Infected Atlantic Salmon Research Articles

Bayesian latent class analysis of diagnostic sensitivity and specificity of tests for surveillance for bacterial kidney disease in Atlantic salmon Salmo salar

Bacterial kidney disease (BKD) diagnosis is often carried out by IFAT, ELISA, and PCR because these tests yield rapid results compared with bacterial culture for the causative agent, Renibacterium salmoninarum. However, the diagnostic sensitivity (DSe) and specificity (DSp) of these tests is generally unknown, particularly in subclinically infected Atlantic salmon. Bayesian models were used to analyse test results from 454 field samples sourced from salmon populations with different infection status and submitted to independent laboratories. Results showed that DSe was highly dependent on the presentation of infection. When applied to fish with BKD-associated gross lesions, the highest to lowest posterior medians for DSe were: PCR2 (0.98), PCR1 (0.97), bacterial culture (0.86), ELISA (0.82) and IFAT (0.75). When applied to fish without lesions, the posterior medians for DSe were: PCR1 (0.82), PCR2 (0.82), bacterial culture (0.29), IFAT (0.29) and ELISA (0.21). The posterior medians for DSp were similar in populations with and without lesions. ELISA had the highest DSp (0.99), followed by culture (0.98–0.99), IFAT (0.96), PCR1 (0.94–0.96) and PCR2 (0.83–0.84), where different numbers reflect the estimates for populations with or without lesions respectively. Among tests that were evaluated for intra-laboratory repeatability (ELISA, PCR1 and IFAT), ELISA had the highest repeatability (kappa=0.82). We concluded that PCR tests were the fittest of the evaluated tests for the purpose of screening subclinically-infected adult Atlantic salmon.

Immunological interactions between Piscine orthoreovirus and Salmonid alphavirus infections in Atlantic salmon

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

Involvement of LuxS in Aeromonas salmonicida metabolism, virulence and infection in Atlantic salmon (Salmo salar L)

Quorum sensing is a bacterial density dependent communication system, which regarded to regulate co-operative behaviors of community and mediated by extracellular signal molecules named autoinducers (AI). Among various signals, autoinducer-2 (AI-2) is believed to be the messengers inter species and produced by LuxS. For Aeromonas salmonicida (A. salmonicida), an opportunistic pathogen to many cold-water teleost, little information has been known about the function of AI-2 and LuxS. Therefore, our aim was to preliminarily clarify the function of LuxS in A. salmonicida. The consequences demonstrated that wild type A. salmonicida exhibited AI-2 activity and luxS defective mutant strain fail to produce AI-2 signals. Furthermore, it was suggested that luxS deficiency could impact bacterial morphology, surface properties and virulence dramatically. Challenge experiment showed a tendency that immune factors expressed earlier when Atlantic salmon was infected with ΔluxS strain. Overall, we hypothesis that AI-2 quorum sensing could regulate the expression of A-layer protein coding gene vapA, and then influence bacterial survival ability when suffered from attack of the host immune system. Though additional studies are warranted, our study will supply a new thinking to control the damage caused by A. salmonicida.

Nutrigenomic effects of glucosinolates on liver, muscle and distal kidney in parasite-free and salmon louse infected Atlantic salmon.

