Tailoring freshwater diets towards boosted immunity and pancreas disease infection robustness in Atlantic salmon post smolts.

The aim of the current study was to investigate how freshwater diets impact on immunity in Atlantic salmon smolts in freshwater, during transfer to seawater and in post smolts during the seawater stage with and without pancreas disease (PD) infection. Three specific freshwater diets were prepared: (i) A diet similar in composition to commercial salmon freshwater diets (Standard diet); (ii) A diet composed of vegetable oils (rapeseed, palm and linseed oils) mimicking the fat composition in aquatic insects - the natural diet of wild salmon in freshwater (Fatty acid diet); (iii) A diet enriched with possible immune modulating amino acids including dl-methionine, l-lysine, l-threonine and taurine (Amino acid diet). After seawater transfer, all fish were fed the same commercial diet. Head kidneys were extracted, and their leukocytes isolated from smolts right before transfer to seawater, from post smolts one and six weeks after transfer to seawater, and from post smolts in seawater after 8 weeks of ongoing PD infection. In addition, to provoke bacterial or virus induced inflammation in vitro, the individual leukocyte suspension from all fish were stimulated by lipopolysaccharide (LPS) or polyinosinic acid: polycytidylic acid (PIC). The transfer of smolts from fresh-to seawater changed the transcription of several types of genes. Particularly in isolates from fish fed the Standard or Fatty acid diet in freshwater, overall gene transcription (IL-1ß, CD83, INF-γ, cox2, cd36, MGAT2, catalase) declined. However, the Amino acid diet stimulated the LPS induced gene transcription of IL-1ß, CD83, Cox2, and INF-γ at this stage. In freshwater smolts, PIC stimulated leukocytes showed higher transcription level of Mx and viperin in the Fatty acid and Amino acid diet groups compared to the Standard diet group. In seawater post smolts, Mx and viperin responded similarly to PIC challenge in all diet groups. Furthermore, leukocytes isolated from PD infected fish, continued responding to PIC, regardless of freshwater diet.

Effect of vaccines against pancreas disease in farmed Atlantic salmon.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) continues to negatively impact salmon farming. To assess the effect on growth and mortality of three vaccines against PD, two controlled field designs were employed: one controlled field study with individual marked fish (PIT tag) assessing three PD vaccines and three controls groups, and a second controlled field study with group marked fish (Maxilla) comparing two PD vaccines against controls. In addition, a descriptive study using whole cages compared fish immunized with two different PD vaccines against controls. The target populations experienced a natural PD outbreak where both SAV 2 and SAV 3 were identified. Only one of the PD vaccines provided statistically significant improvements in harvest weight of 0.43 kg (CI: 0.29-0.57) and 0.51 kg (CI: 0.36-0.65) compared with the control in the PIT tag and the Maxilla study, respectively. In the latter, a significant reduction in mortality of 1.31 (CI:0.8-1.8) per cent points was registered for the same vaccine compared with controls. These results aligned with the growth and PD-specific mortality registered in the descriptive Cage study. The data in this study show a difference in the efficacy of PD vaccines in farmed Atlantic salmon.

[Update 2021: COVID-19 from the perspective of gastroenterology]. / Update 2021: COVID-19 aus Sicht der Gastroenterologie.

During COVID 19 pandemic patients typically present with respiratory symptoms. However, in a significant number of patients the gastrointestinal tract is also involved in the disease. Up to 20 % of patients suffering from gastrointestinal symptoms. New insights in pathophysiological aspects might open new therapeutic concepts. This up-date includes current data regarding epidemiology of gastrointestinal symptoms in COVID 19, its role for prognosis and specific risks in relation to immunosuppressive therapies and underlying diseases.

SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas.

Infection-related diabetes can arise as a result of virus-associated ß-cell destruction. Clinical data suggest that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), impairs glucose homoeostasis, but experimental evidence that SARS-CoV-2 can infect pancreatic tissue has been lacking. In the present study, we show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human ß-cells express viral entry proteins, and SARS-CoV-2 infects and replicates in cultured human islets. Infection is associated with morphological, transcriptional and functional changes, including reduced numbers of insulin-secretory granules in ß-cells and impaired glucose-stimulated insulin secretion. In COVID-19 full-body postmortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the ß-cell marker NKX6.1 and are in close proximity to the islets of Langerhans in all four patients investigated. Our data identify the human pancreas as a target of SARS-CoV-2 infection and suggest that ß-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19.

