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Since the discovery of a non-virulent infectious salmon anaemia virus (ISAV) HPR0 variant, many studies have speculated on the functional role of deletions within the highly polymorphic region (HPR) of genomic segment 6, which codes for the haemagglutinin-esterase (HE) protein. To address this issue, mutant HE proteins with deletions in their HPR were generated from the Scottish HPR0 template (NWM10) and fusion-inducing activity was measured using lipid (octadecyl rhodamine B) and content mixing assays (firefly luciferase). Segment six HPR was found to have a strong influence on ISAV fusion, and deletions in this near-membrane region predominantly increased the fusion-inducing ability of the resulting HE proteins. The position and length of the HPR deletions were not significant factors, suggesting that they may affect fusion non-specifically. In comparison, the amino acid composition of the associated fusion (F) protein was a more crucial criterion. Antibody co-patching and confocal fluorescence demonstrated that the HE and F proteins were highly co-localized, forming defined clusters on the cell surface post-transfection. The binding of erythrocyte ghosts on the attachment protein caused a reduction in the percentage of co-localization, suggesting that ISAV fusion might be triggered through physical separation of the F and HE proteins. In this process, HPR deletion appeared to modulate and reduce the strength of interaction between the two glycoproteins, causing more F protein to be released and activated. This work provides a first insight into the mechanism of virulence acquisition through HPR deletion, with fusion enhancement acting as a major contributing factor.

Saprolegnia infections cause severe economic losses among freshwater fish and their eggs. The banning of malachite green increased the demand for finding effective alternative treatments to control the disease. In the present study, we investigated the ability of boric acid to control saprolegniosis in salmon eggs and yolk sac fry. Under in vitro conditions, boric acid was able to decrease Saprolegnia spore activity and mycelial growth in all tested concentrations above 0.2 g/L, while complete inhibition of germination and growth was observed at a concentration of 0.8 g/L. In in vivo experiments using Atlantic salmon eyed eggs, saprolegniosis was controlled by boric acid at concentrations ranging from 0.2-1.4 g/L during continuous exposure, and at 1.0-4.0 g/L during intermittent exposure. The same effect was observed on salmon yolk sac fry exposed continuously to 0.5 g/L boric acid during the natural outbreak of saprolegniosis. During the experiments no negative impact with regard to hatchability and viability was observed in either eggs or fry, which indicate safety of use at all tested concentrations. The high hatchability and survival rates recorded following the in vivo testing suggest that boric acid is a candidate for prophylaxis and control of saprolegniosis.

Acquired immunity plays an important role in the protection of salmonids vaccinated against infectious pancreatic necrosis virus (IPNV) infections. In recent years, vaccine research has taken a functional approach to find the correlates of protective immunity against IPNV infections. Accumulating evidence suggests that the humoral response, specifically IgM is a correlate of vaccine protection against IPNV infections. The role of IgT on the other hand, especially at the sites of virus entry into the host is yet to be established. The kinetics of CD4+ and CD8+ T-cell gene expression have also been shown to correlate with protection in salmonids, suggesting that other arms of the adaptive immune response e.g. cytotoxic T cell responses and Th1 may also be important. Overall, the mechanisms of vaccine protection observed in salmonids are comparable to those seen in other vertebrates suggesting that the immunological basis of vaccine protection has been conserved across vertebrate taxa., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Batch potency testing of salmonid vaccines is mainly performed by in vivo challenge, which requires a lot of animals and causes severe pain. Due to the animal welfare concerns associated with in vivo immunization challenge tests, methods which could refine, reduce or replace (3Rs) these tests are needed. The aim of this study was to assess the use of serological assay (immunization & antibody estimation with an enzyme-linked immunosorbent assay (ELISA) for batch potency testing of oil adjuvanted, inactivated commercial furunculosis vaccines. In total ten vaccines were included in the study: two commercial multi-component vaccines and two experimental single-component furunculosis vaccines with 5% and 20% antigen content (relative to the commercial vaccine), from two manufacturers. In addition two experimental single component vaccines based on A-layer positive and A-layer negative Aeromonas salmonicida respectively were included. Challenge and blood sampling were conducted 9 weeks post vaccination. There was a correlation between antibody response against A. salmonicida as measured by ELISA and protection in i.p. challenge. This study shows that the ELISA assay can be used for testing different vaccine formulations and can potentially replace in vivo challenge tests for batch potency testing of furunculosis vaccines., (Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Inactivated whole virus vaccines represent the majority of commercial preparations used to prevent infectious pancreatic necrosis (IPN) in salmonids today. The production of these vaccines requires high virus concentrations that are resource-demanding. In this study, we describe the cultivation of high yields of IPN virus in Asian Grouper strain K (AGK) cells. The mechanism by which this is achieved was investigated by comparison with commonly used salmonid cell lines (RTG-2 and CHSE-214 cells). The cells were counted before and sequentially after infection. Thereafter, protein shut down, virus yields and apoptosis were assessed. The effects of poly(I:C) pre-treatment and Mx expression on IPNV concentrations were examined and the results show that high virus yields were associated with high cell numbers per unit volume, delayed cell death and apoptosis inAGKcells while the opposite was observed in RTG-2 cells. Poly(I:C) treatment and Mx expression resulted in a dose-dependent inhibition of virus multiplication. The production capacity of AGK and CHSE-214 cells were compared and higher split ratio and shorter split interval of AGK cells documents dramatic differences in virus antigen production capacity. Collectively, the results suggest that high cell numbers and prolonged survival of AGK cells are responsible for the superior virus yields over RTG-2 and higher split ratio/shorter split interval makes AGK superior over CHSE cells.