BackgroundReduction of Lepeophtheirus salmonis infection in Atlantic salmon achieved by glucosinolates (GLs) from Brassica plants was recently reported. However, wider application of functional feeds based on GLs requires better knowledge of their positive and adverse effects.MethodsLiver, distal kidney and muscle transcriptomes of salmon exposed to the extreme dose of GLs were profiled by microarray, while qPCR analysis followed up selected hepatic and renal responses under the extreme and moderate GLs dose during the L. salmonis challenge. Transcriptional analysis were complemented with measurements of organ indices, liver steatosis and plasma profiling, including indicators of cytolysis and bilirubin. Finally, the third trial was performed to quantify the effect of lower GLs doses on growth.ResultsThe extreme GLs dose caused a decrease in hepatic fat deposition and growth, in line with microarray findings, which suggested tissue remodeling and reduction of cellular proliferation in the skeletal muscle and liver. Lower GLs inclusion levels in a follow-up trial did not show negative effects on growth. Microarray analysis of the distal kidney pointed to activation of anti-fibrotic responses under the overexposure. However, analyses of ALT, CK and AST enzymes in plasma provided no evidence of increased cytolysis and organ damage. Prevalent activation of phase-2 detoxification genes that occurred in all three tissues could be considered part of beneficial effects caused by the extreme dose of GLs. In addition, transcriptomic evidence suggested GLs-mediated iron and heme withdrawal response, including increased heme degradation in muscle (upregulation of heme oxygenase-1), decrease of its synthesis in liver (downregulation of porphobilinogen deaminase) and increased iron sequestration from blood (hepatic induction of hepcidin-1 and renal induction of intracellular storage protein ferritin). This response could be advantageous for salmon upon encountering lice, which depend on the host for the provision of iron carrying heme. Most of the hepatic genes studied by qPCR showed similar expression levels in fish exposed to GLs, lice and their combination, while renal induction of leptin suggested heightened stress by the combination of extreme dose of GLs and lice. High expression of interferon γ (cytokine considered organ-protective in mammalian kidney) was detected at the moderate GLs level. This fish also showed highest plasma bilirubin levels (degradation product of heme), and had lowest number of attached lice, further supporting hypothesis that making heme unavailable to lice could be part of an effective anti-parasitic strategy.ConclusionsModulation of detoxification and iron metabolism in Atlantic salmon tissues could be beneficial prior and during lice infestations. Investigation of anti-lice functional feeds based on low and moderate GLs inclusion levels thus deserves further attention.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1921-7) contains supplementary material, which is available to authorized users.

Iron metabolism modulation in Atlantic salmon infested with the sea lice Lepeophtheirus salmonis and Caligus rogercresseyi: A matter of nutritional immunity?

Sea lice are copepodid ectoparasites that produce high economic losses and environmental issues, thus impacting the salmon aquaculture worldwide. Atlantic salmon (Salmo salar) from Northern and Southern Hemispheres are primarily parasitized by Lepeophtheirus salmonis and Caligus rogercresseyi, respectively. To cope L. salmonis infestation, studies suggest that Atlantic salmon can restrict iron availability as a mechanism of nutritional immunity. However, no molecular studies of iron regulation from salmonids infected with C. rogercresseyi have been reported. The aim of this study was to determine if there are differences in the regulation of iron metabolism in Atlantic salmon infested with L. salmonis or C. rogercresseyi. For comparisons, skin and head kidney were profiled using qPCR of 15 genes related to iron regulation in Atlantic salmons infected with each sea louse species in Norway and Chile, respectively. Prior to infestation, no significant differences were observed between fish group. However, genes involved in iron transport and Heme biosynthesis were highly upregulated in Atlantic salmon infested with L. salmonis. Interestingly, hepcidin and Heme oxygenase, a component of the Heme degradation pathway, were upregulated during C. rogercresseyi infestation. Oxidative stress related genes were also evaluated, showing higher transcription activity in the head kidney than in the skin of Atlantic salmon infested with L. salmonis. These comparative results suggest pathogen-specific responses in infected Atlantic salmon, where iron metabolism is primarily regulated during the infestation with L. salmonis than C. rogercresseyi. Feeding behavior, for instance haematophagy, of the infesting sea lice species in relation to iron modulation is discussed.

Relationship between viral dose and outcome of infection in Atlantic salmon, Salmo salar L., post-smolts bath-challenged with salmonid alphavirus subtype 3.

