Long-term persistence of piscine orthoreovirus-1 (PRV-1) infection during the pre-smolt stages of Atlantic salmon in freshwater.

Piscine orthoreovirus (PRV) causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon. During salmon production cycles, HSMI has predominantly been observed after seawater transfer. More recently, better surveillance and longitudinal studies have detected occurrences of PRV-1 in freshwater broodstock farms and hatcheries. However, very little is known about the viral kinetics of PRV-1 or disease development of HSMI during these pre-smolt stages. In this study, we conducted a long-term PRV-1 challenge experiment to examine the profile of viral load, infectiousness and/or clearance in Atlantic salmon during their development from fry to parr stage. Atlantic salmon fry (mean weight: 1.1 ± 0.19 g) were infected with PRV-1 (high virulent variant) via intraperitoneal (IP) injection. The viral load reached a peak at 2-4 weeks post-challenge (wpc) in heart and muscle tissues. The virus was detected at relatively high levels in whole blood, spleen, and head kidney tissues until 65 wpc. Heart and muscle lesions typical of HSMI were clearly observed at 6 and 8 wpc but then subsided afterwards resolving inflammation. Innate and adaptive immune responses were elicited during the early/acute phase but returned to basal levels during the persistent phase of infection. Despite achieving high viremia, PRV-1 infection failed to cause any mortality during the 65-week virus challenge period. Cohabitation of PRV-1 infected fish (10 and 31 wpc) with naïve Atlantic salmon fry resulted in very low or no infection. Moreover, repeated chasing stress exposures did not affect the viral load or shedding of PRV-1 at 26 and 44 wpc. The present findings provide knowledge about PRV-1 infection in juvenile salmon and highlight the importance of continued monitoring and management to prevent and mitigate the PRV-1 infection in freshwater facilities.

Acute stress response on Atlantic salmon: a time-course study of the effects on plasma metabolites, mucus cortisol levels, and head kidney transcriptome profile.

Farmed Atlantic salmon (Salmo salar) welfare and performance can be strongly influenced by stress episodes caused by handling during farming practices. To better understand the changes occurring after an acute stress response, we exposed a group of Atlantic salmon parr to an acute stressor, which involved netting and transferring fish to several new holding tanks. We describe a time-course response to stress by sampling parr in groups before (T0) and 10, 20, 30, 45, 60, 120, 240, 300, and 330 min post-stress. A subgroup of fish was also subjected to the same stressor for a second time to assess their capacity to respond to the same challenge again within a short timeframe (ReStressed). Fish plasma was assessed for adrenocorticotropic hormone (ACTH), cortisol, and ions levels. Mucus cortisol levels were analyzed and compared with the plasma cortisol levels. At 5 selected time points (T0, 60, 90, 120, 240, and ReStressed), we compared the head kidney transcriptome profile of 10 fish per time point. The considerably delayed increase of ACTH in the plasma (60 min post-stress), and the earlier rise of cortisol levels (10 min post-stress), suggests that cortisol release could be triggered by more rapidly responding factors, such as the sympathetic system. This hypothesis may be supported by a high upregulation of several genes involved in synaptic triggering, observed both during the first and the second stress episodes. Furthermore, while the transcriptome profile showed few changes at 60 min post-stress, expression of genes in several immune-related pathways increased markedly with each successive time point, demonstrating the role of the immune system in fish coping capacity. Although many of the genes discussed in this paper are still poorly characterized, this study provides new insights regarding the mechanisms occurring during the stress response of salmon parr and may form the basis for a useful guideline on timing of sampling protocols.

Protective Immunization of Atlantic Salmon (Salmo salar L.) against Salmon Lice (Lepeophtheirus salmonis) Infestation.

Vaccination against salmon lice (Lepeophtheirus salmonis) is a means of control that averts the negative effects of chemical approaches. Here, we studied the immunogenicity and protective effect of a vaccine formulation (based on a salmon lice-gut recombinant protein [P33]) against Lepeophtheirus salmonis infestation in Atlantic salmon in a laboratory-based trial. Our findings revealed that P33 vaccine can provide a measure of protection against immature and adult salmon lice infestation. This protection seemed to be vaccine dose-dependent, where higher doses resulted in lower parasitic infestation rates. We also provide immunological evidence confirming that P33-specific immune response can be triggered in Atlantic salmon after P33 vaccination, and that production of P33-specific antibodies in blood can be detected in vaccinated fish. The negative correlation between P33-specific IgM in salmon plasma and salmon lice numbers on vaccinated fish suggests that protection against lice can be mediated by the specific antibody in salmon plasma. The success of P33 vaccination in protecting salmon against lice confirms the possibility of employing the hematophagous nature of the parasite to deliver salmon-specific antibodies against lice-gut proteins.

