Effect of pancreas disease vaccines on infection levels and virus transmission in Atlantic salmon (Salmo salar) challenged with salmonid alphavirus, genotype 2.

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.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) 2016/429): infection with Gyrodactylus salaris (GS).

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.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) 2016/429): Bacterial kidney disease (BKD).

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.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU)2016/429): Infection with salmonid alphavirus (SAV).

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.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) 2016/429): Spring Viraemia of Carp (SVC).

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.

Species which may act as vectors or reservoirs of diseases covered by the Animal Health Law: Listed pathogens of molluscs.

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.

Species which may act as vectors or reservoirs of diseases covered by the Animal Health Law: Listed pathogens of crustaceans.

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.

Species which may act as vectors or reservoirs of diseases covered by the Animal Health Law: Listed pathogens of fish.

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.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): infectious pancreatic necrosis (IPN).

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.

Effects of a DNA and multivalent oil-adjuvanted vaccines against pancreas disease in Atlantic salmon (Salmo salar) challenged with salmonid alphavirus subtype 3.

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.

Characterization of early phases of cardiomyopathy syndrome pathogenesis in Atlantic salmon (Salmo salar L.) through various diagnostic methods.

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.

Establishment and Characterization of a Novel Gill Cell Line, LG-1, from Atlantic Lumpfish (Cyclopterus lumpus L.).

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.

Characterization of an outbreak of cardiomyopathy syndrome (CMS) in young Atlantic salmon, Salmo salar L.

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.

Emergence of Salmonid Alphavirus Genotype 2 in Norway-Molecular Characterization of Viral Strains Circulating in Norway and Scotland.

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.

Effect of a novel DNA vaccine against pancreas disease caused by salmonid alphavirus subtype 3 in Atlantic salmon (Salmo salar).

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.

miRNAs Predicted to Regulate Host Anti-viral Gene Pathways in IPNV-Challenged Atlantic Salmon Fry Are Affected by Viral Load, and Associated With the Major IPN Resistance QTL Genotypes in Late Infection.

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.

Detection of specific Atlantic salmon antibodies against salmonid alphavirus using a bead-based immunoassay.

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.

Field Evaluation of Diagnostic Test Sensitivity and Specificity for Salmonid Alphavirus (SAV) Infection and Pancreas Disease (PD) in Farmed Atlantic salmon (Salmo salar L.) in Norway Using Bayesian Latent Class Analysis.

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.

Risk Factors Associated With Outbreaks of Infectious Salmon Anemia (ISA) With Unknown Source of Infection in Norway.

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.