Salmonid alphavirus subtype 3 (SAV3) causes pancreas disease (PD) and adversely affects salmonid aquaculture in Europe. A better understanding of disease transmission is currently needed in order to manage PD outbreaks. Here, we demonstrate the relationship between viral dose and the outcome of SAV3 infection in Atlantic salmon post-smolts using a bath challenge model. Fish were challenged at 12 °C with 3 different SAV3 doses; 139, 27 and 7 TCID50 L−1 of seawater. A dose of as little as 7 TCID50 L−1 of seawater was able to induce SAV3 infection in the challenged population with a substantial level of variation between replicate tanks and, therefore, likely represents a dose close to the minimum dose required to establish an infection in a population. These data also confirm the highly infectious nature of SAV through horizontal transmission. The outcome of SAV3 infection, evaluated by the prevalence of viraemic fish, SAV3-positive hearts, and the virus shedding rate, was positively correlated to the original SAV3 dose. A maximal shedding rate of 2.4 × 104 TCID50 L−1 of seawater h−1 kg−1 was recorded 10 days post-exposure (dpe) from the highest dose group. The method reported here, for the quantification of infectious SAV3 in seawater, could be useful to monitor PD status or obtain data from SAV3 outbreaks at field locations. This information could be incorporated into pathogen dispersal models to improve risk assessment and to better understand how SAV3 spreads between farms during outbreaks. This information may also provide new insights into the control and mitigation of PD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0385-2) contains supplementary material, which is available to authorized users.

Infectious Pancreatic Necrosis Virus Causing Clinical and Subclinical Infections in Atlantic Salmon Have Different Genetic Fingerprints.

Infectious pancreatic necrosis virus (IPNV) is the causative agent of IPN, an important disease of salmonids. IPNV infections result in either sub-clinical or overt disease and the basis of this difference is not well-understood. The objective of the present study was to determine the VP2 gene of the virus associated with the different forms of clinical manifestation. Groups of Atlantic salmon (Salmo salar L.) reared in farms located in different IPN disease pressures were monitored from brood stock until grow-out over a 3 year period. Hatcheries A1 and B1 as well as cooperating seawater farms were located in a low disease risk area while hatcheries A2 and B2 as well as their cooperating seawater farms were in high IPN risk areas. Samples including eggs, milt, whole fry, kidney depending on the stage of production were collected during outbreaks or in apparently healthy populations where no outbreaks occurred. The virus was re-isolated in CHSE cells and the VP2 gene amplified by RT-PCR followed by sequencing. During the freshwater stage, there were no disease outbreaks at hatcheries A1, A2, and B1 (except in one fish group that originated from hatchery B2), although IPNV was isolated from some of the fish groups at all 3 hatcheries. By contrast, all fish groups at hatchery B2 suffered IPN outbreaks. In seawater, only groups of fish originating from hatchery A1 had no IPN outbreaks albeit virus being isolated from the fish. On the other hand, fish originating from hatcheries A2, B1, and B2 experienced outbreaks in seawater. The VP2 amino acid fingerprint of the virus associated with subclinical infections from A1 and co-operating seawater sites was V64A137P217T221A247N252S281D282E319. By contrast, all virus isolates associated with clinical infections had the motif I64T137T217A221T247V252T281N282A319, where underlined amino acids represent the avirulent and highly virulent motif, respectively. Phylogenetic analysis of amino acid sequences showed 2 clades, one of isolates associated with subclinical infections (from A1 and cooperating seawater farms) and the other of isolates from fish with overt disease (all other sites). Furthermore, the clustering pattern of isolates suggests more circulation of virus within fish groups rather than between them.

Effects of lumpfish size on foraging behaviour and co-existence with sea lice infected Atlantic salmon in sea cages

