Establishment of killer whale (Orcinus orca) primary fibroblast cell cultures and their transcriptomic responses to pollutant exposure.

Populations of killer whale (Orcinus orca) contain some of the most polluted animals on Earth. Yet, the knowledge on effects of chemical pollutants is limited in this species. Cell cultures and in vitro exposure experiments are pertinent tools to study effects of pollutants in free-ranging marine mammals. To investigate transcriptional responses to pollutants in killer whale cells, we collected skin biopsies of killer whales from the Northern Norwegian fjords and successfully established primary fibroblast cell cultures from the dermis of 4 out of 5 of them. Cells from the individual with the highest cell yield were exposed to three different concentrations of a mixture of persistent organic pollutants (POPs) that reflects the composition of the 10 most abundant POPs found in Norwegian killer whales (p,p'-DDE, trans-nonachlor, PCB52, 99, 101, 118, 138, 153, 180, 187). Transcriptional responses of 13 selected target genes were studied using digital droplet PCR, and whole transcriptome responses were investigated utilizing RNA sequencing. Among the target genes analysed, CYP1A1 was significantly downregulated in the cells exposed to medium (11.6 µM) and high (116 µM) concentrations of the pollutant mixture, while seven genes involved in endocrine functions showed a non-significant tendency to be upregulated at the highest exposure concentration. Bioinformatic analyses of RNA-seq data indicated that 13 and 43 genes were differentially expressed in the cells exposed to low and high concentrations of the mixture, respectively, in comparison to solvent control. Subsequent pathway and functional analyses of the differentially expressed genes indicated that the enriched pathways were mainly related to lipid metabolism, myogenesis and glucocorticoid receptor regulation. The current study results support previous correlative studies and provide cause-effect relationships, which is highly relevant for chemical and environmental management.

Studies on the effects of LPS, ß-glucan and metabolic inhibitors on the respiratory burst and gene expression in Atlantic salmon macrophages.

Reactive oxygen species (ROS) production in macrophage-like cells is induced as an antimicrobial defence against invading pathogens. In this study, we have explored how different stimuli and metabolic inhibitors affect the level of respiratory burst in Atlantic salmon (Salmo salar L.) head kidney macrophage-like cells. Cells stimulated in vitro by bacterial lipopolysaccharide (LPS) and ß-glucan showed increased production of ROS compared to unstimulated cells. Both stimulation and costimulation by curdlan (ß-glucan) induced a higher production of ROS compared to stimulation and costimulation by LPS. Metabolic inhibitors co-incubated with the stimulants did not, in most cases, perturb the level of ROS generation in the salmon macrophage-like cells. The NAD+ content as well as the NAD+ /NADH ratio increased in curdlan and LPS + curdlan-stimulated cells compared to control cells, which indicated increased metabolic activity in the stimulated cells. Supporting these findings, gene analysis using real-time quantitative PCR showed that arginase-1 and IL-1ß genes were highly expressed in the stimulated cells.

Vaccination of Atlantic lumpfish (Cyclopterus lumpus L.) at a low temperature leads to a low antibody response against Aeromonas salmonicida.

We present a study on the effect of water temperature on immunization of Atlantic lumpfish. In total, 360 fish were vaccinated with either 50 µl of an oil-based injection vaccine (VAX), with Aeromonas salmonicida and Vibrio salmonicida antigens, or PBS. Fish were vaccinated at three different water temperatures, 5°C, 10°C and 15°C, and sorted into six groups (N = 60). Lumpfish were weighed every 3 weeks after vaccination, sampled at 3, 6, 9 and 18 weeks post-immunization (wpi) and evaluated by modified Speilberg score, ELISA and immunoblotting. Vaccinated fish showed low antibody response against V. salmonicida. Fish vaccinated at 5°C showed significantly lower antibody response against A. salmonicida throughout the study. At higher temperatures, vaccinated fish showed significantly increased antibody responses, at 18 wpi for 10°C and at 6 and 18 wpi for 15°C. Immunoblotting demonstrated specific response against the LPS antigen of A. salmonicida in the 10°C and 15°C VAX groups. Mean body weight increased in all groups throughout the study. Vaccinated fish had low Speilberg scores with no melanization of abdominal tissue. Our results show that vaccinating lumpfish at a lower water temperature may lead to a low antibody response against A. salmonicida.

DNA vaccines for fish: Review and perspectives on correlates of protection.

