Accumulation of immunomodulatory laminaran (β(1,3)-D-glucan) in the heart, spleen and kidney of Atlantic cod, Gadus morhua L.

The tissue distribution of ³H- and FITC-lammaran in Atlantic cod, Gadus morhua L., was investigated after intravenous administration. Liquid scintillation counting and whole-body autoradiography revealed a substantial accumulation of ³H-labelled laminaran in the heart throughout the experimental period (1 h to 12 days). High amounts of the immunomodulator were also present in the spleen, anterior kidney and posterior kidney during the study. Renal excretion and intestinal exsorption were the main elimination pathways. Fluorescence microscopic examination revealed accumulation of FITC-labelled laminaran in macrophages in the kidney and ellipsoid sheath cells (macrophages and reticular cells) in the spleen. Furthermore, endothelial entrapment in the heart was pronounced. Endothelial cells in the kidney also contained the FITC-labelled immunomodulator. Immunohistochemical analysis, using anti-β(l,3)-D-glucopyranose antibody, also showed endothelial uptake of laminaran in the heart. The present investigation shows that laminaran accumulates and is retained in immunologically relevant cells in the Atlantic cod.     

The immunomodulatory effect of laminaran [β(1,3)-D-glucan] on Atlantic salmon, Salmo salar L., anterior kidney leucocytes after intraperitoneal, peroral and peranal administration

Anterior kidney leucocytes obtained from Atlantic salmon, Salmo salar L., 2 days after administration of laminaran, were assayed for their capacity to reduce nitroblue tetrazolium to formazan, and for their activity of lysosomal acid phosphatase after intraperitoneal (15 mgkg^?1), peroral (150 mg kg^?1) or peranal (150 mg kg^?1) administration. Leucocytes obtained from salmon treated by an intraperitoneal injection of laminaran produced significantly more superoxide anion than cells obtained from fish treated with dextran or sodium chloride immediately after cell isolation. Immediately after extraction, the activity of acid phosphatase in anterior kidney leucocytes obtained from salmon injected with laminaran was significantly higher than in cells harvested from fish treated with dextran or sodium chloride. Furthermore, cells obtained from salmon treated by peroral instillation of laminaran showed significantly enhanced production of superoxide anion compared with leucocytes from fish treated with either sodium chloride or dextran. The acid phosphatase activity in anterior kidney leucocytes from salmon treated by peroral and peranal instillation of laminaran was significantly higher than in cells from fish treated either with sodium chloride or dextran. Finally, fluorescence microscopic examination of tissue sections from fish treated peranally by intubation with fluorescein labelled laminaran revealed fluorescent vesicles in intestinal epithelial cells and in anterior kidney macrophages.     

The immunomodulatory effect of LPS, laminaran and sulphated laminaran [β(l,3)-D-glucan] on Atlantic salmon, Salmo salar L., macrophages in vitro

Abstract. The stimulatory effect of LPS ( Vibrio anguillarum ), laminaran and sulphated laminaran, aqueous soluble β(1,6)-branched β(l,3)-D-glucan obtained from Laminaria hyperborea , on head kidney macrophages of Atlantic salmon, Salmo salar L., is reported. The macrophages, after stimulation with LPS or laminaran, showed pronounced spreading, membrane ruffling and increased organellc content when examined by light microscopy. LPS stimulation induced enhanced phagocytic and pinocytic activity, higher intraccllular production of superoxide anion, and higher activity of acid phosphatase compared to control cells. Native laminaran stimulated the cells to pinocytose more fluid phase, increase the intracellular production of superoxide anion and to elevate the activity of acid phosphatase. Sulphated laminaran induced higher production of superoxide anion and higher activity of acid phosphatase by macrophages than in control cells.     

Intestinal absorption of immunomodulatory laminaran and derivatives in Atlantic salmon, Salmo salar L.

Abstract. Radiolabelled (¹²⁵I, ³H) immunomodulatory laminaran (isolated from Laminaria hyperborea ), a β(1,6)-branched β(1,3)-D-glucan and different radiolabelled sulphated analogues of laminaran were administered peranally to Atlantic salmon, Salmo salar L. Intestinal absorption and tissue distribution were examined by means of radioactive tracer techniques. The intestinal uptake was highest with native laminaran and laminaran with a low degree of sulphatation, while highly sulphated laminarans were poorly absorbed. The tissue distribution analysis revealed high amounts of radiolabelled compound in the liver, and anterior and posterior kidney, whereas the spleen contained low amounts. Peak serum and organ concentrations were reached about 30 min after administration. The results were confirmed by autoradiography of tissue sections from salmon after peranal administration of radiolabelled laminaran. Finally, the peranal administration of fluorescence labelled laminaran revealed epithelial supranuclear fluorescent vacuoles containing laminaran. It is concluded that native laminaran and slightly sulphated laminaran are absorbed from the posterior intestine and that they are distributed to tissues rich in immunocompetent cells. Thus, these compounds may have potential as immunomodulatory feed additives.     