To assess possible size effects of foraging of lumpfish and co-existence with Atlantic salmon with particular interest to the sea lice grazing efficiency, eight sea cages (5×5×5m) were stocked with 150 Atlantic salmon with a mean (±SD) weight of 538±14g. Six of the cages were stocked with 15 lumpfish each (10% density), with two cages for each of three different size groups of lumpfish. Three duplicate groups of lumpfish with an initial mean (±SD) weight of 22.6±0.7g (small), 77.4±3.6g (medium) and 113.5±2.1g (large) were used in the study. Two cages without lumpfish acted as controls. Sea lice infestation levels were recorded at two to four week intervals for 159days. To determine the diet preferences of lumpfish in the cages gastric lavage was performed at the same time intervals. Behaviour and growth of the lumpfish was assessed throughout the study period and mean weight of the Atlantic salmon measured at the start and end of the study period. From day 35 and onwards growth was higher for the small lumpfish group compared to the two other lumpfish size classes. Lumpfish from the smallest size class had a higher consumption of naturally occurring food items, including sea lice, compared to the other two size classes. Growth stimulation in salmon co-habiting the two smallest lumpfish size groups was observed. Signs of sexual maturation were found in the medium (13%) and the large (20%) size groups. Based on present data small lumpfish (initial size approx. 20g) have a higher overall preference for natural food items compared to larger conspecifics. Although the sea lice infestation rate was low in the study (<0.5licesalmon−1) final lice burden was 40% lower in salmon groups stocked with small lumpfish compared to the control group without lumpfish. Statement of relevanceThe data presented here are highly relevant for Aquaculture as the effective use of lumpfish for biological delousing of salmon is very important for commercial aquaculture of Atlantic salmon.

Yersinia ruckeri Isolates Recovered from Diseased Atlantic Salmon (Salmo salar) in Scotland Are More Diverse than Those from Rainbow Trout (Oncorhynchus mykiss) and Represent Distinct Subpopulations.

Yersinia ruckeri is the etiological agent of enteric redmouth (ERM) disease of farmed salmonids. Enteric redmouth disease is traditionally associated with rainbow trout (Oncorhynchus mykiss, Walbaum), but its incidence in Atlantic salmon (Salmo salar) is increasing. Yersinia ruckeri isolates recovered from diseased Atlantic salmon have been poorly characterized, and very little is known about the relationship of the isolates associated with these two species. Phenotypic approaches were used to characterize 109 Y. ruckeri isolates recovered over a 14-year period from infected Atlantic salmon in Scotland; 26 isolates from infected rainbow trout were also characterized. Biotyping, serotyping, and comparison of outer membrane protein profiles identified 19 Y. ruckeri clones associated with Atlantic salmon but only five associated with rainbow trout; none of the Atlantic salmon clones occurred in rainbow trout and vice versa These findings suggest that distinct subpopulations of Y. ruckeri are associated with each species. A new O serotype (designated O8) was identified in 56 biotype 1 Atlantic salmon isolates and was the most common serotype identified from 2006 to 2011 and in 2014, suggesting an increased prevalence during the time period sampled. Rainbow trout isolates were represented almost exclusively by the same biotype 2, serotype O1 clone that has been responsible for the majority of ERM outbreaks in this species within the United Kingdom since the 1980s. However, the identification of two biotype 2, serotype O8 isolates in rainbow trout suggests that vaccines containing serotypes O1 and O8 should be evaluated in both rainbow trout and Atlantic salmon for application in Scotland. Vaccination plays an important role in protecting Atlantic salmon against the bacterial pathogen Yersinia ruckeri, but, in recent years, there has been an increasing incidence of vaccine breakdown in salmon. This is largely because current vaccines are aimed at rainbow trout and are based on serotypes specific for this species. A wider range of serotypes is responsible for infection in Atlantic salmon, but very little is known about the diversity of these strains and their relationships to those recovered from rainbow trout. In the present study, we demonstrate that Y. ruckeri isolates recovered from diseased Atlantic salmon in Scotland are more diverse than those from rainbow trout; furthermore, isolates from the two species represent distinct subpopulations. In addition, a new O serotype was identified that is responsible for a significant proportion of the disease in Atlantic salmon. Our findings are likely to have important implications for the development of improved vaccines against Y. ruckeri.

Dietary phytochemicals modulate skin gene expression profiles and result in reduced lice counts after experimental infection in Atlantic salmon.