Recently in 2016, the European Medicines Agency (EMA) recommended granting a marketing authorization in the EU for "Clynav," a DNA vaccine against salmon pancreas disease (salmonid alphavirus-3). Generally, DNA vaccines induce both early and late immune responses in fish that may be protective against disease. Several transcriptomic approaches have been performed to map immunome profiles following DNA vaccination, but the precise immune mechanism(s) that is responsible for protection is not known, although reasonable suggestions have been made. The current review includes an overview on main transcriptomic findings from microarray experiments after DNA vaccination against VHSV, IHNV, HIRRV and IPNV-with considerations of what can be considered as correlates of protection (CoP) or merely a surrogate of protection. Identification and use of correlates of protection (COPs) may be a strategic tool for accelerated and targeted vaccine design, testing and licensure. General rules on what can be considered as CoPs can be extracted from past knowledge on protective immune responses following vaccination that induced protection. Lastly, there will be an overview on non-viral molecular adjuvants that have been exploited to obtain higher vaccine potencies and efficacies.

Studies on the antibody response and side effects after intramuscular and intraperitoneal injection of Atlantic lumpfish (Cyclopterus lumpus L.) with different oil-based vaccines.

Atlantic lumpfish (Cyclopterus lumpus L.) is used as a biological delousing agent for sea lice (Lepeophtheirus salmonis K.) infestations in Norwegian aquaculture. Here, we present a study on the antibody response and vaccine side effects after intramuscular and intraperitoneal injection of lumpfish with two vaccines. Both vaccines contained bacterial antigens from atypical Aeromonas salmonicida A-layer types V and VI, Vibrio anguillarum serotype O1 and Moritella viscosa sp., but one vaccine contained a vegetable oil-based adjuvant, while the other contained a mineral oil-based adjuvant. Intramuscular injection of the mineral oil-based vaccine caused a high acute mortality of fish within 48 hr after immunization. Intraperitoneal injection of the mineral oil-based vaccine resulted in a lower severity of intra-abdominal side effects than the vegetable oil-based vaccine. Intramuscular injection of the mineral oil-based vaccine resulted in a significantly higher antibody response against A. salmonicida when compared to controls and the vegetable oil-based vaccine group. The antibody response was poor against V. anguillarum and M. viscosa for all groups. Our results indicate that intramuscular injection of oil-based vaccines might be feasible for providing immunological protection for Atlantic lumpfish against bacterial diseases, especially atypical A. salmonicida, but more work is required to identity optimal adjuvants.

Early immune responses in Atlantic salmon (Salmo salar L.) after immunization with PLGA nanoparticles loaded with a model antigen and ß-glucan.

Polymeric nanoparticles (NPs) of poly (lactic-co-glycolic) acid (PLGA) possess adjuvant properties. To date, there are few studies exploring their application as antigen carriers for vaccination of fish. This study presents a preclinical assessment of the early innate and adaptive immune responses in Atlantic salmon following immunization with PLGA NPs. A model antigen (TNP-LPH) and an immunostimulant (ß-glucan) were entrapped in NPs of 300-400nm either alone or in combination. Both the antigen and the ß-glucan were efficiently entrapped (>50%) in particles and an antigen release study indicated particle stability up to 50 days at 8°C. Spleen and head kidney were analyzed for pro-inflammatory markers (TNF-α, IL-1ß, IL-8, C3a) and T cell cytokines, effector molecules and transcription factors (IFN-γ, T-bet, GATA-3, granzyme A, IL-10, Foxp3) at mRNA transcription levels 2, 4 and 8 days post i.p. immunization. NPs alone were able to moderately up-regulate pro-inflammatory immune responses. Addition of immunogenic cargo, either an antigen or ß-glucan generally increased the gene expression of pro-inflammatory markers, while administering both resulted in the highest gene expression. These findings were also reflected by concurrently increased levels of IL-10. Comparing the treatment groups injected with antigen and ß-glucan co-administered either in NPs or FCA demonstrated that the magnitude of the acute pro-inflammatory responses was equal between the treatments or highest in the NP injected group. Although elevated expression of granzyme A in the NP injected groups (carrying antigen and/or ß-glucan) was observed, PLGA NPs were unable to induce T cell differentiation on mRNA gene expression levels, as increased levels of the indicating cytokines and transcriptions factors failed to occur. In conclusion, this study demonstrates that PLGA NPs have potential as an adjuvant in salmon vaccines as they enhance the early pro-inflammatory responses to immunization.

