Pharmaq (Norway)

Aquaculture is the fastest-growing animal food-producing sector for human consumption globally. Increasing population size, density, and interconnectivity among farms are expected to markedly increase disease outbreak risks, threatening sector sustainability. Infectious diseases cause around a third of observed mortality in farmed salmon in Norway. Cardiomyopathy syndrome (CMS), caused by piscine myocarditis virus (PMCV), is one of the most frequently detected diseases and often causes high mortality in the late phase of salmon production, compromising fish welfare and causing significant economic losses. Despite its impact, the limited understanding of PMCV dispersal and its drivers hampers effective risk mitigation interventions. To address this, we generated one of the largest genomic datasets for any aquatic viral pathogen, focusing on PMCV collected from Scotland (United Kingdom) and Norway. Combined with detailed metadata, this enabled one of the most comprehensive phylodynamic analyses of a fish virus. Phylodynamic analyses reveal that PMCV likely emerged in farmed salmon concurrent with the global expansion of aquaculture, with a most recent common ancestor around the 1970s-1990s. We identify a distinctive national clustering of virus genomes, reflecting intranational spread interspersed with multiple introductions of distinct PMCV lineages into Scotland. We assess factors influencing virus spread, highlighting the importance of geographic proximity and at-sea farm density, and the possible role of boat connectivity in facilitating long-distance dispersal events. Overall, our study emphasizes the importance of viral genomics in understanding the evolutionary and dispersal dynamics of aquatic pathogens and informs targeted biosecurity strategies to mitigate the growing risk of viral disease in expanding aquaculture systems.

The authors declare no conflicts of interest. The data consist of samples (fry, fingerlings and production-size salmon) submitted by fish health services for histological examination in connection with disease investigation of field cases.

Infestations with salmon lice and subsequent salmon lice management is one of the most challenging and costly aspects of marine salmonid aquaculture. Both the handling and treatment, specifically non-medicinal treatment, against salmon lice cause stress and physical injuries to the host, the Atlantic salmon (Salmo salar). This in turn leads to reduced appetite and increased mortality. In this study, we have estimated the short-term growth loss of Atlantic salmon related to treatments (thermal, mechanical, hydrogen peroxide bath, freshwater bath and combination medicinal baths) for removal of salmon lice. To achieve this, we have obtained daily production data at cage-level from 2014 to 2019 from three large Norwegian aquaculture companies. We have used the registered feed-amount, number of fish and seawater temperature at cage level to calculate the thermal growth coefficient (TGC) of 635 fish-groups the week before a pre-treatment starvation period and the week after 2530 different treatments to estimate the reduction in TGC. We modelled this outcome using a mixed effect linear regression model, with treatment method as the main fixed effect of interest and fish weight, seawater temperature, smolt-age and year-class included as fixed effects. Results showed a period of suboptimal feeding and growth after all treatment methods, where non-medicinal treatment methods had a significantly larger negative effect on growth compared to medicinal treatments. The results also showed that timing of treatment played a role in the outcome of a treatment. The short-term biomass-loss in one cage following one non-medicinal treatment was estimated to 31,200 kg (average cage containing 150,000 fish weighing 3 kg, and seawater temperature of 10 °C). Thus, there could exist a potential for increased production in the Atlantic salmon aquaculture industry by reducing the number of delousing operations.

Since the first description of cardiomyopathy syndrome (CMS) in Atlantic salmon, in 1985, the disease caused by piscine myocarditisvirus (PMCV) has become a common problem in Atlantic salmon farming, not only in Norway, but also in other salmon farming countries like Scotland and Ireland. In the last years, CMS has been ranked as the most important salmon viral disease in Norway regarding both mortality and economic losses. Detailed knowledge of infection and pathogenesis is still lacking, a decade after the causal agent was first described, and there is a need for a wider range of methods/tools for diagnostic and research purposes. In this study, we compared the detection of PMCV‐ and CMS‐related tissue lesions using previously used and well‐known methods like histopathology and real‐time RT‐PCR to immunohistochemistry (IHC), a less used method, and a new method, RNAscope in situ hybridization. Tissue samples of three different cardiac compartments, mid‐kidney and skin/muscle tissue were compared with non‐lethal parallel samplings of blood and mucus. The development of pathological cardiac lesions observed in this experiment was in accordance with previous descriptions of CMS. Our results indicate a viremic phase 10‐ to 20‐day post‐challenge (dpc) preceding the cardiac lesions. In this early phase, virus could also be detected in relatively high amount in mid‐kidney by real‐time RT‐PCR. Plasma and/or mid‐kidney samples may, therefore, be candidates to screen for early‐phase PMCV infection. The RNAscope in situ hybridization method showed higher sensitivity and robustness compared with the immunohistochemistry and may be a valuable support to histopathology in CMS diagnostics, especially in cases of untypical lesions or mixed infections.

