Validation of a TaqMan one-step real-time RT-PCR assay targeting ISAV segment 7 spliced mRNA.

Infectious salmon anaemia virus (ISAV) can cause severe systemic infection in Atlantic salmon (Salmo salar L.), and a timely diagnosis is critical. Conventional real-time reverse transcription PCR (RT-qPCR) assays target unspliced RNA from either ISAV segment 7 or 8 and provide data on viral load. Here, we evaluate a TaqMan one-step RT-qPCR assay that detects explicitly a spliced messenger RNA (mRNA) of ISAV segment 7, thus providing evidence of active viral transcription. Assay performance was comparable with existing unspliced segment 7 and segment 8 assays. PCR efficiency as evaluated from dilutions of a synthetic DNA fragment was 98 % (R2 = 1.00). The assay also performed well on clinical heart samples with PCR efficiency of 108 % (R2 = 1.00). Finally, evaluation on kidney samples from experimental infection revealed higher levels of active transcription for high-virulent compared to low-virulent ISAV. At early, peak, and late infection, mean ratios of spliced to unspliced segment 7 RNA were 3.0 % (± 0.7), 1.7 % (± 0.3), and 1.5 % (± 0.1) for the low virulent and 9.4 % (± 2.2), 4.7 % (± 0.8), and 6.2 % (± 0.1) for the high virulent isolate, respectively. By detection and quantification of active ISAV transcription, this assay may provide a more detailed understanding of ISAV infection dynamics.

Tenacibaculosis in Norwegian Atlantic salmon (Salmo salar) cage-farmed in cold sea water is primarily associated with Tenacibaculum finnmarkense genomovar finnmarkense.

Skin conditions associated with Tenacibaculum spp. constitute a significant threat to the health and welfare of sea-farmed Atlantic salmon (Salmo salar L.) in Norway. Fifteen presumptive tenacibaculosis outbreaks distributed along the Norwegian coast during the late winter and spring of 2018 were investigated. Bacteriological culture confirmed the presence of Tenacibaculum spp. Seventy-six isolates cultured from individual fish were selected and subjected to whole-genome sequencing and MALDI-TOF MS analysis. Average nucleotide identity and MALDI-TOF analyses confirmed the presence of T. finnmarkense and T. dicentrarchi, with further division of T. finnmarkense into genomovars (gv.) finnmarkense and ulcerans. Core genome multilocus sequence typing (cgMLST) and single-nucleotide polymorphism (SNP) analyses identified the presence of a genetically conserved cluster of gv. finnmarkense isolates against a background of relatively genetically diverse gv. finnmarkense and gv. ulcerans isolates in 13 of the 15 studied cases. This clustering strongly suggests a link between T. finnmarkense gv. finnmarkense and development of clinical tenacibaculosis in sea-farmed Norwegian salmon in the late winter and spring. Analysis of 25 Tenacibaculum isolates collected during the spring of 2019 from similar cases identified a similar distribution of genotypes. Low water temperatures were common to all cases, and most incidences involved relatively small fish shortly after sea transfer, suggesting that these fish are particularly predisposed to Tenacibaculum infection.

No Evidence of the Vertical Transmission of Non-Virulent Infectious Salmon Anaemia Virus (ISAV-HPR0) in Farmed Atlantic Salmon.

The nonvirulent infectious salmon anaemia virus (ISAV-HPR0) is the putative progenitor for virulent-ISAV, and a potential risk factor for the development of infectious salmon anaemia (ISA). Understanding the transmission dynamics of ISAV-HPR0 is fundamental to proper management and mitigation strategies. Here, we demonstrate that ISAV-HPR0 causes prevalent and transient infections in all three production stages of Atlantic salmon in the Faroe Islands. Phylogenetic analysis of the haemagglutinin-esterase gene from 247 salmon showed a clear geographical structuring into two significantly distinct HPR0-subgroups, which were designated G2 and G4. Whereas G2 and G4 co-circulated in marine farms, Faroese broodfish were predominantly infected by G2, and smolt were predominantly infected by G4. This infection pattern was confirmed by our G2- and G4-specific RT-qPCR assays. Moreover, the HPR0 variants detected in Icelandic and Norwegian broodfish were never detected in the Faroe Islands, despite the extensive import of ova from both countries. Accordingly, the vertical transmission of HPR0 from broodfish to progeny is uncommon. Phylogenetic and statistical analysis suggest that HPR0 persists in the smolt farms as "house-strains", and that new HPR0 variants are occasionally introduced from the marine environment, probably by HPR0-contaminated sea-spray. Thus, high biosecurity-including water and air intake-is required to avoid the introduction of pathogens to the smolt farms.

Two-year surveillance of tilapia lake virus (TiLV) reveals its wide circulation in tilapia farms and hatcheries from multiple districts of Bangladesh.