BackgroundThe use of phytochemicals is a promising solution in biological control against salmon louse (Lepeophtheirus salmonis). Glucosinolates belong to a diverse group of compounds used as protection against herbivores by plants in the family Brassicaceae, while in vertebrates, ingested glucosinolates exert health-promoting effects due to their antioxidant and detoxifying properties as well as effects on cell proliferation and growth. The aim of this study was to investigate if Atlantic salmon fed two different doses of glucosinolate-enriched feeds would be protected against lice infection. The effects of feeding high dose of glucosinolates before the infection, and of high and low doses five weeks into the infection were studied.MethodsSkin was screened by 15 k oligonucleotide microarray and qPCR.ResultsA 25 % reduction (P < 0.05) in lice counts was obtained in the low dose group and a 17 % reduction in the high dose group compared to fish fed control feed. Microarray analysis revealed induction of over 50 interferon (IFN)-related genes prior to lice infection. Genes upregulated five weeks into the infection in glucosinolate-enriched dietary groups included Type 1 pro-inflammatory factors, antimicrobial and acute phase proteins, extracellular matrix remodeling proteases and iron homeostasis regulators. In contrast, genes involved in muscle contraction, lipid and glucose metabolism were found more highly expressed in the skin of infected control fish.ConclusionsAtlantic salmon fed glucosinolates had a significantly lower number of sea lice at the end of the experimental challenge. Feeding glucosinolates coincided with increased expression of IFN-related genes, and higher expression profiles of Type 1 immune genes late into the infection. In addition, regulation of genes involved in the metabolism of iron, lipid and sugar suggested an interplay between metabolism of nutrients and mechanisms of resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1537-y) contains supplementary material, which is available to authorized users.

Atlantic salmon physiological and immune response to amoebic gill disease and insight into the biology of the amoeba

Amoebic gill disease (AGD) is an emerging disease in North European Atlantic salmon (Salmo salar) aquaculture caused by the amoeba Paramoeba perurans. Non-optimal environmental conditions such as increasing water temperature may affect AGD progression. To understand the role of predisposing environmental conditions on the biology of the parasite and on the host physiological and immune response, different clonal cultures of P. perurans and AGD infected Atlantic salmon were exposed, respectively, in vitro and in vivo to two different temperatures, 10°C and 15°C. Outputs from the in vitro experiment are 1) an improved understanding of the impact of temperature on amoebae growth rate during conditions that potentially influence disease development and 2) characterisation of changes in bacterial communities at different temperatures for different clonal cultures. Negative binomial analysis in R (R software, version 3.0.1) showed significant differences in growth rate among clonal cultures over time. Moreover, for each clonal culture, the fraction of amoebae in suspension in seawater showed a higher increase over time at 10°C, whereas the fraction of amoebae attached to the malt yeast agar (MYA) showed a higher increase over time at 15°C. 16S MiSeq analysis was also performed to characterise the changes in bacterial communities present in the cultures at the two different temperatures of the in vitro experiment. The host physiological and immune response to AGD infection during predisposing seasonal weather conditions (summer period) is still not well understood. Atlantic salmon smolts were exposed in vivo to the same temperatures used for the in vitro experiment, 10°C and 15°C, and to an initial concentration of 5000 cells l of a clonal culture of P. perurans for 3 weeks. Analyses of cortisol, glucose, and lactate concentration in plasma samples, white blood cell (wbc) differential count in blood smears, and gene expression have been performed at different time points after the challenge. The present results from the temperature dependent AGD infection and in vitro P. perurans growth rate studies hint that increased AGD outbreaks during summer periods may not be solely due to increased thermal stress in fish but also to increased amoebae attachment at 15°C which cause an increased gill pathology.

Investigations on the role of the salmon louse, Lepeophtheirus salmonis (Caligidae), as a vector in the transmission of Aeromonas salmonicida subsp. salmonicida.

