Immunity In Rainbow Trout Research Articles

Subcellular components of probiotics Kocuria SM1 and Rhodococcus SM2 induce protective immunity in rainbow trout ( Oncorhynchus mykiss, Walbaum) against Vibrio anguillarum

The efficacy of cellular components of probiotics Kocuria SM1 and Rhodococcus SM2 to protect rainbow trout ( Oncorhynchus mykiss, Walbaum) against vibriosis was assessed. Groups of fish (average weight = 10–15 g) were immunized intraperitoneally (i.p.) with 0.1 ml of subcellular materials, i.e., 0.2 ± 0.05 mg protein per fish, comprising extracellular proteins (ECPs), cell wall proteins (CWPs) and whole cell proteins (WCPs) of SM1 and SM2, respectively, or with 0.1 ml of phosphate-buffered saline (PBS) to serve as the control. Seven days after administration, fish from each group were challenged i.p. with 0.1 ml of a suspension in PBS of 3 × 10 5 cells ml −1 per fish of Vibrio anguillarum. Use of CWPs and WCPs demonstrated significantly ( P < 0.05) better protection against V. anguillarum insofar as mortalities were reduced to 11–17% [relative percent survival (RPS) = 80–87%], although ECPs fared less well (mortalities = 33–38%; RPS = 56–62%; P > 0.05), compared to 86% mortalities of the controls. The mode of action reflected activation of innate immune factors by CWPs and WCPs, demonstrating significantly ( P < 0.05) increased expression of respiratory burst (optical density; OD 550nm) from 0.039 to 0.043–0.045, peroxidase (OD 550nm) from 0.26 to 0.37–0.55, and bacterial killing activities (i.e., percentage of surviving bacteria reduced from 79% to 56–57% for SM2). Moreover, an elevation of leucocyte number (from 1.93% to 1.98–2.93%; P > 0.05) and immunoglubolin level (from 27 mg ml −1 to 28.5–33 mg ml −1; P > 0.05) were observed with the experimental groups. These results indicate that cell components of the probiotics stimulate an immune response.

Influence of deltamethrin on nonspecific cellular and humoral defense mechanisms in rainbow trout (Oncorhynchus mykiss)

The influence of deltamethrin on the innate immunity in rainbow trout was examined. Fish were immersed in deltamethrin at doses of 1, 2, and 4 microg/L for 30 min. The results showed that deltamethrin at doses of 2 and 4 microg/L decreased phagocytic activity of spleen macrophages and proliferative response of pronephros lymphocytes at days 1, 2, and 5 after immersion. Deltamethrin at these doses decreased the lysozyme activity, total protein, and immunoglobulin levels in serum. The greatest immunosuppressive influence of deltamethrin at dose 4 microg/L was observed at the end of the study.

Protection of rainbow trout from infectious hematopoietic necrosis (IHN) by injection of infectious pancreatic necrosis virus (IPNV) or Poly(I:C)

It was recently reported that prophylaxis against infectious hematopoietic necrosis virus (IHNV) in fish was induced by pre-exposure to the infectious pancreatic necrosis virus (IPNV). Here the establishment of IHNV immunity in rainbow trout Oncorhynchus mykiss was investigated by IHNV challenge following non-lethal pre-infection with IPNV. Also, synthetic double-stranded RNA polyinosinic polycytidylic acid, Poly(I:C), an inducer for interferon (IFN), was evaluated as a substitute for IPNV induction of the non-specific antiviral state and subsequent IHNV-specific immunity in fish. Rainbow trout pre-infected with IPNV were protected from IHNV challenge 7 d later (relative percentage survival, RPS: 68.8%), and IHNV-specific antibodies were detected in sera from the survivors. Moreover, these surviving fish showed 91.6% RPS when re-challenged with IHNV 28 d after the primary IHNV challenge. Thus, fish appear to acquire IHNV-specific immunity through the IHNV challenge following pre-injection with IPNV. Fish pre-injected with Poly(I:C) were also highly protected from IHNV challenge 2 d later (RPS: 95.2%), and IHNV-specific antibodies were also detected amongst survivors. The survivors showed a 100% survival rate following re-challenge with IHNV both 21 and 49 d after the primary IHNV challenge. Thus, IHNV immunity in rainbow trout is induced by challenge with live IHNV following pre-injection with either IPNV or Poly(I:C). The use of Poly(I:C) to induce an anti-viral state protecting rainbow trout from an otherwise lethal vaccination dose of IHNV may have application to a wider range of fish species and fish pathogenic viruses.

Immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), with a low molecular mass fraction isolated from Flavobacterium psychrophilum

Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome has become a widespread fish pathogen in freshwater aquaculture worldwide. In this study, a low molecular mass fraction (P25-33), with an approximate weight of 25-33 kDa, was identified among F. psychrophilum strains in an immunoblotting analysis with anti-F. psychrophilum sera. The immunogenic efficacy of the isolated and extracted P25-33 was investigated in two intraperitoneal immunization trials with rainbow trout, Oncorhynchus mykiss (Walbaum). The first trial included immunizations using P25-33 with Freund's complete adjuvant (FCA) and the second trial included immunizations using P25-33, formalin-inactivated whole and sonicated F. psychrophilum cell preparations without FCA. In both trials, antibody titres against F. psychrophilum were analysed with an enzyme-linked immunosorbent assay and the efficacy of the immunizations was determined by a challenge with F. psychrophilum. The P25-33 was shown to give rise to a protective immune response in rainbow trout after immunization with FCA, but not without FCA when a low concentration of P25-33 was used. Instead formalin-inactivated whole and sonicated cells of F. psychrophilum were able to protect the immunized fish more effectively when immunized without FCA. The results suggest that whole or sonicated F. psychrophilum cells could be better candidates for a cost-effective water-based injection vaccine than the immunogenic fraction.

Isolation of rifampicin resistant Flavobacterium psychrophilum strains and their potential as live attenuated vaccine candidates

Previous studies have demonstrated that passage of pathogenic bacteria on increasing concentrations of the antibiotic rifampicin leads to the attenuation of virulence and these resistant strains may serve as live attenuated vaccines. Two rifampicin resistant strains of Flavobacterium psychrophilum, 259-93A.16 and 259-93B.17, were generated by passage on TYES plates containing increasing concentrations of rifampicin. Electrophoretic analysis of whole-cell lysates prepared from the parent and resistant strains identified five differentially expressed proteins between the 259-93B.17 strain and parent strain, while there were no differences identified between the 259-93A.16 and parent strain. The LPS banding patterns were identical between all three strains. Bacterial challenges of rainbow trout ( Oncorhynchus mykiss Walbaum) with the resistant strains demonstrated that the 259-93B.17 strain was highly attenuated and the 259-93A.16 strain was modestly attenuated at the challenge doses tested. Immunization of rainbow trout with the live attenuated 259-93B.17 strain by intraperitoneal injection resulted in significant protection against challenge with the virulent parent F. psychrophilum strain at 8 and 15 weeks post-immunization and fish exhibited elevated specific antibody titers. Importantly, immersion delivery of the 259-93B.17 strain stimulated protective immune responses in fish at 10 weeks post-immunization. The results demonstrate that the rifampicin resistant 259-93B.17 strain may serve as an effective live attenuated vaccine for the prevention of F. psychrophilum infections.

Development of adaptive immunity in rainbow trout, Oncorhynchus mykiss (Walbaum) surviving an infection with Yersinia ruckeri

Development of adaptive immunity in rainbow trout ( Oncorhynchus mykiss) surviving a primary infection with 5 × 10 5 CFU Yersinia ruckeri O1 (LD 50 dose) was investigated by transcriptome analysis of spleen tissue. These fish surviving a primary infection showed also a significantly increased survival following a secondary infection (same dose) when compared to naïve trout. The weight of the rainbow trout spleen doubled during the first 14 days of the primary infection but the affected organs subsequently recovered normal weight which remained constant during the re-infection period. Gene transcription in the spleen was measured using Quantitative real-time RT-PCR (qPCR). Samples taken 8 h.p.i., 1, 3, 7, 14 and 28 d.p.i. were compared to PBS-injected control fish sampled at the same time points. The investigated cytokines and chemokines comprised interleukin (IL)-1β, IL-1 receptor antagonist (Ra), IL-6, IL-8, IL-10, IL-11 and IFN-γ, IL-1 receptor I and II (IL-RI and IL-RII). Transcript levels of genes encoding cytokines and receptors were increased during the primary infection but not during the secondary infection. Changes of T cell occurrence or activity in the spleen during the infections were inferred from the transcript level of T cell receptor (TCR), CD4 and CD8α genes. No alteration in the expression of MHC class ll and immunoglobulin (Ig)M and IgT was detected during the experiment. The amount of Y. ruckeri O1 in the spleen was measured with a Y. ruckeri 16S ribosomal RNA specific qPCR and this parameter was correlated to the expression of IL-1β, IL-8 and IL-10 genes with a peak expression at 3 d.p.i. (first infection). The low transcript levels of the bacterial gene and the hosts' immune genes during the re-infection can be interpreted as a result of development of adaptive immunity. This would explain the relatively fast elimination of the bacteria during the secondary infection whereby the activation of cytokines becomes less pronounced.