Quantification of Defensive Proteins in Skin Mucus of Atlantic salmon Using Minimally Invasive Sampling and High-Sensitivity ELISA.

Protocols used to collect fish skin mucus may inadvertently compromise the sampled fish or the resulting sample. Here, we used three methods (wiping, scraping, and absorption) to collect skin mucus from Atlantic salmon and compared their invasiveness on fish skin epithelium. We found that the absorption method was the least invasive. We also compared the abundance of antigen-specific immunoglobulin M subtype A antibodies (IgM-A Ab) and complement component 5 (C5) in mucus samples collected from vaccinated fish by the three methods. An enzyme-cascade-amplification strategy colorimetric immune assay was optimized and used to analyze IgM-A, and ELISA was used to analyze C5. The abundance of antigen-specific IgM-A in skin mucus was comparable between the three methods, but C5 was significantly lower in absorbed mucus in comparison to in the wiped or scraped mucus samples. Absorbed skin mucus samples collected from various body regions of salmon, levels of C5 were comparable, while specific IgM-A amounts varied between the regions. By comparing three mucus-absorbing materials (medical wipe, gauze, and cotton) for their ability to absorb and release IgM-A and C5, medical wipes proved to be ideal for IgM-A analysis, whereas gauze was the best for C5 analysis.

Vaccination with Ectoparasite Proteins Involved in Midgut Function and Blood Digestion Reduces Salmon Louse Infestations.

Infestation with the salmon louse Lepeophtheirus salmonis (Copepoda, Caligidae) affects Atlantic salmon (Salmo salar L.) production in European aquaculture. Furthermore, high levels of salmon lice in farms significantly increase challenge pressure against wild salmon populations. Currently, available control methods for salmon louse have limitations, and vaccination appears as an attractive, environmentally sound strategy. In this study, we addressed one of the main limitations for vaccine development, the identification of candidate protective antigens. Based on recent advances in tick vaccine research, herein, we targeted the salmon louse midgut function and blood digestion for the identification of candidate target proteins for the control of ectoparasite infestations. The results of this translational approach resulted in the identification and subsequent evaluation of the new candidate protective antigens, putative Toll-like receptor 6 (P30), and potassium chloride, and amino acid transporter (P33). Vaccination with these antigens provided protection in Atlantic salmon by reducing adult female (P33) or chalimus II (P30) sea lice infestations. These results support the development of vaccines for the control of sea lice infestations.

Complement factor C5 in Atlantic salmon (Salmo salar): Characterization of cDNA, protein and glycosylation.

Complement component 5 (C5) is an essential factor of the defensive complement system in all vertebrates. We report the characterization of C5 cDNA and protein from Atlantic salmon (Salmo salar), a teleost fish species of high importance in aquaculture. The C5 cDNA cloned from liver is 5079 nucleotides long, whose translation product has a molecular weight of 190 kDa, with the classical ß-α orientation and motifs/sites for ß-α cleavage (678RPKR681) and cleavage by C5 convertases (R758). Mass spectrometric analysis show a single N-linked, biantennary, complex glycan at N1125. Moreover, the N-linked glycan displays an unusual modification in the form of acetylated sialic acid residues. Three anti-C5 antisera produced in mice using purified C5 worked in immunohistochemical analyses of formalin fixed liver tissue. The purification method, whereby inactive and activated (C5b) forms were isolated, opens for interesting studies on the complement function in fish, including possible connection to stress, disease and glycosylation.

Effects of dietary deoxynivalenol or ochratoxin A on performance and selected health indices in Atlantic salmon (Salmo salar).