Immunostimulation of larvae and juveniles of cod, Gadus morhua L.

Cod larval culture is currently hampered by high mortalities in the first 2-3 weeks after hatching, often due to infectious diseases. The immune system of cod is not fully competent until 2-3 months after hatching. Conventional vaccination is, therefore, not of value before this time, and the larvae are wholly reliant on non-specific parameters for their defence against infection. A range of substances, generally derived from bacterial, fungal or plant origin, can activate these non-specific parameters. During three hatching seasons, 2001-2003, at the Marine Institute's Experimental Station, Stadur, Grindavik, Iceland, the effects of several immunostimulants on survival and disease resistance of cod larvae and juveniles were examined. Both bathing treatments and administration in the feed were used. One of these substances, lipopolysaccharide (LPS), isolated from the bacterium Aeromonas salmonicida (ssp. salmonicida or achromogenes), appeared in some instances to improve survival and have a beneficial effect on disease resistance. Other substances tested had limited effects. The results emphasize the need for further work in this field.

Ontogeny of humoral immune parameters in fish.

The first appearance of IgM in lymphocytes varies considerably among fish species. Generally, the first appearance of B-lymphocytes and immunoglobulins is late in marine species compared to fresh water species, and larvae have reached about 20-30 mm in length when IgM is first expressed. Rainbow trout and channel catfish show the first appearances of surface IgM at about 1 week after hatching. Marine species like the sea bass, spotted wolffish and cod show IgM positive lymphocytes 1-10 weeks after hatching. Transfer of maternal antibody to eggs and embryos has been demonstrated in several species. Examples are plaice, tilapia, carp, sea bass and salmon, but not cod. The ontogeny of complement component C3 has been studied in Atlantic halibut (Hippoglossus hippoglossus L.), Atlantic cod (Gadus morhua L.) and the spotted wolffish (Anarhichas minor O.). By Western blotting experiments C3 was found in unfertilised eggs in the spotted wolffish indicating a maternal transfer. RT-PCR analysis revealed C3 mRNA transcripts from 290 degrees in spotted wolffish eggs. Using immunohistochemistry and in situ hybridisation, C3 was found in liver, brain, kidney and muscle of cod larvae 2 days post-hatching and in intestines, pancreas, heart and gills at different stages of larval development. Also, C3 was detectable in halibut larvae in yolk sac, muscle, liver, brain, chondrocytes, spinal chord, eye, heart, intestines and kidney. These studies suggest that complement may play a role in generation of different organs, not only in the defence against invading pathogens. Lysozyme is a bactericidal enzyme present in mucus, lymphoid tissue and serum of most fish species, but not in cod and wolffish. The enzyme has been detected in oocytes, fertilised eggs and larval stages of fish species like coho salmon, sea bass and tilapia. The activity of other enzymes like the cathepsins has been described in eggs and larvae of sea bass, cod and salmonids. Cathepsins may have a bactericidal role in the skin of fish. Lectins are carbohydrate-binding proteins that interact with pathogenic surface structures that result in opsonization, phagocytosis or activation of complement. Lectins have been isolated from the eggs of various fish species.

Isolation and characterisation of spotted wolffish (Anarhichas minor Olafsen) macrophages.

Non-specific mechanisms are important in the defence of all multicellular animals against pathogenic microorganisms. Macrophages and granulocytes play a central role in this respect. It is thus pertinent to develop methods for obtaining and cultivation of macrophages and assessing their functions in the spotted wolffish, a cold water species of current interest for the aquaculture industry. Kidney macrophages from the spotted wolffish (Anarhichas minor Olafsen) were isolated by density sedimentation using Percoll. The cells were highly phagocytic and possessed typical macrophage morphology evaluated by transmission and scanning electron microscopy. Using electron microscopic analysis, the size of the macrophages, collected from the Percoll density interface, was 5-9 microm. The viability in vitro was highest (87.1%) when the cells were kept at 13 degrees C with the addition of synthetic serum replacement (SSR-2) when measured 24 h after seeding. One day old cells were not significantly activated by addition of bacterial lipopolysaccharide (LPS) for 24 h when measured by reduction of nitroblue tetrazolium compared to control cells. The cells were negative in respect to synthesis and contents of complement component C3.