Immunization of Rainbow Trout (Oncorhynchus mykiss) against Lactococcus garvieae Using Vaccine Mixtures

The effectiveness of vaccine mixtures against lactococcosis was tested in rainbow trout (Oncorhynchus mykiss). The M1 strain of Lactococcus garvieae, isolated from a recent outbreak of lactococcosis at a rainbow trout farm in Turkey, was used in a trial comparing five immuniza- tion treatments: (a) formalin inactivated bacterin (vaccine), (b) the above bacterin together with Freund’s Incomplete Adjuvant (FIA), (c) the bacterin combined with β-glucan, (d) β-glucan only, and (e) phosphate buffered saline-PBS (control). Fish were given intrapritoneal injections and challenged by exposure to the bacteria 30, 75, or 125 days after vaccination. In fish exposed to the bacteria 30 days after injection, the relative percent survival (RPS) was 88.89% in the group that received only bacterin and 100% in the group that received the bacterin combined with FIA. Immunity remained high in the bacterin+FIA group, as the RPS in this group remained 100% in fish challenged at 75 days, significantly higher than in all other groups. In fish exposed to the bacteria 125 days after vaccination, the RPS was 54.55% in fish vaccinated with the bacterin only and 84.84% in fish vaccinated with bacterin+FIA. In the group that received only β-glucan, immunity did not improve after vaccination. Micro-agglutination tests of serums showed that immunized fish produced antibodies at high titers within 30 days. In short, the formalin-inacti- vated M1 strain provided longer lasting protection against Lactococcus garvieae in rainbow trout when combined with FIA than when administered alone or with β-glucan.

Cytokine expression in leucocytes and gut cells of rainbow trout, Oncorhynchus mykiss Walbaum, induced by probiotics

Understanding how the various host cells respond to probiotic bacteria in vitro may provide important insight into elaborate immune responses triggered by beneficial bacteria. The aim of this study was to investigate the detailed pattern of the mRNA expression of cytokines (IL-1β, IL-8, TNF-α and TGF-β) in head kidney (HK) leucocytes and gut cells isolated from rainbow trout ( Oncorhynchus mykiss Walbaum) after co-culturing with live probiotics. HK leucocytes and gut cells adjusted to 5 × 10 6 and 2 × 10 6 ml −1, respectively, in L-15 medium containing 25% decomplemented FCS and 300 mg l −1 l-glutamine were co-cultured with Carnobacterium maltaromaticum B26 and C. divergens B33 at an multiplicity of infection of 25 for 6 and 12 h. Quantitative real-time reverse transcriptase polymerase chain reaction using SYBR Green I was employed to determine the mRNA expression of studied genes. Although neither probiotic strains significantly induced mRNA of the cytokines in gut cells, expression ratios of IL-1β and TNF-α of HK cells were significantly higher, suggesting that these bacteria can stimulate innate immunity in rainbow trout.

Immunity in rainbow trout, Oncorhynchus mykiss, against the monogenean Discocotyle sagittata following primary infection.