Implications of SARS-CoV-2 infection for neurogastroenterology.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with gastrointestinal and hepatic manifestation in up to one fifth of patients. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of COVID-19, infects gastrointestinal epithelial cells expressing angiotensin-converting enzyme 2 (ACE2) receptors triggering a cascade of events leading to mucosal and systemic inflammation. Symptomatic patients display changes in gut microbiota composition and function which may contribute to intestinal barrier dysfunction and immune activation. Evidence suggests that SARS-CoV-2 infection and related mucosal inflammation impact on the function of the enteric nervous system and the activation of sensory fibers conveying information to the central nervous system, which, may at least in part, contribute symptom generation such as vomiting and diarrhea described in COVID-19. Liver and pancreas dysfunctions have also been described as non-respiratory complications of COVID-19 and add further emphasis to the common view of SARS-CoV-2 infection as a systemic disease with multiorgan involvement. PURPOSE: The aim of this review was to highlight the current knowledge on the pathophysiology of gastrointestinal SARS-CoV-2 infection, including the crosstalk with the gut microbiota, the fecal-oral route of virus transmission, and the potential interaction of the virus with the enteric nervous system. We also review the current available data on gastrointestinal and liver manifestations, management, and outcomes of patients with COVID-19.

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.

Pancreatic Injury in COVID-19 Patients.

BACKGROUND AND AIM: Coronavirus disease 2019 (COVID 2019) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may cause multisystem dysfunction. We studied pancreatic injury (serum amylase and serum lipase levels) in COVID-19 patients. METHODS: A retrospective study involving 42 COVID-19 patients (diagnosed by real-time PCR) admitted to a tertiary care hospital was conducted. Serum amylase and serum lipase levels were analysed in relation to severity of COVID-19 and mortality. RESULTS: Mean age of patients was 50 ± 16 years, with male to female ratio of 3.7:1. Serum amylase was elevated in 14 patients (33%). Serum lipase was elevated in 7 out of 29 patients (24.1%). Mortality was seen in 18 patients (42.8%). Serum amylase or lipase did not correlate with severity of COVID-19 or its mortality. However, both patients who had high lipase (>3times) died. CONCLUSION: The prevalence of hyperamylasemia in patients of COVID-19 was 33%, while that of elevated lipase was 24.1%. Pancreatic injury failed to show any statistically significant relation to severity or outcome of COVID-19.

Pancreatic injury in COVID-19: pathogenesis and challenges.

Apart from the mechanisms reported by Fernandes et al, the thromboembolic pathogenesis should also be taken into account in patients with severe COVID-19 and prophylaxis with low molecular weight heparin should be implemented.

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.

Identification of genetic loci associated with higher resistance to pancreas disease (PD) in Atlantic salmon (Salmo salar L.).

BACKGROUND: Pancreas disease (PD) is a contagious disease caused by salmonid alphavirus (SAV) with significant economic and welfare impacts on salmon farming. Previous work has shown that higher resistance against PD has underlying additive genetic components and can potentially be improved through selective breeding. To better understand the genetic basis of PD resistance in Atlantic salmon, we challenged 4506 smolts from 296 families of the SalmoBreed strain. Fish were challenged through intraperitoneal injection with the most virulent form of the virus found in Norway (i.e., SAV3). Mortalities were recorded, and more than 900 fish were further genotyped on a 55 K SNP array. RESULTS: The estimated heritability for PD resistance was 0.41 ± 0.017. The genetic markers on two chromosomes, ssa03 and ssa07, showed significant associations with higher disease resistance. Collectively, markers on these two QTL regions explained about 60% of the additive genetic variance. We also sequenced and compared the cardiac transcriptomics of moribund fish and animals that survived the challenge with a focus on candidate genes within the chromosomal segments harbouring QTL. Approximately 200 genes, within the QTL regions, were found to be differentially expressed. Of particular interest, we identified various components of immunoglobulin-heavy-chain locus B (IGH-B) on ssa03 and immunoglobulin-light-chain on ssa07 with markedly higher levels of transcription in the resistant animals. These genes are closely linked to the most strongly QTL associated SNPs, making them likely candidates for further investigation. CONCLUSIONS: The findings presented here provide supporting evidence that breeding is an efficient tool for increasing PD resistance in Atlantic salmon populations. The estimated heritability is one of the largest reported for any disease resistance in this species, where the majority of the genetic variation is explained by two major QTL. The transcriptomic analysis has revealed the activation of essential components of the innate and the adaptive immune responses following infection with SAV3. Furthermore, the complementation of the genomic with the transcriptomic data has highlighted the possible critical role of the immunoglobulin loci in combating PD virus.