A bacteria-parasite challenge model was used to study the role of sea lice, Lepeophtheirus salmonis (Copepoda), as a vector of Aeromonas salmonicida subsp. salmonicida. Three hypotheses were tested: (i) L.salmonis can acquire A.salmonicida subsp. salmonicida via water bath exposure; (ii) L.salmonis can acquire the bacteria via parasitizing infected Atlantic salmon, Salmo salar; and (iii) L.salmonis can transmit the bacteria to naïve Atlantic salmon via parasitism. Adult L.salmonis exposed to varying A.salmonicida subsp. salmonicida suspensions (10(1) -10(7) cellsmL(-1) ) for 1.0, 3.0 or 6.0h acquired the bacteria externally (12.5-100%) and internally (10.0-100%), with higher prevalences associated with the highest concentrations and exposures. After exposure to 10(7) cellsmL(-1) , viable A.salmonicida subsp. salmonicida could be isolated from the external carapace of L.salmonis for 120h. Lepeophtheirus salmonis also acquired the bacteria externally and internally from parasitizing infected fish. Bacterial transmission was observed only when L.salmonis had acquired the pathogen internally via feeding on 'donor fish' and then by parasitizing smaller (<50g) 'naive' fish. Under specific experimental conditions, L.salmonis can transfer A.salmonicida subsp. salmonicida via parasitism; however, its role as a mechanical or biological vector was not defined.

Infectious salmon anaemia virus (ISAV) mucosal infection in Atlantic salmon.

All viruses infecting fish must cross the surface mucosal barrier to successfully enter a host. Infectious salmon anaemia virus (ISAV), the causative agent of the economically important infectious salmon anaemia (ISA) in Atlantic salmon, Salmo salar L., has been shown to use the gills as its entry point. However, other entry ports have not been investigated despite the expression of virus receptors on the surface of epithelial cells in the skin, the gastrointestinal (GI) tract and the conjunctiva. Here we investigate the ISAV mucosal infection in Atlantic salmon after experimental immersion (bath) challenge and in farmed fish collected from a confirmed outbreak of ISA in Norway. We show for the first time evidence of early replication in several mucosal surfaces in addition to the gills, including the pectoral fin, skin and GI tract suggesting several potential entry points for the virus. Initially, the infection is localized and primarily infecting epithelial cells, however at later stages it becomes systemic, infecting the endothelial cells lining the circulatory system. Viruses of low and high virulence used in the challenge revealed possible variation in virus progression during infection at the mucosal surfaces.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0265-1) contains supplementary material, which is available to authorized users.

Correction: Characterization of a Novel Calicivirus Causing Systemic Infection in Atlantic Salmon (Salmo salar L.): Proposal for a New Genus of Caliciviridae

Correction: Characterization of a Novel Calicivirus Causing Systemic Infection in Atlantic Salmon (Salmo salar L.): Proposal for a New Genus of Caliciviridae

Antiviral and metabolic gene expression responses to viral infection in Atlantic salmon (Salmo salar)

Salmonid alphavirus (SAV), the aetiological agent of pancreas disease, is recognized as a serious pathogen of farmed Atlantic salmon. This disease results in loss of weight followed by poor growth of surviving fish, as such it is viewed as a wasting disease. SAV and other chronic disease causing viruses affect the heart and skeletal muscle tissues, at present the mechanisms by which pathology occurs is unknown. The relationship between antiviral activity and other physiological parameters especially in skeletal muscle are currently not examined in depth in fish. An experimental SAV (isotype 3) infection was carried out using a cohabitation approach, from which samples were collected at 0, 4, 8 & 12 week post challenge. Maximum viral load in the muscle tissue was 4 weeks post infection which was reduced at 8 weeks and undetectable by 12 weeks. Histopathology score peaked at 4 weeks post infection in pancreas and heart whereas there was maximum damage in skeletal muscle at 8 weeks. The peak expression of antiviral immune genes coincided with the viral load. Several genes involved in protein degradation were increased following infection including atrogin-1 and cathepsin D, at 4 weeks post challenge suggesting reallocation of amino acid reserves. Taken together, these observations increase our understanding of salmon poor growth during viral infection, and will serve as a basis to develop strategies to manage this viral wasting disease.

Characterization of a novel calicivirus causing systemic infection in atlantic salmon (Salmo salar L.): proposal for a new genus of caliciviridae.