Immunization by an antigen-encoding DNA was approved for commercial sale in Canada against a Novirhabdovirus infection in fish. DNA vaccines have been particularly successful against the Novirhabdoviruses while there are reports on the efficacy against viral pathogens like infectious pancreatic necrosis virus, infectious salmon anemia virus, and lymphocystis disease virus and these are inferior to what has been attained for the novirhabdoviruses. Most recently, DNA vaccination of Penaeus monodon against white spot syndrome virus was reported. Research efforts are now focused on the development of more effective vectors for DNA vaccines, improvement of vaccine efficacy against various viral diseases of fish for which there is currently no vaccines available and provision of co-expression of viral antigen and immunomodulatory compounds. Scientists are also in the process of developing new delivery methods. While a DNA vaccine has been approved for commercial use in farmed salmon in Canada, it is foreseen that it is still a long way to go before a DNA vaccine is approved for use in farmed fish in Europe., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Salmon pancreas disease virus (SPDV) also referred to as salmonid alphavirus (SAV) is a virus causing pancreas disease in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss). Although the virus causes an economically important disease, relatively few full-length genome sequences of SAV strains are currently available. Here, we report full-length genome sequences of nine SAV3 strains from sites farming Atlantic salmon geographically spread along the Norwegian coastline. The virus genomes were sequenced directly from infected heart tissue, to avoid culture selection bias. Sequence analysis confirmed a high level of sequence identity within SAV3 strains, with a mean nucleotide diversity of 0.11 %. Sequence divergence was highest in 6K and E2, while lowest in the capsid protein and the non-structural proteins (nsP4 and nsP2). This study reports for the first time that numerous defective viruses containing genome deletions are generated during natural infection with SAV. Deletions occurred in all virus strains and were not distributed randomly throughout the genome but instead tended to aggregate in certain areas. We suggest imprecise homologous recombination as an explanation for generation of defective viruses with genome deletions. The presence of such viruses, provides a possible explanation for the difficulties in isolating SAV in cell culture. Primary virus isolation was successfully achieved for only two of eight strains, despite extensive attempts using three different cell lines. Both SAV isolates were easily propagated further and concomitant viral deletion mutants present in clinically infected heart tissue were maintained following serial passage in CHH-1 cells.

Heart and Skeletal Muscle Inflammation (HSMI) is an emerging viral disease caused by a novel Atlantic salmon reovirus (ASRV) affecting farmed fish. Primary symptoms associated with HSMI include myocardial and skeletal muscle necrosis indicating a severe inflammatory process. Recently, we applied the concept of clinical nutrition to moderate the long-term inflammatory process associated with HSMI in salmon subjected to experimental ASRV challenge. The use of functional feeds with lower lipid (hence energy) content reduced the inflammatory response to ASRV infection and the severity of associated heart lesions. The aim of the present study was to elucidate possible mechanisms underpinning the observed effects of the functional feeds, focussing on eicosanoid and fatty acid metabolism in liver and head kidney. Here we show that liver was also a site for histopathological lesions in HSMI showing steatosis reflecting impaired lipid metabolism. This study is also the first to evaluate the expression of a suite of key genes involved in pathways relating diet and membrane phospholipid fatty acid compositions, and the inflammatory response after ASRV infection. The expression of hepatic Δ6 and Δ5 desaturases was higher in fish fed the functional feeds, potentially increasing their capacity for endogenous production and availability of anti-inflammatory EPA. Effects on mobilization of lipids and changes in the LC-PUFA composition of membrane phospholipids, along with significant changes in the expression of the genes related to eicosanoid pathways, showed the important role of the head kidney in inflammatory diseases caused by viral infections. The results from the present study suggest that clinical nutrition through functional feeding could be an effective complementary therapy for emerging salmon viral diseases associated with long-term inflammation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Saprolegnia spp. can cause mortality and economic losses in freshwater fish and eggs. Biofilm formation is generally regarded as a virulence factor, and biofilms can be an important cause of infection recurrence. Evidence of persistent sources of Saprolegnia infections on fish and eggs in fish farms support the assumption that Saprolegnia spp. might be able to form biofilms. In this study, we aimed to test the ability of Saprolegnia to form biofilms where it can survive, reproduce and resist different chemicals used for its control. Naturally formed biofilms were obtained from laboratory aquaria. Saprolegnia growth within these biofilms was demonstrated with light microscopy and confirmed by isolation. Isolates were identified morphologically and molecularly on the basis of ITS-sequences. Two isolates were identified as Saprolegnia parasitica, a species known to be highly pathogenic for fish, while the other belonged to S. australis. Selected Saprolegnia strains obtained from natural biofilms were then used to establish simple methods for in vitro induction of Saprolegnia biofilm. The ability of Saprolegnia isolates to form biofilms with subsequent production of infective motile zoospores within the biofilm was documented by light and confocal laser scanning microscopy. We demonstrate for the first time that isolates of S. parasitica and S. australis can form biofilm communities together with multiple microorganisms, wherein they grow and reproduce. It is therefore likely that natural biofilms constitute incessant Saprolegnia reservoirs in nature and aquaculture., (Copyright © 2012 Elsevier B.V. All rights reserved.)

An enduring challenge in the vaccinology of infectious pancreatic necrosis virus (IPNV) is the lack of correlation between neutralizing antibodies and protection against mortality. To better understand the immunological basis of vaccine protection, an efficacy trial including Atlantic salmon (Salmo salar L.) vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine was carried out in a cohabitation challenge model using the highly virulent Norwegian Sp strain NVI015. To pinpoint the immunological basis of vaccine protection, pathogenic mechanisms of IPNV were unraveled in control fish while obtaining feedback on mechanisms of protection in the vaccinated fish. During the incubation period, infection rates were highest in control fish, followed by the LoAg group with the lowest infections being in the HiAg group. Although both the liver and pancreas are target organs prone to tissue damage, infection in the liver was delayed until acute infection in most fish. A correlate of pathology determined as the cutoff threshold of viral copy numbers linked to tissue damage in target organs was estimated at ≥ 107.0, which corresponded with an increase in mortality. The kinetics of IFNα and Mx expression suggests that these genes can be used as biomarkers of IPNV infection progression. Mechanisms of vaccine protection involved reducing infection rates, preventing infection of the liver and reducing virus replication in target organs to levels below the correlate of pathology. Overall, the study shows that antigen dose corresponds with vaccine efficacy and that antibody levels can be used as a signature of protective immunity against pathological disease and mortality.

