Atlantic Salmon Head Kidney Research Articles

Rapid sequence modification in the highly polymorphic region (HPR) of the hemagglutinin gene of the infectious salmon anaemia virus (ISAV) suggests intra-segmental template switching recombination.

The ISAV has a genome composed of eight segments of (-)ssRNA, segment 6 codes for the hemagglutinin-esterase protein, and has the most variable region of the genome, the highly polymorphic region (HPR), which is unique among orthomyxoviruses. The HPR has been associated with virulence, infectivity and pathogenicity. The full length of the HPR is called HPR0 and the strain with this HPR is avirulent, in contrast to strains with deleted HPR that are virulent to varying degrees. The molecular mechanism that gives rise to the different HPRs remains unclear. Here, we studied in vitro the evolution of reassortant recombinant ISAV (rISAV) in Atlantic salmon head kidney (ASK) cells. To this end, we rescued and cultivated a set of rISAV with different segment 6-HPR genotypes using a reverse genetics system and then sequencing HPR regions of the viruses. Our results show rapid multiple recombination events in ISAV, with sequence insertions and deletions in the HPR, indicating a dynamic process. Inserted sequences can be found in four segments of the ISAV genome (segments 1, 5, 6, and 8). The results suggest intra-segmental heterologous recombination, probably by class I and class II template switching, similar to the proposed segment 5 recombination mechanism.

Characterization of Differentially Expressed miRNAs and Their Predicted Target Transcripts during Smoltification and Adaptation to Seawater in Head Kidney of Atlantic Salmon.

Smoltification and early seawater phase are critical developmental periods with physiological and biochemical changes in Atlantic salmon that facilitates survival in saltwater. MicroRNAs (miRNAs) are known to have important roles in development, but whether any miRNAs are involved in regulation of gene expression during smoltification and the adaption to seawater is largely unknown. Here, small RNA sequencing of materials from head kidney before, during smoltification and post seawater transfer were used to study expression dynamics of miRNAs, while microarray analysis was applied to study mRNA expression dynamics. Comparing all timepoints, 71 miRNAs and 2709 mRNAs were identified as differentially expressed (DE). Hierarchical clustering analysis of the DE miRNAs showed three major clusters with characteristic expression changes. Eighty-one DE mRNAs revealed negatively correlated expression patterns to DE miRNAs in Cluster I and III. Furthermore, 42 of these mRNAs were predicted as DE miRNA targets. Gene enrichment analysis of negatively correlated target genes showed they were enriched in gene ontology groups hormone biosynthesis, stress management, immune response, and ion transport. The results strongly indicate that post-transcriptional regulation of gene expression by miRNAs is important in smoltification and sea water adaption, and this study identifies several putative miRNA-target pairs for further functional studies.

Characterization of miRNAs in Cultured Atlantic Salmon Head Kidney Monocyte-Like and Macrophage-Like Cells.

Macrophages are among the first cells to respond to infection and disease. While microRNAs (miRNAs) are involved in the process of monocyte-to-macrophage differentiation in mammals, less is known in teleost fish. Here, Atlantic salmon head kidney leukocytes (HKLs) were used to study the expression of miRNAs in response to in vitro culture. The morphological analysis of cultures showed predominantly monocyte-like cells on Day 1 and macrophage-like cells on Day 5, suggesting that the HKLs had differentiated from monocytes to macrophages. Day 5 HKLs also contained a higher percentage of phagocytic cells. Small RNA sequencing and qPCR analysis were applied to examine the miRNA diversity and expression. There were 370 known mature Atlantic salmon miRNAs in HKLs. Twenty-two miRNAs (15 families) were downregulated while 44 miRNAs (25 families) were upregulated on Day 5 vs. Day 1. Mammalian orthologs of many of the differentially expressed (DE) miRNAs are known to regulate macrophage activation and differentiation, while the teleost-specific miR-2188, miR-462 and miR-731 were also DE and are associated with immune responses in fish. In silico predictions identified several putative target genes of qPCR-validated miRNAs associated with vertebrate macrophage differentiation. This study identified Atlantic salmon miRNAs likely to influence macrophage differentiation, providing important knowledge for future functional studies.