The Caliciviridae is a family of viruses infecting humans, a wide range of animals, birds and marine fish and mammals, resulting in a wide spectrum of diseases. We describe the identification and genetic characterization of a novel calicivirus replicating in Atlantic salmon. The virus has a high prevalence in farmed salmon and is found in fish suffering from several diseases and conditions and also in presumable healthy fish. A challenge and vaccination trial shows that the calicivirus replicates in Atlantic salmon and establishes a systemic infection, which can be reduced by vaccination with formalin-inactivated virus preparation. The virus, named Atlantic salmon calicivirus (ASCV), is found in two genetically distinct variants, a cell culture isolated and a variant sequenced directly from field material. The genomes are 7,4 kb and contain two open reading frames where typical conserved amino acid motifs and domains predict a gene order reminiscent of calicivirus genomes. Phylogenetic analysis performed on extracted capsid amino acid sequences segregated the two ASCV variants in a unique cluster sharing root with the branch of noroviruses infecting humans and the unassigned Tulane virus and St-Valérien like viruses, infecting rhesus monkey and pig, respectively, with relatively large distance to the marine calicivirus subgroup of vesiviruses. Based on the analyses presented, the ASCV is predicted to represent a new genus of Caliciviridae for which we propose the name Salovirus.

Comparative transcriptomics of Atlantic Salmo salar, chum Oncorhynchus keta and pink salmon O. gorbuscha during infections with salmon lice Lepeophtheirus salmonis

BackgroundSalmon species vary in susceptibility to infections with the salmon louse (Lepeophtheirus salmonis). Comparing mechanisms underlying responses in susceptible and resistant species is important for estimating impacts of infections on wild salmon, selective breeding of farmed salmon, and expanding our knowledge of fish immune responses to ectoparasites. Herein we report three L. salmonis experimental infection trials of co-habited Atlantic Salmo salar, chum Oncorhynchus keta and pink salmon O. gorbuscha, profiling hematocrit, blood cortisol concentrations, and transcriptomic responses of the anterior kidney and skin to the infection.ResultsIn all trials, infection densities (lice per host weight (g)) were consistently highest on chum salmon, followed by Atlantic salmon, and lowest in pink salmon. At 43 days post-exposure, all lice had developed to motile stages, and infection density was uniformly low among species. Hematocrit was reduced in infected Atlantic and chum salmon, and cortisol was elevated in infected chum salmon. Systemic transcriptomic responses were profiled in all species and large differences in response functions were identified between Atlantic and Pacific (chum and pink) salmon. Pink and chum salmon up-regulated acute phase response genes, including complement and coagulation components, and down-regulated antiviral immune genes. The pink salmon response involved the largest and most diverse iron sequestration and homeostasis mechanisms. Pattern recognition receptors were up-regulated in all species but the active components were often species-specific. C-type lectin domain family 4 member M and acidic mammalian chitinase were specifically up-regulated in the resistant pink salmon.ConclusionsExperimental exposures consistently indicated increased susceptibility in chum and Atlantic salmon, and resistance in pink salmon, with differences in infection density occurring within the first three days of infection. Transcriptomic analysis suggested candidate resistance functions including local inflammation with cytokines, specific innate pattern recognition receptors, and iron homeostasis. Suppressed antiviral immunity in both susceptible and resistant species indicates the importance of future work investigating co-infections of viral pathogens and lice.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-200) contains supplementary material, which is available to authorized users.

PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture. To prevent and control outbreaks, a rapid, reproducible, sensitive, and effective diagnostic method is needed. We evaluated a new conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) protocol using a primer set (VohB_Fw-VohB_Rv) designed to amplify a 112 bp fragment flanking the vohB gene (coding for hemolysin production), against 24 V. ordalii strains isolated from different fish species, the V. ordalii type strain, and 42 representative related and unrelated bacterial species. The primer set was species-specific, recognizing all V. ordalii strains evaluated, with no cross-reaction with the other bacterial species. A sensitivity of 103 copies of the vohB gene was obtained with a standard curve. When the VohB_Fw-VohB_Rv qPCR protocol was applied to Atlantic salmon seeded tissues (kidney, liver, spleen, and muscle), the detection limit ranged from 5.27 × 102 to 4.13 × 103 V. ordalii CFU ml-1, i.e. 62 to 145 copies of the vohB gene, using the previously calculated standard curve. The conventional PCR also detected V. ordalii, but the total reaction time was 1 h longer. When the qPCR protocol was applied to naturally infected cage-cultured Atlantic salmon samples, 5 of 8 fish tested positive for V. ordalii, but only one of them was diagnosed as positive by direct cultivation on agar. We conclude that the PCR protocol evaluated is fast, specific, and sensitive enough to detect V. ordalii in infected tissues and is an important tool for secure diagnosis of atypical vibriosis, and is therefore helpful for the control of the disease through the prompt detection within fish populations.

