Fish handling and injection-based vaccination is one of the biggest causes of stress for fish and always results in increased mortality due to post-vaccination outbreaks of saprolegniosis and bacterial infections. In Scotland alone, millions of pre-smolt (10% of all hatched salmon) die due to Saprolegnia infections after receiving an injection-based vaccine. Therefore, there is an urgent need to develop better vaccination practices to increase animal welfare and reduce mortalities. Immersion vaccination of fish was the first method used to demonstrate effective protection against bacterial diseases, but was overtaken by injection vaccination due to the need for inclusion of vaccine adjuvants. Should enhanced delivery of antigens be possible by the immersion route clearly this holds enormous promise to completely obviate fish handling, thus reducing stress and significantly lowering mortality. The central hypothesis of our programme is that immersion vaccination with recombinant SpHtp1-coupled antigens can be used successfully to protect fish against diseases. Our overall objective is to develop and commercialise the SpHtp1 translocation mechanism into an immersion vaccine delivery method for salmonids and other farmed fish. We have already demonstrated that our patented method can be used successfully and effectively in directing proteins into live fish. Furthermore, we have observed specific antibody production in trout that were immersed in a solution of SpHtp1 fused to mRFP. Therefore, our main outputs will be: 1) Evidence that the translocating peptide of SpHtp1 coupled to selected antigens give protection against disease of concern in salmonid aquaculture, in immersion vaccination trial experiments. 2) Optimisation of the immune response in fish. 3) A highly efficient and productive SpHtp1-recombinant protein overexpression method for large-scale production and future commercialisation of the vaccine method by our commercial partners BAHL and Pulcea Ltd. 4) A method to recycle and inactivate the immersion vaccine that can be employed on site in the farm. All outputs will be exploited by BAHL and Pulcea Ltd. in obtaining licenses and approvals for commercialisation and eventually full use by the aquaculture industry. Antigens of essential pathogens are already available through BAHL, and further antigens will be sought in future projects by the UoA and BAHL. The current application is ambitious but achievable and will take 24 months to complete because of the significant amount of work that is proposed. In summary, the potential impact of an immersion vaccine for finfish is "aquaculture industry-transformational" as fish vaccinated by injection could be eliminated, which would greatly increase the welfare of the fish and minimise fish losses and thus financial losses in the aquaculture business worldwide.

Vaccines for chronic viral pathogens in salmon- generation of interferon attenuated cell lines. Acroym: SalVacCell Project goal / key aims. There is a major requirement to generate new vaccines for viral pathogens in the salmon aquaculture industry. Viruses represent one of the major economic losses to the salmon industry, which is a direct reflection of the lack of highly protective vaccines. In order to improve vaccine design and testing high quantities of viruses are required, but at present this is not possible. This project will use cell lines that have been edited by CRISPR/cas9 to be highly permissive for growth of difficult to grow viruses. We will knockout the type I interferon pathway which is the major antiviral mechanism in animals. Specifically we will target key genes that regulate interferon induced cellular responses to viral infection. These cell lines will additionally be used to explain the underlying function of genes associated with natural viral resistance / susceptibility in salmon that is of direct importance to breeding programs. We have secured Industrial partners Benchmark (vaccine company) and Landcatch (fish breeding). Consortium agreements will clearly define roles and confidentiality of IP. OBJECTIVES 1. Development of IFN-deficient fish cell lines by knock-out of IFN function 2. Evaluation KO cell lines to mount an antiviral response to confirm phenotype 3. Use the cell lines to explain naturally occurring resistance / susceptibility to fish viruses 4. Comparison of yield for viral particle production and viral diagnostic turn-over time between traditional cell lines and newly developed cell lines KEY CHALLENGES The overarching challenge is to be able to produce high titres of virus for vaccine companies to be able to improve design and protection to economically important chronic viral pathogens. Many labs have attempted to knock down genes in salmonid cells, but to date this has not been achieved. To