Experimental Piscine orthoreovirus infection mediates protection against pancreas disease in Atlantic salmon (Salmo salar).
ABSTRACT: Viral diseases are among the main challenges in farming of Atlantic salmon (Salmo salar). The most prevalent viral diseases in Norwegian salmon aquaculture are heart and skeletal muscle inflammation (HSMI) caused by Piscine orthoreovirus (PRV), and pancreas disease (PD) caused by Salmonid alphavirus (SAV). Both PRV and SAV target heart and skeletal muscles, but SAV additionally targets exocrine pancreas. PRV and SAV are often present in the same locations and co-infections occur, but the effect of this crosstalk on disease development has not been investigated. In the present experiment, the effect of a primary PRV infection on subsequent SAV infection was studied. Atlantic salmon were infected with PRV by cohabitation, followed by addition of SAV shedder fish 4 or 10 weeks after the initial PRV infection. Histopathological evaluation, monitoring of viral RNA levels and host gene expression analysis were used to assess disease development. Significant reduction of SAV RNA levels and of PD specific histopathological changes were observed in the co-infected groups compared to fish infected by SAV only. A strong correlation was found between histopathological development and expression of disease related genes in heart. In conclusion, experimentally PRV infected salmon are less susceptible to secondary SAV infection and development of PD.
Project description: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 (?340 ). Application of the SPR assay to serum samples from a cohabitation trial of SAV infection in salmon showed that the ?340 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.
Project description:Triploid Atlantic salmon (Salmo salar L.) may play an important role in the sustainable expansion of the Norwegian aquaculture industry. Therefore, the susceptibility of triploid salmon to common infections such as salmonid alphavirus (SAV), the causative agent of pancreas disease (PD), requires investigation. In this study, shortly after seawater transfer, diploid and triploid post-smolts were exposed to SAV type 3 (SAV3) using a bath challenge model where the infectious dose was 48 TCID50 ml-1 of tank water. Copy number analysis of SAV3 RNA in heart tissue showed that there was no difference in viral loads between the diploids and triploids. Prevalence reached 100% by the end of the 35-day experimental period in both infected groups. However, prevalence accumulated more slowly in the triploid group reaching 19% and 56% at 14 and 21 days post exposure (dpe) respectively. Whereas prevalence in the diploid group was 82% and 100% at the same time points indicating some differences between diploid and triploid fish. Both heart and pancreas from infected groups at 14 dpe showed typical histopathological changes associated with pancreas disease. Observation of this slower accumulation of prevalence following a natural infection route was possible due to the early sampling points and the exposure to a relatively low dose of virus. The triploid salmon in this study were not more susceptible to SAV3 than diploid salmon indicating that they could be used commercially to reduce the environmental impact of escaped farmed fish interbreeding with wild salmon. This is important information regarding the future use of triploid fish in large scale aquaculture where SAV3 is a financial threat to increased production.
Project description:Infectious hematopoietic necrosis virus (IHNV) is endemic in farmed rainbow trout in continental Europe and in various salmonid fish species at the Pacific coast of North America. IHN has never occurred in European Atlantic salmon (Salmo salar) farms, but is considered as a major threat for the European salmon industry. Another virus, Piscine orthoreovirus (PRV), is widespread in the sea phase of Atlantic salmon, and is identified as the causative agent of heart and skeletal muscle inflammation. The aim of this study was to investigate the interactions between a primary PRV infection and a secondary IHNV infection under experimental conditions. A PRV cohabitation challenge was performed with Atlantic salmon. At peak of PRV viremia the fish were challenged by immersion with an IHNV genogroup E isolate. Clinical signs and morbidity were monitored. Target organs were sampled at selected time points to assess viral loads of both pathogens. Antiviral immune response and presence of histopathological findings were also investigated. Whereas the PRV-negative/IHNV positive group suffered significant decrease in survival caused by IHNV, the PRV infected groups did not suffer any morbidity and showed negligible levels of IHNV infection. Antiviral response genes were induced, as measured in spleen samples, from PRV infected fish prior to IHNV challenge. In conclusion, PRV-infection protects Atlantic salmon against IHNV infection and morbidity, most likely by inducing a protective innate antiviral response.
Project description:Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-?, Mx and ISG15. IFN-? and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-?, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3?, CD4, CD8, TCR-? and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.
