Development of in vitro assays to determine vaccine efficacy in fish

Fish farming in the UK has increased dramatically over the last two decades. Scotland currently produces over 130,000 tonnes of Atlantic salmon and is the third largest producer of farmed salmon globally. Around 16,000 tonnes of rainbow trout are also produced in the UK annually. Consumer consumption of farmed fish has trebled in the last ten years, with 1 million salmon meals now eaten each day in the UK. Of various factors that impact on the sustainability of the industry, fish health is key with a number of disease issues of particular concern. To obtain a sustainable aquaculture industry health management is thus necessary and at the heart of this management is the development of new vaccines, which require thorough testing before they can be marketed. This involves large numbers of fish exposed to disease agents, where mortality is used as the end point to test vaccine efficacy. The development of a method that would allow researchers to establish vaccine efficacy more humanely, would significantly reduce the suffering of fish in these trials.
Protection by vaccination relies on the production of specific antibodies and the activation of T cells that mediate specific cell-mediated responses, capable of killing infected host cells and activating non-specific defences. The study of these cellular responses is key to reducing the suffering of fish during vaccine trials. Some T cells (T-helper cells) release mediators called cytokines that initiate and regulate the host responses. Different responses can be generated dependent upon the cytokines released. The measurement of these molecules after vaccination can be correlated with the protection conferred by immunisation, a well established method used with reasonable success in mammals.
In this study we will determine the numbers of cells producing two cytokines (IL-17 and IL-22) in trout, following vaccination against one of two bacterial pathogens (Yersinia ruckeri or Aeromonas salmonicida) and exposure to the homologous or heterologous pathogen. IL-17 and IL-22 were chosen as they are associated with T-helper cell subpopulations responsible for protection against these types of pathogens in mammals. Their production will be assessed as an in vitro correlate of protection, and the results will serve more generally as a proof of principle for fish, with a view to develop a means to undertake a great deal of vaccine optimisation without the need for lethal challenge experiments. In this way this work will help to reduce fish suffering in future vaccine development studies.

Fish farming in the UK has increased dramatically over the last two decades, with salmonid fish the focal point of this development. Of various factors that impact on the sustainability of the industry, fish health is key with a number of disease issues of particular concern. The development of fish vaccines is at the heart of fish management but new vaccines require thorough testing before they can be marketed, and this is currently reliant upon mortality testing where fish are exposed to virulent pathogens and survival is measured. This programme will explore the potential to use an in vitro assay as a correlate of disease resistance following vaccination of fish, using two well known fish Gram negative bacterial pathogens as models, Aeromonas salmonicida the causative agent of Furunculosis and Yersinia ruckeri the causative agent of Enteric Redmouth disease. Thus this proposal aims to significantly reduce the numbers of animals suffering from lethal infection during future vaccine development. On the basis that vaccination relies on the activation of T cells to effect specific cell-mediated responses, we aim to study these cellular responses and specifically develop assays to determine the numbers of cells producing and secreting cytokines (in this case IL-17 and IL-22) associated with Th cell protective responses to extracellular bacterial pathogens. The production of these key cytokines will be studied using two tools, flow cytometry and real-time PCR, which will complement each other and give a clear picture of the magnitude and specificity of the responses elicited in trout given efficacious vaccines and then exposed to the homologous or a heterologous pathogen challenge. Although we are concentrating on bacterial diseases in trout, the data obtained would serve more generally as a proof in principle of this approach for fish. By allowing the measurement of relevant protective immune responses we believe it should be possible to undertake a great deal of vaccine optimisation without the need for lethal challenge experiments using virulent pathogens, and therefore this study will help reduce fish suffering during future vaccine development programmes.