Comparison of Host Selection and Gene Expression of Adult Lepeophtheirus Salmonis and Salmo Salar During a Cohabitation of Initially Infected and Uninfected Fish

Lepeophtheirus salmonis is a common parasite of salmonid fish and has a significant economic impact on Atlantic salmon (Salmo salar) fish farms. Over time L. salmonis has developed resistance to a number of chemotherapeutants, making the discovery of new treatments important to maintain a profitable farming industry. Determining processes in both L. salmonis and Atlantic salmon important to host selection and colonization may provide new targets for treatment development. During a two week cohabitation of L. salmonis infected and uninfected Atlantic salmon, we were able to collect information on the ability of L. salmonis to switch hosts, and preference for infected or uninfected fish. Whole L. salmonis and Atlantic salmon tissues were collected at 2 and 14 days post cohabitation to determine if differential gene expression was occurring during this process. At 2 days post cohabitation there was no significant difference in the number of male lice on the initially infected and uninfected fish. Eight L. salmonis genes putatively associated with various facets of lice survival (CYP18 A1-like, cytochrome p450 Isoform 1-like protein, glycene receptor α-2-like, leukocyte receptor cluster member 9-like, nicotinic acetylcholine receptor subunit-like, tissue plasminogen activator precursor-like, peroxinectin-like, and Trypsin-1) were analysed in both adult male and female lice, as well as five genes indicating immune status in Atlantic salmon. Comparisons were made to look for differential gene regulation as well as correlation between expression of L. salmonis genes and Atlantic salmon genes. Only MMP9 expression in salmon spleen was differentially regulated during the study period, however, correlations between the expression of several louse and salmon genes were found. Notably, the expression of a peroxinectin-like gene in male and female L. salmonis was correlated with the expression of IL-1, IL-12, IgT and matrix metalloproteinase 9 intermittently in salmon. This paper provides new insight into the interactions between L. salmonis and S. salar during infection.

Estimation of Parameters Influencing Waterborne Transmission of Infectious Hematopoietic Necrosis Virus (IHNV) in Atlantic Salmon (Salmo salar)

Understanding how pathogenic organisms spread in the environment is crucial for the management of disease, yet knowledge of propagule dispersal and transmission in aquatic environments is limited. We conducted empirical studies using the aquatic virus, infectious hematopoietic necrosis virus (IHNV), to quantify infectious dose, shedding capacity, and virus destruction rates in order to better understand the transmission of IHN virus among Atlantic salmon marine net-pen aquaculture. Transmission of virus and subsequent mortality in Atlantic salmon post-smolts was initiated with as low as 10 plaque forming units (pfu) ml−1. Virus shedding from IHNV infected Atlantic salmon was detected before the onset of visible signs of disease with peak shed rates averaging 3.2×107 pfu fish−1 hour−1 one to two days prior to mortality. Once shed into the marine environment, the abundance of free IHNV is modulated by sunlight (UV A and B) and the growth of natural biota present in the seawater. Virus decayed very slowly in sterilized seawater while rates as high as k = 4.37 d−1 were observed in natural seawater. Decay rates were further accelerated when exposed to sunlight with virus infectivity reduced by six orders of magnitude within 3 hours of full sunlight exposure. Coupling the IHNV transmission parameter estimates determined here with physical water circulation models, will increase the understanding of IHNV dispersal and provide accurate geospatial predictions of risk for IHNV transmission from marine salmon sites.