Project description:Heart and skeletal muscle inflammation (HSMI) is associated with Piscine orthoreovirus (PRV) infection and is an important disease in Atlantic salmon (Salmo salar) aquaculture. Since PRV infects erythrocytes and farmed salmon frequently experience environmental hypoxia, the current study examined mutual effects of PRV infection and hypoxia on pathogenesis and fish performance. Furthermore, effects of HSMI on hypoxia tolerance, cardiorespiratory performance and blood oxygen transport were studied. A cohabitation trial including PRV-infected post-smolts exposed to periodic hypoxic stress (4 h of 40% O2; PRV-H) at 4, 7 and 10 weeks post-infection (WPI) and infected fish reared under normoxic conditions (PRV) was conducted. Periodic hypoxic stress did not influence infection levels or histopathological changes in the heart. Individual incipient lethal oxygen saturation (ILOS) was examined using a standardized hypoxia challenge test (HCT). At 7 WPI, i.e. peak level of infection, both PRV and PRV-H groups exhibited reduced hypoxia tolerance compared to non-infected fish. Three weeks later (10 WPI), during peak levels of pathological changes, reduced hypoxia tolerance was still observed for the PRV group while PRV-H performed equal to non-infected fish, implying a positive effect of the repeated exposure to hypoxic stress. This was in line with maximum heart rate (fHmax) measurements, showing equal performance of PRV-H and non-infected groups, but lower fHmax above 19°C as well as lower temperature optimum (Topt) for aerobic scope for PRV, suggesting reduced cardiac performance and thermal tolerance. In contrast, the PRV-H group had reduced hemoglobin-oxygen affinity compared to non-infected fish. In conclusion, Atlantic salmon suffering from HSMI have reduced hypoxia tolerance and cardiac performance, which can be improved by preconditioning fish to transient hypoxic stress episodes.
Project description:Viral diseases pose a significant threat to the productivity in aquaculture. Heart- and skeletal muscle inflammation (HSMI) is an emerging disease in Atlantic salmon (Salmo salar) farming. HSMI is associated with Piscine orthoreovirus (PRV) infection, but PRV is ubiquitous in farmed Atlantic salmon and thus present also in apparently healthy individuals. This has brought speculations if additional etiological factors are required, and experiments focusing on the causal relationship between PRV and HSMI are highly warranted. A major bottleneck in PRV research has been the lack of cell lines that allow propagation of the virus. To bypass this, we propagated PRV in salmon, bled the fish at the peak of the infection, and purified virus particles from blood cells. Electron microscopy, western blot and high-throughput sequencing all verified the purity of the viral particles. Purified PRV particles were inoculated into naïve Atlantic salmon. The purified virus replicated in inoculated fish, spread to naïve cohabitants, and induced histopathological changes consistent with HSMI. PRV specific staining was demonstrated in the pathological lesions. A dose-dependent response was observed; a high dose of virus gave earlier peak of the viral load and development of histopathological changes compared to a lower dose, but no difference in the severity of the disease. The experiment demonstrated that PRV can be purified from blood cells, and that PRV is the etiological agent of HSMI in Atlantic salmon.
Project description:Aquaculture is the fastest growing food production sector in the world. However, the increased production has been accompanied by the emergence of infectious diseases. Heart and skeletal muscle inflammation (HSMI) is one example of an emerging disease in farmed Atlantic salmon (Salmo salar L). Since the first recognition as a disease entity in 1999 it has become a widespread and economically important disease in Norway. The disease was recently found to be associated with infection with a novel reovirus, piscine reovirus (PRV). The load of PRV, examined by RT-qPCR, correlated with severity of HSMI in naturally and experimentally infected salmon. The disease is characterized by epi-, endo- and myocarditis, myocardial necrosis, myositis and necrosis of the red skeletal muscle. The aim of this study was to investigate the presence of PRV antigens in heart tissue of Atlantic salmon and monitor the virus distribution in the heart during the disease development. This included target cell specificity, viral load and tissue location during an HSMI outbreak. Rabbit polyclonal antisera were raised against putative PRV capsid proteins ?1C and ?1 and used in immunohistochemical analysis of archived salmon heart tissue from an experimental infection. The results are consistent with the histopathological changes of HSMI and showed a sequential staining pattern with PRV antigens initially present in leukocyte-like cells and subsequently in cardiomyocytes in the heart ventricle. Our results confirm the association between PRV and HSMI, and strengthen the hypothesis of PRV being the causative agent of HSMI. Immunohistochemical detection of PRV antigens will be beneficial for the understanding of the pathogenesis of HSMI as well as for diagnostic purposes.