Imaging Flow Cytometry Protocols for Examining Phagocytosis of Microplastics and Bioparticles by Immune Cells of Aquatic Animals.

Imaging flow cytometry (IFC) is a powerful tool which combines flow cytometry with digital microscopy to generate quantitative high-throughput imaging data. Despite various advantages of IFC over standard flow cytometry, widespread adoption of this technology for studies in aquatic sciences is limited, probably due to the relatively high equipment cost, complexity of image analysis-based data interpretation and lack of core facilities with trained personnel. Here, we describe the application of IFC to examine phagocytosis of particles including microplastics by cells from aquatic animals. For this purpose, we studied (1) live/dead cell assays and identification of cell types, (2) phagocytosis of degradable and non-degradable particles by Atlantic salmon head kidney cells and (3) the effect of incubation temperature on phagocytosis of degradable particles in three aquatic animals–Atlantic salmon, Nile tilapia, and blue mussel. The usefulness of the developed method was assessed by evaluating the effect of incubation temperature on phagocytosis. Our studies demonstrate that IFC provides significant benefits over standard flow cytometry in phagocytosis measurement by allowing integration of morphometric parameters, especially while identifying cell populations and distinguishing between different types of fluorescent particles and detecting their localization.

Differential Effects of Dietary Supplementation of Krill Meal, Soybean Meal, Butyrate, and Bactocell® on the Gene Expression of Atlantic Salmon Head Kidney.

The head kidney is a key organ that plays a fundamental role in the regulation of the fish immune response and in the maintenance of endocrine homeostasis. Previous studies indicate that the supplementation of exogenous dietary components, such as krill meal (KM), soybean meal (SM), Bactocell® (BA), and butyrate (BU), can have a significant effect on the immune function of the head kidney. The aim of this study was to investigate the differential effect of these four dietary ingredients on the transcriptional profiles of the head kidney of the Atlantic salmon. This study revealed that just a small number of genes were responsive to the feeding regime after a long-term (12 weeks) treatment, and evidenced that the most significant alterations, both in terms of the number of affected genes and magnitude of changes in gene expression, were detectable in the BU- and KM-fed groups compared with controls, while the SM diet had a nearly negligible effect, and BA had no significant effects at all. Most of the differentially expressed genes were involved in the immune response and, in line with data previously obtained from pyloric caeca, major components of the complement system were significantly affected. These alterations were accompanied by an increase in the density of melanomacrophage centers in the KM- and SM-fed group and their reduction in the BU-fed group. While three types of dietary supplements (BU, KM, and SM) were able to produce a significant modulation of some molecular players of the immune system, the butyrate-rich diet was revealed as the one with the most relevant immune-stimulating properties in the head kidney. These preliminary results suggest that further investigations should be aimed towards the elucidation of the potential beneficial effects of butyrate and krill meal supplementation on farmed salmon health and growth performance.

Transcriptomic Changes of Piscirickettsia salmonis During Intracellular Growth in a Salmon Macrophage-Like Cell Line.

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, a systemic infection of salmonid fish species. P. salmonis infects and survives in its host cell, a process that correlates with the expression of virulence factors including components of the type IVB secretion system. To gain further insights into the cellular and molecular mechanism behind the adaptive response of P. salmonis during host infection, we established an in vitro model of infection using the SHK-1 cell line from Atlantic salmon head kidney. The results indicated that in comparison to uninfected SHK-1 cells, infection significantly decreased cell viability after 10 days along with a significant increment of P. salmonis genome equivalents. At that time, the intracellular bacteria were localized within a spacious cytoplasmic vacuole. By using a whole-genome microarray of P. salmonis LF-89, the transcriptome of this bacterium was examined during intracellular growth in the SHK-1 cell line and exponential growth in broth. Transcriptome analysis revealed a global shutdown of translation during P. salmonis intracellular growth and suggested an induction of the stringent response. Accordingly, key genes of the stringent response pathway were up-regulated during intracellular growth as well as at stationary phase bacteria, suggesting a role of the stringent response on bacterial virulence. Our results also reinforce the participation of the Dot/Icm type IVB secretion system during P. salmonis infection and reveals many unexplored genes with potential roles in the adaptation to intracellular growth. Finally, we proposed that intracellular P. salmonis alternates between a replicative phase and a stationary phase in which the stringent response is activated.

