Molecular cloning of MDA5, phylogenetic analysis of RIG-I-like receptors (RLRs) and differential gene expression of RLRs, interferons and proinflammatory cytokines after in vitro challenge with IPNV, ISAV and SAV in the salmonid cell line TO.

The RIG-I receptors RIG-I, MDA5 and LGP2 are involved in viral recognition, and they have different ligand specificity and recognize different viruses. Activation of RIG-I-like receptors (RLRs) leads to production of cytokines essential for antiviral immunity. In fish, most research has focused on interferons, and less is known about the production of proinflammatory cytokines during viral infections. In this study, we have cloned the full-length MDA5 sequence in Atlantic salmon, and compared it with RIG-I and LGP2. Further, the salmonid cell line TO was infected with three fish pathogenic viruses, infectious pancreatic necrosis virus (IPNV), infectious salmon anaemia virus (ISAV) and salmonid alphavirus (SAV), and differential gene expression (DEG) analyses of RLRs, interferons (IFNa-d) and proinflammatory cytokines (TNF-α1, TNF-α2, IL-1ß, IL-6, IL-12 p40s) were performed. The DEG analyses showed that the responses of proinflammatory cytokines in TO cells infected with IPNV and ISAV were profoundly different from SAV-infected cells. In the two aforementioned, TNF-α1 and TNF-α2 were highly upregulated, while in SAV-infected cells these cytokines were downregulated. Knowledge of virus recognition by the host and the immune responses during infection may help elucidate why and how some viruses can escape the immune system. Such knowledge is useful for the development of immune prophylactic measures.

Comparative assessment of Vibrio virulence in marine fish larvae.

Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single-egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty-nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake has been related to V. anguillarum virulence; however, the presence or absence of the plasmid pJM1 encoding anguibactin did not correlate with virulence. The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high-virulent strains had acquired virulence genes from other pathogenic Vibrio.

Differential expression of immune genes in Atlantic salmon (Salmo salar L.) challenged intraperitoneally or by cohabitation with IPNV.

Atlantic salmon smolts challenged intraperitoneally (ip) and by cohabitation with a highly virulent strain of infectious pancreatic necrosis virus showed strong activation of important immune genes in spleen, liver, head-kidney and gill measured by real-time quantitative PCR. The genes investigated were IL-1beta, IL-10, IFN-alpha, IFN-gamma, Mx, MHC-I, MHC-II, TCR-alpha, CD8-alpha and mIgM. A low final cumulative mortality of about 10% was seen in the ip-challenged group, while more than 40% of the cohabitants died in the sampling period. Sampling was performed at day 15, 24 and 37 post ip-challenge. Overall, the expression of the investigated genes varied highly. The expression of IL-1beta, IL-10, MHC-II, TCR-alpha, CD8-alpha and mIgM showed more or less the same patterns between the two groups of fish by being significantly upregulated at day 24 post ip-challenge. However, the degree of regulation varied a lot among the genes. A pattern showing differences between ip-challenged and cohabitants were seen for IFN-gamma and especially IFN-alpha, where the upregulation seemed to last longer for the cohabitants. The Mx gene was the most induced gene, but also the one with highest individual variance. Mx but also MHC-I were both still highly upregulated at the last sampling point within both groups of fish. The results seem to indicate that the differences in expression pattern(s) could reflect the different routes of entrance of the virus into the fish. This could maybe explain the different kinetics in the onset and the degree of mortality or the potential different molecular mechanisms used for combating the virus.

Cloning and expression of TNF-alpha, IL-1beta and COX-2 in an anadromous and landlocked strain of Atlantic salmon (Salmo salar L.) during the smolting period.

The parr-smolt transformation involves complex modulation of immune parameters, affecting both cell populations and humoral factors. The expression of cytokines was studied in salmon cells and tissues during this period using an anadromous and a landlocked freshwater resident dwarf strain of Atlantic salmon (Salmo salar L.). The constitutive activity of three immunoregulatory genes encoding the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) and the cyclo-oxygenase (COX) isoform COX-2 was investigated in head kidney, spleen and gill tissue from healthy, unvaccinated fish by real-time PCR. The TNF-alpha gene was generally lower expressed than COX-2 and IL-1beta1, which were approximately expressed at equal levels and constitutive expression was seen for COX-2 and IL-1beta1 in all tissues examined and at all sampling dates. The expression of all three genes in head kidney and spleen tissue seemed to be highest at the sampling in May for both strains around the time of seawater transfer suggesting an influence of smolting related hormones on cytokine expression. The gill tissue experienced the highest expression of IL-1beta1 and COX-2 at all sampling dates indicating that this organ is immunologically important.

Peripheral blood and head kidney leucocyte populations during out-of-season (0+) parr-smolt transformation and seawater transfer of Atlantic salmon (Salmo salar L.).

