Search

Finfish aquaculture is one of the fastest-growing food production sectors in the world, and numerous infectious diseases are a constant challenge to the fish farming industry, causing decreased fish health and, consequently, economic losses. Specific and sensitive tools for pathogen detection are crucial for the surveillance of environmental samples to prevent the spread of fish pathogens in farms. Monitoring of waterborne pathogens through filtration of water and subsequent molecular detection of target-specific DNA or RNA sequence motifs is an animal-friendly method. This approach could reduce or even replace the sacrifice of fish for monitoring purposes in aquaculture and allow earlier implementation of disease control measures. Sampling methods might be a bottleneck, and there is a need for simple sampling methods that still ensure the best detection probability. In this study, we tested different filtration methods with spiked freshwater and seawater for a panel of fish pathogens to discern a suitable procedure that can be easily applied on-site by farm personnel without compromising detection probability. Specifically, we tested combinations of different filtration flow rates, lysis buffers, and filters for the detection of some of the pathogens relevant to the aquaculture industry. The results showed that a "sandwich" filtration method using two different filters and a flow rate of up to 4.0 L/min ensured good pathogen detection. The filters, consisting of a hydrophilic glass fibre filter with binder resin on the top and a hydrophilic mixed cellulose esters membrane at the bottom, achieved the best concentration and qPCR detection of both viral and bacterial fish pathogens. This up-and-coming tool allows the detection of very different fish pathogens during a single filtration step, and it can be combined with one single automated total nucleic acid extraction step for all the investigated pathogens, reducing both analysis costs and time., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Benedicenti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aquaculture sludge (uneaten feed and faeces) is nutrient rich and has potential as feed for insects. The aim of this study was to investigate the transfer of chemical and biological contaminants, as well as nutrients, from aquaculture sludge to black soldier fly larvae. The larvae were reared on a sludge mixture made of different sludges collected from Norwegian freshwater salmonid facilities. The sludge was spiked with four common salmon pathogens: Infectious Pancreatic Necrosis Virus, Infectious Salmon Anemia virus, Yersinia ruckeri or Mycobacterium salmoniphilum. During the 15 days of growth on sludge, the black soldier fly larvae accumulated valuable nutrients including protein, fat, eicosapentaenoic acid, iron, manganese, zinc and selenium. The larvae also accumulated undesirable substances including cadmium, mercury, dioxins and polychlorinated biphenyls. The concentrations of dioxins exceeded the EU maximum level set for animal feed. None of the salmon pathogens that were spiked to the sludge were detected in the black soldier fly larvae. This study reports low risk of transfer of salmon pathogens from sludge to insect larvae, and showed that the transfer of heavy metals, minerals and metalloids are in accordance with earlier studies. The large variations in levels of heavy metals between batches of sludge can cause levels in BSF exceeding the EU maximum levels, and thus indicate a need for monitoring of the proposed value chain. The transfer of dioxins from sludge to insects, reported for the first time in this paper, would be of special interest for future research, with special focus on risk mitigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Side streams from aquaculture production such as fish sludge poses ample opportunities for biological upcycling, as the sludge contains high amounts of nutrients, energy and valuable biochemicals, making it an ideal food for extractive species. Sludge has been proposed as a feed stock for polychaete production, which in turn can be utilized live in shrimp aquaculture or as an aquafeed ingredient. However, the biosafety of such value chains has not yet been addressed. We conducted an experiment exposing the polychaete Hediste diversicolor to aquaculture sludge spiked with four different fish pathogens (Mycobacterium salmoniphilum, Yersinia ruckeri, Infectious Pancreatic Necrosis (IPN) and Infectious Salmon Anaemia (ISA)) known to cause diseases in Atlantic salmon (Salmo salar L.). Moreover, we assessed whether heavy metals and other potentially hazardous elements present in fish sludge bioaccumulates in the polychaetes. Neither of the bacteria nor viruses could be detected in the polychaetes after 14 days of continuous exposure. Seven of the 15 elements we analysed showed bioaccumulation factors significantly below one, meaning biodilution, while the other eight did not differ from one, meaning no bioaccumulation. None of the elements showed a significant bioaccumulation. Further on, none of the heavy metals found in the polychaetes at the end of our experiment exceeded the EU regulatory maximum levels for fish feed ingredients. The current results suggest that a H. diversicolor can reared on aquaculture sludge, and aquaculture sludge may serve as feed stock for polychaete production without the product exceeding EU regulations for contaminants in animal feed., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Arne M. Malzahn reports financial support was provided by Norwegian Seafood Research Fund. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A total of 47 fish sludge samples from commercial land-based Atlantic salmon (Salmo salar) farms in Norway were assessed for their nutrient composition, presence of various legacy contaminants and a wide spectrum of contaminants of emerging concern, veterinary medicines as well as selected salmonid pathogenic bacteria and virus. The aim was to document the levels of desirable and undesirable components in fish sludge in relation to a potential future use of sludge as invertebrate feed. The samples had variable, but relatively high protein and fat contents, indicating a high load of undigested feed in some of the sludge samples. Fatty acid analysis showed the presence of essential omega-3 fatty acids. In terms of undesirable substances, 43% and 84% of the sludge samples contained levels of arsenic and cadmium, respectively, which exceeded the EU Maximum Levels established for complete animal feed. The concentrations of copper, zinc, iron and aluminum were highly variable in the sludge samples. The concentrations of dioxins, sum PCB6, and chlorinated pesticides were all below the Maximum Levels for animal feed. Of the 18 per- and polyfluoroalkyl substances (PFAS) only one compound (L-PFOS) was present at measurable levels. None of the samples had detectable levels of veterinary medicines, salmonid virus or bacteria. Performing a suspect and non-target screening of the sludge samples identified 18 compounds, including four pharmaceuticals, plastic-related products and the UV filter benzophenone, warranting further investigations. Overall, the results from this study show that fish sludge is a nutrient-rich resource; however, undesirable substances, originating from the feed or from treatment of sludge may be present., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Veronika Sele reports financial support was provided by Norwegian Seafood Research Fund. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Salmonid alphavirus (SAV) causes pancreas disease (PD), which negatively impacts farmed Atlantic salmon. In this study, fish were vaccinated with a DNA-PD vaccine (DNA-PD) and an oil-adjuvanted, inactivated whole virus PD vaccine (Oil-PD). Controls were two non-PD vaccinated groups. Fish were kept in one tank and challenged by cohabitation with SAV genotype 2 in seawater. Protection against infection and mortality was assessed for 84 days (Efficacy study). Nineteen days post challenge (dpc), subgroups of fish from all treatment groups were transferred to separate tanks and cohabited with naïve fish (Transmission study 1) or fish vaccinated with a homologous vaccine (Transmission study 2), to evaluate virus transmission for 26 days (47 dpc). Viremia, heart RT-qPCR and histopathological scoring of key organs affected by PD were used to measure infection levels. RT-droplet digital PCR quantified shedding of SAV into water for transmission studies. The Efficacy study showed that PD associated growth-loss was significantly lower and clearance of SAV2 RNA significantly higher in the PD-DNA group compared to the other groups. The PD-DNA group had milder lesions in the heart and muscle. Cumulative mortality post challenge was low and not different between groups, but the DNA-PD group had delayed time-to-death. In Transmission study 1, the lowest water levels of SAV RNA were measured in the tanks containing the DNA-PD group at 21 and 34 dpc. Despite this, and irrespective of the treatment group, SAV2 was effectively transmitted to the naïve fish during 26-day cohabitation. At 47 dpc, the SAV RNA concentrations in the water were lower in all tanks compared to 34 dpc. In Transmission study 2, none of the DNA-PD immunized cohabitants residing with DNA-PD-vaccinated, pre-challenged fish got infected. In contrast, Oil-PD immunized cohabitants residing with Oil-PD-vaccinated, pre-challenged fish, showed infection levels similar to the naïve cohabitants in Transmission study 1. The results demonstrate that the DNA-PD vaccine may curb the spread of SAV infection as the DNA-PD vaccinated, SAV2 exposed fish, did not spread the infection to cohabiting DNA-PD vaccinated fish. This signifies that herd immunity may be achieved by the DNA-PD vaccine, a valuable tool to control the PD epizootic in farmed Atlantic salmon., Competing Interests: Author RT was employed by company Elanco Animal Health. Author AR was employed by company VESO Aqualab. Author JCW was employed by company Experimental Pathology Laboratories Inc. Author JFR was employed by company Elanco Canada Ltd. Author ES provided the statistical analysis of all the study data the capacity of a private consultant. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Thorarinsson, Ramstad, Wolf, Sindre, Skjerve, Rimstad, Evensen and Rodriguez.)