Post-smolt Atlantic salmon (Salmo salar) were fed with standard feed added one of five concentrations of either pure deoxynivalenol (DON; 0.5-6 mg/kg) or pure ochratoxin A (OTA; 0.2-2.4 mg/kg), or no added toxins for up to 8 weeks. Performance effects (feed intake, feed efficiency, gain, length and condition factor), various clinical biochemical parameters, packed cell volume and vaccination response against Aeromonas salmonicidae were all inversely correlated with DON dose, whereas relative liver weight increased with DON dose. In fish fed OTA, however, the effects at the doses tested were rather small. We observed no effects of OTA exposure on performance parameters, but some clinical biochemical parameters tended to increase with OTA dose primarily at 3 weeks, and compared with controls OTA exposure caused increased mRNA expression of two immune markers in the spleen. No liver histopathological effects were found from DON or OTA exposure. For DON, we derived a BMDL20 of 0.3 mg/kg feed for reduced total protein in plasma, a BMDL5 of 0.5 mg/kg feed for reduced condition factor, and a NOAEL of 1 mg/kg feed for DON. For OTA, a BMDL or NOAEL could not be derived (>2.4 mg/kg).

Piscine orthoreovirus can infect and shed through the intestine in experimentally challenged Atlantic salmon (Salmo salar L.).

Piscine orthoreovirus (PRV) is a ubiquitous virus in Norwegian salmon farms associated with the disease heart and skeletal muscle inflammation (HSMI). Experimental challenge has shown that the virus replicates in circulating red blood cells of Atlantic salmon prior to infecting heart myocytes. The infection route from water to blood is however still unknown. The related mammalian orthoreovirus primarily infects the lungs and gastrointestinal (GI) tract and is proposed to spread mainly through the faecal-oral route. To investigate the role of the salmonid GI tract in PRV-infection, oral and anal administration of virus was compared to intraperitoneal (i.p.) injection. When administered anally, PRV was transferred to blood 4 days post challenge (dpc) and levels peaked at 42 dpc, similar to i.p. injected fish. PRV was detected in heart and faeces with corresponding kinetics, and inflammatory heart lesions consistent with HSMI were observed from 49 dpc. The orally intubated group showed slower virus kinetics in both blood and heart, and no signs of HSMI. Compared to the oral and i.p. administration routes, leakage of virus inoculate by anal intubation was minor and challenge was restricted to the mid- and distal intestine. These findings show that anal intubation is an efficacious method for PRV delivery to the GI tract and demonstrates that PRV can establish infection through the intestine with the potential for transmission via faeces.

Vaccine Adjuvants in Fish Vaccines Make a Difference: Comparing Three Adjuvants (Montanide ISA763A Oil, CpG/Poly I:C Combo and VHSV Glycoprotein) Alone or in Combination Formulated with an Inactivated Whole Salmonid Alphavirus Antigen.

Most commercial vaccines offered to the aquaculture industry include inactivated antigens (Ag) formulated in oil adjuvants. Safety concerns are related to the use of oil adjuvants in multivalent vaccines for fish, since adverse side effects (e.g., adhesions) can appear. Therefore, there is a request for vaccine formulations for which protection will be maintained or improved, while the risk of side effects is reduced. Here, by using an inactivated salmonid alphavirus (SAV) as the test Ag, the combined use of two Toll-like receptor (TLR) ligand adjuvants, CpG oligonucleotides (ODNs) and poly I:C, as well as a genetic adjuvant consisting of a DNA plasmid vector expressing the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) was explored. VHSV-G DNA vaccine was intramuscularly injected in combination with intraperitoneal injection of either SAV Ag alone or combined with the oil adjuvant, Montanide ISA763, or the CpG/polyI:C combo. Adjuvant formulations were evaluated for their ability to boost immune responses and induce protection against SAV in Atlantic salmon, following cohabitation challenge. It was observed that CpG/polyI:C-based formulations generated the highest neutralizing antibody titres (nAbs) before challenge, which endured post challenge. nAb responses for VHSV G-DNA- and oil-adjuvanted formulations were marginal compared to the CpG/poly I:C treatment. Interestingly, heat-inactivated sera showed reduced nAb titres compared to their non-heated counterparts, which suggests a role of complement-mediated neutralization against SAV. Consistently elevated levels of innate antiviral immune genes in the CpG/polyI:C injected groups suggested a role of IFN-mediated responses. Co-delivery of the VHSV-G DNA construct with either CpG/polyI:C or oil-adjuvanted SAV vaccine generated higher CD4 responses in head kidney at 48 h compared to injection of this vector or SAV Ag alone. The results demonstrate that a combination of pattern recognizing receptor (PRR) ligands, such as CpG/polyI:C, increases both adaptive and innate responses and represents a promising adjuvant strategy for enhancing the protection of future viral vaccines.

Sequence analysis of the genome of piscine orthoreovirus (PRV) associated with heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar).

