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Previous studies have shown that intraperitoneal (i.p.) injection of a yeast cell wall glucan into Atlantic slamon, Salmo salar L., resulted in increased resistance to bacterial pathogens and that the glucan enhanced the protective effect of an i.p.-administered furunculosis vaccine in salmon. In the present work, the peritoneal cell responses to yeast glucan, glycogen, Freund's incomplete adjuvant (FIA) and saline were compared. Whereas saline gave no detectable peritoneal cell response, injection of the three other agents resulted in accumulation of macrophages, neutrophils and thrombocytes in the peritoneal cavity 2–22 days later. Glucan and glycogen gave maximum leucocyte responses 2 days after injection, whereas the number of leucocytes after FIA injection showed a continuous increase during the 22-day experimental period. Two days after injection, macrophages dominated in the leucocyte response to yeast glucan, whereas neutrophils dominated in response to glycogen and FIA. At 8 and 22 days post-injection of yeast glucan and FIA, thrombocytes represented the largest fraction of peritoneal cells. Except for an elevated number of neutrophils 3 weeks after injection, i.p. administration of yeast glucan gave no significant change in leucocyte numbers from the head kidney. On the other hand, head kidney macrophages from glucan-treated fish showed an increased ability to kill a virulent strain of Aeromonas salmonicida.

Bactericidal activity and O-2 production of head kidney macrophages were investigated 1, 2 and 3 weeks after intraperitoneal injection of a β-1,3 and β-1,6 linked yeast glucan into rainbow trout (Oncorhynchus mykiss). Macrophages from glucan-treated fish showed increased ability to kill both an avirulent and a virulent strain of the salmonid pathogen Aeromonas salmonicida. The difference in bactericidal activity between macrophages from control and glucan-treated fish was largest 2 weeks after injection. Upon phorbol myristate acetate (PMA) stimulation, 3 h post-harvest head kidney macrophages from glucan-treated fish showed increased O-2 production 2 and 3 weeks after glucan injection but not at week 1. At 24 h post-harvest, however, only week 2 glucan macrophages retained this activity. Thus, there is a correlation between the enhanced bactericidal activity by the macrophages from glucan-treated fish and their capacity to produce reactive oxygen species at week 2, but this does not exclude the possibility of other mechanisms being involved. Injection of glucan into rainbow trout also resulted in enhanced serum lysozyme activity throughout the 3-week experimental period.

Background Type I and type II interferons (IFNs) exert their effects mainly through the JAK/STAT pathway, which is presently best described in mammals. STAT1 is involved in signaling pathways induced by both types of IFNs. It has a domain-like structure including an amino-terminus that stabilizes interaction between STAT dimers in a promoter-binding situation, a coiled coil domain facilitating interactions to other proteins, a central DNA-binding domain, a SH2 domain responsible for dimerization of phosphorylated STATs and conserved phosphorylation sites within the carboxy terminus. The latter is also the transcriptional activation domain. Results A salmon (Salmo salar) STAT1 homologue, named ssSTAT1a, has been identified and was shown to be ubiquitously expressed in various cells and tissues. The ssSTAT1a had a domain-like structure with functional motifs that are similar to higher vertebrates. Endogenous STAT1 was shown to be phosphorylated at tyrosine residues both in salmon leukocytes and in TO cells treated with recombinant type I and type II IFNs. Also ectopically expressed ssSTAT1 was phosphorylated in salmon cells upon in vitro stimulation by the IFNs, confirming that the cloned gene was recognized by upstream tyrosine kinases. Treatment with IFNs led to nuclear translocation of STAT1 within one hour. The ability of salmon STAT1 to dimerize was also shown. Conclusions The structural and functional properties of salmon STAT1 resemble the properties of mammalian STAT1.

Toll-like receptor 8 (TLR 8) belongs to a subgroup of the TLR family that recognizes nucleic acids and that is involved in the protection against viruses. In mammals, TLR7 and 8 have been characterized as receptors for viral and synthetic single-stranded RNA. Here we describe the cloning of a TLR8 homolog in Atlantic salmon (Salmo salar) and its proximal adaptor protein MyD88. The mRNA expression of SsTLR8 was tissue-restricted and its highest level was detected in the spleen while SsMyD88 was expressed in all of the tested organs. SsTLR8 and SsMyD88 mRNAs were up-regulated in TO cells treated with recombinant IFN alpha1 and IFN gamma. In vivo, the expression of SsTLR8 was not significantly affected following challenge with salmon alphavirus subtype 3 (SAV3). By contrast, infection with SAV3 up-regulated SsMyD88 transcripts on day 7 post-challenge and the expression remained elevated at day 28. The SsMyD88 expression in vivo paralleled type I IFN expression. In vitro stimulation of salmon head kidney leukocytes with CpG ODNs and IFN gamma also up-regulated SsMyD88 mRNA. Furthermore, ectopic expression of SsMyD88 in HEK cells was able to activate a NF-kappaB reporter construct indicating that the cloned salmon molecule was a functional MyD88 homologue.