Salmon farming has increased dramatically over last thirty years and a key to the success is the introduction of protective vaccines. In Norway, almost 100% of all Atlantic salmon are vaccinated prior to sea transfer. This extensive use of vaccines demands use of a lot of resources in production and quality control of vaccines, and fish are now one of the most widely used laboratory animal species in Norway, since all batch testing today is performed by challenge experiments. With an increasing focus on the 3 R's (Replacement, Reduction and Refinement), new methods are needed. The aim of this study was to assess the use of different vaccine evaluation methods to identify furunculosis vaccines of different "potency", using ELISA as in vitro assay and intraperitoneal and cohabitation challenge as in vivo assays. Eleven vaccines with different antigen content (0, 2, 5, 10, 20, 40, 80, 100 and 200%) and different antigen qualities were included in the study. Challenge and blood sampling for the ELISA assay were conducted 9 weeks post vaccination. The results from this study indicated that there is a close correlation between the antigen dose in the vaccine and the antibody response against Aeromonas salmonicida as measured by ELISA. There is also a close correlation between the antibody response and protection for both i.p. and cohabitation challenge models. The ELISA method identified sub-potent batches better than currently used in vivo assay (i.p challenge) and seems to be the best method of performing a batch potency test of furunculosis vaccines particularly when taking the 3R's principles into account., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Infectious pancreatic necrosis virus (IPNV) is a member of the family Birnaviridae that has been linked to high mortalities in juvenile salmonids and postsmolt stages of Atlantic salmon (Salmo salar L.) after transfer to seawater. IPN vaccines have been available for a long time but their efficacy has been variable. The reason for the varying immune response to these vaccines has not well defined and studies on the importance of using vaccine trains homologous to the virulent field strain has not been conclusive. In this study we prepared one vaccine identical to the virulent Norwegian Sp strain NVI-015 (NCBI: 379740) (T(217)A(221)T(247) of VP2) and three other vaccine strains developed using the same genomic backbone altered by reverse genetics at three residues yielding variants, T(217)T(221)T(247), P(217)A(221)A(247), P(217)T(221)A(247). These 4 strains, differing in these three positions only, were used as inactivated, oil-adjuvanted vaccines while two strains, T(217)A(221)T(247) and P(217)T(221)A(247), were used as live vaccines. The results show that these three residues of the VP2 capsid play a key role for immunogenicity of IPNV vaccines. The virulent strain for inactivated vaccines elicited the highest level of virus neutralization (VN) titers and ELISA antibodies. Interestingly, differences in immunogenicity were not reflected in differences in post challenge survival percentages (PCSP) for oil-adjuvanted, inactivated vaccines but clearly so for live vaccines (TAT and PTA). Further post challenge viral carrier state correlated inversely with VN titers at challenge for inactivated vaccines and prevalence of pathology in target organs inversely correlated with protection for live vaccines. Overall, our findings show that a few residues localized on the VP2-capsid are important for immunogenicity of IPNV vaccines.

Atlantic salmon post-smolts were exposed to either chronic hypoxic (Hy) or normal oxygen (No) conditions in seawater tanks for 58 days, mimicking conditions typical of sea cages for farmed salmon at some periods of the year. By day 29 head kidney macrophages were isolated and subjected to in vitro poly I:C stimulation to simulate viral infection, and samples were collected over 48 h. By day 58 fish were subjected to in vivo stimulation using poly I:C or a Vibrio water-based vaccine to simulate viral or bacterial infection, respectively. The fish were monitored for stress responses and expression of several pro-inflammatory genes in head kidney and intestinal tissue up to five days post-injection. Stress load was monitored by plasma cortisol estimation at days 29 and 58, and on days 1, 2, 3 and 5 post-injection in the in vivo trial. Hy exposure resulted in elevated plasma cortisol levels on day 29 compared to No, while on day 58 cortisol levels were higher in the control group. Additionally, both poly I:C and the Vibrio vaccine gave significantly increased cortisol levels one day post-injection compared to PBS treated controls, irrespective of previous oxygen exposure. In vitro stimulation of macrophages with poly I:C revealed higher IFNα mRNA levels at 6, 12 and 24 h and for Mx at 12 and 24 h post-stimulation, for both No and Hy individuals. Moreover, IFNα levels were higher in No than in Hy individuals at all time points, and a similar difference was seen in Mx at 48 h. In vivo stimulation with poly I:C elicited strong elevation of the IL-1β, IFNγ, Mx and IP10 mRNA transcripts in head kidney, while TNFα1 and IFNα were found unaffected. The Vibrio vaccine elicited a strong up regulation of IL-1β, IFNγ and IP10 mRNA, whereas Mx, TNFα1 and IFNα appeared unchanged. Significant differences in expression between different oxygen exposure groups were found for all genes and both stimuli. The overall trend suggests that long-term hypoxia either reduces or delays the expression of these genes in head kidney. Expression of IFNγ and Mx in intestinal tissues also showed a strong up regulation of the genes following poly I:C stimulation, and also here the overall trend suggests that chronic hypoxia results in a lower or delayed expression of the measured genes. In summary, our results indicate that chronic hypoxia modulates the expression of important immune related genes putatively altering the immune response. As the effect is present in isolated macrophages as well as head kidney and intestinal tissue the modulation appears to be affecting local as well as systemic responses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We have studied stress-induced reversion to virulence of infectious pancreatic necrosis virus (IPNV) in persistently infected Atlantic salmon (Salmo salar L.) fry. Naïve fry were persistently infected with a virulent strain (T(217)A(221) of major structural virus protein 2, VP2) or a low virulent (T(217)T(221)) variant of IPNV. The fry were infected prior to immunocompetence as documented by lack of recombination activating gene-1, T-cell receptor and B-cell receptor mRNA expression at time of challenge. The fish were followed over 6 months and monitored monthly for presence of virus and viral genome mutations. No mutation was identified in the TA or TT group over the 6 months period post infection. Six months post infection TA and TT infected groups were subject to daily stress for 7 days and then sampled weekly for an additional period of 28 days post stress. Stress-responses were documented by down-regulation of mRNA expression of IFN-α1 and concomitant increase of replication levels of T(217)T(221) infected fish at day 1 post stress. By 28 days post stress a T221A reversion was found in 3 of 6 fish in the T(217)T(221) infected group. Sequencing of reverted isolates showed single nucleotide peaks on chromatograms for residue 221 for all three isolates and no mix of TA and TT strains. Replication fitness of reverted (TA) and non-reverted (TT) variants was studied in vitro under an antiviral state induced by recombinant IFN-α1. The T(217)A(221) reverted variant replicated to levels 23-fold higher than the T(217)T(221) strain in IFN-α1 treated cells. Finally, reverted TA strains were virulent when tested in an in vivo trial in susceptible salmon fry. In conclusion, these results indicate that stress plays a key role in viral replication in vivo and can facilitate conditions that will allow reversion from attenuated virus variants of IPNV.