Bacterial kidney disease (BKD) was assessed according to the criteria of the Animal Health Law (AHL), in particular the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as laid out in Article 9 and Article 8 for listing animal species related to BKD. The assessment was performed following the ad hoc method on data collection and assessment developed by AHAW Panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to this assessment, BKD can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (66-90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that BKD does not meet the criteria in Sections 1, 2 and 3 (Categories A, B and C; 1-5%, 33-66% and 33-66% probability of meeting the criteria, respectively) but meets the criteria in Sections 4 and 5 (Categories D and E; 66-90% and 66-90% probability of meeting the criteria, respectively). The animal species to be listed for BKD according to Article 8 criteria are provided., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Infection with Gyrodactylus salaris was assessed according to the criteria of the Animal Health Law (AHL), in particular, the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as laid down in Article 9 and Article 8 for listing animal species related to infection with G. salaris . The assessment was performed following the ad hoc method for data collection and assessment previously developed by AHAW panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment here performed, it is uncertain whether infection with G. salaris can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (33-70% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that Infection with G. salaris does not meet the criteria in Section 1 and 3 (Category A and C; 1-5% and 10-33% probability of fulfilling the criteria, respectively) and it is uncertain whether it meets the criteria in Sections 2, 4 and 5 (Categories B, D and E; 33-80%, 33-66% and 33-80% probability of meeting the criteria, respectively). The animal species to be listed for infection with G. salaris according to Article 8 criteria are provided., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Spring Viraemia of Carp (SVC) was assessed according to the criteria of the Animal Health Law (AHL), in particular the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as in Article 9 and Article 8 for listing animal species related to SVC. The assessment was performed following the ad hoc method for data collection and assessment previously developed by the AHAW panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment performed here, it is uncertain whether SVC can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (45-90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that SVC does not meet the criteria in Section 1 (Category A; 5-33% probability of meeting the criteria) and it is uncertain whether it meets the criteria in Sections 2, 3, 4 and 5 (Categories B, C, D and E; 33-66%, 10-66%, 45-90% and 45-90% probability of meeting the criteria, respectively). The animal species to be listed for SVC according to Article 8 criteria are provided., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Infection with salmonid alphavirus (SAV) was assessed according to the criteria of the Animal Health Law (AHL), in particular the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as laid out in Article 9 and Article 8 for listing animal species related to infection with SAV. The assessment was performed following the ad hoc method on data collection and assessment developed by AHAW Panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment, it was uncertain whether infection with salmonid alphavirus can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (50-80% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that infection with salmonid alphavirus does not meet the criteria in Section 1 (Category A; 5-10% probability of meeting the criteria) and it is uncertain whether it meets the criteria in Sections 2, 3, 4 and 5 (Categories B, C, D and E; 50-90%, probability of meeting the criteria). The animal species to be listed for infection with SAV according to Article 8 criteria are provided., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Vector or reservoir species of three diseases of crustaceans listed in the Animal Health Law were identified based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Crustacean species on or in which Taura syndrome virus (TSV), Yellow head virus (YHV) or White spot syndrome virus (WSSV) were identified, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected crustaceans was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90-100%) that WSSV, TSV and YHV will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or aquaculture establishments or by water supply can possibly transmit WSSV, TSV and YHV., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Vector or reservoir species of five mollusc diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Mollusc species on or in which Mikrocytos mackini , Perkinsus marinus , Bonamia exitiosa , Bonamia ostreae and Marteilia refringens were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, this studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected molluscs was not found, these were defined as reservoir. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir mollusc species during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90-100%) that M. mackini, P. marinus, B. exitiosa B. ostreae and M. refringens will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or at aquaculture establishments or through contaminated water supply can possibly transmit these pathogens. For transmission of M. refringens, the presence of an intermediate host, a copepod, is necessary., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Vector or reservoir species of five fish diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review (ELR), to support a possible updating of Regulation (EU) 2018/1882. Fish species on or in which highly polymorphic region-deleted infectious salmon anaemia virus (HPR∆ ISAV), Koi herpes virus (KHV), epizootic haematopoietic necrosis virus (EHNV), infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV) were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms or reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected fish was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir fish species during transport was collected from scientific literature. For VHSV, IHNV or HPR∆ ISAV, it was concluded that under transport conditions at temperatures below 25°C, it is likely (66-90%) they will remain infective. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild, aquaculture establishments or through water supply can possibly transmit VHSV, IHNV or HPR∆ ISAV into a non-affected area when transported at a temperature below 25°C. The conclusion was the same for EHN and KHV; however, they are likely to remain infective under all transport temperatures., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Infectious pancreatic necrosis (IPN) was assessed according to the criteria of the Animal Health Law (AHL), in particular, the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as in Article 9, and Article 8 for listing animal species related to IPN. The assessment was performed following a methodology previously published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment here performed, it is uncertain whether IPN can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (50-90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that IPN does not meet the criteria in Section 1 (Category A; 0-1% probability of meeting the criteria) and it is uncertain whether it meets the criteria in Sections 2, 3, 4 and 5 (Categories B, C, D and E; 33-66%, 33-66%, 50-90% and 50-99% probability of meeting the criteria, respectively). The animal species to be listed for IPN according to Article 8 criteria are provided., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