Totiviridae is a virus family well known to infect uni-cellular organisms like fungi and protozoa. In more recent years, viruses characterized as toti-like viruses, have been found in primarily arthropods, but also a couple in planarians and piscine species. These toti-like viruses share phylogenetic similarities to totiviruses; however, their genomes also includes additional coding sequences in either 5′ or 3′ ends expected to relate to more advanced infection mechanisms in more advanced hosts. Here, we applied next generation sequencing (NGS) technologies and discovered three new toti-like viruses, one in wild common carp and one in bluegill from the USA and one in farmed lumpsucker from Norway. These are named common carp toti-like virus 1 (CCTLV-1), bluegill toti-like virus 1 (BGTLV-1), and Cyclopterus lumpus toti-like virus (CLuTLV), respectively. The genomes of these viruses have been characterized and compared to the three previously known piscine toti-like viruses, piscine myocarditis virus (PMCV) found in Atlantic salmon and the two from golden shiner, now named golden shiner toti-like virus 1 and 2 (GSTLV-1 and -2), and also to totiviruses and other toti-like viruses. We found that four piscine toti-like viruses had additional gene(s) in the 3′ end of the genome, and also clustered phylogenetically based on both capsid and RdRp-genes. This cluster constituted a distant branch in the Totiviridae, and we suggest this should be defined as a separate genus named Pistolvirus, to reflect this major cluster of piscine toti-like viruses. The remaining two piscine toti-like viruses differentiated from these by lacking any additional 3′ end genes and also by phylogenetical relation, but were both clustering with arthropod viruses in two different clusters.

We report the development of a multigene gene expression assay on the BioMark HD platform for the evaluation of immune competence (ImCom) in farmed Atlantic salmon. The first version of the assay included 92 genes selected on the basis of transcriptome analyses in 54 trials that challenged the immune system; annotations were taken into account to represent the key pathways of innate and adaptive immunity. ImCom was tested on samples collected from seven independent projects. Fish were reared from the start feeding to eight months in the sea at eight units in different parts of Norway. Several tissues were analyzed. Linear discriminant analysis (LDA) showed that no more than 10 genes were required to separate groups, and a set of 46 immune genes was sufficient for any task. The second version of the assay was tested in the gills of two groups of high-performing healthy smolts and in groups with intermediate and high mortality rates (IM and HM, respectively). A set of 645 gill samples from clinically healthy Atlantic salmon was used as a reference. The IM group showed general suppression of immunity. All HM group salmon were above the threshold by the squared deviation from the reference. This group showed marked upregulation of genes involved in acute stress and inflammation: mmp-9, mmp-13, hsp70, il-1b, lect2, and cathelicidin. Further work will clarify the boundaries of the norm and explore various cases of impaired immunity.

Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (~10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish.

The Norwegian salmonid alphavirus (NSAV) infects farmed Atlantic salmon and rainbow trout, causes pancreas disease and leads to economic losses and fish health issues for the aquaculture. Vaccines are available, but recurring infection outbreaks at Norwegian fish farms have led to endeavours in finding solutions for increased prevention. The NSAV E1 and E2 envelope proteins are potential targets for production of recombinant subunit vaccines and for generation of antibodies for diagnostics. Efficient expression of target proteins is necessary for these applications, and here we present a new strategy for expressing this kinds of viral proteins. We show that 5′-terminal fusion of signal sequences OmpA and CSP to the e1 and e2 genes and removal of the C-terminal hydrophobic interaction and transmembrane domains of E1 and E2 leads to significantly increased expression levels. Recombinant Escherichia coli strains for high-level production of E1 and E2 harbouring these modifications were established using the inducible XylS/Pm expression cassette. Furthermore, reduction of temperature to 16°C after induction leads to 4-fold increase in production for E1, and under high-cell-density cultivations we obtained production levels up to 2.3 g/L. We also show that these proteins can be purified from inclusion bodies by affinity chromatography. This demonstrates the present approach as promising for large scale production of such viral proteins.

Cardiomyopathy syndrome (CMS) in Atlantic salmon, Salmo salar L., is a severe cardiac disease characterized by a necrotizing myocarditis involving the atrium and the spongious part of the ventricle. The disease is caused by piscine myocarditis virus (PMCV), a double-stranded RNA virus likely belonging to the family Totiviridae. The objective of this study was to evaluate the genetic variation in Norwegian PMCV isolates focusing on the putative structural proteins encoded by open reading frames (ORFs) 1 and 3. The virus isolates were sampled from a total of 36 farms along the Norwegian coastline. This study represents the first investigation of PMCV genome variation and shows that Norwegian isolates are highly similar, with the most divergent isolates sharing 98.6% nucleotide identity. Interestingly, amino acid sequence diversity within ORF3 is approximately threefold higher than for ORF1. While phylogenetic analysis based on concatenated nucleotide data covering ORF1 and ORF3 revealed four main clusters, the maximum sequence variation of 1.4% at the nucleotide level suggests that all Norwegian isolates belong to a single genogroup. Substantial sequence variation within farms was also observed, which may complicate future molecular epidemiological investigations. The genetic homogeneity among the Norwegian isolates might facilitate development of both diagnostic tools and an efficient vaccine against CMS in the future.