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，影响着澳大利亚和塔斯马尼亚州海洋养殖的大西洋鲑鱼（Salmo salar L.）行业，仅在治疗方面，该行业每年就花费高达2300万美元。最近，AGD在欧洲和美洲成为一种新出现的疾病，在欧洲的某些情况下（例如2012年在设得兰群岛）造成的损失高达收成的三分之一。AGD的特征是鳃表面出现多灶性白色斑块，引起上皮细胞和黏液细胞增生，可导致板层融合区域，导致呼吸衰竭死亡。AGD的病原体是秘鲁新拟阿米巴，这是一种自由生活的原生动物，在鳃上定居。该项目将是Cefas和埃克塞特大学之间的合作项目，将实现以下目标：致病性和非致病性阿米巴原虫分子宿主病原体相互作用的特征-学生将进行一项时间感染研究，在该研究中，鲑鱼鳃细胞将感染致病性N. perurans，通过多次体外传代获得的N. perurans的减毒克隆和非致病性阿米巴（密切相关但非致病性新阿米巴pemaquidensis）。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），并对三种实验方案进行比较，以确定致病性N. Perurans的分子靶标，这些靶标在感染的早期阶段表达，与其他形式的阿米巴原虫不同。目标2：鉴定与葡萄球菌致病性有关的基因-学生将进行RNAi实验，以确定从上述鉴定的基因中确定哪些基因与葡萄球菌致病性有关。双链RNA （dsRNA）将被设计用于候选基因，并在体外传递给培养的变形虫。RNAi递送的有效性将通过使用致病性生物标志物（例如il -1 β）进行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。

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.