Project description:Piscine orthoreovirus (PRV) mediated diseases have emerged throughout salmonid aquaculture. Three PRV subtypes are currently reported as causative agents of or in association with diseases in different salmonid species. PRV-1 causes heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) and is associated with jaundice syndrome in farmed chinook salmon (Oncorhynchus tshawytscha). PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon in Japan. PRV-3 has recently been associated with a disease in rainbow trout (Oncorhynchus mykiss) characterized by anaemia, heart and red muscle pathology; to jaundice syndrome in coho salmon (Oncorhynchus kisutch). In this study, we conducted a 10-week long experimental infection trial in rainbow trout with purified PRV-3 particles to assess the causal relationship between the virus and development of heart inflammation. The monitoring the PRV-3 load in heart and spleen by RT-qPCR shows a progressive increase of viral RNA to a peak, followed by clearance without a measurable change in haematocrit. The development of characteristic cardiac histopathological findings occurred in the late phase of the trial and was associated with increased expression of CD8+, indicating cytotoxic T cell proliferation. The findings indicate that, under these experimental conditions, PRV-3 infection in rainbow trout act similarly to PRV-1 infection in Atlantic salmon with regards to immunological responses and development of heart pathology, but not in the ability to establish a persistent infection.
Project description:Piscine orthoreovirus (PRV) is a reovirus that has predominantly been detected in Atlantic salmon (Salmo salar L.). PRV is associated with heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon, and recently erythrocytes were identified as major target cells. The study of PRV replication and pathogenesis of the infection has been impeded by the inability to propagate PRV in vitro. In this study we developed an ex vivo cultivation system for PRV in Atlantic salmon erythrocytes. PRV was successfully passaged to naïve erythrocytes using lysates of blood cells from infected salmon. During cultivation a significant increase in viral load was observed by RT-qPCR and flow cytometry, which coincided with the formation of cytoplasmic inclusions. The inclusions resembled viral factories and contained both PRV protein and dsRNA. In addition, the erythrocytes generated an antiviral immune gene activation after PRV infection, with significant up-regulation of IFN-?, RIG-I, Mx and PKR transcripts. Supernatants from the first passage successfully transmitted virus to naïve erythrocytes. This study demonstrates that PRV replicates in Atlantic salmon erythrocytes ex vivo. The ex vivo infection model closely reflects the situation in vivo and can be used to study the infection and replication mechanisms of PRV, as well as the antiviral immune responses of salmonid erythrocytes.
Project description:BACKGROUND:Piscine orthoreovirus (PRV) is an emergent virus in salmon aquaculture belonging to the family Reoviridae. PRV is associated with a growing list of pathological conditions including heart and skeletal inflammation (HSMI) of farmed Atlantic salmon. Despite widespread PRV infection in commercially farmed Atlantic salmon, information on PRV prevalence and on the genetic sequence variation of PRV in Atlantic salmon on the north Pacific Coast is limited. METHODS:Feral Atlantic salmon caught in Washington State and British Columbia following a large containment failure at a farm in northern Puget Sound were sampled. Fish tissues were tested for PRV by RT-qPCR assay for segment L1 and conventional RT-PCR for PRV segment S1. The PCR products were sequenced and their relationship to PRV strains in GenBank was determined using phylogenetic analysis and nucleotide and amino acid homology comparisons. RESULTS:Following the escape of 253,000 Atlantic salmon from a salmon farm in Washington State, USA, 72/73 tissue samples from 27 Atlantic salmon captured shortly after the escape tested PRV-positive. We estimate PRV-prevalence in the source farm population at 95% or greater. The PRV found in the fish was identified as PRV sub-genotype Ia and very similar to PRV from farmed Atlantic salmon in Iceland. This correlates with the source of the fish in the farm. Eggs of infected fish were positive for PRV indicating the possibility of vertical transfer and spread with fish egg transports. CONCLUSIONS:PRV prevalence was close to 100% in farmed Atlantic salmon that were caught in Washington State and British Columbia following a large containment failure at a farm in northern Puget Sound. The PRV strains present in the escaped Atlantic salmon were very similar to the PRV strain reported in farmed Atlantic salmon from the source hatchery in Iceland that was used to stock commercial aquaculture sites in Washington State. This study emphasizes the need to screen Atlantic salmon broodstock for PRV, particularly where used to supply eggs to the global Atlantic salmon farming industry thereby improving our understanding of PRV epidemiology.