Profiling the transcriptome response of Atlantic salmon head kidney to formalin-killed Renibacterium salmoninarum

Renibacterium salmoninarum is a Gram-positive, intracellular bacterial pathogen that causes Bacterial Kidney Disease (BKD) in Atlantic salmon (Salmo salar). The host transcriptomic response to this immune-suppressive pathogen remains poorly understood. To identify R. salmoninarum-responsive genes, Atlantic salmon were intraperitoneally injected with a low (5 × 105 cells/kg, Low-Rs) or high (5 × 107 cells/kg; High-Rs) dose of formalin-killed R. salmoninarum bacterin or phosphate-buffered saline (PBS control); head kidney samples were collected before and 24 h after injection. Using 44K microarray analysis, we identified 107 and 345 differentially expressed probes in response to R. salmoninarum bacterin (i.e. High-Rs vs. PBS control) by Significance Analysis of Microarrays (SAM) and Rank Products (RP), respectively. Twenty-two microarray-identified genes were subjected to qPCR assays, and 17 genes were confirmed as being significantly responsive to the bacterin. There was an up-regulation in expression of genes playing putative roles as immune receptors and antimicrobial effectors. Genes with putative roles as pathogen recognition (e.g. clec12b and tlr5) or immunoregulatory (e.g. tnfrsf6b and tnfrsf11b) receptors were up-regulated in response to R.salmoninarum bacterin. Also, chemokines and a chemokine receptor showed opposite regulation [up-regulation of effectors (i.e. ccl13 and ccl) and down-regulation of cxcr1] in response to the bacterin. The present study identified and validated novel biomarker genes (e.g. ctsl1, lipe, cldn4, ccny) that can be used to assess Atlantic salmon response to R. salmoninarum, and will be valuable in the development of tools to combat BKD.

Visualization of CCL19-like transcripts in the ILT, thymus and head kidney of Atlantic salmon (Salmo salar L.)

Visualization of CCL19-like transcripts in the ILT, thymus and head kidney of Atlantic salmon (Salmo salar L.)

Effect of dietary replacement of fish meal with insect meal on in vitro bacterial and viral induced gene response in Atlantic salmon (Salmo salar) head kidney leukocytes

With the fast growth of today's aquaculture industry, the demand for aquafeeds is expanding dramatically. Insects, which are part of the natural diet of salmonids, could represent a sustainable ingredient for aquaculture feed. The aim of the current study was to test how a partial or total replacement of dietary fishmeal with insect meal affect gene responses involved in inflammation, the eicosanoid pathway and stress response in Atlantic salmon (Salmo salar L.) in isolated head kidney leukocytes after exposure to bacterial or viral mimic.Insect meal (IM) was produced from black soldier fly (BSF, Hermetia illucens) larvae. Seawater Atlantic salmon were fed three different diets for 8 weeks; a control diet (IM0, protein from fishmeal and plant based ingredients (25:75) and lipid from fish oil and vegetable oil (33:66); and two insect-meal containing diets, IM66 and IM100, where 66 and 100% of the fishmeal protein was replaced with IM, respectively. Leukocytes were isolated from the head kidney of fish (n = 6) from each of the three dietary groups. Isolated leukocytes were seeded into culture wells and added either a bacterial mimic (lipopolysaccharide, LPS) or a viral mimic (polyinosinic acid: polycytidylic acid, poly I: C) to induce an inflammatory response. Controls (Ctl) without LPS and poly I: C were included.The transcription of interleukins IL-1β, IL-8, IL-10 and TNF-α were elevated in LPS treated leukocytes isolated from salmon fed the three dietary groups (IM0, IM66 and IM100). The inflammatory-related gene expression in head kidney cells were, however, not affected by the pre-fed substitution of fish meal with IM in the diet of salmon. Gene transcriptions of PTGDS and PTGES were neither affected by LPS, poly I: C or the experimental diets fed prior to cell isolation, while salmon fed with IM showed a lower expression of LOX5. The gene expression of TLR22 and C/EBP-β were down-regulated by the LPS treatment in the cells isolated from salmon fed insect-based diets (IM66 and IM100) compared to fish fed the IM0. Similarly, the leukocytes challenged with LPS and isolated from fish fed with IM66 and IM100 down-regulated the expression of Mn-SOD, GPx1, HSP27 and HSP70 compared to salmon fed IM0. In general, these results suggested that replacement of fishmeal with IM in the diets of Atlantic salmon had no effect on the transcription of pro-inflammatory genes in the head kidney cells. There was, however, an effect of dietary IM on the transcription of antioxidant and stress related genes in the leukocytes.