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., (© 2022 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon ( Salmo salar ). In seawater-farmed salmonids in the southern part of Norway SAV subtype 3 (SAV3) is dominating. PD continues to cause significant economic and fish health concerns in this region despite years of extensive use of oil-adjuvanted vaccines (OAVs) containing inactivated whole virus (IWV) antigens. In the current study, three commercially available PD vaccines were tested. Group A got a DNA vaccine (DNAV) injected intramuscularly (i.m.) plus an OAV without a PD component injected intraperitoneally (i.p.). Groups B and C got different OAV IWV vaccines injected i.p., respectively. The control group was i.p. injected with saline. Approximately 12 weeks after vaccination, the post smolt groups were challenged in seawater with SAV3 by cohabitation. Samples were collected pre-challenge, and at 19, 54 and 83 days post-challenge (dpc). There were no differences in growth or visible intraperitoneal side effects between the immunized groups prior to challenge. Fish in group A had significantly higher SAV3 neutralizing antibody titers than the other groups pre-challenge and significantly lower SAV3 viremia levels than the control group at 19 dpc. Fish in group A had significantly more weight gain than the other groups measured at 54 and 83 dpc. Prevalence and severity of heart necrosis at 19 dpc and loss of exocrine pancreas tissue at 54 and 83 dpc were significantly lower in groups A and B compared to group C and controls. The cumulative mortality in the control group during the challenge period was 10.5%. Group A experienced the lowest mortality (6.4%) albeit not statistically different from the controls. The results suggest that DNAV may reduce the clinical and economic impact of PD by improved protection against SAV3-induced changes in pancreas tissue and growth impairment., Competing Interests: Ragnar Thorarinsson is employed by Elanco Animal Health which funded this study and is the marketing authorization holder of the DNAV. The remaining authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Elanco Animal Health Inc. Published by Elsevier Ltd.)