Rainbow trout (Oncorhynchus mykiss) were experimentally infected by continuous or single exposure with the monogenean Discocotyle sagittata. To determine whether immunity follows primary infection, fish were exposed to a secondary challenge by one of two modes: (1) primary infections were cleared with praziquantel (PZQ) and hosts re-infected with 100 oncomiracidia; (2) parasites were allowed to reach maturity and hosts super-infected with 100 oncomiracidia. Fish challenged after initial continuous exposure developed significant partial resistance to re-infection, carrying burdens 35% lower than controls. PZQ treatment controls demonstrated that the drug did not account for the protection observed. Single exposure did not significantly modify the outcome of secondary challenges. Super-infection experiments suggested that no concomitant immunity develops. No correlation was found between initial burdens and the outcome of secondary challenges in the same individual. Significantly elevated anti-D. sagittata antibodies were detected in infected fish, but there was no correlation between immunoglobulin levels and parasite burdens.

Enhancement of innate immunity in rainbow trout ( Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products

The effects of orally administered carotenoids from natural sources on the non-specific defense mechanisms of rainbow trout were evaluated in a nine-week feeding trial. Fish were fed four diets containing either β-carotene or astaxanthin at 100 and 200 mg kg −1 from the marine algae Dunaliella salina and red yeast Phaffia rhodozyma, respectively, and a control diet containing no supplemented carotenoids. Specific growth rate and feed:gain ratio were not affected by dietary carotenoid supplementation. Among the humoral factors, serum alternative complement activity increased significantly in all carotenoid supplemented groups when compared to the control. On the other hand, serum lysozyme activity increased in the Dunaliella group but not in the Phaffia group, whereas plasma total immunoglobulin levels were not altered by the feeding treatments. As for the cellular responses, the superoxide anion production from the head kidney remained unchanged while the phagocytic rate and index in all supplemented groups were significantly higher than those of the control. These findings demonstrate that dietary carotenoids from both D. salina and P. rhodozyma can modulate some of the innate defense mechanisms in rainbow trout.

Bacterial kidney disease as a model for studies of cell mediated immunity in rainbow trout (Oncorhynchus mykiss)

A cell mediated immune (CMI) response was measured in vitro to heat-killed and to paraformaldehyde fixed Renibacterium salmoninarum (Rs) in rainbow trout (Oncorhynchus mykiss) experimentally challenged with live Rs. The mitogenic response to the T lymphocyte mitogen Concanavalin A (Con A) was reduced during samplings 4 to 6 weeks after immersion, but no effect of the response to the B lymphocyte mitogen lipopolysaccharide (LPS) was detected. The subpopulation of lymphocytes, detected by the monoclonal antibody 1C2, was decreased from the 4th week to the 5th week of infection, and remained at the decreased level up to 10 weeks post immersion. The proportion of Immunoglobulin (Ig) bearing lymphocytes was not affected during the Rs infection period. The humoral antibody level to heat-stable Rs-antigens was increased up to 10 weeks after immersion but after 27 weeks was reduced to a level similar to that of the non-challenged fish. An anamnestic response was demonstrated in challenged fish, as intraperitoneal injection of heat-treated Rs bacteria into Rs challenged fish elicited a stronger humoral antibody response compared with injection into non-challenged fish.

Bacterial kidney disease as a model for studies of cell mediated immunity in rainbow trout (Oncorhynchus mykiss)

A cell mediated immune (CMI) response was measured in vitro to heat-killed and to paraformaldehyde fixed Renibacterium salmoninarum (Rs) in rainbow trout (Oncorhynchus mykiss) experimentally challenged with live Rs. The mitogenic response to the T lymphocyte mitogen Concanavalin A (Con A) was reduced during samplings 4 to 6 weeks after immersion, but no effect of the response to the B lymphocyte mitogen lipopolysaccharide (LPS) was detected. The subpopulation of lymphocytes, detected by the monoclonal antibody 1C2, was decreased from the 4th week to the 5th week of infection, and remained at the decreased level up to 10 weeks post immersion. The proportion of Immunoglobulin (Ig) bearing lymphocytes was not affected during the Rs infection period. The humoral antibody level to heat-stable Rs-antigens was increased up to 10 weeks after immersion but after 27 weeks was reduced to a level similar to that of the non-challenged fish. An anamnestic response was demonstrated in challenged fish, as intraperitoneal injection of heat-treated Rs bacteria into Rs challenged fish elicited a stronger humoral antibody response compared with injection into non-challenged fish.

