Use and development of fish cell lines as in vitro models for the study of intracellular and extracellular protozoan pathogens

鱼类和贝类疾病一直是渔业和水产养殖业关注的一个问题，因为它会造成经济损失，并对人类造成疾病威胁。病原体，无论是病毒、细菌还是寄生虫，在水生环境中自然存在，通常受到活体动物体内平衡机制的控制。当环境条件由于诸如全球变暖、污染、拥挤压力、接触外来分子等事件而发生变化时，现有的平衡机制就会发生变化，动物就会变得容易患病。我们对哺乳动物的疾病进展、预防、控制和治疗了解甚多，但对鱼类和贝类的疾病进展知之甚少。本研究项目旨在研究两种模式原生动物寄生虫：一种是海洋变形虫，新拟变形虫，被认为是海洋鱼类和贝类几种疾病的病原体；淡水微孢子虫，鳗异孢子虫，一种鳗鱼的细胞内寄生虫，它也被证明会影响其他物种，如鲈鱼，白眼鱼，鳟鱼，最近有报道称它发生在五大湖的物种中。体外细胞培养系统已被证明在帮助重要的哺乳动物和昆虫原生动物病原体的生长和研究方面是成功的。由于方便的鱼类细胞系相对缺乏，这种方法尚未在鱼类中充分发挥其潜力。本研究的目的是利用体外方法阐明鱼体内病原体和宿主相互作用的机制，目的如下：开发相对简单和容易获得的检测系统，用于研究病原体-宿主细胞相互作用；开发准确的定性和定量手段，使用新的方法来辨别疾病进展的早期变化，例如分析寄生原生动物暴露的鱼细胞中的蛋白质表达特征，3。研究时间和物理化学因素调节病原体-细胞相互作用和发展或预防毒力，3。比较上述方法在水生原生动物疾病的预防、诊断和治疗中的实用性和准确性。

Fish and shellfish diseases have been a cause of concern in fisheries and aquaculture because of economic losses as well as the threat of disease to humans.  Pathogens, whether viruses, bacteria or parasites, occur naturally in the aquatic environment and are usually kept under control by the homeostatic mechanisms of living animals. When environmental conditions change due to events such as global warming, pollution, stress of crowding, exposure to foreign molecules, etc, the balancing mechanisms in place change, and the animals become susceptible to disease. Much is known about disease progression, prevention, control and treatment in mammals, but comparatively little is known about these events in fish and shellfish. This research program aims to study two model protozoan parasites: a marine amoeba, Neoparamoeba pemaquidensis, believed to be the causative agent for several diseases in marine finfish and shellfish; and the freshwater microsporidian, Heterosporis anguillarum, an intracellular parasite of eels that has also been shown to affect other species such as perch, walleye, trout, and that has been recently reported to occur in species within the Great Lakes. In vitro cell culture systems have proven successful for aiding in the growth and research of important mammalian and insect protozoan pathogens. This approach has not been exploited to its full potential with fish species due to the relative paucity of convenient fish cell lines. The objective of this proposal is to elucidate the mechanisms of pathogen and host interactions in fish using in vitro approaches with the following goals: 1. Develop relatively simple and readily accessible assay systems for studying pathogen-host cell interactions, 2. Develop accurate qualitative and quantitative means to discern early changes in disease progression using novel methodologies such as analyzing protein expression signatures in parasitic protozoa exposed fish cells, 3. Investigate temporal and physicochemical factors modulating pathogen-cell interactions and the development or prevention of virulence, 4. Compare the utility and accuracy of above approaches in the prevention, diagnosis and treatment of aquatic protozoan diseases.