Cardiomyopathy syndrome (CMS) is the most common viral cardiac disease in Norwegian Atlantic salmon farming and typically affects large, market size fish. Only six months after seawater transfer, Atlantic salmon were diagnosed with CMS at a fish farm in the south-western part of Norway. Due to the unexpected young age and the remarkable large amounts of virus-specific RNA (Ct <10), the fish group was monitored with five additional samplings until slaughtered almost 10 months later. At three weeks after the first CMS diagnosis (weeks post-diagnosis, wpd) and at slaughter (39 wpd), more comprehensive samplings were performed of the study cage, with specific focus on three different cardiac compartments. The clinical, autopsy and histopathological findings at first diagnosis and at all succeeding samplings were similar to previous descriptions of typical CMS. A slightly elevated mortality was observed in the cage with diseased fish at the time of the first CMS diagnosis and continued throughout the study. The prevalence and load of PMCV-specific RNA in the fish remained high until slaughtering, with similar amounts in all sampled cardiac compartments. No fish from the other five cages at the site were diagnosed with CMS, until fish sampled from the last cage at the site were diagnosed 10 weeks after slaughtering of the study cage (49 wpd). Sequence analysis of the PMCV on the site showed that the outbreak virus was similar to PMCV variants previously sequenced from Norwegian field outbreaks. In conclusion, CMS in young Atlantic salmon had clinical signs and histopathological cardiac lesions typical for the disease, and diseased fish could be found in the study cage until slaughtering., (© 2021 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

The use of lumpfish ( Cyclopterus lumpus ) as a cleaner fish to fight sea lice infestation in farmed Atlantic salmon has become increasingly common. Still, tools to increase our knowledge about lumpfish biology are lacking. Here, we successfully established and characterized the first Lumpfish Gill cell line (LG-1). LG-1 are adherent, homogenous and have a flat, stretched-out and almost transparent appearance. Transmission electron microscopy revealed cellular protrusions and desmosome-like structures that, together with their ability to generate a transcellular epithelial/endothelial resistance, suggest an epithelial or endothelial cell type. Furthermore, the cells exert Cytochrome P450 1A activity. LG-1 supported the propagation of several viruses that may lead to severe infectious diseases with high mortalities in fish farming, including viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV). Altogether, our data indicate that the LG-1 cell line originates from an epithelial or endothelial cell type and will be a valuable in vitro research tool to study gill cell function as well as host-pathogen interactions in lumpfish.