Antibody–antigen kinetics following immunization of rainbow trout ( Oncorhynchus mykiss) with a T-cell dependent antigen

Enhancement of the immune response through affinity maturation of the antibody response is a feature of the mammalian immune system and has important implications with respect to development of vaccination strategies. However, an absence of germinal centres and apparent lack of somatic hypermutation of immunoglobulin V genes suggests that this phenomenon does not occur in fish. We investigated the question of affinity maturation in rainbow trout ( Oncorhynchus mykiss) by measuring antibody–antigen binding kinetics using a BIAcore biosensor. Following immunization with a T-cell dependent antigen (FITC-KLH), relative binding affinities of serum and mucosal antibodies were assessed based on their dissociation rate constants ( k diss.). A detectable serum anti-FITC response developed by 4 weeks post-immunization, and a consistent shift to higher affinity antibody production (i.e. a decrease in k diss.) was observed over the ensuing course of the immune response. An average k diss. of 3.5×10 −4±0.27×10 −4 sec −1 was observed during early stages of the response (4 weeks), while by 6 weeks this decreased significantly ( p<0.05). Further reduction in k diss. was observed, with a low of 1.2×10 −4±0.06×10 −4 sec −1 being observed by week 12. Analysis of the anti-FITC response in skin-derived mucus revealed a similar pattern of decreasing k diss. as the immune response progressed. While these data clearly demonstrate a 2–3 fold increase in antibody–antigen binding during the course of the immune response in trout, the magnitude of this increase is much less than that seen in the mammalian immune response. This may reflect differences in the mechanisms underpinning this phenomenon in divergent species.

Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination

A DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was shown to provide significant protection as soon as 4 d after intramuscular vaccination in 2 g rainbow trout ( Oncorhynchus mykiss) held at 15°C. Nearly complete protection was also observed at later time points (7, 14, and 28 d) using a standardized waterborne challenge model. In a test of the specificity of this early protection, immunization of rainbow trout with a DNA vaccine against another fish rhabdovirus, viral hemorrhagic septicemia virus, provided a significant level of cross-protection against IHNV challenge for a transient period of time, whereas a rabies virus DNA vaccine was not protective. This indication of distinct early and late protective mechanisms was not dependent on DNA vaccine doses from 0.1 to 2.5 μg.

Partial cross protection against Ichthyophthirius multifiliis in Gyrodactylus derjavini immunized rainbow trout

Partial cross protection against a skin-parasitic ciliate has been recorded in rainbow trout previously immunized with an ectoparasitic platyhelminth. The susceptibility to infection by Ichthyophthirius multifiliis differed significantly between naive and Gyrodactylus derjavini immunized rainbow trout. Fish partly immune to the ectoparasitic monogenean G. derjavini became less infected and experienced lower mortality than naive fish when exposed to I. multifiliisinfections. In vitro studies on immobilization of theronts using decomplemented (heat-inactivated) serum from G. derjavini immune or non-immune hosts showed no immobilization. However, untreated serum from both immune and non-immune fish containing intact complement immobilized theronts (titre 128–256). In addition, non-specific priming of the host response with interleukin (IL-1), bacterial lipopolysaccharide (LPS), concanavalin A (Con A) or mannan did confer a partial resistance to I. multifiliis infection. This will suggest that non-specific factors including complement could be partly responsible for the host response against infections with this ciliate.

Partial cross protection against Ichthyophthirius multifiliis in Gyrodactylus derjavini immunized rainbow trout.

Partial cross protection against a skin-parasitic ciliate has been recorded in rainbow trout previously immunized with an ectoparasitic platyhelminth. The susceptibility to infection by Ichthyophthirius multifiliis differed significantly between naive and Gyrodactylus derjavini immunized rainbow trout. Fish partly immune to the ectoparasitic monogenean G. derjavini became less infected and experienced lower mortality than naive fish when exposed to I. multifiliis infections. In vitro studies on immobilization of theronts using decomplemented (heat-inactivated) serum from G. derjavini immune or non-immune hosts showed no immobilization. However, untreated serum from both immune and non-immune fish containing intact complement immobilized theronts (titre 128-256). In addition, non-specific priming of the host response with interleukin (IL-1), bacterial lipopolysaccharide (LPS), concanavalin A (Con A) or mannan did confer a partial resistance to I. multifiliis infection. This will suggest that non-specific factors including complement could be partly responsible for the host response against infections with this ciliate.

Recombinant infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout (Oncorhynchus mykiss).

