Genetics of multi-pathogen resistance in Atlantic salmon

Summary single pathogen challenge models salmonid alphavirus 3 (SAV-3) Chinook salmon embryonic cells (CHSE-214) were used to propagate SAV-3 virus (GenBank: JQ799139). Approximately 70 healthy Atlantic salmon pre-smolts, weighing 35 ± 10 g, originated from Sørsmolts AS and were transported to the wetlab. After a week of acclimatization and formalin treatment against ectoparasites, the experiment began. The fish were injected intramuscularly with 0.1 mL of virus (1 × 10^6 TCID50/mL). A control group of 15 fish, injected with L-15 medium, was kept separately. At 2, 4, and 8 weeks post-infection, samples were collected from SAV-3 injected fish and controls, targeting tissues like the heart, pancreas, and muscle. Formalin-fixed tissues were used for histopathology, while RNAlater-preserved samples were stored at -20°C for estimation of the viral genome in the heart. Standard histology methods, including paraffin embedding and H&E staining, were followed. RNA was isolated using the RNeasy kit, and viral genome was detected using a commercial kit (AAT, Taiwan). Results Salmon challenged with SAV showed high viral replication in heart, starting from 2 weeks post-challenge, peaking at 4 weeks and declining by 8 wpc. Histopathological changes were evident from 2 wpc, peaking at 4 and with initial regeneration in the pancreas by 8 wpc. A challenge model for SAV3 in Atlantic salmon has been established with a defined challenge dose and time course. Challenge study with PMCV (piscine myocarditis virus) The PMCV inoculum, originating from an Atlantic salmon CMS outbreak, was prepared by homogenizing heart tissue, centrifuging to remove debris, and aliquoting the supernatant into 2 mL tubes, which were stored at -80°C. Fish were injected intraperitoneally with a 1:500 dilution, after undergoing anesthesia, with random allocation to tanks. Results Heart and kidney samples for histopathology and PCR were collected from 24 fish at each time post-challenge, 4, 6, 8, and 12 weeks. At each sampling, blood samples were taken, followed by a dissection of the head kidney for qPCR analysis, and heart samples were taken for histopathology and qPCR analysis. Histopathology samples were fixed in formalin, paraffin-embedded, sectioned at 2-3 µm, and stained with H&E. Histopathological scoring was done on atrium and ventricle using a 0-4 scale. Inflammatory foci in the heart were scored on a scale from 0 to 4. Heart changes are found at 4 wpc, increasing by 6 wpc, with a moderate increase from 6 to 8 wpc, peaking at 12 wpc. At the final collection time, 95% of the fish had scores >0, with an average of 2.6 for atrium and ventricle. A challenge model for PMCV in Atlantic salmon has been established with defined challenge dose and time course. Sea lice challenge A trial was conducted using Atlantic salmon, 3 parallel tanks, 25 fish in each tank, water temperature of 10-11 °C. The challenge was done using copepodids obtained from Industry Laboratory, (ILAB), and fish were exposed to 30 copepodids per fish for 30 minutes in stagnant water while maintaining oxygen saturation above 80%. This was done by flushing oxygen into the tanks to maintain oxygen saturation. Water flow was resumed after the exposure period of 30 minutes. Fish were monitored daily, and first lice counts were performed on 9 randomly selected fish (3 from each tank) 11 days post-challenge (dpc). The lice on the remaining fish were counted at 30 dpc, where different developmental stages of lice were determined. The infection level was 66-67% with little variation between tanks, and with a relatively uniform development of lice, chalimus II – preadult, and an even distribution of sexes. The challenge was repeated twice, and the data was comparable between the experiments. A challenge method has been established for sea lice with consistent outcomes of infection level and development stages. AGD samples from natural outbreak Mucus swabs from the gills of 201 fish during a natural AGD outbreak were collected for 16S rRNA microbiome sequencing. The fish were also scored for gill condition on both the left and right sides. The sequence reads were processed using the DADA2 pipeline, with truncation lengths set at 240bp for forward reads and 220bp for reverse reads. Only amplicon sequencing variants (ASVs) within the expected length range of 400 to 410bp were retained. After the taxonomy assignment, ASVs classified as chloroplasts and mitochondria were removed. Results: We observed no significant differences between gill scores and the abundance or variation of microbes. This could be attributed to data loss and an uneven distribution of data across gill scores, which reduced the statistical power, or it might indicate that the microbial community does not influence the gill scores measured. Additionally, we specifically examined bacteria known to impact gill health from other studies, but these also showed no significant differences across gill score categories.

In aquaculture, disease resistance plays an important role in the overall sustainability and profitability of the industry. Resistance to specific diseases, evaluated based on survival under a challenge test with a specific pathogen, is part of the breeding objectives for many Atlantic salmon breeding programs. However, when there is an outbreak, it is common for fish to be infected by multiple pathogens simultaneously, and genetic correlations of resistance to different diseases are low to none. Selective breeding to increase host resistance to multiple disease pathogens in interaction with each other will be very important for mitigating the impact of diseases. Furthermore, the current disease challenge models are designed with a high dose of pathogens to achieve at least a 50% mortality rate. This is far from what the fish experience in the production environment. The project will develop and optimize a multi-pathogen challenge test for CMS, PD, AGD, and Sea lice. Further, the proposal will study the genetics of multiple disease resistance in salmon and determine both specific resistance to one disease when other pathogens are present and the general coping of each fish with a complex pathogen load. This paves the way for the development of coinfection-resistant fish through genetic improvement. The mechanisms and gene markers of resistance to multiple infections will be studied by gene expression analyses. The ethical and social implications of establishing and implementing a multi-pathogen challenge test will be determined through a dynamic RRI (responsible research and innovation) process. The project will create a knowledge base and example for the industry regarding mitigation of the impact of disease through genetic improvement for coinfection resistance, this will greatly improve the bio-economy and welfare of Atlantic salmon production.