Salmon cells SHK-1 internalize infectious pancreatic necrosis virus by macropinocytosis.

We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE-214 by macropinocytosis. In this study, we have extended our investigation into SHK-1 cells, a macrophage-like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK-1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK-1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK-1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis-mediated entry of IPNV into its target cells.

Surplus arginine reduced lipopolysaccharide induced transcription of proinflammatory genes in Atlantic salmon head kidney cells

In aquaculture production, studies of salmon health and interaction between pathogens and nutrition are of high importance. This study aimed to compare genes and pathways involved in salmon head kidney cells and liver cells, isolated from the same fish, towards polyinosinic acid: polycytidylic acid (poly I:C) and lipopolysaccharide (LPS), with and without addition of surplus arginine. Selected transcriptional responses of genes involved in inflammation, polyamine synthesis, oxidation and apoptosis were elucidated.For the genes related to inflammation, viperin, Mx and Toll like receptor 3 (TLR3), transcription were significantly upregulated by poly I:C in head kidney cells, while viperin was upregulated in liver cells. Surplus arginine did not affect poly I:C induced responses with the exception of reducing poly I:C induced Mx transcription in head kidney cells. Gene transcription of Interleukin 1β (IL-1β), Interleukin-8 (IL-8) and cyclooxygenase 2 (Cox2) were elevated during LPS treatment in all liver and head kidney cell cultures. In addition, LPS induced significantly, CD83 transcription in liver cells and TNF-α transcription in head kidney cells. Surplus arginine significantly reduced IL-8, Cox2 and TNF-α transcription in head kidney cells. LPS upregulated arginase in head kidney cells while poly I:C upregulated S-adenosyl methionine decarboxylase (SAMdc) transcription in liver cells. This suggests that LPS and poly I:C modulates genes involved in polyamine synthesis. In addition, in head kidney cells, surplus arginine, when cultured together with LPS, increased the transcription of ornithine decarboxylase (ODC) the limiting enzyme of polyamine synthesis. The genes involved with oxidation and apoptosis were not affect by any of the treatments in liver cells, while LPS decreased caspase 3 transcription in head kidney cells. In liver cells, protein expression of catalase was reduced by surplus arginine alone and when challenged with poly I:C. Both liver cells and head kidney cells isolated from the same individual fish responded to LPS and poly I:C, depending on the gene analyzed. Additionally, arginine could modulate transcription of pro-inflammatory genes induced by LPS in salmon immune cells, thus affecting salmon immunity.

Studies on the effects of LPS, ß-glucan and metabolic inhibitors on the respiratory burst and gene expression in Atlantic salmon macrophages.

Reactive oxygen species (ROS) production in macrophage-like cells is induced as an antimicrobial defence against invading pathogens. In this study, we have explored how different stimuli and metabolic inhibitors affect the level of respiratory burst in Atlantic salmon (Salmo salar L.) head kidney macrophage-like cells. Cells stimulated invitro by bacterial lipopolysaccharide (LPS) and ß-glucan showed increased production of ROS compared to unstimulated cells. Both stimulation and costimulation by curdlan (ß-glucan) induced a higher production of ROS compared to stimulation and costimulation by LPS. Metabolic inhibitors co-incubated with the stimulants did not, in most cases, perturb the level of ROS generation in the salmon macrophage-like cells. The NAD+ content as well as the NAD+ /NADH ratio increased in curdlan and LPS+curdlan-stimulated cells compared to control cells, which indicated increased metabolic activity in the stimulated cells. Supporting these findings, gene analysis using real-time quantitative PCR showed that arginase-1 and IL-1ß genes were highly expressed in the stimulated cells.

