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

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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.