our knowledge we are the first to create such cell lines and as such our unfunded preliminary work has already overcome a major hurdle. The challenge of this project is to fully exploit our cell line technology for both disease management and also to greatly improve the basis of genetic selection. Deliverables- to be used by partners a. Engineered cell lines that are deficient in antiviral responses. b. Cells lines that can be used to produce high titre of viruses for vaccines c. Capacity to upscale viral production in industrial environment. d. Improved precision of selection for disease resistance in salmon breeding Project duration: 24 months Total project cost: £354622 Contribution requested from (100%) BBSRC/NERC £249,622 (£199697.60 80%) Contribution from MSS: secured £65K Contribution from Benchmark secured £20K Contribution from Landcatch secured £20K

Farmed salmon is a major source of high quality protein and fatty acids essential for human health. Salmon aquaculture is worth approximately £1Bn to the UK economy, and supports many rural and coastal communities. However, disease outbreaks have a major negative effect on salmon production and animal welfare. Infectious salmon anaemia (ISA) is one such disease, and is sometimes dubbed 'salmon flu' because it is caused by a virus (ISAV) that is similar in to influenza. At present, ISA is a notifiable disease in the UK, meaning farmers are obliged to cull their stock in the event of an outbreak. Vaccination and biosecurity cannot fully prevent outbreaks, and developing disease resistant strains of salmon is high priority. Selective breeding can result in moderate improvements in disease resistance of salmon stocks and may take many generations. However, a revolutionary approach known as genome editing has potential to rapidly increase the rate at which disease resistant salmon can be produced. Genome editing involves the use of "gene scissors" to precisely cut the genome at a specific location, leading to small-scale targeted changes in the DNA sequence. In this proposal, genome editing technology will be used to investigate genes underlying resistance to ISAV, and potentially to produce a disease-resistant salmon. The first stage of the project is to identify target genes that will be edited. This will be achieved by measuring the ISAV resistance in a selective breeding program. Genetic markers dispersed throughout the salmon genome will then be used to map individual genes that contribute to variation in resistance in the population. Salmon from resistant and susceptible families will also be sequenced and to identify candidate genes and mutations causing this genetic effect on resistance to ISAV. In parallel to the 'forward genetic' approach described above, a 'reverse genetic' approach to identifying ISAV resistance candidates will be employed using cell culture models. A genome editing method known as CRISPR-Cas9 will be applied to destroy the function of key candidate ISAV resistance genes in the cell lines. Two methods of choosing candidate genes will be used. The first is based on prior knowledge of the biology of the interaction between the virus and the host cell, partly harnessing extensive research which has been performed on ISAV's close relative influenza. The second is to use the genes affecting natural resistance identified in the forward genetic screen described above. These edited cell lines will be infected with ISAV, and the impact of the edited gene on ISAV resistance and cellular response to infection will be assessed. This will build on an ongoing project to develop genome editing for salmon cell lines. Finally, genome editing will be used in Atlantic salmon embryos to test the highest priority ISAV resistance genes, especially where knockout of the gene has an impact on resistance in cell culture. Targeted editing of the genes will be performed by microinjecting newly fertilised embryos, which will be reared until the freshwater fry stage. These edited embryos, and unedited controls from the same family, will be challenged with ISAV. The nature and frequency of the edited genes in the resistant and susceptible salmon will be measured. This proposal has potential to create Atlantic salmon with resistance to a problematic viral disease (ISA) using a novel breeding technology. As such, it could have major animal welfare and economic impacts via prevention of outbreaks and subsequent culling of stocks. The approaches will be directly relevant to other viral disease in fish aquaculture. While the regulatory landscape for application of edited animals in food production is uncertain, a successful outcome of this proposal will provide a high profile example of the power of this technology to understand biology and to improve food security and animal health.