In vitro modulation of Drimys winteri bark extract and the active compound polygodial on Salmo salar immune genes after exposure to Saprolegnia parasitica

The rapid development of the aquaculture industry has global concerns with health management and control strategies to prevent and/or treat diseases and increase sustainability standards. Saprolegniosis is a disease caused by Saprolegnia parasitica, and is characterized by promoting an immunosuppression in the host. This study evaluated in vitro the extract and one active compound (polygodial) of Drimys winteri, a Chilean medicinal tree as a potential early immunostimulatory aid in Saprolegniosis control. Atlantic salmon (Salmo salar) head kidney cells (ASK-1) were incubated with both extract and pure polygodial before exposure to S. parasitica mycelium, and the expression of the immune-related genes interleukin 1β (IL-1β), interferon α (IFNα), and major histocompatibility complex II (MHCII) was evaluated. Both evidenced immunomodulatory capacities by increasing gene expressions. This immunomodulation related to a mitigatory action counteracting the immunosuppressing effects of S. parasitica. Despite that most immune-related genes were up-regulated, the down-regulation of MHCII, characteristic of S. parasitica infection, was lessened by pre-incubation with the compounds. This study provides the first insight on the potential of D. winteri bark extract as a possible immunomodulatory and defensive strategy against this oomycete infection in fish.

Expression kinetics of interferon stimulated genes in Atlantic salmon head kidney cells (ASK) nucleofected with Poly(I:C)

Viral diseases are of great concern in fish farming. Close to 20% of salmon put out to sea are lost during production and a large part of this is due to infections. Oil-adjuvented vaccines are available against some virus, but the efficiency under field conditions have been questioned. Our goal is to test known agonists for human Toll-like receptors (TLRs) in modified nanobeads carriers as adjuvants in different vaccines formulations, against fish viruses. The primary early response to viruses is mediated by interferons (IFNs) and interferon stimulated genes (ISGs). The interferon system is activated through the recognition of pathogen associated molecular patterns (PAMP) by conserved pattern recognition receptors (PRRs), such as TLRs. Poly (I:C) is a synthetic analog of double-stranded RNA (dsRNA) that mimics a viral infection in mammals and it is one of the most thoroughly studied PAMPs with respect to immunostimulatory potential in therapeutics or vaccines. Before developing a vaccine is necessary to understand more about how the immune system responds to these ligands. We therefore analyzed the expression of some interferon stimulated genes at different time points in Atlantic salmon head kidney cells (ASK) transfected by electroporation with Poly(I:C). Cells were transfected using Amaxa Nucleofector Kit with 5μg of Poly (I:C) or mock transfected with only buffer. After transfection, cells were plated and incubated for different time points (0, 6, 12, 24, 48, 72 hours) when samples of RNA were extracted for cDNA synthesis. Our results shows that there are large differences in the temporal pattern of IFN and ISG expression in salmon cells. QPCR analysis revealed strong upregulation of IFN alpha and TNF alpha during the first 6 hours after stimulation. Both Isg15 and Mx1 showed higher levels, comparing with control, in the first 12 hours and reached a peak at 24 hours after transfection. Mock transfections using only buffer without adding RNA or DNA also induced upregulation of some of the genes tested, especially TNF alpha. Endoplasmic reticulum (ER) stress induced by transfections was analyzed by a XBP-1 splicing assay analysis, but the results were not consistent. Lipofections and nucleofections are popular techniques for introducing RNA and DNA into cells to study both protein expression and immunity to cytoplasmic nucleic acids. Further studies are therefore necessary to quantify the magnitude and duration of cellular stress caused by these methods to separate their impact on effects mediated via PPRs .