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in µ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-ß signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins µ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in µ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.

Immune parameters correlating with reduced susceptibility to pancreas disease in experimentally challenged Atlantic salmon (Salmo salar).

Two strains of Atlantic salmon (Salmon salar) with different susceptibility to infectious salmon anaemia (ISA) were challenged with salmon pancreas disease virus (SPDV), the etiological agent of salmon pancreas disease (PD), by cohabitation. Serum and tissues were sampled at 0, 1, 3, 6 and 8 weeks post-challenge. Experimental challenge with SAV did not cause mortality, but virus loads and assessment of histopathology indicated that the fish more resistant to ISAV (ISAHi) also was more resistant to PD. Eight weeks post-challenge, the ISAHi strain had higher titres of SAV-neutralising antibodies than the less resistant strain (ISALo). Transcript levels of four adaptive and six innate immune parameters were analysed by real-time RT-PCR in heart, head kidney (HK) and gills of both strains. Secretory IgM (sIgM) and CD8 levels differed most between the two salmon strains. The ISAHi strain had significantly higher levels of sIgM in HK at all samplings, and significantly higher CD8 levels in gills at most samplings. In heart, both sIgM and CD8 levels increased significantly during the challenge, but the increase appeared earlier for the ISALo strain. By hierarchical clustering analysis of mRNA levels, a clear segregation was observed between the two strains prior to the virus challenge. As the viral infection developed, the clustering divide between fish strains disappeared, first for innate and later for adaptive parameters. At eight weeks post-challenge, the divide had however reformed for adaptive parameters. Possible pair-wise correlation between transcript levels of immune parameters was evaluated by a non-parametric statistical test. For innate parameters, the extent of correlation peaked at 3 wpc in all tissues; this came rapidly for ISALo and more gradual for ISAHi. The ISAHi strain tended to show higher correlation for innate parameters in heart and gill than ISALo at early sampling times. For adaptive immune parameters, little correlation was observed in general, except for ISAHi in heart at 6 wpc. Overall, the observed differences in immune parameters may provide important clues to the causes underlying the observed difference in susceptibility to PD.

Immune responses in Atlantic salmon (Salmo salar) following protective vaccination against infectious salmon anemia (ISA) and subsequent ISA virus infection.

Infectious salmon anemia (ISA) is an orthomyxoviral disease that has had devastating effects on farmed Atlantic salmon. ISA is still a disease resulting in continued loss of revenues and therefore development of effective vaccines is of great importance. Commercial vaccines against ISA are available, but the efficacy is poorly described. There is little information about vaccine-induced immune factors preventing ISA virus (ISAV) infection today. In this study we assessed the protective effects and immunogenicity of vaccines containing three different quantities of the inactivated ISAV antigen. Our findings indicated that immunization induced effective protection in Atlantic salmon with a relative percent survival (RPS) as high as 86. The level of protection was correlated to the amount of ISAV antigen in the vaccine, and fish immunized with high antigen amounts produced detectable ISAV-specific and neutralizing antibodies. While ISAV infection was detectable in non-vaccinated control fish challenged by cohabitation, no infection was detected in fish immunized with high antigen amounts. After challenge, transcriptional analysis of selected immune-related genes demonstrated activation of innate immune responses in ISAV-infected control fish, but not in vaccine protected fish. This study furthers the knowledge about vaccine efficacy and vaccine-induced immunity to ISAV challenge in Atlantic salmon.

Putative virulence genes in Moritella viscosa: activity during in vitro inoculation and in vivo infection.

Moritella viscosa is considered to be the main aetiological agent of winter ulcer disease, primarily affecting farmed salmonid fish in cold marine waters. Transcription profiles of twelve M. viscosa genes, potentially involved in the pathogenesis, were studied during the course of an in vitro cell culture infection assay. Transcription of the same genes was compared in vivo, in head kidney and ulcer tissues of Atlantic salmon challenged with M. viscosa. During the in vitro infection, three putative toxins: a putative repeats in toxin gene (rtxA), a putative cytotoxic necrotizing factor (cnf) and a putative hemolysin increased their transcription significantly with time and coincident with cell rounding. Furthermore, the majority of the genes were stimulated by presence of fish cells and showed higher activity when adhered to fish cells compared to their planktonic counterpart. In vivo gene transcription studies revealed an up-regulation of a putative lateral flagellin in ulcer compared to head kidney tissues in the same individual. A similar trend was seen for cnf and a gene encoding a putative protease, indicating a role for these factors in colonization and tissue damage.

