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

由于经济损失以及疾病对人类的威胁，鱼类和贝类疾病一直是渔业和水产养殖业关注的一个原因。 病原体，无论是病毒、细菌还是寄生虫，都在水生环境中自然发生，通常受到活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、暴露于外来分子等事件而改变时，适当的平衡机制发生变化，动物变得易受疾病影响。人们对哺乳动物的疾病进展、预防、控制和治疗了解很多，但对鱼类和贝类的这些事件了解相对较少。这项研究计划旨在研究两种模式原生动物寄生虫：一种海洋变形虫，Neoparamoeba pemaquarensis，被认为是海洋鱼类和贝类几种疾病的病原体;以及淡水微孢子虫，鳗异孢子虫，一种鳗鱼的细胞内寄生虫，也被证明影响其他物种，如鲈鱼，海象，鳟鱼，最近有报道称，这种情况发生在五大湖的物种中。体外细胞培养系统已被证明成功地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。由于相对缺乏方便的鱼类细胞系，这种方法尚未在鱼类物种中充分发挥其潜力。本论文的主要目的是利用体外方法研究鱼类病原菌与宿主相互作用的机制。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，2。开发准确的定性和定量方法，使用新的方法来识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.调查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，4。比较上述方法在水生原生动物疾病预防、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.