Jarl Bøgwald

Growth and survival of Atlantic salmon (Salmo salar) fry given diets supplemented with fish protein hydrolysate and lactic acid bacteria during a challenge trial with Aeromonas salmonicida

Abstract Atlantic salmon fry were reared ad libitum on a commercial fish feed supplemented with 10% cod muscle protein (A), 10% hydrolyzed cod muscle protein (B) or 10% of a hydrolysate based culture of lactic acid bacteria isolated from salmon intestines. The fry grew well, and equally so, on each of the three diets (specific growth rate, 2.5% day −1 ). Very low numbers of intestinal bacteria were detected in fish given diets A and B, whereas a considerable colonization of the intestine by lactic acid bacteria was achieved in fish given diet C. After 5 weeks of feeding, the fish were challenged with cohabitants infected with Aeromonas salmonicida . Four weeks after infection, a cumulative mortality between 42 and 75% was recorded in the different tanks. Unexpectedly the highest mortality was observed with fish given the diet containing lactic acid bacteria, whereas no significant difference was observed between fish given feed supplemented with cod muscle protein and hydrolyzed cod muscle protein.

What happens to the DNA vaccine in fish? A review of current knowledge.

The primary function of DNA vaccines, a bacterial plasmid DNA containing a construct for a given protective antigen, is to establish specific and long-lasting protective immunity against diseases where conventional vaccines fail to induce protection. It is acknowledged that less effort has been made to study the fate, in terms of cellular uptake, persistence and degradation, of DNA vaccines after in vivo administration. However, during the last year some papers have given new insights into the fate of DNA vaccines in fish. By comparing the newly acquired information in fish with similar knowledge from studies in mammals, similarities with regard to transport, blood clearance, cellular uptake and degradation of DNA vaccines have been found. But the amount of DNA vaccine redistributed from the administration site after intramuscular administration seems to differ between fish and mammals. This review presents up-to-date and in-depth knowledge concerning the fate of DNA vaccines with emphasis on tissue distribution, cellular uptake and uptake mechanism(s) before finally describing the intracellular hurdles that DNA vaccines need to overcome in order to produce their gene product.

Adjuvants and immunostimulants in fish vaccines: Current knowledge and future perspectives.

Vaccination is the most adequate method to control infectious diseases that threaten the aquaculture industry worldwide. Unfortunately, vaccines are usually not able to confer protection on their own; especially those vaccines based on recombinant antigens or inactivated pathogens. Therefore, the use of adjuvants or immunostimulants is often necessary to increase the vaccine efficacy. Traditional adjuvants such as mineral oils are routinely used in different commercial bacterial vaccines available for fish; however, important side effects may occur with this type of adjuvants. A search for alternative molecules or certain combinations of them as adjuvants is desirable in order to increase animal welfare without reducing protection levels. Especially, combinations that may target specific cell responses and thus a specific pathogen, with no or minor side effects, should be explored. Despite this, the oil adjuvants currently used are quite friendlier with respect to side effects compared with the oil adjuvants previously used. The great lack of fish antiviral vaccines also evidences the importance of identifying optimal combinations of a vaccination strategy with the use of a targeting adjuvant, especially for the promising fish antiviral DNA vaccines. In this review, we summarise previous studies performed with both traditional adjuvants as well as the most promising new generation adjuvants such as ligands for Toll receptors or different cytokines, focussing mostly on their protective efficacies, and also on what is known concerning their effects on the fish immune system when delivered in vivo.

Isolation of acid peptide fractions from a fish protein hydrolysate with strong stimulatory effect on atlantic salmon (Salmo salar) head kidney leucocytes

Abstract Medium size (3000 d > Mw > 500 d) peptides from a hydrolysate of emptied stomachs from Atlantic cod (Gadus morhua) were fractionated on an S-Sepharose cation exchange chromatography column. Four distinctly separated acid peptide fractions were used in in vitro stimulatory experiments with head kidney leucocytes from Atlantic salmon (Salmo salar). All four acid peptide fractions promoted strongly elevated oxidative burst reactions in the leucocytes after 2 and 7 days of incubation at concentrations from 1 to 25 μg/ml. The stimulation was equally good, and in most cases better than the stimulation achieved with similar concentrations of lipopolysaccharides from the fish pathogen Aeromonas salmonicida. Visual inspection and pictures of peptide stimulated cells showed strongly enhanced vacuolisation and formation of long stretched out pseudopodes after 7 days of incubation. Acid peptide fractions from fish protein hydrolysate may be useful as adjuvants in fish vaccine and as an immune stimulant in fish feed.

A soluble β-1,3-D-glucan derivative potentiates the cytostatic and cytolytic capacity of mouse peritoneal macrophages in vitro

Abstract An aminated β-1,3- D -glucan derivative of curdlan is reported to render macrophages cytostatic to L-929 cells and to potentiate macrophage cytotoxicity to the tumor cells in vitro.