Pancreas disease (PD) and sleeping disease (SD), caused by an alphavirus, are endemic in European salmonid aquaculture, causing significant mortality, reduced growth and poor flesh quality. In 2010, a new variant of salmonid alphavirus emerged in Norway, marine salmonid alphavirus genotype 2 (SAV2). As this genotype is highly prevalent in Scotland, transmission through well boat traffic was hypothesized as one possible source of infection. In this study, we performed full-length genome sequencing of SAV2 sampled between 2006 and 2012 in Norway and Scotland, and present the first comprehensive full-length characterization of Norwegian marine SAV2 strains. We analyze their relationship with selected Scottish SAV2 strains and explore the genetic diversity of SAV. Our results show that all Norwegian marine SAV2 share a recent last common ancestor with marine SAV2 circulating in Scotland and a higher level of genomic diversity among the Scottish marine SAV2 strains compared to strains from Norway. These findings support the hypothesis of a single introduction of SAV2 to Norway sometime from 2006-2010, followed by horizontal spread along the coast.

Pancreas disease (PD) caused by salmonid alphavirus subtype 3 (SAV3) is a serious disease with large economic impact on farmed Norwegian Atlantic salmon production despite years of use of oil-adjuvanted vaccines against PD (OAVs). In this study, two commercially available PD vaccines, a DNA vaccine (DNAV) and an OAV, were compared in an experimental setting. At approximately 1040° days (dd) at 12 °C post immunization, the fish were challenged with SAV3 by cohabitation 9 days after transfer to sea water. Sampling was done prior to challenge and at 19, 54, and 83 days post-challenge (dpc). When compared to the OAV and control (Saline) groups, the DNAV group had significantly higher SAV3 neutralizing antibody titers after the immunization period, significantly lower SAV3 viremia levels at 19 dpc, significantly reduced transmission of SAV3 to naïve fish in the latter part of the viremic phase, significantly higher weight gain post-challenge, and significantly reduced prevalence and/or severity of SAV-induced morphologic changes in target organs. The DNAV group had also significantly higher post-challenge survival compared to the Saline group, but not to the OAV group. The data suggest that use of DNAV may reduce the economic impact of PD by protecting against destruction of the pancreas tissue and subsequent growth impairment which is the most common and costly clinical outcome of this disease., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Salmonid alphavirus (SAV) is the etiological cause of pancreas disease (PD) in Atlantic salmon (Salmo salar). Several vaccines against SAV are in use, but PD still cause significant mortality and concern in European aquaculture, raising the need for optimal tools to monitor SAV immunity. To monitor and control the distribution of PD in Norway, all salmonid farms are regularly screened for SAV by RT-qPCR. While the direct detection of SAV is helpful in the early stages of infection, serological methods could bring additional information on acquired SAV immunity in the later stages. Traditionally, SAV antibodies are monitored in neutralization assays, but they are time-consuming and cumbersome, thus alternative assays are warranted. Enzyme-linked immunosorbent assays (ELISAs) have not yet been successfully used for anti-SAV antibody detection in aquaculture. We aimed to develop a bead-based immunoassay for SAV-specific antibodies. By using detergent-treated SAV particles as antigens, we detected SAV-specific antibodies in plasma collected from both a SAV challenge trial and a field outbreak of PD. Increased levels of SAV-specific antibodies were seen after most fish had become negative for viral RNA. The bead-based assay is time saving compared to virus neutralization assays, and suitable for non-lethal testing due to low sample size requirements. We conclude that the bead-based immunoassay for SAV antibody detection is a promising diagnostic tool to complement SAV screening in aquaculture., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Infectious pancreatic necrosis virus (IPNV) infection has been a major problem in salmonid aquaculture. Marker-assisted selection of individuals with resistant genotype at the major IPN quantitative trait locus (IPN-QTL) has significantly reduced mortality in recent years. We have identified host miRNAs that respond to IPNV challenge in salmon fry that were either homozygous resistant (RR) or homozygous susceptible (SS) for the IPN-QTL. Small RNA-sequenced control samples were compared to samples collected at 1, 7, and 20 days post challenge (dpc). This revealed 72 differentially expressed miRNAs (DE miRNAs). Viral load (VL) was lower in RR vs. SS individuals at 7 and 20 dpc. However, analysis of miRNA expression changes revealed no differences between RR vs. SS individuals in controls, at 1 or 7 dpc, while 38 "high viral load responding" miRNAs (HVL-DE miRNAs) were identified at 20 dpc. Most of the HVL-DE miRNAs showed changes that were more pronounced in the high VL SS group than in the low VL RR group when compared to the controls. The absence of differences between QTL groups in controls, 1 and 7 dpc indicates that the QTL genotype does not affect miRNA expression in healthy fish or their first response to viral infections. The miRNA differences at 20 dpc were associated with the QTL genotype and could, possibly, contribute to differences in resistance/susceptibility at the later stage of infection. In silico target gene predictions revealed that 180 immune genes were putative targets, and enrichment analysis indicated that the miRNAs may regulate several major immune system pathways. Among the targets of HVL-DE miRNAs were IRF3, STAT4, NFKB2, MYD88, and IKKA. Interestingly, TNF-alpha paralogs were targeted by different DE miRNAs. Most DE miRNAs were from conserved miRNA families that respond to viral infections in teleost (e.g., miR-21, miR-146, miR-181, miR-192, miR-221, miR-462, miR-731, and miR-8159), while eight were species specific. The miRNAs showed dynamic temporal changes implying they would affect their target genes differently throughout disease progression. This shows that miRNAs are sensitive to VL and disease progression, and may act as fine-tuners of both immediate immune response activation and the later inflammatory processes., (Copyright © 2020 Woldemariam, Agafonov, Sindre, Høyheim, Houston, Robledo, Bron and Andreassen.)