A novel totivirus and piscine reovirus (PRV) in Atlantic salmon (Salmo salar) with cardiomyopathy syndrome (CMS).

BACKGROUND: Cardiomyopathy syndrome (CMS) is a severe disease affecting large farmed Atlantic salmon. Mortality often appears without prior clinical signs, typically shortly prior to slaughter. We recently reported the finding and the complete genomic sequence of a novel piscine reovirus (PRV), which is associated with another cardiac disease in Atlantic salmon; heart and skeletal muscle inflammation (HSMI). In the present work we have studied whether PRV or other infectious agents may be involved in the etiology of CMS. RESULTS: Using high throughput sequencing on heart samples from natural outbreaks of CMS and from fish experimentally challenged with material from fish diagnosed with CMS a high number of sequence reads identical to the PRV genome were identified. In addition, a sequence contig from a novel totivirus could also be constructed. Using RT-qPCR, levels of PRV in tissue samples were quantified and the totivirus was detected in all samples tested from CMS fish but not in controls. In situ hybridization supported this pattern indicating a possible association between CMS and the novel piscine totivirus. CONCLUSIONS: Although causality for CMS in Atlantic salmon could not be proven for either of the two viruses, our results are compatible with a hypothesis where, in the experimental challenge studied, PRV behaves as an opportunist whereas the totivirus might be more directly linked with the development of CMS.

Quantitative investigation of antigen and immune response in nervous and lymphoid tissues of Atlantic halibut (Hippoglossus hippoglossus) challenged with nodavirus.

The present study reports the quantitative analysis of the spatio-temporal development of nodavirus infection and corresponding immune response in juvenile Atlantic halibut (Hippoglossus hippoglossus) challenged by intramuscular injection of nodavirus. Novel quantitative real-time RT-PCR protocols were applied to evaluate the absolute copy numbers of nodavirus RNA2 (RNA2) and secretory-IgM mRNA (sec-igmicro) in the eye, brain, mid/posterior kidney and spleen sampled over a period of 81 days. In the eye and brain, levels of both RNA2 and sec-igmicro increased significantly early in the infection. In the spleen and mid/posterior kidney, both RNA2 and sec-igmicro were detected but the levels remained unchanged during the experimental period. The levels of RNA2 and sec-igmicro in the eye and brain were strongly correlated (P<0.0001). Nodavirus antigen was demonstrated by immunohistochemistry (IHC) in the retina of eyes from a relatively few fish from day 34 post challenge (brain not examined), but not at any time in the spleen and anterior kidney. By IHC, IgM+ cells were observed in conjunction with nodavirus positive IHC labelling in the retina. In both the spleen and anterior kidney, the number of IgM+ cells increased from day 3 post challenge. By conventional real-time RT-PCR, RNA2 was only sporadically demonstrated in the posterior intestine, heart and gills. ELISA analysis revealed a nodavirus specific antibody response in serum that was significant from day 18 post challenge. No clinical signs or mortality related to nodavirus infection were observed in the challenged halibut. The results suggest that the nodavirus infection induced a significant antibody response through activation of B-cells in the kidney and spleen, and involved a substantial migration of antibody-secreting cells to infected peripheral tissues.

Serum immunoglobulin M in Atlantic halibut (Hippoglossus hippoglossus): characterisation of the molecule and its immunoreactivity.

Three preparations of purified immunoglobulin (IgM) were isolated from serum of Atlantic halibut (Hippoglossus hippoglossus) by means of three different methods, and each of the three IgM preparations was used to produce a polyclonal rabbit anti-halibut IgM antiserum. One of the IgM preparations was employed in the characterisation of halibut serum immunoglobulin. Halibut IgM was shown to consist of two subunits, compatible with heavy (mu) and light (L) chains. A single mu chain at approximately 76 kDa, and six possible molecular weight (MW) variants of L chain were found (range approximately 25 to approximately 28.5 kDa). IgM was glycosylated on the heavy chain and N-linked carbohydrate constituted approximately 10.3% (w/w) of the total MW of IgM. The dominant form of non-reduced IgM had a MW of approximately 780 kDa, suggesting a tetrameric structure. Non-reduced IgM also showed a number of minor protein bands. Based on estimated MW, the relative carbohydrate content and the reactivity with all three anti-halibut IgM antisera, mono-, di- and trimeric redox forms of IgM were identified. The three antisera were characterised as to specificity and reactivity by means of enzyme linked immuno-sorbent assay (ELISA), crossed immuno-electrophoresis (CIE), and immunoblotting methods. The antisera showed a considerable diversity in their specificity to the suggested MW variants of halibut Ig light chain. A method for immunohistochemical detection of IgM in tissue was established. Protein A or protein G affinity for the IgM was not detectable.

