Investigating virulence factors in Neoparamoeba perurans causing amoebic gill disease (AGD) in Atlantic salmon

阿米巴鳃病(AGD)是一种严重的疾病，影响澳大利亚和塔斯马尼亚的海洋养殖大西洋鲑鱼Salmo salar L.，在那里，仅治疗一项就花费该行业高达2300万美元。最近，AGD在欧洲和美洲成为一种新出现的疾病，在某些情况下，导致欧洲高达三分之一的收成损失(例如2012年在设得兰)。AGD的特点是鳃表面有多处白色斑块，导致上皮细胞和粘液细胞增殖，导致板层融合区，导致呼吸衰竭死亡。AGD的病原体是Perurans新阿米巴，一种在鳃上定居的自由生活的原生动物。这个项目将是CEFA和埃克塞特大学的合作项目，并将解决以下目标：目标1：表征致病性和非致病性阿米巴中的分子宿主病原体相互作用-学生将进行一项暂时感染研究，其中鲑鱼鳃细胞将感染致病性Perurans，通过多代体外传代获得的Perurans减毒克隆和一种非致病性阿米巴(亲缘关系密切但非致病的新阿米巴)。将进行RNA-Seq分析，以量化基因表达随时间的变化(来自宿主和病原体)，三种实验情景之间的比较将允许识别致病性Perurans的分子靶标，这些靶标在感染的早期阶段表达，并与其他形式的阿米巴不同。目的2：鉴定与美洲斑潜蝇致病相关的基因--学生随后将进行RNAi实验，以确定上述被鉴定的基因中哪些基因与美洲斑潜蝇致病有关。双链RNA(DsRNA)将被设计用于候选基因，并在体外传递给培养的阿米巴。RNAi传递的有效性将通过使用致病性生物标记物(例如IL-1β)的基因表达分析来测试。利用这个体外平台，一系列基因将被筛选出来。

Amoebic gill disease (AGD) is a serious disease affecting the marine-farmed Atlantic salmon, Salmo salar L., industry in Australia and Tasmania, where it costs the industry up to $23M a year in treatment alone. Recently, AGD became an emerging disease in Europe and America accounting for losses of up to one third of the harvest in some cases in Europe (for example in Shetland in 2012). AGD is characterised by multifocal white patches on the gill surface and causes hyperplasia of the epithelial and mucous cells, which can result in areas of lamellar fusion, leading to death due to respiratory failure. The causative agent of AGD is Neoparamoeba perurans, a free living protozoan which colonises the gills. This project will be a collaborative project between Cefas and the University of Exeter and will address the following objectives:Objective 1: Characterisation of the molecular host pathogen interactions in pathogenic and non-pathogenic amoeba - The student will conduct a temporal infection study in which salmon gill cells will be infected with pathogenic N. perurans, an attenuated clone of N. perurans obtained through multiple in vitro passages and a non-pathogenic amoeba (the closely related but non-pathogenic Neoparamoeba pemaquidensis). RNA-Seq analysis will be conducted to quantify changes in gene expression (from both the host and pathogen) over time and comparisons between the three experimental scenarios will allow for the identification of molecular targets from the pathogenic N. Perurans that are expressed during the early stages of infection and differ from the other forms of amoeba. Objective 2: Identification of the genes responsible for the pathogenicity of N perurans - The student will then perform RNAi experiments to determine which genes from those identified above are responsible for the pathogenicity of N. perurans. Double-stranded RNA (dsRNA) will be designed for candidate genes and delivered in vitro to cultured amoebae. The efficacy of RNAi delivery will be tested by gene expression analyses using biomarkers of pathogenicity (for example IL-1beta). Using this in vitro platform, a range of genes will be screened.