Fragments of the glycoprotein genes of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) were cloned into a bacterial broad-host-range expression vector under the control of the plac promoter. Western blot (immunoblot) analysis with monoclonal antibodies specific to the glycoproteins demonstrated the inducible expression of the fusion proteins in Escherichia coli. Aeromonas salmonicida is the causative agent of furunculosis in salmonid fish. It was confirmed that an avirulent strain of A. salmonicida, A440, which contains a deletion in the structural gene for the paracrystalline surface protein array, will provide protective immunity against furunculosis when used as a live attenuated vaccine. The plasmid-encoded viral epitopes were then mobilized into A440 for use as a shuttle system for the expression of fragments of the glycoprotein genes of IHNV and VHSV. Vaccination of rainbow trout with A440 containing the viral epitopes resulted in the development of protective immunity against both VHSV and IHNV. This indicates that the use of cloned fragments of the glycoproteins and the use of A. salmonicida as a shuttle system constitute a feasible approach to fish vaccine development.

A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout.

Viral haemorrhagic septicaemia (VHS) is a fish rhabdovirus infection of world-wide importance. Control policies have been established but the disease still causes heavy losses in fish farming. The development of a recombinant subunit vaccine was initiated to produce a safe and effective vaccine to protect fish against VHS. The VHS virus (VHSV) glycoprotein, which induces neutralizing antibodies in rainbow trout, was chosen for expression in insect cells using a baculovirus vector. The M(r) of the recombinant protein estimated by SDS-PAGE was slightly lower than that of the native viral protein. The recombinant protein displayed different degrees of glycosylation and was recognized in ELISA by neutralizing antibodies. It was transported to the plasma membrane of insect cells where its ability to induce membrane fusion was preserved. The efficacy of the recombinant protein as a vaccine was compared with those of an inactivated and an attenuated vaccine. When injected intraperitoneally into rainbow trout, the baculovirus-encoded protein was shown (i) to induce the synthesis of VHSV-neutralizing antibodies and (ii) to confer protection against virus challenge. Immunization performed by immersion failed. This is the first report of a recombinant vaccine that protects fish against VHSV.

Immunization of rainbow trout ( Oncorhynchus mykiss) against gonadotropin-releasing hormone (GnRH): a potential anti-maturation vaccine?

Rainbow trout were injected with conjugates of gonadotropin-releasing hormone (GnRH) coupled to various protein carriers in an attempt to elicit an antibody response to GnRH. Glutaraldehyde-formed conjugates of GnRH injected in Freund's complete adjuvant (FCA) promoted some anti-GnRH antibody formation in trout but relied upon multiple boosting injections. Immunization of rainbow trout with an extended form of the releasing hormone (GnRH-Gly-Cys), coupled to the carrier proteins via a cysteine extension of the peptide, greatly enhanced antibody formation to GnRH. The purified protein derivative (PPD) of tuberculin was considered to be the most effective carrier tested for GnRH in rainbow trout, with just a single injection of GnRH-PPD conjugate eliciting good antibody production (log 2 serum titre=15). Such anti-GnRH antibody formation may be of value in salmonid culture to prevent fish maturation by neutralization of endogenous GnRH.

Molecular cloning and expression in Escherichia coli of the glycoprotein gene of VHS virus, and immunization of rainbow trout with the recombinant protein.

The gene encoding the envelope glycoprotein of a recent Danish isolate of a salmonid rhabdovirus, viral haemorrhagic septicaemia virus (VHSV) has been cloned and sequenced at the cDNA level. When compared with the deduced sequence of a French isolate of VHSV, it was noted that there were 13 amino acid substitutions in the Danish virus. Amino acid homologies with the glycoprotein of a North American salmonid rhabdovirus (infectious haematopoietic necrosis virus) indicate a high degree of structural similarity between the two fish rhabdovirus glycoproteins. Results from partial enzymatic deglycosylation of the viral protein indicate that all four NXT/S sites found in the sequence are N-glycosylated in the virus. The glycoprotein, without the N-terminal leader sequence and C-terminal hydrophobic anchor segment, was expressed in Escherichia coli as a factor Xa protease-cleavable fusion protein. The purified and renatured viral part of the recombinant protein was able to elicit VHSV-specific antibodies and neutralizing antibody activity in serum when injected into rainbow trout.