Polyclonal hypergammaglobulinemia and autoantibody production induced by vaccination in farmed Atlantic salmon.

The introduction of oil-adjuvanted vaccines in salmon aquaculture made large-scale production feasible by reducing the impact of infections. Vaccines given intraperitoneally (ip) contain oil adjuvant such as mineral oil. However, in rodents, a single ip injection of adjuvant hydrocarbon oil induces lupus-like systemic autoimmune syndrome. We have recently reported that autoimmune disease in farmed salmon, characterized by production of various autoantibodies, immune complex glomerulonephritis, liver thrombosis, and spinal deformity, are previously unrecognized side effects of vaccination. In the present study, we examined whether vaccination-induced autoantibody production in farmed Atlantic salmon is a mere result of polyclonal B-cell activation. Sera were collected from 205 vaccinated and unvaccinated Atlantic salmon (experimental, 7 farms) and wild salmon. Total IgM levels and autoantibodies to salmon blood cell (SBC) extract in sera were measured by ELISA and the relationship between hypergammaglobulinemia and autoantibody production was analyzed. Comparison of endpoint titers vs levels/units using a single dilution of sera in detection of autoantibodies to SBC showed near perfect correlation, justifying the use of the latter for screening. Both total IgM and anti-SBC antibodies are increased in vaccinated salmon compared with unvaccinated controls, however, they do not always correlate well when compared between groups or between individuals, suggesting the involvement of antigen-specific mechanisms in the production of anti-SBC autoantibodies. The primary considerations of successful vaccine for aquaculture are cost-effectiveness and safety. Vaccination-induced autoimmunity in farmed Atlantic salmon may have consequences on future vaccine development and salmon farming strategy. Evaluation for polyclonal hypergamamglobulinemia and autoimmunity should be included as an important trait when vaccine efficacy and safety are evaluated in future.

Vaccination-induced systemic autoimmunity in farmed Atlantic salmon.

Over half of the salmon consumed globally are farm-raised. The introduction of oil-adjuvanted vaccines into salmon aquaculture made large-scale production feasible by preventing infections. The vaccines that are given i.p. contain oil adjuvant such as mineral oil. However, in rodents, a single i.p. injection of adjuvant hydrocarbon oil induces lupus-like systemic autoimmune syndrome, characterized by autoantibodies, immune complex glomerulonephritis, and arthritis. In the present study, whether the farmed salmon that received oil-adjuvanted vaccine have autoimmune syndrome similar to adjuvant oil-injected rodents was examined. Sera and tissues were collected from vaccinated or unvaccinated Atlantic salmon (experimental, seven farms) and wild salmon. Autoantibodies (immunofluorescence, ELISA, and immunoprecipitation) and IgM levels (ELISA) in sera were measured. Kidneys and livers were examined for pathology. Autoantibodies were common in vaccinated fish vs unvaccinated controls and they reacted with salmon cells/Ags in addition to their reactivity with mammalian Ags. Diffuse nuclear/cytoplasmic staining was common in immunofluorescence but some had more specific patterns. Serum total IgM levels were also increased in vaccinated fish; however, the fold increase of autoantibodies was much more than that of total IgM. Sera from vaccinated fish immunoprecipitated ferritin and approximately 50% also reacted with other unique proteins. Thrombosis and granulomatous inflammation in liver, and immune-complex glomerulonephritis were common in vaccinated fish. Autoimmunity similar to the mouse model of adjuvant oil-induced lupus is common in vaccinated farmed Atlantic salmon. This may have a significant impact on production loss, disease of previously unknown etiology, and future strategies of vaccines and salmon farming.

Monitoring viral-induced cell death using electric cell-substrate impedance sensing.