Quantitative trait loci and genes associated with salmonid alphavirus load in Atlantic salmon: implications for pancreas disease resistance and tolerance.

Salmonid alphavirus infection results in pancreas disease causing severe economic losses for Atlantic salmon aquaculture. Knowledge about genes and pathways contributing to resistance is limited. A 54 K SNP panel was used to genotype 10 full-sibling families each consisting of ~ 110 offspring challenged with salmonid alphavirus subtype 3. Relative heart viral load was assessed at 4- and 10-weeks post-infection using quantitative PCR. A moderate genomic heritability of viral load at 4 weeks (0.15-0.21) and a high positive correlation with survival (0.91-0.98) were detected. Positions of QTL detected on chromosome 3 matched those for survival detected by other studies. The SNP of highest significance occurred in the 3' untranslated region of gig1, a fish-specific antiviral effector. Locus B of immunoglobulin heavy chain mapped to an area containing multiple SNPs with genome-wide association. Heart mRNA-seq comparing parr from families with high- versus low-genomic breeding value, and matching sample genotypes for SNPs, identified two eQTL for salmonid alphavirus load. Immune genes associated with trans-eQTL were numerous and spread throughout the genome. QTL regions contained several genes with known or predicted immune functions, some differentially expressed. The putative functional genes and variants identified could help improve marker-based selection for pancreas disease resistance.

Response of the Salmon Heart Transcriptome to Pancreas Disease: Differences Between High- and Low-Ranking Families for Resistance.

Pancreas disease caused by salmonid alphaviruses leads to severe losses in Atlantic salmon aquaculture. The aim of our study was to gain a better understanding of the biological differences between salmon with high and low genomic breeding values (H-gEBV and L-gEBV respectively) for pancreas disease resistance. Fish from H- and L-gEBV families were challenged by intraperitoneal injection of salmonid alphavirus or co-habitation with infected fish. Mortality was higher with co-habitation than injection, and for L- than H-gEBV. Heart for RNA-seq and histopathology was collected before challenge and at four- and ten-weeks post-challenge. Heart damage was less severe in injection-challenged H- than L-gEBV fish at week 4. Viral load was lower in H- than L-gEBV salmon after co-habitant challenge. Gene expression differences between H- and L-gEBV manifested before challenge, peaked at week 4, and moderated by week 10. At week 4, H-gEBV salmon showed lower expression of innate antiviral defence genes, stimulation of B- and T-cell immune function, and weaker stress responses. Retarded resolution of the disease explains the higher expression of immune genes in L-gEBV at week 10. Results suggest earlier mobilization of acquired immunity better protects H-gEBV salmon by accelerating clearance of the virus and resolution of the disease.

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.

Effect of pancreas disease caused by SAV 2 on protein and fat digestion in Atlantic salmon.

Salmonid alphavirus (SAV) causes pancreas disease (PD) in farmed Atlantic salmon (Salmo salar L.), and exocrine pancreas tissue is a primary target of the virus. Digestive enzymes secreted by the exocrine pancreas break down macromolecules in feed into smaller molecules that can be absorbed. The effect of SAV infection on digestion has been poorly studied. In this study, longitudinal observations of PD outbreaks caused by SAV subtype 2 (SAV2) in Atlantic salmon at two commercial sea sites were performed. The development of PD was assessed by measurement of SAV2 RNA load and evaluation of histopathological lesions typical of PD. Reduced digestion of both protein and fat co-varied with the severity of PD lesions and viral load. Also, the study found that during a PD outbreak, the pen population comprise several subpopulations, with different likelihoods of being sampled. The body length of sampled fish deviated from the expected increase or steady state over time, and the infection status in sampled fish deviated from the expected course of infection in the population. Both conditions indicate that disease status of the individual fish influenced the likelihood of being sampled, which may cause sampling bias in population studies.