Pancreas disease (PD) in salmonid fish is caused by an infection with Salmonid alphavirus (SAV) and remains as one of the major health problems in the European fish farming industry. Sequence studies have revealed a genetic diversity among viral strains. A subtype of SAV (SAV3) is causing an epizootic in farmed salmonids in Norway. Here we evaluate efficacy and safety of an inactivated virus vaccine based on ALV405, a strain of SAV3 that was isolated from Norwegian salmon. The vaccine provided an average relative percent survival (RPS) of 98.5 in an intraperitoneal challenge model, and induced nearly total protection against PD in a cohabitant challenge model. It provided significant protection against SAV-induced mortality also in a field trial under industrial conditions. Local reactions seen as melanization and adhesions in the visceral cavity were less severe than those induced by two commercial vaccines. Finally, we demonstrated that the protection is not impaired when the ALV405 antigen is combined with other viral or bacterial antigens in a polyvalent vaccine. The results confirm that efficient and safe protection against SAV infection and development of PD is possible using an inactivated virus vaccine, both alone and as a component in a polyvalent vaccine., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Two strains of IPNV made by reverse genetics on the Norwegian Sp strain NVI-015 (GenBank AY379740) backbone encoding the virulent (T(217)A(221)) and avirulent (P(217)T(221)) motifs were used to prepare inactivated whole virus (IWV), nanoparticle vaccines with whole virus, Escherichia coli subunit encoding truncated VP2-TA and VP2-PT, VP2-TA and VP2-PT fusion antigens with putative translocating domains of Pseudomonas aeruginosa exotoxin, and plasmid DNA encoding segment A of the TA strain. Post challenge survival percentages (PCSP) showed that IWV vaccines conferred highest protection (PCSP=42-53) while nanoparticle, sub-unit recombinant and DNA vaccines fell short of the IWV vaccines in Atlantic salmon (Salmo salar L.) postsmolts challenged with the highly virulent Sp strain NVI-015 (TA strain) of IPNV after 560 degree days post vaccination. Antibody levels induced by these vaccines did not show antigenic differences between the virulent and avirulent motifs for vaccines made with the same antigen dose and delivery system after 8 weeks post vaccination. Our findings show that fish vaccinated with less potent vaccines comprising of nanoparticle, DNA and recombinant vaccines got infected much earlier and yielded to higher infection rates than fish vaccinated with IWV vaccines that were highly potent. Ability of the virulent (T(217)A(221)) and avirulent (P(217)T(221)) motifs to limit establishment of infection showed equal protection for vaccines made of the same antigen dose and delivery systems. Prevention of tissue damage linked to viral infection was eminent in the more potent vaccines than the less protective ones. Hence, there still remains the challenge of developing highly efficacious vaccines with the ability to eliminate the post challenge carrier state in IPNV vaccinology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Salmonid alphavirus 3 (SAV-3) is an emerging pathogen in Norwegian salmon farming and causes severe annual losses. We studied the immunogenicity and protective ability of subunit and DNA vaccines based on E1 and E2 spike proteins of salmonid alphavirus subtype 3 (SAV-3), and compared these to an experimental inactivated, whole virus (IWV) vaccine in Atlantic salmon. The antigens were delivered as water-in-oil emulsions for the subunit and inactivated vaccines and non-formulated for the DNA vaccines. The IWV and the E2 subunit prime-boost groups had circulating neutralizing antibodies at challenge, correlating with high protection against lethal challenge and 3-log(10) reduction of virus titer in heart for the IWV group. Prime-boost with E1 subunit vaccine also conferred significant protection against mortality, but did not correlate with neutralizing antibody levels. Protection against pathology in internal organs was only seen for the IWV group. Prime-boost with E1 and E2 DNA vaccines showed marginal protection in terms of reduction of viral replication in target organs and protection against mortality was not different from controls. The IWV group showed significant upregulation of IFNγ and IL2 mRNA expression at 4 weeks post challenge possibly indicating that other mechanisms in addition to antibody responses play a role in mediating protection against infection. This is the first report comparing the immunogenicity and protection against mortality for IWV vaccines and spike protein subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon. The IWV vaccine has superior immunogenicity over sub-unit and DNA vaccines., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Stress can affect the immune system and increase susceptibility to various diseases but knowledge of the underlying mechanisms is scarce. There is a complex interaction between the immune system and the endocrine system of vertebrates. In fish, cortisol is a key hormone regulating stress response and recent studies have also suggested that this hormone can affect the immune system, where cortisol is mainly regarded as an immunosuppressive factor. The aim of the present study was to examine the impact of chronically elevated levels of cortisol on the immune response and susceptibility to experimental infection with infectious pancreatic necrosis virus (IPNV). Further, the effect of IPNV challenge on circulating levels of cortisol was investigated. Atlantic salmon parr were implanted intraperitoneally with sustained-release implants of bovine of cortisol (50 μg cortisol g(-1) body weight in an implant based on vegetable lipids). Vehicle implants were used as control (sham-injected). At 45 days after implantation (DAI), fish were challenged with a low virulent isolate of IPNV (by immersion). Samples of plasma, liver and head kidney was taken from fish before and 24 h, 48 h, 7 days week and 21 days post infection (DPI). Cortisol level in plasma was measured using radioimmunoassay and gene expression in liver and head kidney was analyzed with real-time PCR (RT-PCR). Infection prevalence in infected fish was assessed by virus culture and RT-PCR of head kidney samples. Cortisol implantation compared with sham-implanted fish had increased levels of plasma cortisol at 45 DAI. The relative expression of Interferon alpha-1 (IFNα-1), Myxo virus-1 Mx, Heat-shock protein 70 (HSP70), Serum amyloid A (SAA), Glucocorticoid receptor (GR) and Heat-shock protein 90 (HSP90) tends to be down-regulated by cortisol implantation. There was a higher prevalence of fish with detectable levels of IPNV, as measured by cell culture and RT-PCR, in the cortisol-implanted group challenged with IPNV (0 = 0.0305) relative to the group that received a sham implantation. Further, cortisol seems to delay the induction of the antiviral IFNα-1 pathway and Mx mRNA expression. This study shows that elevated plasma cortisol level leads to an impaired innate immune response, and higher virus (IPNV) prevalence in Atlantic salmon parr., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Heart and Skeletal Muscle Inflammation (HSMI), recently associated with a novel Atlantic salmon reovirus (ASRV), is currently one of the most prevalent inflammatory diseases in commercial Atlantic salmon farms in Norway. Mortality varies from low to 20%, but morbidity can be very high, reducing growth performance and causing considerable financial impact. Clinical symptoms, including myocarditis, myocardial and red skeletal muscle necrosis, correlate with the intensity of the inflammatory response. In the present study, the effects of two functional feeds (FF1 and FF2) were compared to a standard commercial reference feed (ST) in Atlantic salmon subjected to an ASRV challenge. The functional feeds had reduced levels of total lipid and digestible energy, and different levels and proportions of long-chain polyunsaturated fatty acids (LC-PUFA). The objective was to determine whether these feeds could provide effective protection by decreasing the inflammatory response associated with HSMI. Histopathology, viral load, fatty acid composition and gene expression of heart tissue were assessed over a period of 16 weeks post-infection with ASRV. The viral load and histopathology scores in heart tissue in response to ASRV infection were reduced in fish fed both functional feeds, with FF1 showing the greatest effect. Microarray hierarchical cluster analysis showed that the functional feeds greatly affected expression of inflammation/immune related genes over the course of the ASRV infection. Viral load correlated with up-regulation of pro-inflammatory genes at the early-mid stages of infection in fish fed the ST diet. Expression of inflammatory genes 16-weeks after ASRV challenge reflected the difference in efficacy between the functional feeds, with fish fed FF1 showing lower expression. Thus, severity of the lesions in heart tissue correlated with the intensity of the innate immune response and was associated with tissue fatty acid compositions. The present study demonstrated that dietary modulation through clinical nutrition had major influences on the development and severity of the response to ASRV infection in salmon. Thus, HSMI was reduced in fish fed the functional feeds, particularly FF1. The modulation of gene expression between fish fed the different feeds provided further insight into the molecular mechanisms and progression of the inflammatory and immune responses to ASRV infection in salmon.

