Development of a non-lethal sampling method to monitor immune response and disease progression in salmonid fish

Evaluation of pathogen virulence, testing the efficacy of vaccines or examining the immune response of fish, are traditionally carried out through infection experiments whereby animals are sacrificed at regular intervals and the pathogen load and/or immune parameters are measured. However, because the precise stage of the infection is unknown in the sampled fish using this model, the status of the immune response and its efficacy against the pathogen are poorly described. We propose to develop a non-lethal experimental infection model in order to reduce the number of fish required in experimentation by up to 80%, and improve the scientific output from infectivity studies. This project will be divided into three work packages WP1, WP2 and WP3. WP1 will focus on the development of a non-lethal experimental infection model to generate a collection of blood and tissue samples from Atlantic salmon (Salmo salar) infected with either viral (Infectious Salmon Anaemia Virus [ISAV], Salmon Alphavirus [SAV], totivirus, the causative agent of the cardiomyopathy syndrome [CMS]) or bacterial (Yersinia ruckeri) pathogens that cause significant economic losses to the UK fish farming industry. Welfare parameters will be measured to evaluate the severity of the repeated blood sampling procedure. The type of immune response induced by each category of pathogens will be characterised in WP2. Expression levels of key cytokine genes, indicative of T helper cell subset differentiation, will be measured by qPCR in blood leukocytes. The change in blood leukocyte composition during the infection will be analysed by FACS. At the final sampling point or in moribund fish the histopathological status of the individuals will be evaluated. For each pathogen, the correlation between the type of immune response and it kinetics, the viraemia/bacteraemia, route of infection, terminal pathological status will be established. In WP3, novel tools will be developed to measure immune parameters non-destructively, together with the adaptation of existing tools. Cell-based in vitro system for Th2 and pro-inflammatory responses will be established using key transcription factors and gene promoters. Antibody-based detection methods (Luminex) will be developed to measure several parameters in the same serum sample. Besides the immediate 80% reduction in the number of animals required for experimentation, the synthesis and correlation between the kinetics of the immune parameters in blood with the outcome of the disease (death, severity of pathology, complete recovery) will provide valuable information to help prevent and/or predict the risk of pathogen infections in fish farms.

• Research Review 2013: Minimising fish use in immune studies
• Further Funding: NC3Rs Pilot Study Grant, Novel approaches to immortalise salmon fish cells,  December 2013, £74,443