Summary Members of the Toll-like receptor family 9 (TLR9) subfamily sense viral and bacterial DNA present in the endosomal compartment. Here we describe the cloning and regulation of a TLR9 gene from Atlantic salmon ( Salmo salar ). The salmon TLR9 cDNA encodes 1075 amino acids and analysis of the inferred protein sequence shows that several amino acid residues known to be important for the functions of TLR9 in mammals are conserved in salmon. Furthermore, TLR9 expression was elevated in head kidney leukocytes after in vitro treatment with CpG ODNs and recombinant trout interferon (IFN)- γ . IFN- γ was the strongest inducer of TLR9 expression. Together, the results indicate that the structure, the expression and possibly the function of TLR9 are conserved across the teleost and mammalian lineages.

Infectious pancreatic necrosis virus (IPNV) is a bisegmented, double-stranded RNA (dsRNA) virus of the Birnaviridae family that causes widespread disease in salmonids. Its two genomic segments are encapsulated together with the viral RNA-dependent RNA polymerase, VP1, and the assumed internal protein, VP3, in a single-shell capsid composed of VP2. Major aspects of the molecular biology of IPNV, such as particle assembly and interference with host macromolecules, are as yet poorly understood. To understand the infection process, analysis of viral protein interactions is of crucial importance. In this study, we focus on the interaction properties of VP3, the suggested key organizer of particle assembly in birnaviruses. By applying the yeast two-hybrid system in combination with coimmunoprecipitation, VP3 was proven to bind to VP1 and to self-associate strongly. In addition, VP3 was shown to specifically bind to dsRNA in a sequence-independent manner by in vitro pull-down experiments. The binding between VP3 and VP1 was not dependent on the presence of dsRNA. Deletion analyses mapped the VP3 self-interaction domain within the 101 N-terminal amino acids and the VP1 interaction domain within the 62 C-terminal amino acids of VP3. The C-terminal end was also crucial but not sufficient for the dsRNA binding capacity of VP3. For VP1, the 90 C-terminal amino acids constituted the only dispensable part for maintaining VP3-binding ability. Kinetic analysis revealed the presence of VP1-VP3 complexes prior to the formation of mature virions in IPNV-infected CHSE-214 cells, which indicates a role in promoting the assembly process.

A transgenic cell line for the detection of salmon interferons (IFNs) has been established. It is based on a CHSE-214 cell line containing a reporter construct expressing firefly luciferase under the control of the rainbow trout promoter for the IFN-induced Mx1 gene. This cell line, named CHSE-Mx10, showed IFN-induced luciferase expression after more than 80 passages, confirming the stability of this cell line. Interestingly, the Mx promoter was shown to respond to both salmon IFN-alpha/beta and trout IFN-gamma in a dose-dependent manner, while there was no response to TNF-alpha and IL-1beta. IFN-alpha/beta activity could be measured at a range of 9-150 U/ml, and IFN-gamma showed activity between 10 and 100 ng/ml. The reproducibility of both responses was good. The CHSE-Mx10 reporter system constitutes a versatile tool to study the induction and regulation of IFN signaling in teleost fish. A preliminary study presented herein suggests that both infectious pancreas necrosis virus (IPNV) and salmon pancreas disease virus (SPDV) may block activation of the Mx promoter in CHSE-Mx10 stimulated with IFN-alpha/beta.