Effects of a peptide derived from the extreme carboxyl terminal of IL-8 on immune response genes in the SHK-1 cell line

Interleukin (IL)-8 is a chemokine principally characterized for being a leukocyte chemotactic. Recently, our laboratory chemically synthesized an alpha-helical peptide derived from the extreme carboxyl terminal of IL-8 that, due to physicochemical characteristics and functions, also evidences an antimicrobial effector function against Gram-negative bacteria such as Aeromona salmonicidina, Pseudomona aeruginosa, and Escherichia coli. In higher vertebrates, there is ample evidence for the role that antimicrobial peptides play in regulating the immune response. Therefore, the objective of our study was to determine if the peptide derived from IL-8 affects relevant immune genes. For this, the transcriptional expression of immune response genes were assessed in the Atlantic salmon (Salmo salar) head kidney (SHK-1) cell line when stimulated with the synthetic antimicrobial peptide derived from IL-8. Our results evidenced that the expression of IL-6 slightly increased, while the expressions of iNOS and IL-1β decreased. Moreover, this peptide did not appear to affect the expressional levels of IL-8 or TNF. Our results suggest that this peptide could directly influence the genetic expression of leukocytes, such as macrophages, through a downor upregulation of immune genes. In conclusion, the peptide derived from IL-8 would not only contribute towards limiting bacterial infection, but it would also have the ability to block macrophage activity and thereby likely participate in the end of the immune response. This studied was financed through Proyecto FONDECYT N° 3150678

Patterns of Piscirickettsia salmonis load in susceptible and resistant families of Salmo salar

The pathogen Piscirickettsia salmonis produces a systemic aggressive infection that involves several organs and tissues in salmonids. In spite of the great economic losses caused by this pathogen in the Atlantic salmon (Salmo salar) industry, very little is known about the resistance mechanisms of the host to this pathogen. In this paper, for the first time, we aimed to identify the bacterial load in head kidney and muscle of Atlantic salmon exhibiting differential familiar mortality. Furthermore, in order to assess the patterns of gene expression of immune related genes in susceptible and resistant families, a set of candidate genes was evaluated using deep sequencing of the transcriptome. The results showed that the bacterial load was significantly lower in resistant fish, when compared with the susceptible individuals. Based on the candidate genes analysis, we infer that the resistant hosts triggered up-regulation of specific genes (such as for example the LysC), which may explain a decrease in the bacterial load in head kidney, while the susceptible fish presented an exacerbated innate response, which is unable to exert an effective response against the bacteria. Interestingly, we found a higher bacterial load in muscle when compared with head kidney. We argue that this is possible due to the availability of an additional source of iron in muscle. Besides, the results show that the resistant fish could not be a likely reservoir of the bacteria.

De novo assembly and transcriptome analysis of Atlantic salmon macrophage/dendritic-like TO cells following type I IFN treatment and Salmonid alphavirus subtype-3 infection.

BackgroundInterferons (IFN) are cytokines secreted by vertebrate cells involved in activation of signaling pathways that direct the synthesis of antiviral genes. To gain a global understanding of antiviral genes induced by type I IFNs in salmonids, we used RNA-seq to characterize the transcriptomic changes induced by type I IFN treatment and salmon alphavirus subtype 3 (SAV-3) infection in TO-cells, a macrophage/dendritic like cell-line derived from Atlantic salmon (Salmo salar L) head kidney leukocytes.ResultsMore than 23 million reads generated by RNA-seq were de novo assembled into 58098 unigenes used to generate a total of 3149 and 23289 differentially expressed genes (DEGs) from TO-cells exposed to type I IFN treatment and SAV-3 infection, respectively. Although the DEGs were classified into genes associated with biological processes, cellular components and molecular function based on gene ontology classification, transcriptomic changes reported here show upregulation of genes belonging to the canonical type I IFN signaling pathways together with a broad spectrum of antiviral genes that block virus replication in host cells. In addition, the transcriptome shows a profile of genes associated with apoptosis as well as genes that activate adaptive immunity. Further, our findings show that the profile of genes expressed by TO-cells is comparable to orthologous genes expressed by mammalian macrophages and dendritic cells in response to type I IFNs. Twenty DEGs randomly selected for qRT-PCR confirmed the validity of the transcriptomic changes detected by RNA-seq by showing that the genes upregulated by RNA-seq were also upregulated by qRT-PCR and that genes downregulated by RNA-seq were also downregulated by qRT-PCR.ConclusionsThe de novo assembled transcriptome presented here provides a global description of genes induced by type I IFNs in TO-cells that could serve as a repository for future studies in fish cells. Transcriptome analysis shows that a large proportion of IFN genes expressed in this study are comparable to IFNs genes expressed in mammalia. In addition, the study shows that SAV-3 is a potent inducer of type I IFNs and that the responses it induces in TO-cells could serve as a model for studying IFN responses in salmonids.