Furunculosis (Aeromonoas salmonicida) is an important disease in Atlantic salmon (Salmo salar) farming. Vaccination and selective breeding for increased resistance to the disease on the basis of challenge tests of unvaccinated fish are used as complementary prophylactic methods. An important issue is whether genetic predisposition to infection is consistent across vaccinated and unvaccinated fish. Hence, the main objective of this study was to determine the magnitude of the genetic associations (correlations) between resistance to furunculosis in vaccinated and unvaccinated fish, and to estimate the magnitude of the correlation of resistance to furunculosis with resistance to the viral diseases infectious pancreatic necrosis (IPN) and infectious salmon anaemia (ISA). Sub-samples of unvaccinated and vaccinated salmon from 150 full-sib families were subjected to separate cohabitation challenge tests. Substantial genetic variation was found in resistance to furunculosis in both the unvaccinated (heritabilities of 0.51 ± 0.05) and vaccinated (0.39 ± 0.06) fish. However, the genetic correlation between resistance to furunculosis in the two groups was low (0.32 ± 0.13), indicating a weak genetic association between resistance in the two groups. Hence, the current selection strategy on the basis of challenge tests of unvaccinated fish is likely to produce low genetic improvement in resistance to furunculosis under field conditions, where fish are vaccinated with an effective vaccine. Evidence was found of significantly favourable genetic associations of resistance to furunculosis in unvaccinated (but less so for vaccinated) fish with resistance to both IPN and ISA (unvaccinated fish), indicating that vaccination 'mask' genetic associations between resistance to different diseases.

The Chilean salmonid industry was developed by importing breeding materials, a practice still in effect due to deficits in the national supply of roe. Importation of breeding materials is often associated with the transmission of pathogens. The objectives of this study were to compare the infectious pancreatic necrosis virus (IPNV) isolates from Chile to those of European origin and to determine the diversity of the Chilean IPNV. The VP2 genes of IPNV from Chilean fish (whose eggs originated from Scotland, Iceland and Norway) were compared to isolates from fish in Norway and Ireland. The results show that the isolates are identical (97-99%) and cluster into one genogroup. Our findings support previous reports of association between the trade-in breeding materials and transmission of pathogens. Furthermore, our results demonstrate the genotypic diversity of Chilean IPNV isolates. These findings have important implications for IPNV disease diagnosis and control in Chile.

Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-alpha)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-gamma in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-alpha [rIFN-alpha] and rIFN-gamma) and used for in vitro studies. SAV-3 infection in a permissive salmon cell line (TO cells) results in IFN-alpha and IFN-stimulated gene (ISG) mRNA upregulation. Preinfection treatment (4 to 24 h prior to infection) with salmon rIFN-alpha induces an antiviral state that inhibits the replication of SAV-3 and protects the cells against virus-induced cytopathic effects (CPE). The antiviral state coincides with a strong expression of Mx and ISG15 mRNA and Mx protein expression. When rIFN-alpha is administered at the time of infection and up to 24 h postinfection, virus replication is not inhibited, and cells are not protected against virus-induced CPE. By 40 h postinfection, the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) is phosphorylated concomitant with the expression of the E2 protein as assessed by Western blotting. Postinfection treatment with rIFN-alpha results in a moderate reduction in E2 expression levels in accordance with a moderate downregulation of cellular protein synthesis, an approximately 65% reduction by 60 h postinfection. rIFN-gamma has only a minor inhibitory effect on SAV-3 replication in vitro. SAV-3 is sensitive to the preinfection antiviral state induced by rIFN-alpha, while postinfection antiviral responses or postinfection treatment with rIFN-alpha is not able to limit viral replication.

Background: Two decades after the introduction of oil-based vaccines in the control of bacterial and viral diseases in farmed salmonids, the mechanisms of induced side effects manifested as intra-abdominal granulomas remain unresolved. Side effects have been associated with generation of auto-antibodies and autoimmunity but the underlying profile of inflammatory and immune response has not been characterized. This study was undertaken with the aim to elucidate the inflammatory and immune mechanisms of granuloma formation at gene expression level associated with high and low side effect (granuloma) indices.Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscosa antigens in order to induce polarized (severe and mild) granulomatous reactions. The established granulomatous reactions were confirmed by gross and histological methods at 3 months post vaccination when responses were known to have matured. The corresponding gene expression patterns in the head kidneys were profiled using salmonid cDNA microarrays followed by validation by real-time quantitative PCR (qPCR). qPCR was also used to examine the expression of additional genes known to be important in the adaptive immune response., Results: Granulomatous lesions were observed in all vaccinated fish. The presence of severe granulomas was associated with a profile of up-regulation of innate immunity-related genes such as complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and metallothionein. In addition, TGF-beta (p = 0.001), IL-17A (p = 0.007) and its receptor (IL-17AR) (p = 0.009) representing TH17 were significantly up-regulated in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of T(H)1 T cell lineage (IFN-gamma, CD4) or T(H)2 (GATA-3) responses were differentially expressed., Conclusions: Granulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon have the profile of strong expression of genes related to innate immune responses. The expression of TGF-beta, IL-17A and its receptor suggests an involvement of T(H)17 T cell lineage and is in conformity with strong infiltration of neutrophils and macrophages into inflamed areas. Arginase upregulation shows that macrophages in these reactions are alternatively activated, indicating also a T(H)2-profile. To what extent the expression of IL-17A and its receptor reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon when vaccinated with oil-based vaccines.