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.

Molecular tracing confirms that infection with infectious pancreatic necrosis virus follows the smolt from hatchery to grow-out farm.

Infectious pancreatic necrosis (IPN) is an important restraint to production of salmonids in aquaculture globally. In order to implement efficacious mitigation strategies for control of this disease, it is important to understand infection routes under current production systems. IPN virus has been shown to be transmitted vertically in Rainbow trout, from broodstock to fingerlings in hatcheries, and there is circumstantial evidence suggesting that vertical transmission can also occur in Atlantic salmon, in addition to horizontal transmission between grow-out fish in farms. In this study, we show that the smolt carries infection with IPN from hatchery to the marine farm. We do this by comparing sequences from fish groups taken both in hatcheries and on corresponding marine grow-out farms. We use statistical analysis to prove that sequences obtained from the same fish group in both hatchery and marine farm are more similar than sequences obtained from random fish groups on hatcheries and marine farms.

Type 1 Diabetes and Type 1 Interferonopathies: Localization of a Type 1 Common Thread of Virus Infection in the Pancreas.

Type 1 diabetes (T1D) has been associated with both genetic and environmental factors. Increasing incidence of T1D worldwide is prompting researchers to adopt different approaches to explain the biology of T1D, beyond the presence and activity of autoreactive lymphocytes. In this review, we propose inflammatory pathways as triggers for T1D. Within the scope of those inflammatory pathways and in understanding the pathogenesis of disease, we suggest that viruses, in particular Coxsackieviruses, act by causing a type 1 interferonopathy within the pancreas and the microenvironment of the islet. As such, this connection and common thread represents an exciting platform for the development of new diagnostic, treatment and/or prevention options.

Immune gene profiles in Atlantic salmon (salmo salar L.) post-smolts infected with SAV3 by bath-challenge show a delayed response and lower levels of gene transcription compared to injected fish.

Salmonid alphavirus (SAV) causes pancreatic disease (PD) in salmonids in Northern Europe which results in large economic losses within the aquaculture industry. In order to better understand the underlying immune mechanisms during a SAV3 infection Atlantic salmon post-smolts were infected by either i.m.-injection or bath immersion and their immune responses compared. Analysis of viral loads showed that by 14 dpi i.m.-injected and bath immersion groups had 95.6% and 100% prevalence respectively and that both groups had developed the severe pathology typical of PD. The immune response was evaluated by using RT-qPCR to measure the transcription of innate immune genes involved in the interferon (IFN) response as well as genes associated with inflammation. Our results showed that IFNa transcription was only weakly upregulated, especially in the bath immersion group. Despite this, high levels of the IFN-stimulated genes (ISGs) such as Mx and viperin were observed. The immune response in the i.m.-injected group as measured by immune gene transcription was generally faster, and more pronounced than the response in the bath immersion group, especially at earlier time-points. The response in the bath immersion group started later as expected and appeared to last longer often exceeding the response in the i.m-injected fish at later time-points. High levels of transcription of many genes indicative of an active innate immune response were present in both groups.

Selective precipitation reaction: a novel diagnostic test for tissue pathology in Atlantic salmon, Salmo salar, infected with salmonid alphavirus (SAV3).

While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancreas disease (PD), a selective precipitation reaction (SPR) has been discovered in serum which could be an on-farm qualitative test and an in-laboratory quantitative assay for health assessments in aquaculture. Mixing serum from Atlantic salmon, Salmo salar, with SAV infection with a sodium acetate buffer caused a visible precipitation which does not occur with serum from healthy salmon. Proteomic examination of the precipitate has revealed that the components are a mix of muscle proteins, for example enolase and aldolase, along with serum protein such as serotransferrin and complement C9. The assay has been optimized for molarity, pH, temperature and wavelength so that the precipitation can be measured as the change in optical density at 340 nm (Δ340 ). Application of the SPR assay to serum samples from a cohabitation trial of SAV infection in salmon showed that the Δ340 in infected fish rose from undetectable to a maximum at 6 weeks post-infection correlating with histopathological score of pancreas, heart and muscle damage. This test may have a valuable role to play in the diagnostic evaluation of stock health in salmon.