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

Fish farming in the UK and around the world faces serious economical threats from viruses and bacteria causing outbreaks and loss of valuable livestock. Vaccines and immunostimulants are often administered to fish to prevent these outbreaks. Continuous research is required to verify whether new products are effective. In order to do so, research worldwide is routinely carried out whereby large groups of animals are experimentally infected with a pathogen. The level of mortalities in groups of fish treated with such substances are then compared with a group of untreated fish. Other researchers have focused on understanding how the fish immune system works and how it combats invasion by bacteria and viruses. For this, experiments are undertaken whereby a group of fish is experimentally infected with viral or bacterial pathogens, then at regular intervals, at least 5 fish are killed and analysed. Both methods are very costly in terms of the number of animals used, and we propose to reduce this from the work carried out during this 2 year project. Instead of killing fish at regular intervals, we propose to take small volumes of blood repeatedly during the course of the infection without harming the fish. The number of animals required for this experiment design represents only 20 % of the number of fish required using traditional sampling methods. In addition, following the same fish during the course of the infection will allow a better understanding of the immune response elicited by the fish, and the outcome of this response i.e. death or survival. Part of the project will be dedicated to improving the analysis method. Because only a small volume of blood is repeatedly sampled over the course of the infection, novel methods are required to measure and describe the immune response. Some of the tools to be includes are antibodies, specifically recognise individual immune molecules, information on fish immune genes and the existence of immortal fish cells that can be cultivated in vitro. These methods will be adapted for use with the small volumes of blood collected and will be used to understand which blood cells, and serum molecules are important in combating the pathogen. The relation between the cell types, the molecules involved, the type of pathogen and the final outcome of the infection will be very important in predicting the severity of infection, determining the appropriate immunostimulant to be used and improving existing fish vaccines.

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 i) reduce the number of fish required in experimentation by up to 80 %, and ii) improve the scientific output from infectivity studies. This project will be divided into three work packages WP1, 2 and 3. 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.