Using monoclonal antibodies (MAb) and flow cytometry, Atlantic salmon neutrophils and Ig+ cells in blood and head kidney were studied in under-yearling out-of-season (0+) smolts, and 2 and 4 weeks after transfer to seawater. The parr-smolt transformation was induced using a phase advanced simulated natural photoperiod regime, and sampling of four fish was performed at regular intervals, starting on the date of the photoperiod initiation. During the freshwater period the proportion of neutrophils in the head kidney leucocytes (HKL) remained quite stable and only gradual changes in Ig+ cells were observed. In the peripheral blood leucocytes (PBL), the proportion of neutrophils markedly increased during the last month prior to seawater transfer. The most notable changes in the proportions of MAb+ leucocytes were observed in PBL after seawater transfer, with a significant increase in Ig+ cells and a significant decrease in neutrophils after two weeks in seawater. In the freshwater samples, although there were fluctuations, a decrease in the numbers of total leucocytes per millilitre blood and per gram head kidney during parr-smolt transformation was observed. The number of MAb+ cells in blood appeared to be relatively stable, while the number in head kidney tended to decrease. Following seawater transfer, the numbers of total and MAb+ leucocytes in both blood and head kidney increased markedly. The results suggest that changes in both distribution and numbers of leucocytes in peripheral blood and head kidney take place during parr-smolt transformation, and that marked changes are associated with seawater transfer. Some mechanisms possibly involved are indicated.

Susceptibility of three different strains of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at two different temperatures to Vibrio anguillarum and temperature effect on antibody response.

Three geographically distinct-reared strains (Canadian, Icelandic, Norwegian) of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at optimal and super-optimal growth temperatures (12 and 18 degrees C respectively), were challenged with a virulent isolate of Vibrio anguillarum by injection. The halibut were injected intraperitoneally with 100 microl of the bacterial suspension (1 x 10(6) cells per fish). After challenge, temperature and strain-related differences in survival were observed. Canadian and Icelandic halibut cultured at the super-optimal temperature of 18 degrees C were significantly more susceptible to infection than those strains cultured at 12 degrees C. Total mortality at 18 degrees C for the Canadian and Icelandic strains was 56.4 and 61.85% respectively, compared to 32 and 26.6% respectively at 12 degrees C. Norwegian halibut were significantly more resistant to infection with V. anguillarum at 18 degrees C compared to the other strains, with total mortality of 13.3%. There was no significant difference in total mortality of Norwegian halibut at 18 or 12 degrees C (13.3, 25% respectively). The specificity of the antibodies in sera from challenged halibut cultured at 18 degrees C was primarily to LPS. Immunoblots showed the presence of antibodies against O-side chain antigens. This reaction was strongest in sera from the Norwegian halibut strain compared with the Canadian and Icelandic halibut, which suggests that the difference in resistance to challenge may be ascribable to the presence of antibodies to LPS. Specific antibody levels, as measured by ELISA, increased with increasing temperature and strain differences were apparent, however these did not relate to disease resistance.

A salmonid cell line (TO) for production of infectious salmon anaemia virus (ISAV).

A new cell line designated TO which provides a high yield of infectious salmon anaemia virus (ISAV) has been established. The cells originate from head kidney leukocytes isolated from Atlantic salmon and grow well at 20 degrees C in EMEM with 5% CO2 and without CO2 supplement in HMEM. The cells have at present been passed more than 150 times and no changes in morphology, growth or virus production have been observed. The virus infection results in cytopathic effects (CPE) within 9 d, and the virus titre obtained from centrifuged and filtrated cell lysates, measured as TCID50, was about 10(9.1) ml(-1). The virus isolated from lysates of infected cells by a sucrose gradient provided purified ISAV when examined by silver stained SDS-PAGE. Salmon injected with diluted virus supernatant showed mortalities, hematocrit values and clinical signs in accordance with infectious salmon anaemia.

Antibodies against Vibrio salmonicida lipopolysaccharide (LPS) and whole bacteria in sera from Atlantic salmon (Salmo salar L.) vaccinated during the smolting and early post-smolt period.

The antibody response to Vibrio salmonicida LPS was studied by ELISA and immunoblot after vaccination of salmon with an aqueous vaccine containing the bacterium. The vaccination of the groups took place from February to July. Antibodies to LPS were present in all sera. Sera from unvaccinated fish and samples collected a short time after vaccination contained low antibody levels. The antibody levels in vaccinated groups increased with time after vaccination except for fish vaccinated during the smolting period. For these fish groups decreasing levels were found in the autumn. Vaccination provided high levels of antibodies to LPS at least 6 months later, even with low water temperatures at primary and secondary vaccination. Fish vaccinated during smolting at higher water temperatures reached high antibody levels a short time after vaccination, but for these groups a decrease took place resulting in the lowest antibody levels of the vaccinated groups in September. Immunoblots showed that antibody reacted with low molecular weight components corresponding to the O-side chain. Immunoblots using whole bacteria as antigen showed a few immunoreactive bands and individual reaction patterns with respect to location of the bands and immunodominance.