Using an electrical measurement known as electric cell-substrate impedance sensing (ECIS), we have recorded the dynamics of viral infections in cell culture. With this technique, cells are cultured on small gold electrodes where the measured impedance mirrors changes in attachment and morphology of cultured cells. As the cells attach and spread on the electrode, the measured impedance increases until the electrode is completely covered. Viral infection inducing cytopathic effect results in dramatic impedance changes, which are mainly due to cell death. In the current study, two different fish cell lines have been used: chinook salmonid embryonic (CHSE-214) cells infected with infectious pancreatic necrosis virus (IPNV) and epithelioma papulosum cyprini (EPC) carp cells infected with infectious hematopoeitic necrosis virus (IHNV). The impedance changes caused by cell response to virus are easily measured and converted to resistance and capacitance. An approximate linear correlation between log of viral titer and time of cell death was determined.

Manifestations of systemic autoimmunity in vaccinated salmon.

The development of systemic autoimmunity may result as an undesired side-effect following vaccination, and this condition was recently shown to occur in farmed salmon (Salmo salar). Several of previously reported side-effects following vaccination of fish should therefore be reviewed in the light of this condition. Here, organs and pathological changes in three separate groups of fish severely affected by vaccination were investigated by different morphological methods (n=84). Granulomas or microgranulomas were observed at the injection site and in several organs. Mott cells were observed in all tissues examined. Pannus-like changes with lymphocyte infiltrates were observed in spines. In conclusion, the reactions following vaccination were of a systemic nature that may be explained by a pathogenetic mechanism caused by systemic autoimmunity.

Identification and characterization of a novel intraepithelial lymphoid tissue in the gills of Atlantic salmon.

In addition to being the respiratory organ in fish, the gills form a barrier against the external milieu. Innate and adaptive immune system components have been detected in the gills, but lymphoid cell accumulations similar to that seen in the mammalian mucosa have not been described. The present investigations revealed cell accumulations on the caudal edge of interbranchial septum at the base of the gill filaments in the Atlantic salmon. Cytokeratin immunohistochemical staining and identification of a basal membrane and desmosome cell junctions by electron microscopy showed that the cell accumulation was located intraepithelially. Major histocompatibility complex (MHC) class II+ cells were detected by immunohistochemistry, and laser capture micro-dissection and subsequent RT-PCR analysis revealed expression of T-cell receptor transcripts in the investigated tissue, suggesting the presence of T cells. The intraepithelial tissue reported here may be a suitable location for immune surveillance of gill infections, as well as a target site for new vaccine approaches and investigations of epithelial immunity. This is the first description of a lymphocyte cell aggregation within a teleostian gill epithelium network, illustrating a phylogenetically early form of leukocyte accumulations in a respiratory organ.

Melanogenesis and evidence for melanosome transport to the plasma membrane in a CD83 teleost leukocyte cell line.

Visceral organs of ectothermic vertebrates harbour melanin-containing leukocytes termed melanomacrophages. These cells are thought to participate in immune reactions and free-radical trapping. In teleosts, the melanin-producing ability of melanomacrophages has hitherto not been confirmed by molecular techniques. Here, a leukocyte marker and the apparatus for melanosome production and transport were investigated in an Atlantic salmon (Salmo salar) pronephros-derived mononuclear leukocyte (SHK-1) cell line. The SHK-1 cells expressed transcripts specific for a mammalian CD83 homologue, a standard surface marker for activated or differentiated dendritic cells, and dopachrome tautomerase/tyrosinase-related protein-2, a melanocyte specific enzyme essential for melanin production. Reduction potential of melanin or its precursors was demonstrated histochemically after prolonged cultivation. Ultrastructural investigations revealed tyrosinase and acid phosphate activity in identical organelles and BSA-gold co-localized with multilamellar melanosomes after 2 h internalization. Apparently, melanosomes were transported and released through periodically occurring tubules fusing with the plasma membrane. Video monitoring revealed filopodia and macropinocytosis. These results showed that the SHK-1 cell line is capable of melanogenesis and melanosome secretion. Melanin-producing cells in teleost pronephros may represent a distinct CD83(+) leukocyte population consisting of phylogenetically relict multifunctional cells. This is the first report of a melanin-producing leukocyte cell-line.