Tilapia lake virus (TiLV) is an emerging pathogen in aquaculture, reportedly affecting farmed tilapia in 16 countries across multiple continents. Following an early warning in 2017 that TiLV might be widespread, we executed a surveillance programme on tilapia grow-out farms and hatcheries from 10 districts of Bangladesh in 2017 and 2019. Among farms experiencing unusual mortality, eight out of 11 farms tested positive for TiLV in 2017, and two out of seven tested positive in 2019. Investigation of asymptomatic broodstock collected from 16 tilapia hatcheries revealed that six hatcheries tested positive for TiLV. Representative samples subjected to histopathology confirmed pathognomonic lesions of syncytial hepatitis. We recovered three complete genomes of TiLV from infected fish, one from 2017 and two from 2019. Phylogenetic analyses based on both the concatenated coding sequences of 10 segments and only segment 1 consistently revealed that Bangladeshi TiLV isolates formed a unique cluster within Thai clade, suggesting a close genetic relation. In summary, this study revealed the circulation of TiLV in 10 farms and six hatcheries located in eight districts of Bangladesh. We recommend continuing TiLV-targeted surveillance efforts to identify contaminated sources to minimize the countrywide spread and severity of TiLV infection.

Mapping the knowledge of the main diseases affecting sea bass and sea bream in Mediterranean.

Good knowledge on the disease situation and its impact on production is a base mechanism for designing health surveillance, risk analysis and biosecurity systems. Mediterranean marine fish farming, as any aquaculture production, is affected by various infectious diseases. However, seabass and seabream, the main produced species, are not listed as susceptible host species for the notifiable pathogens listed in the current EU legislation, which generates a lack of systematic reporting. The results presented in this study come from a survey directly to fish farms (50 hatchery and on-growing units from 10 Mediterranean countries), with data from 2015 to 2017, conducted by the H2020 project MedAID. Seabass showed a higher survival rate (85%) through a production cycle than seabream (80%) in spite of equal mortality due to pathogen infections (10%). The differences in survival may be explained by mortality 'of other causes'. Seabream and seabass have different disease profiles, and the profile is slightly different between geographical regions. Among the most important diseases, tenacibaculosis and vibriosis were identified in seabass and Sparicotyle chrysophrii (a gill fluke) and nodavirus in seabream. Correlating mortality data to management variables showed that increasing density, buying fingerlings from external sources and treatments due to disease are factors that negatively influence mortality rate. Most of the surveyed farms did not keep sufficient quality data to implement good health status reports and perform detailed impact studies, which shows the necessity of updating the current legislative framework to provide the basis for better reporting of relevant pathogens in the Mediterranean basin.

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.

Pancreas disease caused by Salmonid alphavirus subtype 2 reduces growth and feed conversion in farmed Atlantic salmon.

Pancreas disease (PD), caused by several subtypes of salmonid alphavirus (SAV), is associated with significant economic losses in European salmonid aquaculture. In this retrospective cohort study, we investigate the impact of PD caused by SAV subtype 2 (SAV2) on growth, feed conversion, and mortality in farmed Atlantic salmon (Salmo salar L.). The study was based on harvest data from a large salmon farming company operating in the SAV2 endemic area of Norway. Mixed-effect regression analyses showed a severe impact on both growth and feed conversion when PD appeared late in the production cycle. In a scenario with fixed slaughter time the estimated impact corresponded to a growth reduction of 0.7 kg and 0.07 points increase in feed conversion ratio. No effect on mortality was observed in this data set. In conclusion, the most important consequences of PD caused by SAV2 infection is reduced growth and feed conversion in large Atlantic salmon. The lack of effect on mortality in this study may be due to other factors overshadowing the impact of PD.

A stochastic network-based model to simulate the spread of pancreas disease (PD) in the Norwegian salmon industry based on the observed vessel movements and seaway distance between marine farms.

Pancreas disease (PD) is a viral disease of economic importance affecting farmed Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhyncus mykiss (Walbaum)) in the seawater phase in Ireland, Norway and Scotland. In this study we used a stochastic network-based disease spread model to better understand the role of vessel movements and nearby seaway distance on PD spread in marine farms. We used five different edge's definitions and weights for the network construction: high-risk vessel movements, high-risk wellboat movements and high-risk nearby seaway distance at <20 km, <10 km or <5 km, respectively. Models were used to simulate PD spread in marine farms as well as to simulate the spread of marine SAV2 and SAV3 subtypes independently and results were compared with the observed PD, marine SAV2 and SAV3 cases in Norway in 2016. Results revealed that the model that provided the best fit of the observed data and, therefore, the one considered more biologically plausible, was the one using high-risk wellboat movements. The marine SAV2, SAV3 and PD models using wellboat movements were able to correctly simulate the farms status (PD positive or PD negative) with the sensitivity of 84%, 85%, 84% and Specificity of 98%, 97% and 94%, respectively. These results should contribute to inform more cost-effective prevention and control policies to mitigate PD spread and to improve the sustainability and long-term profitability of the salmon industry in Norway.

