标记。PCR引物将允许实验室快速表征分离物，并帮助我们了解菌株类型在鱼类种群中的分布。我们寻找标记的方法是基因组减法杂交，其中从两个菌株分离的单链DNA混合并允许重新退火为双链。从测试菌株中没有找到匹配的DNA片段，即使从第二（驱动）菌株中有过量的DNA，也将被克隆并用于筛选从许多来源收集的细菌菌株。鱼类对病原体菌株的反应最好在鱼类挑战实验中进行研究，但鱼类反应的高度个体差异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一个有用的模型，以表明细菌的基因，影响毒力。原生动物，如阿米巴原虫，以细菌为食，但有些会抵抗消化。耐药细菌通常是病原体，也许是因为它们抵抗变形虫的特性也能有效地抵抗宿主巨噬细胞。我们将使用培养的变形虫作为一种比较细菌菌株（以及在所研究基因中构建的突变体）存活的方法。对阿米巴-拉克氏耶尔森菌相互作用的研究也可以表明阿米巴在水生环境中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。这可以为检测环境和水产养殖循环系统中的重要鱼类病原体提供新的方法。

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.