Studies of Atlantic salmon (Salmo salar L.) blood, spleen and head kidney leucocytes using specific monoclonal antibodies, immunohistochemistry and flow cytometry.

Monoclonal antibodies (mabs) raised against Atlantic salmon serum IgM (C7G7 and G2H3) and isolated peripheral blood leucocytes (PBL) (E3D9, C4B6 and D8B3) were applied in this study. Using immunoenzymehistochemistry, immunofluorescence and flow cytometry, the distribution of mab+ cells in blood, spleen and head kidney from Atlantic salmon were studied. Immunostaining on cytospin preparations and flow cytometry of isolated PBL showed that the Ig+ cells recognised by C7G7 and G2H3 were mononuclear leucocytes (MNL). The cytospin preparations showed some Ig+ cells with strong cytoplasmic staining, most likely plasma cells. The salmon blood neutrophils were the only E3D9+ cells in cytospin preparations of PBL, and E3D9 recognised about 94% of the defined neutrophil fraction in flow cytometry. The reactivities of C4B6 and D8B3 were to a large degree similar in both immunoenzymehistochemistry and flow cytometry, recognising both MNL and blood neutrophils. Immunofluorescence double staining of PBL with C4B6 and D8B3 showed double staining of all mab+ cells and D8B3 was apparently not able to block the binding of C4B6 to PBL. Immunofluorescence double staining of PBL also revealed more E3D9+ than C4B6+ neutrophils. In immunostaining on cryostat sections of spleen and head kidney, staining of cells was observed with all the mabs, the head kidney generally containing more positive cells than the spleen. Some potential applications for immunological studies using these mabs are suggested.

Antibodies to staphylococcal peptidoglycan and its peptide epitopes, teichoic acid, and lipoteichoic acid in sera from blood donors and patients with staphylococcal infections.

Antibodies to the staphylococcal antigens peptidoglycan, beta-ribitol teichoic acid, and lipoteichoic acid, as well as to the peptidoglycan epitopes L-Lys-D-Ala-D-Ala, L-Lys-D-Ala, and pentaglycine, were found over a wide range of concentrations in sera from both blood donors and patients with verified or suspected staphylococcal infections. The patient group was heterogeneous with regard to both age and type of staphylococcal infections, being representative for sera sent to our laboratory. In single-antigen assays antibodies to pentaglycine had the highest predictive positive value (67%), although only 32% of the patients had elevated levels of such antibodies. Combinations of test antigens could yield positive predictive values as high as 100%, but then the fraction of positive sera was low. Indeed, the fraction of patient sera which was positive in multiple-antigen tests never exceeded 61%. The clinical usefulness of these seroassays for identifying Staphylococcus aureus as a causative agent was limited, owing to the considerable overlap in the range of antibody concentrations between patient and blood donor sera.

Monoclonal antibodies evoked by the free oligopeptide (Gly)5 reacting specifically with peptidoglycan from staphylococci.

Murine monoclonal antibodies reactive with the Staphylococcus aureus peptidoglycan (PG) epitope (Gly)5 were obtained using the synthetic oligopeptide (Gly)5 in its free form as immunogen. The selected monoclonal antibodies were of the IgM kappa isotype and reacted specifically with PG from S. aureus and Staphylococcus epidermidis, but gave no reaction with PG from Streptococcus pyogenes, Bacillus subtilis and Micrococcus lysodeikticus. Affinity chromatography showed that the antibodies were reactive with the N-terminus of the (Gly)5 peptide. These monoclonal antibodies can be used for the detection of staphylococcal PG in solution.

Antibodies to various bacterial cell wall peptidoglycans in human and rabbit sera.

Sera from patients with verified systemic staphylococcal infection contained antibodies reactive with peptidoglycan (PG) from Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus lysodeikticus, Bacillus subtilis, and Escherichia coli. The presence of anti-PG cross-reactive antibodies was verified in patient sera by inhibition studies with the various bacterial PGs. Antibodies to nonstaphylococcal PGs were also elevated in sera from rabbits immunized with S. aureus PG. Antibodies to S. aureus PG were removed with the synthetic peptide analogs of S. aureus PG, the L-Lys-D-Ala-D-Ala, L-Lys-D-Ala, and (Gly)5 determinants, as well as with an S. aureus PG peptide fragment containing the determinants D-Ala-D-Ala and L-Lys-D-Ala. Isolated antibodies to the PG peptides, both synthetic and native, were reactive with S. aureus and S. epidermidis PGs. The antibodies to the D-Ala-D-Ala and the L-Lys-D-Ala determinants were also reactive with S. pyogenes PG, but not with PGs from M. lysodeikticus, B. subtilis, and E. coli.