Persistent infections with infectious pancreatic necrosis virus (IPNV) of different virulence in Atlantic salmon, Salmo salar L.

Infectious pancreatic necrosis virus (IPNV) is a prevalent pathogen in fish worldwide. The virus causes substantial mortality in Atlantic salmon juveniles and smolts when transferred to sea water and persistent infection in surviving fish after disease outbreaks. Here, we have investigated the occurrence of the virus as well as the innate immune marker Mx in the head kidney (HK) of Atlantic salmon throughout an experimental challenge covering both a fresh and a seawater phase. The fish were challenged with a high (HV) and low virulence (LV) IPNV. Both isolates caused mortality due to reactivation of the virus after transfer to sea water. In the freshwater phase, higher levels of virus transcripts were detected in the HK of fish infected with LV IPNV compared to HV, suggesting that the HV isolate is able to limit its own replication to a level where the innate immune system is not alerted. Further, ex vivoHK leucocytes derived from fish infected with the two isolates were stimulated with CpG DNA. Significantly, higher IFN levels were found in the LV compared to the HV group in the freshwater phase. This suggests that the viruses attenuate the antiviral host immune response at different levels which may contribute to the observed differences in disease outcome.

The impact of exogenous ω-6 and ω-3 polyunsaturated fatty acids on the induced production of pro- and anti-inflammatory prostaglandins and leukotrienes in Atlantic salmon head kidney cells using a full factorial design and LC–MS/MS

The production of prostaglandins (PGE2, PGE3) and leukotrienes (LTB4, LTB5) in salmon head kidney cell cultures, exposed to different combinations of 20:4ω-6, 20:5ω-3 and 22:6ω-3 polyunsaturated fatty acids (PUFAs), was evaluated by means of a two level factorial design and LC-MS/MS. The method was selective for the pro- and anti-inflammatory analytes and their corresponding stable-isotope labelled internal standards. The regression models were linear over the concentration range 0.5-150 ng/ml with limits of detection of 0.25 ng/ml and quantification of 0.40 ng/ml for the analysed metabolites. The recovery ranged from 78 to 107% for prostaglandins and 73 to 115% for leukotrienes. The analysis of the samples exposed to different combinations of PUFAs revealed that the presence of single ω-3 PUFAs brought an enhancement of the metabolites from the lipooxygenase pathway, specially LTB4, and a reduction of the metabolites from the cyclooxygenase pathway (PGE2 and PGE3), while the two-term interactions generated the opposite effect (high concentration of prostaglandins and low concentrations of leukotrienes). To our knowledge, this is the first implementation of a fully crossed design for investigating the impact of ω-6 and ω-3 PUFAs on the production of eicosanoids not only through their individual but also through their combined effects on Atlantic salmon head kidney cells.

Interferon-mediated host response in experimentally induced salmonid alphavirus 1 infection in Atlantic salmon (Salmo salar L.)

Salmonid alphavirus (SAV) infection in cultured salmonids causes severe economic losses across Europe. Immune protection and antiviral mechanisms of the host against SAV are poorly characterised in vivo. Analysis of immune gene expression in head kidney of Atlantic salmon (Salmo salar L.) experimentally infected with SAV 1, using a quantitative reverse transcription polymerase chain reaction (qRT-PCR), revealed rapid induction of interferon I (INF-I), interferon II (INF-II) and INF-I associated Mx genes in SAV 1 infected fish compared to control fish injected with tissue culture supernatant. Mx protein was found to be highly expressed in the heart and mucosal membranes of infected fish by immunohistochemistry (IHC). Interestingly, the pathological changes that were observed in the target tissues of the virus became visible some time after peak expression of genes associated with the INF-I-pathway in head kidney tissue. These findings suggest that a non-specific antiviral immune response is rapidly induced during the early stages of SAV infection in salmon.