Bath immunostimulation of rainbow trout (Oncorhynchus mykiss) fry induces enhancement of inflammatory cytokine transcripts, while repeated bath induce no changes

The application of immunostimulants may be a cost-effective practice in production of delicate and fragile fish fry since it may confer disease resistance. Bath administration to fish fry is considered an ideal delivery route for mass manipulation since there is no need for individual handling. In the current study, rainbow trout fry were bathed with three different immunostimulants: Plasmid DNA, lactoferrin and beta-glucan at two different doses (0.1 microM and 1.0 microM) for 45 min, four times with an interval of 1 week. Ten fish per treatment group were sampled, and RNA of pooled tissues consisting of eye, tongue, skin, gill, thymus, spleen, head kidney, liver, small and large intestine were isolated from the fish obtained at first, second and fourth bathing (24 and 72 h post-bathing). Before cDNA transcription, two parallel samples were pooled giving a total of 5 parallels in each treatment group. Results showed that plasmid DNA and lactoferrin as well as beta-glucan treated fish possessed higher gene expression with regard to the pro-inflammatory cytokines IL-1beta, TNF-alpha, IL-6 and the anti-inflammatory cytokines IL-10 and TGF-beta after the first bathing, especially 24 h post-bathing in the high-dose groups. This indicates that bath delivery of immunostimulants can induce pro-inflammatory responses. No significant changes of the pro-inflammatory cytokine IL-17A transcripts, compared to the respective controls, were observed. Gene expression levels in the immunostimulated fish after the fourth bathing did not show significant differences compared to controls.

Structural studies of the Vibrio salmonicida lipopolysaccharide

The oligosaccharide part of the Vibrio salmonicida (strain NCMB 2262) lipopolysaccharide was isolated by mild acid hydrolysis followed by gel-permeation chromatography. The structure was established mainly by methylation analysis, mass spectrometry, and NMR spectroscopy. It is concluded that the oligosaccharide has the following structure, in which L-alpha-D-Hep p is L-glycero-alpha-D-manno-heptopyranose, D-alpha-D-Hepp is D-glycero-alpha-D-manno-heptopyranose, alpha-D-Fuc p4N is 4-amino-4,6-dideoxy-alpha-D-galactopyranose, alpha-NonA is 5-acetamidino-7-acetamido-3,5,7, 9-tetradeoxy-L-glycero-alpha-D-galacto-nonulosonic acid, BA is (R)-3-hydroxybutanoyl, and PEA is phosphoethanolamine. The substitution pattern of the branching heptosyl residue was deduced from 1H NMR chemical shifts and conformations of the branching region, obtained by molecular modelling. The absolute configuration for NonA was determined by NMR spectroscopy from NOE correlations to the neighbouring sugar and 13C NMR chemical shift data. It could also be shown that assignments of nonulosonic acids with the D-glycero-L-galacto configuration, reported by previous investigators, are erroneous and should be changed to L-glycero-D-galacto. The oligosaccharide is assumed to be linked to the 5-position of a Kdo residue, phosphorylated in the 4-position as observed for other lipopolysaccharides from Vibrionaceae. [formula: see text]

Coupling of polysaccharides activated by means of chloroacetaldehyde dimethyl acetal to amines or proteins by reductive amination

Abstract A novel method has been developed for the coupling of modified polysaccharides to proteins or other amines. Chloroacetaldehyde dimethyl acetal has been used for the introduction of O -(2,2-dimethoxyethyl) groups into amylose, dextran, and a linear (1→3)-β- d -glucan. In amylose and the (1→3)-β- d -glucan, these groups were attached preponderantly at O-6 and in dextran at O-2. Mild treatment with acid then gave polysaccharide derivatives substituted with aldehyde groups which were coupled in good yields to proteins and other amines by reductive amination with sodium cyanoborohydride in aqueous solution at pH 7. An aminated (1→3)-β- d -glucan derivative that induced antitumor activity in mouse macrophages in vitro is reported.

Oral administration of lipopolysaccharide to Atlantic salmon (Salmo salar L.) fry. Uptake, distribution, influence on growth and immune stimulation

Abstract Atlantic salmon fry were fed Aeromonas salmonicida lipopolysaccharide (LPS)-coated feed for 62 days and then challenged with virulent A. salmonicida bacteria. The fry were fed LPS-coated feed also after challenge. Fry that were fed LPS feed (0.1% LPS) showed a higher mortality throughout the challenge period (accumulated mortality of 57% after 42 days) compared with fry fed control feed (accumulated mortality of 36.5% after 42 days). Fry receiving LPS-coated feed showed an increase in mean weight of 10.7% at day 62 compared with fry receiving control feed. The increase was, however, not statistically significant. Sixty days of feeding with LPS-coated feed did not result in measurable amounts of specific antibodies against A. salmonicida LPS in the homogenised fry. The total amount of immunoglobulins (Igs) was, however, slightly increased. Studies of immunohistochemical localisation and radioactive LPS distribution revealed high levels of LPS in the intestinal epithelial cells. Head kidney, liver and heart showed low levels of radioactivity and no immunohistochemical staining. In another set of experiments, Atlantic salmon fry were fed LPS-coated feed (0.03% and 0.01%, respectively) for 64 days and then challenged with A. salmonicida and Vibrio anguillarum . The accumulated mortality in fry challenged with A. salmonicida was 40.9%, 34.3% and 30.8% after feeding 0.03% and 0.01% LPS and control feed, respectively. The accumulated mortality in fry challenged with V. anguillarum was 47.0%, 55.0% and 55.1% after feeding 0.03% and 0.01% LPS and control feed, respectively. Fry receiving 0.03% and 0.01% LPS-coated feed showed a statistically significant increase in mean weight at day 64 compared with fry receiving control feed.

Early immune responses in Atlantic salmon (Salmo salar L.) after immunization with PLGA nanoparticles loaded with a model antigen and β-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 (β-glucan) were entrapped in NPs of 300-400nm either alone or in combination. Both the antigen and the β-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-1β, 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 β-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 β-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 β-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.