Farmed salmon is a major source of high quality protein and fatty acids essential for human health. Salmon aquaculture is worth approximately £1Bn to the UK economy, and supports many rural and coastal communities. However, sea lice are a major perennial problem for salmon aquaculture worldwide. These parasites attach to the skin of salmon and feed on tissue, mucus and blood. Infected fish show impaired growth and increased occurrence of secondary infections. They cause significant negative impacts on salmonid health and welfare, while lice prevention and treatment costs are a large economic burden for salmon farming, over £800M per annum. Encouragingly there is substantial genetic variation in resistance to sea lice both within and across salmonid species. While the commonly farmed Atlantic salmon are generally susceptible to infection, other salmonid species such as coho salmon are fully resistant. Improving the innate genetic resistance of the farmed salmon to sea lice is an environmentally friendly, but underexploited approach to lice control. Incremental improvements have been achieved via selective breeding of Atlantic salmon, but their long generation interval slows progress. Genome editing raises the possibility of rapidly increasing the resistance of salmon via precise targeted changes to their genomes; the key is knowing which specific genes to target. This project focusses on understanding the genetic mechanisms underlying resistance to sea lice, and identifying gene targets for genome editing to develop lice-resistant Atlantic salmon. To identify target lice resistance genes for editing, several different approaches will be taken, each exploiting the latest genomic technologies. Firstly, whole genome sequences will be obtained from a large population of farmed salmon on which sea lice counts following challenge have been collected. These will be used to map individual genes that contribute to variation in resistance in the commercial Atlantic salmon population. Secondly, it is known that the mechanisms underlying resistance to sea lice are due to a successful localised immune response close to the attachment site of the louse. Therefore, a detailed gene expression comparison of the immune response of Atlantic and coho salmon in the first four days following a lice challenge will be undertaken, using single cell sequencing approaches to highlight different responses in distinct cell populations at louse attachment sites. This will be complemented by profiling of the gene expression of the lice, and identification of potential immunomodulatory proteins and their targets in the host. Thirdly, genome editing approaches will be used to assess the impact of perturbing candidate resistance genes on response to sea lice both in cell culture and in the fish themselves. The former will be used to assess the cellular response to proteins secreted from the sea lice, and the consequences of knocking out each of the target genes on that response. This will lead to a final set of target genes for editing in salmon embryos, after which the edited fish will be challenged with sea lice. The resistance of the edited fish compared to full sibling control fish will then be assessed. The scientific programme of the project will be complemented by co-development of a strategy for the breeding and dissemination of edited lice resistant salmon, together with industrial partner Benchmark PLC. Furthermore, public and stakeholder engagement events are planned to communicate the research plans and outcomes, with a particular focus on the benefits and risks of genome editing in aquaculture. A successful outcome of lice resistant salmon would have major animal welfare and economic impacts via prevention of outbreaks, and removal of the need for chemical treatments. It would also provide a high profile example of the power of genome editing technology to understand biology and to improve food security and animal health.

Fish diseases are a huge threat for the aquaculture industry and for global food security. Some of the most important disease-causing organisms in aquaculture are part of the oomycetes or watermoulds, in particular Saprolegnia parasitica, Saprolegnia diclina and Saprolegnia australis are causing serious fish losses. Collectively, these fungal-like organisms are responsible for at least 10% annual mortalities in most salmon hatcheries and freshwater sites. Consequently, Saprolegnia ranks among the most important pathogens of Atlantic salmon. Unfortunately, over the last few years the incidences of saprolegniosis outbreaks in Scottish farms have significantly increased. Indeed, some sites have had very high losses due to saprolegniosis. Whereas other farms have remained largely disease free. The reasons as to why some farms are badly affected and others seem to avoid disease outbreaks, with apparent identical welfare standards and husbandry management practises, are at present completely unclear and form the main rational for the current application. Our hypothesis is that several risk factors (pertaining to fish, pathogens and the environment) are playing a synergistic role in suppressing immunity in fish towards Saprolegnia, which lead to outbreaks of saprolegniosis. Therefore, we propose a concerted industry-wide, industry-led and industry-supported research programme to discover, map, model and understand the main drivers, risk factors, that allow saprolegniosis outbreaks. A "big data" resource will be created that will be scrutinised with statistical methods to identify the main risk factors and conditions for outbreaks of saprolegniosis. Undoubtedly, identifying the main, or a combination of, risk factors will greatly aid the salmon aquaculture industry to pre-empt any future outbreaks and would lead to an integrated approach to saprolegniosis management, which would result in increased welfare standards, improved fish health, fewer losses and a reduction in production and treatment costs.