Absolute quantitation of RNA by a competitive real-time RT-PCR method using piscine nodavirus as a model.

The development and validation of a novel competitive real-time RT-PCR assay for the absolute quantitation of RNA from a piscine nodavirus are described. The assay utilises simultaneous amplification of target RNA and a recombinant RNA competitor in a single reaction, using the same pair of primers. The target and competitor products are distinguished by the use of two specific double-dye probes. The recombinant RNA competitor was designed to obtain a maximum sequence similarity with the target sequence, and the RT-PCR amplification efficiency of the competitor and target RNA was found to be identical. The intra-assay variation was 15-24% depending on the specific protocol for quantitation. The lower quantitation limit was estimated to 980 copies of RNA/reaction. The assay was used to evaluate the temporal development of the virus titre in an in vitro experiment, in which SSN-1 cell cultures were inoculated with nodavirus.

Immune- and enzyme histochemical characterisation of leukocyte populations within lymphoid and mucosal tissues of Atlantic halibut (Hippoglossus hippoglossus).

Leukocyte populations within the kidney, spleen, posterior intestine and gills of Atlantic halibut were investigated using a panel of histological, enzyme- and immunohistochemical methods. In the kidney and spleen, a diverse population of leukocytes was associated with the extensive network of sinusoids and larger blood vessels present in these tissues. IgM+ cells (B-cells, plasma cells and IgM-bearing macrophages) and large mononuclear cells showing reactivity for non-specific esterase (NSE) and acid phosphatase (ACP), representing macrophage populations, were often associated with vessel walls that were also the site of trapping of fluorescent microspheres. In the kidney, trapping of 0.1 and 0.5 microm diameter microspheres occurred at these sites but in the spleen, the 0.1 microm diameter microspheres were retained in ellipsoids. The lymphoid tissues of the kidney and spleen possessed a spread population of 5'-nucleotidase+ (5'N+) cells but compartmentalisation of the splenic white pulp was suggested by an absence of these 5'N+ reticular cells in areas associated with melanomacrophage accumulations and in areas rich in IgM+ cells. A striking feature of the mucosal tissues was the diversity of leukocyte populations within the epithelium particularly of the posterior intestine, including IgM+ cells and NSE+, ACP+ and 5'N+ mononuclear cells. Although limited in numbers in the posterior intestine, IgM+ cells were more common in the epithelium than in the lamina propria. In the gills, leukocytes as detected by enzymatic reactivity were scarce, but IgM+ cells were very abundant in the stratified epithelium of the gill arch and filaments. The difference in distribution of these leukocyte populations between the intestines and gills suggested a compartmentalisation of the mucosal immune system and the need to assess the immunological competence of mucosal tissues in Atlantic halibut.

Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination.

Fish nodaviruses (betanodaviruses) are small, non-enveloped icosahedral single-stranded positive-sense RNA viruses that can cause viral encephalopathy and retinopathy (VER) in a number of cultured marine teleost species, including Atlantic halibut (Hippoglossus hippoglossus). A recombinant protein vaccine and a DNA vaccine were produced, based on the same capsid-encoding region of the Atlantic halibut nodavirus (AHNV) genome, and tested for protection in juvenile turbot (Scophthalmus maximus). Vaccine efficacy was demonstrated in the fish vaccinated with recombinant capsid protein but not in the DNA-vaccinated fish, despite the fact that in vivo expression of the DNA vaccine-encoded antigen was confirmed by RNA in situ hybridisation and immunohistochemistry. Combined DNA and recombinant vaccine administration did not improve the effect of the latter. Surprisingly, fish vaccinated with 50 microg recombinant protein demonstrated a threefold lower survival rate than the two groups that received 10 microg recombinant protein. Neither the recombinant protein vaccine nor the DNA vaccine induced anti-viral antibodies 9 weeks after immunisation, while antibodies reactive with the recombinant protein were detectable mainly in fish vaccinated with 50 microg recombinant protein. The study also demonstrates evidence of viral replication inside the myocytes of intramuscularly challenged fish.