p38 mitogen-activated protein kinase is activated by environmental stress and cytokines and plays a role in transcriptional regulation and inflammatory responses. In this study, three distinct Atlantic salmon p38 (As-p38) cDNAs were cloned, which all translated into 361 amino acid proteins. The As-p38 protein sequences possessed showed85% identity to the mammalian homolog, p38alpha. All three contained the conserved phosphorylation motif TGY located in the activation loop of the kinase. Salmon p38 showed ubiquitous tissue distribution, including expression in the immune organs head kidney and spleen. A higher p38 mRNA expression was detected in the ovary compared to other organs suggesting that p38 may perform specific functions within this organ. Western blot analysis with an antibody specific for phosphorylated p38 showed that ectopically expressed As-p38 variants were activated in CHSE-214 cells in response to chemical stress. Furthermore, lipopolysaccharide, CpG oligonucleotides and recombinant trout IL-1beta induced endogenous phosphorylation of p38 in salmon head kidney macrophages in a dose-dependent manner. The importance of p38 for regulation of salmon innate immunity was further demonstrated by the ability of the p38 specific inhibitor SB203580 to completely abolish LPS-stimulated TNF-2 and IL-1beta mRNA expression in the macrophages.

Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are in humans divided into three distinct classes (A, B and C). The CpG ODNs, like baterial DNA, are recognized by the vertebrate immune system and are known to stimulate immune responses. The characterization of the different classes is based on their structure and biological activity. In this study, we report the effects of these classes of CpG ODNs as they are defined in humans on IFN alpha/beta production and cell proliferation in Atlantic salmon spleen, head kidney and blood leukocytes. These studies revealed that CpG A together with CpG C are strong inducers of IFN alpha/beta in spleen and head kidney leukocytes, whilst CpG B and CpG C had the highest capacity to stimulate cell proliferation in all three cell populations. These findings are the first to establish that the effects of the different CpG classes are comparable between fish and man.

Unmethylated CpG motifs in DNA are recognised by vertebrate immune cells as pathogen signatures. Consequently, oligodeoxynucleotides containing CpG motifs (CpG ODNs) are able to enhance and direct immune responses. Recent studies have demonstrated that CpG ODNs activate antiviral immune responses in Atlantic salmon (Salmo salar L.) leukocytes, and are therefore promising agents as vaccine adjuvants or immunostimulants in fish. In this work, we report synergy of CpG ODN and cationic proteins in the stimulation of type I IFN activity in Atlantic salmon leukocytes. Different fractions of cationic histone proteins derived from cod milt or poly-l-arginine and poly-l-lysine were screened for their ability to enhance CpG ODN induced type I IFN activity in Atlantic salmon leukocytes. Optimal ratio of histones to CpG ODN was identified, and effects on transcription of type I IFN and antiviral Mx genes were studied. Delivery of CpG ODN with cationic proteins enhanced the production of type I IFN and succeeding Mx transcripts after two and five days of stimulation at substimulatory concentrations of CpG ODN. These results indicate that co-delivery of CpG ODN and cationic proteins enhance antiviral mechanisms in Atlantic salmon leukocytes as compared to CpG ODN alone.

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs are known to stimulate immune responses and are potent adjuvants in higher vertebrates, but so far the effects in fish are poorly described. We here report that CpG ODNs induce IL-1β expression and production of interferon-like cytokines in rainbow trout head-kidney macrophages, whereas ODNs with an inverted motif (GpC) have a much less stimulatory effect. We further demonstrate that endosomal maturation is essential for CpG signalling, as chloroquine, a compound known to block endosomal acidification, inhibits cytokine expression in the macrophages.

Serum amyloid A (A-SAA) has previously been reported to be an acute-phase protein in salmonids. Hepatocytes represent a major source of A-SAA in salmonids, but nothing is known about hepatocyte SAA synthesis in fish. In the present work, the expression of A-SAA transcripts in primary cultures of Atlantic salmon hepatocytes in response to macrophage derived cytokines, human recombinant cytokines and bacterial lipopolysaccharide (LPS) was studied by Northern blot analysis. The macrophage supernatants were prepared by stimulating Atlantic salmon head kidney macrophages with LPS, yeast glucan or a leukocyte derived macrophage activating factor (MAF). The supernatants from glucan- or MAF-stimulated macrophages had no effect on A-SAA expression of the hepatocytes, while supernatants from LPS-stimulated macrophages gave about a 2-fold increase in expression. The combination of either glucan and MAF, or LPS and MAF were more effective and these supernatants gave a 3.4- and 5.2-fold increase in A-SAA expression, respectively. The hepatocytes were also treated with the human recombinant cytokines TNFalpha, IL-1beta and IL-6, alone or in combination. The A-SAA response to each of them alone was modest, but TNFalpha and IL-6 or IL-1beta and IL-6 in combination gave a higher response than each cytokine alone. These data suggest that the expression of A-SAA by hepatocytes from Atlantic salmon is induced by cytokine-like molecules. Interestingly, hepatocytes treated directly with LPS gave a more than 10-fold increase in SAA mRNA expression, but it is not known if this is a direct effect of LPS on the hepatocytes or if it is mediated by other contaminating cell types.