Antigen uptake and immunoglobulin production in Atlantic cod (Gadus morhua L.) after intraperitoneal injection of Vibrio anguillarum.

Atlantic cod (Gadus morhua L.) were injected intraperitoneally with formalin-killed Vibrio anguillarum bacteria. Immunostaining revealed uptake of V. anguillarum antigens especially in the spleen after intraperitoneal (i.p.) administration. The uptake was time dependent in the interval 1-24 h. Most of the antigen uptake in the spleen was concentrated in areas around small blood vessels, while immunoglobulin producing cells were localised to some thick walled arteries. There was apparently little or no co-localisation of antigens and antibody producing cells. In the heart, some of the high endocardial endothelial cells of the atrium contained bacterial antigens and in head kidney some macrophage-like cells were stained. Very little antigen was found in the pigmented loose connective tissues of the peritoneum. In contrast, endothelial cells of the underlying blood vessels contained substantial amounts. In the heart, peritoneum and anterior kidney the number of antigen positive cells did not seem to change in the time interval 1-24 h. After i.p. immunisation with a mixture of V. anguillarum and Freunds complete adjuvant, the humoral immune response in Atlantic cod was low when tested 21, 42 and 105 days later. There was apparently no enhanced number of immunoglobulin synthesising cells caused by the antigen stimulation.

Scavenger-receptor-mediated endocytosis of lipopolysaccharide in Atlantic cod (Gadus morhua L.).

The mechanism of elimination of blood-borne Vibrio salmonicida lipopolysaccharide (LPS) from Atlantic cod (Gadus morhua L.) was studied. The anatomical distribution of LPS was determined using both morphological and radiotracing methods. Immunohistochemistry performed on tissue specimens after injection of LPS disclosed that the endocardial endothelial cells (EECs) represented the cellular site of uptake in heart. Co-injection of trace amounts of [(125)I]LPS together with excess amounts of formaldehyde-treated albumin (FSA), a ligand for the scavenger receptor, significantly inhibited the accumulation of the radiotracer in heart only. Studies on purified monolayer cultures of atrial EECs showed that fluorescein-labelled LPS was taken up in structures reminiscent of endosomal/lysosomal vesicles. Incubation of cultures with [(125)I]LPS together with excess amounts of FSA, fucoidan and dextran sulphate, molecules known to compete for endocytosis via the scavenger receptor, reduced uptake of the probe by 80 %. Mannan, a ligand for the mannose receptor, did not compete for uptake. Kinetic studies on the uptake and degradation of [(125)I]LPS in cultured atrial endocardial cells revealed no degradation after 48 h of culture. In conclusion, we have shown that the EECs of cod remove V. salmonicida LPS from the circulation by scavenger-receptor-mediated endocytosis.

Bath exposure of Atlantic halibut (Hippoglossus hippoglossus L.) yolk sac larvae to bacterial lipopolysaccharide (LPS): absorption and distribution of the LPS and effect on fish survival.

Radiolabelled bacterial lipopolysaccharide (3H-LPS) obtained from Aeromonas salmonicida subsp. salmonicida was added to the petri dishes containing yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). The larvae were exposed either to 6.25, 12.5, 25, 50 or 100 micrograms 3H-LPS ml-1. The uptake was both dependent on the LPS concentration and the time of exposure. After 5 days of exposure, each larva contained 1.8-7.4 ng 3H-LPS dependent on the initial concentration. After 10 days of exposure each larva contained 7.0-12.4 ng LPS and after 15 days they contained 18.3-34.9 ng 3H-LPS. Fluorescence microscopic analysis of sections obtained from larvae exposed to FITC-LPS (25, 50 and 100 micrograms ml-1) for 5, 10 and 15 days, revealed fluorescence in intestinal epithelial cells, cells in the connective tissue adjacent to the intestine, in cells located between the integumental layer and yolk sac, and in some epithelial cells in the integument. By use of immunohistochemical techniques, LPS was confined to intestinal epithelial cells, lumen of excretory duct and in numerous cells in the epidermal layer. Control specimens did not contain fluorescence or were immunohistochemically negative for LPS. In groups of larvae exposed to 12.5, 25, 50 and 100 micrograms LPS ml-1, the survival was significantly increased after exposure to 50 and 100 micrograms LPS ml-1 from day 20 (96 d degree) and throughout the yolk sac period compared to untreated larvae.