Salmonid alphavirus (SAV) is the OIE-listed, viral cause of pancreas disease (PD) in farmed Atlantic salmon. SAV is routinely detected by PCR-methods while typical histopathological lesions are additionally used to confirm the diagnosis. Field evaluation of diagnostic test performance is essential to ensure confidence in a test's ability to predict the infection or disease status of a target animal. For most tests used in aquaculture, characteristics like sensitivity (Se) and specificity (Sp) at the analytical level may be known. Few tests are, however, evaluated at the diagnostic level according to the OIE standard. In the present work, we estimated diagnostic test sensitivity (DSe) and diagnostic test specificity (DSp) for five laboratory tests used for SAV detection. As there is no gold standard, the study was designed using Bayesian latent class analysis. Real-time RT-PCR, cell culture, histopathology, virus neutralization test, and immunohistochemistry were compared using samples taken from three different farmed Atlantic salmon populations with different infection status; one population regarded negative, one in an early stage of infection, and one in a later stage of infection. The average fish weight in the three populations was 2.0, 1.6, and 1.5 kg, respectively. The DSe and DSp of real-time RT-PCR is of particular interest due to its common use as a screening tool. The method showed high DSe (≥0.977) and moderate DSp (0.831) in all 3-populations models. The results further suggest that a follow-up test of serum samples in real-time RT-PCR negative populations may be prudent in cases where epidemiological information suggest a high risk of infection and where a false negative result is of high consequence. This study underlines the need to choose a test appropriate for the purpose of the testing. In the case of a weak positive PCR-result, a follow-up test should be conducted to verify the presence of SAV. Cell culture showed high DSe and DSp and may be used to verify viral presence., (Copyright © 2019 Jansen, Guarracino, Carson, Modahl, Taksdal, Sindre, Brun and Tavornpanich.)

The occurrence of infectious salmon anemia (ISA) outbreaks in marine farmed Atlantic salmon constitutes a recurring challenge in Norway. Here, we aim to identify risk factors associated with ISA outbreaks with an unknown source of infection (referred to as primary ISA outbreaks). Primary ISA outbreaks are here defined by an earlier published transmission model. We explored a wide range of possible risk factors with logistic regression analysis, trying to explain occurrence of primary ISA with available data from all Norwegian farm sites from 2004 to June 2017. Explanatory variables included site latitude and a range of production and disease data. The mean annual risk of having a primary outbreak of ISA in Norway was 0.7% during this study period. We identified the occurrence of infectious pancreatic necrosis (IPN), having a stocking period longer than 2 months, having the site located at high latitude and high fish density (biomass per cage volume) in the first six months after transfer to sea site as significant risk factors ( p < 0.05). We have identified factors related to management routines, other disease problems, and latitude that may help to understand the hitherto unidentified drivers behind the emergence of primary ISA outbreaks. Based on our findings, we also provide management advice that may reduce the incidence of primary ISA outbreaks.