The mechanisms of viral persistence of infectious pancreatic necrosis virus (IPNV) are not well understood. In this study we have used a model of IPNV persistently infected CHSE (Chinook salmon embryonic) cells as correlate of persistent infection in fish focusing on differentially expressed genes using subtractive hybridization (SSH). Selected genes were also analyzed by quantitative real-time PCR (qPCR) in persistently infected parr of Atlantic salmon. Persistent infection was established by growing CHSE cells surviving an IPNV infection. Infection in rescued cells was non-lytic with a virus yield of 10(3)-10(5) TCID(50)/ml of supernatant, resembling what can be found during a persistent infection in vivo. By comparing gene expression in persistently infected cell vs. non-infected cells we found an upregulation of genes involved in direct interaction or degradation of viral proteins, proteasome activating subunit 3, and of ATRX (X-linked alpha-thalassemia/mental retardation syndrome), a transcription repressor, which may indicate a repression of viral replication through reduced transcription. Further ephrin B1 (signal-transduction group) was found strongly up-regulated, and receptors for various ephrins are used for cell interaction and as entry points for other viruses in higher vertebrates. Endonuclease/reverse transcriptase 1 (RVT1) was also found highly up-regulated in persistently infected cells. The comparison of persistently infected cells to in vivo infected fish showed that the expression profiles found in CHSE cells give corresponding results for selected genes, as ATRX, ephrin B1 and RVT-1. We observed similar results by use of two independent methods (SSH and qPCR) for 8 out of 15 genes analyzed and the transcript profile of persistently IPNV-infected cells involve upregulation of genes encoding proteins involved in viral protein degradation and translation inhibition. The understanding is that this may contribute to keep the number of virus particles low during viral persistence., (2010 Elsevier Ltd. All rights reserved.)

Existing methods for enumeration of viable Saprolegniaceae propagules in water are laborious, time consuming and prevent examination of large numbers of samples or samples with high spore loads. In the present study a microwell plate (MWP) assay for estimation of Saprolegniaceae in water samples, modified from Hagen (1992), was evaluated. The ability of the assay to recover Saprolegniaceae was assessed by applying it to spore suspensions with four predetermined concentrations, 500-10,000 spores per liter of samples tested. The method also was used to analyze a set of field samples and compare it to a standard filtration method to ascertain its practicability. The MWP assay underestimated the number of spores in the test suspensions with predetermined concentrations. The accuracy of the assay varied with spore concentration, giving the lowest recovery (62.5%) at low spore numbers and the highest (86%) at intermediate concentrations (1000-5000 spores/L) for both isolates and growth media. The findings indicate that spores aggregate with increasing concentration. When applied to field samples the assay clearly distinguished among samples with presumptive differences in spore load and yielded significantly higher counts than the filtration method. The results justify the MWP method foruse in estimation of Saprolegniaceae in water bodies particularly relevant for monitoring of spore load in aquaculture as well as in ecological studies.

In high intensive fish production systems, hyperoxygenation and reduced flow are often used to save water and increase the holding capacity. This commonly used husbandry practice has been shown to be stressful to fish and increase mortality after infectious pancreatic necrosis virus (IPNV) challenge, but the cause and effect relationship is not known. Salmonids are particularly sensitive to stress during smoltification and the first weeks after seawater (SW) transfer. This work aimed at investigating the impact of hyperoxygenation combined with reduced flow in fresh water (FW), on the intestinal barrier in FW as well as during later life stages in SW. It further aims at investigating the role of the intestinal barrier during IPNV challenge and possible secondary infections. Hyperoxygenation in FW acted as a stressor as shown by significantly elevated plasma cortisol levels. This stressful husbandry condition tended to increase paracellular permeability (P(app)) as well as translocation of Aeromonas salmonicida in the posterior intestine of Atlantic salmon. After transfer to SW and subsequent IPNV challenge, intestinal permeability, as shown by P(app), and translocation rate of A. salmonicida increased in the anterior intestine, concomitant with further elevation in plasma cortisol levels. In the anterior intestine, four of five fish displayed alterations in intestinal appearance. In two of five fish, IPNV caused massive necrosis with significant loss of cell material and in a further two fish, IPNV caused increased infiltration of lymphocytes into the epithelium and granulocytes in the lamina propria. Hyperoxygenation and reduced flow in the FW stage may serve as stressors with impact mainly during later stages of development. Fish with an early history of hyperoxygenation showed a higher stress response concomitant with a disturbed intestinal barrier function, which may be a cause for the increased susceptibility to IPNV infection and increased susceptibility to secondary infections.

DNA vaccines encoding the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV are very efficient in eliciting protective immune responses against their respective diseases in rainbow trout (Oncorhynchus mykiss). The early anti-viral response (EAVR) provides protection by 4 days post vaccination and is non-specific and transient while the specific anti-viral response (SAVR) is long lasting and highly specific. Since both VHSV and IHNV are endemic in rainbow trout in several geographical regions of Europe and Atlantic salmon (Salmo salar) on the Pacific coast of North America, co-vaccination against the two diseases would be a preferable option. In the present study we demonstrated that a single injection of mixed DNA vaccines induced long-lasting protection against both individual and a simultaneous virus challenge 80 days post vaccination. Transfected muscle cells at the injection site expressed both G proteins. This study confirms the applied potential of using a combined DNA vaccination for protection of fish against two different rhabdoviral diseases.

Two strains of viral hemorrhagic septicemia virus (VHSV) with known different virulence characteristics in vivo were studied (by a time course approach) for their abilities to infect and translocate across a primary culture of gill epithelial cells (GEC) of rainbow trout (RBT; Oncorhynchus mykiss). The strains included one low-virulence marine VHSV (ma-VHSV) strain, ma-1p8, and a highly pathogenic freshwater VHSV (fw-VHSV) strain, fw-DK-3592B. Infectivities toward trout head kidney macrophages were also studied (by a time course method), and differences in in vivo virulence were reconfirmed, the aim being to determine any correlation between in vivo virulence and in vitro infectivity. The in vitro studies showed that the fw-VHSV isolate infected and caused a cytotoxic effect in monolayers of GEC (demonstrating virulence) at an early time point (2 h postinoculation) and that the same virus strain had translocated over a confluent, polarized GEC layer by 2 h postinoculation. The marine isolate did not infect monolayers of GEC, and delayed translocation across polarized GEC was seen by 48 h postinoculation. Primary cultures of head kidney macrophages were also infected with fw-VHSV, with a maximum of 9.5% virus-positive cells by 3 days postinfection, while for the ma-VHSV strain, only 0.5% of the macrophages were positive after 3 days of culture. In vivo studies showed that the fw-VHSV strain was highly virulent for RBT fry and caused high mortality, with classical features of viral hemorrhagic septicemia. The ma-VHSV showed a very low level of virulence (only one pool of samples from the dead fish was VHSV positive). This study has shown that the differences in virulence between marine and freshwater strains of VHSV following the in vivo infection of RBT correlate with in vitro abilities to infect primary cultures of GEC and head kidney macrophages of the same species.