Genetic variants in loci 1p13 and 9p21 and fatal coronary heart disease in a Norwegian case-cohort study.

Single nucleotide polymorphisms (SNPs) in loci 1p13 and 9p21 have previously been found to be associated with incident coronary heart disease (CHD). This study aimed to investigate whether these SNPs show associations with fatal CHD in a population-based cohort study after adjustment for socioeconomic- and lifestyle-related CHD risk factors not commonly included in genetic association studies. Using the population-based Cohort of Norway (CONOR), a nested case-cohort study was set up and DNA from 2,953 subjects (829 cases and 2,124 non-cases) were genotyped. The association with fatal CHD was estimated for four SNPs, three from locus 1p13 and one from locus 9p21. Multivariable Cox regression was used to estimate unstratified and gender-stratified hazard ratios while adjusting for major CHD risk factors. The associations between three SNPs from locus 1p13 and non-HDL cholesterol levels were also estimated. Men homozygous for the risk alleles on rs1333049 (9p21) and rs14000 (1p13) were found to have significantly increased hazard ratios in crude and adjusted models, and the hazard ratios remained statistically significant when both genders were analyzed together. Adjustment for additional socioeconomic- and lifestyle-related CHD risk factors influenced the association estimates only slightly. No significant associations were observed between the other two SNPs in loci 1p13 (rs599839 and rs646776) and CHD mortality in either gender. Both rs599839 and rs646776 showed significant, gradual increases in non-HDL cholesterol levels with increasing number of risk alleles. This study confirms the association between 9p21 (rs1333049) and fatal CHD in a Norwegian population-based cohort. The effect was not influenced by several socioeconomic- and lifestyle-related risk factors. Our results show that 1p13 (rs14000) may also be associated with fatal CHD. SNPs at 1p13 (rs599839 and rs646776) were associated with non-HDL cholesterol levels.

Is ADH1C genotype relevant for the cardioprotective effect of alcohol?

The cardioprotective effect of ethanol has been suggested to be linked to one of the ethanol metabolizing enzymes (ADH1C), which constitutes a high V(max) and a low V(max) variant. This has been demonstrated in some studies, while others have not been able to replicate the findings. The aim of the present study was to investigate the relation between the different ADH1C genotypes, death from coronary heart disease (CHD) and alcohol in a material larger than the previously published studies. Eight hundred CHD deaths as well as 1303 controls were genotyped for the high V(max) (γ1) and the low V(max) (γ2) ADH1C variant. Information of alcohol use was available for all subjects. Multiple logistic regression analyses was used to study if the decreased risk of death from CHD in alcohol consuming subjects was more pronounced in subjects homozygous for the γ2 allele (γ2γ2 subjects) compared to γ1γ1 and γ1γ2 subjects. The odds ratio (OR) for death from CHD in alcohol consumers compared to abstainers was similar in the genotype groups, i.e., 0.62 (95% CI: 0.43-0.88) in γ1γ1 subjects and 0.62 (95% CI: 0.42-0.91) in γ2γ2 subjects. Also when stratifying the results by gender and when dividing alcohol consumers into different alcohol consumption groups, there was no difference in the OR between the different genotype groups. This study, which included the largest study group published so far, failed to find any link between the ADH1C genotype and the cardioprotective effects of alcohol.

Divergent associations of drinking frequency and binge consumption of alcohol with mortality within the same cohort.

BACKGROUND: Observational studies show beneficial effects of moderate alcohol drinking on all-cause and cardiovascular disease (CVD) mortality, while binge drinking has been linked with increased mortality. The aim of this study was to assess the associations of alcohol use with mortality in a population with a hybrid of drinking patterns. METHOD: Participants in a population based cardiovascular health survey in Finnmark county in 1987-1988, aged 20-62 years, constituted the study cohort. Alcohol use was self-reported by use of questions on frequency of beer, wine and liquor intake, and one question on intake of around five drinks or more per occasion (binge drinking). Information on education, income and use of alcohol in an earlier and in a later survey was linked to the file. Mortality was assessed throughout 2009 by Cox regression, with adjustment for potential confounding factors. In the analysis of mortality by frequency of any alcohol use, we adjusted for binge consumption and vice versa. RESULTS: Two opposite trends appeared: a higher all-cause mortality in both sexes, and higher CVD mortality in men, with increasing frequency of binge drinking, compared with non-bingers. Second, in both sexes low-frequent use of any alcohol was associated with lower all-cause and CVD mortality, compared with abstention. The combination of any use of alcohol at least weekly and binge consumption at least monthly was common, particularly in men. CONCLUSIONS: Questions on drinking frequency and a specific question on binge drinking capture different effects of alcohol use on all-cause and CVD mortality.