Background: MicroRNAs (miRNAs) control multiple biological processes including the innate immune responses by negative post-transcriptional regulation of gene expression. As there were no studies on the role(s) of miRNAs in viral diseases in Atlantic salmon, we aimed to identify miRNAs responding to salmonid alphavirus (SAV) infection. Their expression were studied at different time points post infection with SAV isolates associated with different mortalities. Furthermore, the genome sequences of the identified miRNAs were analysed to reveal putative cis-regulatory elements, and, finally, their putative target genes were predicted., Results: Twenty differentially expressed miRNAs (DE miRNAs) were identified. The expression of the majority of these increased post infection with maximum levels reached after the viral load were stabilized or decreasing. On the other hand, some miRNAs (e.g. the miRNA-21 family) showed decreased expression at the early time points post infection. There were significant differences in the temporal expression of individual miRNA associated with different SAV isolates. Target gene prediction in SAV responsive immune network genes showed that seventeen of the DE miRNAs could target 24 genes (e.g. IRF3, IRF7). Applying the Atlantic salmon transcriptome as input 28 more immune network genes were revealed as putative targets (e.g. IRF5, IRF4). The majority of the predicted target genes promote inflammatory response. The upstream sequences of the miRNA genes revealed a high density of cis-regulatory sequences known as binding sites for immune network transcription factors (TFs). A high expression in the late phase could therefore be due to increased transcription promoted by immune response activated TFs. Based on the in silico target predictions, we discuss their putative roles as early promotors or late inhibitors of inflammation. We propose that the differences in expressions associated with different SAV isolates could contribute to their differences in mortality rates., Conclusions: This study represents the first steps in exploring miRNAs important in viral-host interaction in Atlantic salmon. We identified several miRNAs responding to SAV infection. Some likely to prohibit harmful inflammation while other may promote an early immune response. Their predicted functions need to be validated and further studied in functional assays to fully understand their roles in immune homeostasis.

Heart and skeletal muscle inflammation (HSMI) and pancreas disease (PD) cause substantial losses in Atlantic salmon (Salmo salar) aquaculture. The respective causative agents, Piscine orthoreovirus (PRV) and Salmonid alphavirus (SAV), are widespread and often concurrently present in farmed salmon. An experimental infection in Atlantic salmon was conducted to study the interaction between the two viruses, including the immunological mechanisms involved. The co-infected fish were infected with PRV four or ten weeks before they were infected with SAV. The SAV RNA level and the PD specific lesions were significantly lower in co-infected groups compared to the group infected by only SAV. The expression profiles of a panel of innate antiviral response genes and the plasma SAV neutralization titers were examined. The innate antiviral response genes were in general upregulated for at least ten weeks after the primary PRV infection. Plasma from co-infected fish had lower SAV neutralizing titers compared to the controls infected with only SAV. Plasma from some individuals infected with only PRV neutralized SAV, but heat treatment removed this effect. Field studies of co-infected fish populations indicated a negative correlation between the two viruses in randomly sampled apparently healthy fish, in line with the experimental findings, but a positive correlation in moribund or dead fish. The results indicate that the innate antiviral response induced by PRV may temporary protect against a secondary SAV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pancreas disease (PD) is a viral disease caused by Salmonid alphavirus (SAV) that affects farmed Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss (Walbaum)) in the seawater phase. Since its first description in Scotland in 1976, a large number of studies have been conducted relating to the disease itself and to factors contributing to agent spread and disease occurrence. This paper summarizes the currently available, scientific information on the epidemiology of PD and its associated mitigation and control measures. Available literature shows infected farmed salmonids to be the main reservoir of SAV. Transmission between seawater sites occurs mainly passively by water currents or actively through human activity coupled with inadequate biosecurity measures. All available information suggests that the current fallowing procedures are adequate to prevent agent survival within the environment through the fallowing period and thus that a repeated disease outbreak at the same site is due to a new agent introduction. There has been no scientific evaluation of currently used on-site biosecurity measures, and there is limited information on the impact of available mitigation measures and control strategies., (© 2016 John Wiley & Sons Ltd.)