The objective of the present study was to investigate the development of intimal changes of coronary arteries over the lifetime of farmed Atlantic salmon, Salmo salar L., fed either a 100% fish oil or a 100% vegetable oil blend. The study was performed as a randomized observer blinded controlled trial with parallel group design. At the start of the project, the fish were divided in two groups and sampled at five different time points throughout their life span. The total study sample consisted of 259 healthy fish. Serial sections were taken from the coronary artery lying on the bulbus arteriosus for histopathological evaluation and for area measurements using semi-quantitative and quantitative methods. The earliest onset of vascular changes was detected in fish from both groups in the freshwater stage prior to smoltification. The mean range lesion (MRL), used to describe the severity of the lesions observed, increased significantly for both groups from sea transfer throughout the study period. Comparison of the two groups based on the overall material corrected for time of sampling did not show any difference (P = 0.20) between the two groups with regard to MRL. The percentage lumen loss (PLL) measured by a quantitative method and used as a measure to indicate lesion severity showed an incremental, non-significant increase from week 72 to week 92 and further to week 115 in both diet groups during the seawater phase. Comparison of the groups corrected for time of sampling indicated a difference of PLL in favour of VO (P = 0.02). Heart weight, body weight and body length were all positively and significantly correlated to Log MRL. The partial correlation analysis indicated that heart weight was the most dominant variable in the set. Early vascular changes were found in the major bifurcation of the coronary artery at the apex and beyond the flow divider into the daughter branches. The latter represented the dominant changes and were found throughout the entire lifecycle of the fish. Increasing in size over time they formed pads or cushions that were regularly located close to the outer walls of the bifurcation. The origin of the cells forming the intimal thickening has not been conclusively determined, but immunohistochemical findings indicate a smooth muscle cell origin, possibly of a myointimal type. Our findings suggest there is no correlation between diet and intimal changes. The severity of the changes, MRL and PLL, of the coronary vessels correlate with heart weight and fish weight growth and growth rate and mechanical factors are implicated in intimal development, but rather than being induced by external injury due to the location of the coronaries, haemodynamic factors and low shear stress are proposed as the main mechanism behind these changes.

Infectious pancreatic necrosis virus (IPNV), the causative agent of a highly infectious disease in salmonid fish, encodes a small non-structural protein designated VP5. This protein contains Bcl-2 homologous domains and inhibits apoptosis when expressed in cell culture. We have previously reported the generation of three VP5 mutants of IPNV-Sp serotype, using reverse genetics (Santi, N., Song, H., Vakharia, V.N., Evensen, Ø., 2005. Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence. J. Virol. 79 (14), 9206-9216). The wild-type rNVI15 virus encodes a truncated 12-kDa VP5 protein, rNVI15-15K encodes a full-length 15-kDa VP5, whereas rNVI15-DeltaVP5 is deficient in VP5 expression. In the present report, the role of VP5 in apoptosis was assessed both in vitro and in vivo, using the recombinant IPNV strains. Apoptosis was observed in hepatocytes of Atlantic salmon post-smolts challenged with all three VP5 mutant viruses. Using a double-labeling technique to detect apoptotic cells and IPNV antigens, we found that viral antigen and apoptotic cells co-distributed. In addition, numerous double-positive cells were seen. The recombinant viruses also induced apoptosis in infected cell cultures, and the morphology and membrane integrity of infected cells at different time points was similar. In summary, these results indicate that IPNV induces apoptosis in infected cell cultures and in fish, independent of VP5 expression. However, substitutions of putative functionally important amino acids in the BH2 domain of VP5 of IPNV-Sp strains were identified, which might influence the anti-apoptosis effect of the protein, and partly explain the apparent absence of this specific function.

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.

Infectious pancreatic necrosis virus (IPNV) is the causative agent of infectious pancreatic necrosis (IPN) disease in salmonid fish. Recent studies have revealed variation in virulence between isolates of the Sp serotype, associated with certain residues of the structural protein VP2. The isolates are also highly heterogenic in the coding region of the nonstructural VP5 protein. To study the involvement of this protein in the pathogenesis of disease, we generated three recombinant VP5 mutant viruses using reverse genetics. The "wild-type" recombinant NVI15 (rNVI15) virus is virulent, having a premature stop codon at nucleotide position 427, putatively encoding a truncated 12-kDa VP5 protein, whereas rNVI15-15K virus encodes a 15-kDa protein. Recombinant rNVI15-deltaVP5 virus contains a mutation in the initiation codon of the VP5 gene that ablates the expression of VP5. Atlantic salmon postsmolts were challenged to study the virulence characteristics of the recovered viruses in vivo. The role of VP5 in persistent infection was investigated by challenging Atlantic salmon fry with the recovered viruses, as well as with the low-virulence field strain Sp103 and a naturally occurring VP5-deficient mutant of Sp103. The results show that VP5 is not required for viral replication in vivo, and its absence does not alter the virulence characteristics of the virus or the establishment of persistent IPNV infection.

The transfection efficiency of liposome-based DNA formulations was studied in different salmonid cell lines of hepatocyte and macrophage origin. Parallel assessment of cell viability was carried out to define the balance between transfection efficiency and toxicity. For all cell lines, transfection efficiency varied with the lipoplex charge ratio and the amount of DNA added to the liposomes. The hepatocyte-derived cell line was most readily transfected while lower transfection efficiency was observed for the macrophage cell lines. The cationic liposomes showed a dose-dependent toxicity and were found to be most toxic for cells of macrophage origin. This was in line with the observation that higher amounts of lipids were associated with the cells of macrophage origin than the hepatocytes. Complexing DNA with the liposomes reduced the toxicity for all three cell lines, most markedly, however, for macrophage cell lines. The differences in the transfection and toxicity patterns between the cell lines are probably caused by differences in membrane composition as well as differences in phagocytic activity and processing of the liposomes/lipoplexes.

The persistence of antigens at the injection site (area around pyloric caeca and spleen), concomitant inflammatory reaction and granuloma development were monitored at 3, 6 and 12 months following intraperitoneal injection with multivalent, oil-adjuvanted vaccines in Atlantic salmon. Parallel assessment of side-effect profiles and growth rate were also performed. Antigen persistence was examined by use of a monoclonal antibody that recognises Aeromonas salmonicida lipopolysaccharide in an immunohistochemical method for in situ identification of bacteria or bacterial fragments. The inflammatory reaction was monitored using standard histological techniques. The amount of persistent antigens and size of inflammation/adhesions were estimated semi-quantitatively. A steady decrease in the quantity of antigens at the injection site was observed from 3 to 12 months. Antigens were consistently found in inflamed tissues located in the pancreatic region. The size of inflammation increased during the first 6 months but declined thereafter. These findings suggest that persistent antigens at the injection site may act as inflammatory stimulants that induce and perpetuate the inflammatory reaction, eventually leading to adverse side-effects.