Previous studies have shown that head kidney macrophages isolated from glucan injected rainbow trout (Oncorhychus mykiss) and Atlantic salmon (Salmo salar L.) have increased ability to kill Aeromonas salmonicida. The present work was aimed at investigating the in vitro effects of glucan on the respiratory burst and bactericidal potential of Atlantic salmon head kidney macrophages. Salmon macrophages were incubated for 1-7 days with various concentrations of yeast beta-glucan (MacroGard) and tested for respiratory burst activity by the reduction of nitroblue-tetrazolium (NBT) after exposure to phorbol myristate acetate (PMA) or opsonized zymosan. The macrophages showed a marked increase in respiratory burst activity 4 to 7 days after addition of glucan. Macrophages treated with 0.1-1 microgram mL-1 gave a maximum respiratory burst response, whereas 10 micrograms mL-1 had no effect and 50 micrograms mL-1 was inhibitory. The glucan also triggered respiratory burst activity directly, but this occurred only at relative high concentrations with a maximal effect at > or = 200 micrograms mL-1. The validity of using the NBT-assay as a measure of respiratory burst activity was confirmed by using inhibitors of O2- production (superoxide dismutase, trifluoperazine and diphenylene iodonium). Despite the stimulatory effect of glucan on the respiratory burst activity of salmon macrophages, these cells did not show increased bactericidal activity against the avirulent and virulent strain of A. salmonicida. Upregulation of burst activity alone is thus apparently not sufficient to enhance bactericidal activity against this pathogen by Atlantic salmon macrophages.

Background Infectious pancreatic necrosis virus (IPNV) is an aquatic member of the Birnaviridae family that causes widespread disease in salmonids. IPNV is represented by multiple strains with markedly different virulence. Comparison of isolates reveals hyper variable regions (HVR), which are presumably associated with pathogenicity. However little is known about the rates and modes of sequence divergence and molecular mechanisms that determine virulence. Also how the host response may influence IPNV virulence is poorly described. Methods In this study we compared two field isolates of IPNV (NFH-Ar and NFH-El). The sequence changes, replication and mortality were assessed following experimental challenge of Atlantic salmon. Gene expression analyses with qPCR and microarray were applied to examine the immune responses in head kidney. Results Significant differences in mortality were observed between the two isolates, and viral load in the pancreas at 13 days post infection (d p.i.) was more than 4 orders of magnitude greater for NFH-Ar in comparison with NFH-El. Sequence comparison of five viral genes from the IPNV isolates revealed different mutation rates and Ka/Ks ratios. A strong tendency towards non-synonymous mutations was found in the HRV of VP2 and in VP3. All mutations in VP5 produced precocious stop codons. Prior to the challenge, NFH-Ar and NFH-El possessed high and low virulence motifs in VP2, respectively. Nucleotide substitutions were noticed already during passage of viruses in CHSE-214 cells and their accumulation continued in the challenged fish. The sequence changes were notably directed towards low virulence. Co-ordinated activation of anti-viral genes with diverse functions (IFN-a1 and c, sensors - Rig-I, MDA-5, TLR8 and 9, signal transducers - Srk2, MyD88, effectors - Mx, galectin 9, galectin binding protein, antigen presentation - b2-microglobulin) was observed at 13 d p.i. (NFH-Ar) and 29 d p.i. (both isolates). Conclusions Mortality and expression levels of the immune genes were directly related to the rate of viral replication, which was in turn associated with sequences of viral genes. Rapid changes in the viral genome that dramatically reduced virus proliferation might indicate a higher susceptibility to protective mechanism employed by the host. Disease outbreak and mortality depend on a delicate balance between host defence, regulation of signalling cascades and virus genomic properties.

Relative quantification using RT-qPCR is a widely used method for transcription profiling. Transcript levels of target genes in fish after experimental infection is often reported without documentation of stably transcribed reference genes. We present results demonstrating that transcription of typically used reference genes in Atlantic salmon is not stable during experimental infection with salmon pancreas disease virus (SPDV). Transcript levels 0 to 6 weeks after challenge revealed statistically significant changes between time-points that corresponded with a peak in viral load 3 weeks after challenge. The results emphasize the need for thorough method validation prior to transcriptional studies during viral infections.