Pancreas disease (PD), caused by salmonid alphavirus subtype 3 (SAV3), emerged in Norwegian aquaculture in the 1980s and is now endemic along the south-western coast. In 2011, the first cases of PD caused by marine salmonid alphavirus subtype 2 (SAV2) were reported. This subtype has spread rapidly among the fish farms outside the PD-endemic zone and is responsible for disease outbreaks at an increasing numbers of sites. To describe the geographical distribution of salmonid alphavirus (SAV), and to assess the time and site of introduction of marine SAV2 to Norway, an extensive genetic characterization including more than 200 SAV-positive samples from 157 Norwegian marine production sites collected from May 2007 to December 2012 was executed. The first samples positive for marine SAV2 originated from Romsdal, in June 2010. Sequence analysis of the E2 gene revealed that all marine SAV2 included in this study were nearly identical, suggesting a single introduction into Norwegian aquaculture. Further, this study provides evidence of a separate geographical distribution of two subtypes in Norway. SAV3 is present in south-western Norway, and marine SAV2 circulates in north-western and Mid-Norway, a geographical area which since 2010 constitutes the endemic zone for marine SAV2., (© 2015 John Wiley & Sons Ltd.)

Pancreas disease (PD) caused by salmonid alphavirus (SAV) has a significant negative economic impact in the salmonid fish farming industry in northern Europe. Until recently, only SAV subtype 3 was present in Norwegian fish farms. However, in 2011, a marine SAV 2 subtype was detected in a fish farm outside the PD-endemic zone. This subtype has spread rapidly among fish farms in mid-Norway. The PD mortality in several farms has been lower than expected, although high mortality has also been reported. In this situation, the industry and the authorities needed scientific-based information about the virulence of the marine SAV 2 strain in Norway to decide how to handle this new situation. Atlantic salmon post-smolts were experimentally infected with SAV 2 and SAV 3 strains from six different PD cases in Norway. SAV 3-infected fish showed higher mortality than SAV 2-infected fish. Among the SAV 3 isolates, two isolates gave higher mortality than the third one. At the end of the experiment, fish in all SAV-infected groups had significantly lower weight than the uninfected control fish. This is the first published paper on PD to document that waterborne infection produced significantly higher mortality than intraperitoneal injection., (© 2014 John Wiley & Sons Ltd.)

Uncultivable HPR0 strains of infectious salmon anaemia viruses (ISAVs) infecting gills are non-virulent putative precursors of virulent ISAVs (vISAVs) causing systemic disease in farmed Atlantic salmon (Salmo salar). The transition to virulence involves two molecular events, a deletion in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) gene and a Q266→L266 substitution or insertion next to the putative cleavage site (R267) in the fusion protein (F). We have performed ultra-deep pyrosequencing (UDPS) of these gene regions from healthy fish positive for HPR0 virus carrying full-length HPR sampled in a screening program, and a vISAV strain from an ISA outbreak at the same farming site three weeks later, and compared the mutant spectra. As the UDPS data shows the presence of both HE genotypes at both sampling times, and the outbreak strain was unlikely to be directly related to the HPR0 strain, this is the first report of a double infection with HPR0s and vISAVs. For F amplicon reads, mutation frequencies generating L266 codons in screening samples and Q266 codons in outbreak samples were not higher than at any random site. We suggest quasispecies heterogeneity as well as RNA structural properties are linked to transition to virulence. More specifically, a mechanism where selected single point mutations in the full-length HPR alter the RNA structure facilitating single- or sequential deletions in this region is proposed. The data provides stronger support for the deletion hypothesis, as opposed to recombination, as the responsible mechanism for generating the sequence deletions in HE.

Background: The aquatic birnavirus infectious pancreatic necrosis virus (IPNV) causes infectious pancreatic necrosis (IPN), a severe disease in farmed salmonid fish. IPNV has a very broad host range and infects many different species of fish as well as molluscs and crustaceans. Investigation of the host reservoir of a virus may reveal important molecular mechanisms governing the infection processes such as receptors and entry mechanisms. In the present work we have studied whether IPNV is able to infect cells with different mammalian origin., Results: IPNV bound in a specific manner to a membrane protein of the rabbit kidney cell line RK-13 as shown by the use of a virus overlay protein binding assay (VOPBA). Six different mammalian cell lines were inoculated with IPNV and incubated in parallels at different temperatures. At 7 days post inoculation (dpi), IPNV was detected by indirect immunofluorescent antibody test (IFAT) in all the cell lines. Confocal microscopy confirmed intracellular presence of the virus. No apparent cytopathic effect (cpe) was observed in any of the cultures, and no viral replication was demonstrated with real-time RT-PCR., Conclusion: Our results show that IPNV is able to enter into a wide range of mammalian cells, and virus entry is most likely receptor mediated. We found no indication of IPNV replication in any of the mammalian cell lines tested.