Infectious pancreatic necrosis viruses (IPNVs) belonging to the family Birnaviridae display a high degree of antigenic variability, pathogenicity, and differences in outbreak mortality in salmonid species. To determine if virus isolates of Sp serotype differ in virulence, fry of Atlantic salmon (Salmo salar L.) were challenged with nine different field strains. These viruses caused either high mortality and severe pathological changes or low mortality and no lesions. To study the molecular basis for the variation in virulence of IPNV, complete nucleotide sequences of segment A of all these strains as well as segment B of three selected strains were determined. All viruses tested had a unique genome sequence. Only minor differences were noted in the genes encoding VP1, VP3, and VP4 proteins, whereas most changes were observed in the gene encoding the VP2 protein. A high level of variation was found in the small open reading frame (ORF), which encodes a 15-kDa nonstructural (NS) polypeptide also known as VP5. One of the strains lacked the initiation codon for this protein, whereas the other four could encode a truncated version of the NS protein. Additional data obtained by sequencing of the NS and VP2 genes directly from diseased fish demonstrated changes in the VP2 gene after two passages in cell culture, which could possibly be associated with attenuation. Comparison of the deduced amino acid sequences of the NS and VP2 genes reveals that the virulent strains possess a 12-kDa coding NS gene and have residues Thr, Ala, Thr/Ala, and Tyr/His at positions 217, 221, 247, and 500 of the VP2 gene, respectively-the motifs identified in this study to be involved in the virulence of IPNV.

In the present work, the expression of luciferase in selected organs following administration of DNA delivered as naked, liposome-formulated or chitosan-formulated by different routes of administration (intramuscular, intraperitoneal and intravenous injection, immersion and anal intubation) was studied in rainbow trout (Oncorhynchus mykiss). The different formulations and routes of administration both influenced in which organs luciferase was expressed and the magnitude of expression. The highest expression levels of luciferase in the head kidney and liver were found after an intraperitoneal injection of lipoplex 2. In the spleen, the highest levels were detected after injection of naked DNA (intraperitonal or intramuscular) and lipoplex 2 (intraperitoneal). Following intravenous injection, naked DNA gave higher expression levels in the organs than the formulated plasmids and immersion and anal intubation were not effective routes of delivery as no expression of luciferase could be detected in any of the organs tested. Additionally, PCR using a primer specific for a 600 bp region of the luciferase gene pcDNA3-luc was used to assess the distribution of the plasmid itself after intramuscular and intraperitoneal injection. Positive amplification was obtained in spleen, head kidney, liver and muscle at the injection site following injection of formulated plasmids, while only muscle tissue from the injection site was positive when naked DNA was used.

In this study we report the differences in distribution and retention of Aeromonas salmonicida antigens after vaccination with two different vaccines. Parr of Atlantic salmon (Salmo salar) were given intraperitoneal injections of either a commercial, monovalent furunculosis vaccine (Apoject) or live, attenuated A. salmonicida (DeltaaroA). Fish were sampled at weeks 2, 4 and 12 post-vaccination and head kidney and spleen were collected. Presence of LPS and 16S rDNA in isolated leukocytes were investigated by immunocytochemistry and polymerase chain reaction (PCR).16S rDNA was detected in head kidney and spleen of all DeltaaroA vaccinated and most Apoject-vaccinated fish at weeks 2 and 4. At week 12, 16S rDNA was detected in none of the DeltaaroA vaccinated fish, but it was detected in head kidney of 75% of Apoject-vaccinated fish. LPS was detected in both vaccination groups at all sampling times, but most frequently in the DeltaaroA vaccinated fish (in head kidney 75-83% vs. 50%, in spleen 58-67% vs. 17-25%).

The aim of this study was to investigate how a selection of formulation variables affects the in vitro transfection efficiency and physicochemical properties (particle size, zetapotential and chitosan-plasmid association) of chitosan-based polyplexes. Experimental designs in combination with multivariate data analysis were applied to reveal the effects of the formulation variables on the responses. The following formulation variables were studied: molecular weight and degree of acetylation of chitosan, pH and ionic strength of the buffer in which chitosan was dissolved, charge ratio of polyplexes, plasmid concentration and inclusion of a coacervation agent in the plasmid solution. The in vitro transfection efficiency in Epithelioma papulosum cyprini (EPC) cells was affected by the polyplex charge ratio, the DNA concentration in the complexes as well as the molecular weight and degree of acetylation of the chitosans. Two favourable formulations were identified in a more thorough investigation. These formulations were made of SC113 (theoretical charge ratio 10) and SC214 (theoretical charge ratio 3). The size of the complexes was affected by the degree of acetylation, concentration of DNA, pH, inclusion of a coacervation agent and the charge ratio. The charge ratio, pH and ionic strength determined the zetapotential of the particles, while the charge ratio was important for the association between the plasmid and chitosan.

A successful regime for intramuscular injection of naked DNA is developed in fish, but the exploration of other ways of administration has not yet been studied in any detail. Immersion is a delivery route offering many advantages compared to conventional ways of administration. Applying cationic liposomes as a delivery system for DNA by this route, however, is met with severe toxicity problems. In this report, the underlying mechanisms of the acute toxicity were investigated in vivo and in vitro. The most critical factor was found to be the charge of the liposomes. Cationic liposomes above a certain threshold concentration had a lethal effect in rainbow trout fry. In contrast, similar concentrations of neutral or anionic liposomes were not toxic. Furthermore, large liposome-mucin complexes were formed upon addition of mucin to cationic liposomes. This was not observed with neutral or anionic liposomes. Lipoplexes were less toxic and interacted less with mucin compared to cationic liposomes. Hence, the mechanism of the acute toxicity in rainbow trout fry is suggested to be an interaction between the cationic liposomes and anionic components of gill mucin. The consequence is hypoxia and this is most likely the cause of acute toxicity observed in rainbow trout fry.

The possibility of utilising DNA vaccines for aquaculture fish has been of growing interest in recent years and novel methods to deliver DNA to the fish are under investigation. One of the delivery methods of interest is immersion. Due to the favourable properties of chitosan in gene delivery and bioadhesion, chitosan-DNA formulations have been investigated for use for immersion delivery to fish. Initial studies on this system, however, revealed an acute toxic effect of the formulations. In this study, factors important for the acute toxicity of chitosan and chitosan formulations are identified and attempts are made to explain the underlying mechanisms for the toxicity. In vivo methods revealed that the toxicity mainly was dependent on the concentration of chitosan, but also the molecular weight and the degree of acetylation of the chitosans were of importance. Noteworthy, the toxicity of the polymer decreased dramatically when the chitosan was 'decharged' by complexation with DNA. In vitro experiments supported the in vivo observations. Most likely, the observed toxicity is caused by an electrostatic interaction between the cationic polymer and the anionic parts of the gill mucus. The result is obstructed oxygen diffusion over the gills and the fish are killed by acute hypoxia. Careful selection of chitosan type and charge of the particles may result in a potential for chitosan based immersion delivery of plasmid DNA.