Infectious Salmon Anemia (ISA) is a viral infectious disease with potentially major economic burden on the Norwegian salmon industry. This is a disease that devastated the salmon farming in the Faroe Islands and in Chile during 2003 and 2008 respectively and it is raising major concerns across Norway, as over the past few years farmers have witnessed a significant increase in the number of its outbreaks throughout the country. In this project, using a multidisciplinary approach, we combine genomic information with new methods to detect and quantify viral load and immune response in heart and skin mucus to develop new phenotypes for ISA robustness. This aims to complement and enhance the current practices for identifying and selecting the best candidates while breeding against ISA virus. A challenge test was performed in 2018 and repeated in 2019, using 2000 fish from 15 families. In addition to record mortality, heart samples were taken from the survivors and skin mucus samples from random fish 3 and 6 days post infection and from the survivors. A third of the fish were vaccinated.
The mucus and heart samples were analysed by PCR to estimate genetic variation for viral load in these tissues. Mucus samples from before and after infection, from vaccinated and unvaccinated fish, have been analysed to study immune response. All fish were also genotyped. Quantitative genetic analyses revealed significant genetic variation in survival and heart viral load for unvaccinated fish, but lower genetic variation for the same traits in vaccinated fish. Correlations between same trait in vaccinated and unvaccinated fish were low, indicating different genes to affect resistance in vaccinated and unvaccinated fish. A marker-trait association study to search for single genes with major effect on resistance to ISA, identified no such genes.
Methods for analyzing metabolites in mucus (i.e. metabolomics) have been established and optimized, including the implementation of a new kit (AbsoluteIDQ p400 HR, Biocrates, Austria) for so-called targeted analysis of 408 metabolites. The mucus samples from the 2018 trial have been analyzed for their metabolite content, partly with a 'targeted' method and partly with two 'untargeted' methods (normal and reverse phase mass-spectrometric chromatography). The results of the targeted assay show that acylcarnitine and carnitine may be (a) metabolite marker(s) that differentiate between the experimental groups. The mass-spectrometric analysis of the proteome in selected mucus samples illustrated the need for improved handling and preparation of samples from exposure studies in salmon. As a result, novel purification methodologies were explored. Immunological methodology for the analysis of ILA-specific antibodies in blood and mucus samples has been improved. The ELISA methodology published in the literature was found to provide significant background/noise and a substantially optimized method has therefore been developed based on purified viral protein fractions. Development of a similar immunological method using fluorescent beads and BioPlex instrumentation has been initiated. This method is expected to increase sensitivity and thus facilitate analysis of mucus samples.
We use RNA sequencing to identify molecular signatures that can serve as new phenotype(s) while selecting for increased resistance to ISA. To do so, we first compared the gene expression profiles of resistant animals versus susceptible fish. The comparative analyses were performed based on the expression of thousands of genes, identified in the heart and the mucus. The first important discovery was the finding that the more susceptible fish shed more virus to the environment. This finding has important implications for animal welfare and also for helping to reduce the number of ISA outbreaks. Secondly, we found irrespective of the tissue investigated, either heart or mucus, a clear difference in resistant and susceptible fish gene expression patterns. We further selected several of these markers, developed assays, and tested them on many challenged fish. All the assays could clearly distinguish between resistant and susceptible animals. We have further tested the assays following a field outbreak, where we screened hundreds of fish. Same as before, we could differentiate between the two groups.
In 2019, an epidemiology trial was conducted, where naïve fish were distributed to 18 tanks and infected by diseased fish with varying resistance against ISA. This study is designed to show if infectivity is affected by resistance. It was shown that both vaccination and selection for resistance reduced ISAV transmission, but neither of these prevented ISAV transmission. Genetic selection seemed to be more powerful than vaccination in reducing transmission, but this difference was not statistically significant.
The project has provided new insight into immune responses and infectivity of ISAV infection and identified useful biomarkers for existing and new resistance traits.
Benchmark Genetics has already implemented some of the key findings of this study in their breeding program. In particular, by utilising the developed assays, they are now screening the immune response of fish following ISAV infection.
Infectious diseases continue to pose major problems for aquaculture in Norway, resulting in large economic losses and negatively impacting animal welfare and their living habitats. Selective breeding to enhance pathogen resistance has been a successful strategy, although for some diseases, such as chronic or subclinical infections, the best selection criteria or phenotypes are yet to be defined. Recent developments of various genomic resources in Atlantic salmon has now provided us with the opportunity to exploit these advances to both improve our understanding of the underlying genetic basis of disease resistance, and to provide tools to more accurately and efficiently select them in breeding programs. In this application, using a multidisciplinary approach, we aim to use these advances to complement and enhance the current practices for identifying and selecting the best candidates while breeding against infectious salmon anaemia, a deadly contagious disease of farmed Atlantic salmon. We expect selection based on the newly defined phenotype(s) and the tools and methodologies that will be developed for measuring them, to provide the supporting industry partner, SalmoBreed, with a highly valued product and the underlying idea to further be applied to many other diseases of economic importance.