Prophylactic effect of β(1,3)-D-glucan (laminaran) against experimental Aeromonas salmonicida and Vibrio salmonicida infections

Immersion and intraperitoneal vaccination are the two most common and successful methods for vaccination of fish (Holm & Júrgensen 1987; Lillehaug 1989). Effective vaccines and the improvement of environmental conditions have dramatically reduced the amount of antibiotics used in aquaculture in Norway from 48.5 t in 1987 to 1.03 t in 1996. Nevertheless, the labour intensive process of vaccination and the often undesirable side-effects of the adjuvants incorporated in injectable vaccines (Anderson 1992; Mulvey, Landolt & Busch 1995; Midtlyng, Reitan, Speilberg 1996), have resulted in a search for alternative approaches to achieve immune stimulation. Hence. there is growing interest in the development of immunomodulators that do not cause or have only minor unwanted effects on the host and preferably can be given as a feed additive. It is widely acknowledged that the beneficial (or in some instances undesirable) effects of immunomodulators such as b-glucans and bacterial products in an infection are mainly a result of their stimulatory effects on macrophages (Johnston 1978; Chung & Secombes 1988; Júrgensen, Lunde & Robertsen 1993).     

Haemorrhagic kidney syndrome of Atlantic salmon, Salmo salar L.

This report describes a new syndrome affecting farmed Atlantic salmon on the Canadian east coast that has resulted in increased morbidity and mortality in affected stocks. The major pathological findings are apparent only microscopically and include renal interstitial haemorrhage and acute tubular necrosis and tubular casting. As a result, the disease has become known as haemorrhagic kidney syndrome (HKS). Affected fish are lethargic and anorectic, and lack external lesions. Clinically, HKS fish are anaemic, hypoproteinaemic and hyperosmolalic, with increased serum concentrations of sodium and chloride. At necropsy, internal changes ranged from apparently normal to include one or several of the following: swelling and/or patchy reddening of the kidney, pale gills, exophthalmos, serosanguinous ascites, darkening of the posterior intestine and splenomegaly. Ultrastructurally, viral inclusions were found in the cytoplasm of erythrocytes of HKS fish, and there were unusual electron‐dense inclusions within the tips of renal tubular microvilli of HKS fish. The significance and relevance of the ultrastructural findings to HKS are unknown. Virus isolation was attempted using CHSE, RTG‐2, FH‐10, BB and EPC cell lines; no virus was isolated. Bacteriological analysis failed to reveal significant pathogens. Analysis of tissues for heavy metals and pesticides was negative. Assays for clostridial toxins, lipopolysaccharide and verotoxins were negative. The aetiology of HKS remains unresolved.     

Mitochondrial and peroxisomal β-oxidation capacities in various tissues from Atlantic salmon Salmo salar

In order to investigate the capacities of different tissues to oxidize fatty acids, total β-oxidation (mitochondrial and peroxisomal) of [1–¹⁴C]palmitoyl-CoA was determined in liver and red- and white muscle from adult and juvenile Atlantic salmon Salmo salar. By including potassium cyanide (KCN) in the assay medium, it was possible to differentiate between mitochondrial and peroxisomal β-oxidation capacities. Mitochondrial β-oxidation dominated in all tissues except in livers from juvenile fish where the peroxisomal β-oxidation dominated. In general, the red muscle possesses the highest fatty acid oxidation capacity, however, by taking into consideration the fact that white muscle occupies approximately 60% of the total body weight, this study demonstrates that the white muscle is an important tissue in the overall fatty acid catabolism.     

Localization of purified surface molecules (lipopolysaccharide and A-protein) from Aeromonas salmonicida in the kidney and spleen from Atlantic salmon, Salmo salar L.

Lipopolysaccharide (LPS) and A-layer protein purified from Aeromonas salmonicida were administered intravenously in Atlantic salmon, Salmo salar L., either alone or in combination. Tissues from each organ were examined by immunohistochemical techniques, using a polyclonal antiserum against A-protein and a monoclonal antibody against LPS. When given simultaneously, the antigens seemed to be taken up by different cells in both the head and trunk kidney. The most striking finding was that A-protein was located in epithelial cells in renal proximal tubules, in contrast to LPS, which was not detected in this location. The amount of A-protein in the tissue increased with time until 2 h after injection. Autoradiography of SDS polyacrylamide gel electrophoresis of head kidney homogenates showed that in vivo processing of A-protein coupled to iodinated tyramine cellobiose (¹²⁵I-TC-A-protein) was completed within 24 h, in contrast to LPS, which was maintained in tissues. The findings of the present study suggest that cells of the head kidney of Atlantic salmon are capable of taking up and processing the A-protein.     

The immunomodulatory effects of levamisole on the nonspecific immune system of Atlantic salmon, Salmo salar L.

Sea water-adapted Atlantic salmon, Salmo salar L., were given a 2-h bath in a 2.5 mg L⁻¹ levamisole (as levamisole hydrochloride) solution in fresh-water. Following bathing, the fish were held in full salinity sea water for 2 weeks before being subjected to a number of immunological assays. Heightened activity of the nonspecific defence system was demonstrated by increases in phagocytic index, phagocytic capacity and phagocytic activity, increased levels of the reactive oxygen intermediate, superoxide anion, and an increased lytic activity of both the mucus and the serum. These results indicate that levamisole is effective in augmenting parts of the nonspecific defence system of Atlantic salmon. This is the first record of the use and efficacy of levamisole as an immunomodulator in Atlantic salmon.     

Reproductive strategies in Atlantic salmon, Salmo salar L.

Abstract. Life-history strategies are means by which animals solve the problems of successful reproduction in varying environments. Their development patterns are consequences of responses to the opportunities the environment offers them. Understanding them requires an understanding of the way they evolved, their ontogenetic development, their physiological control, and their adaptive value. The present paper views the salmonids as marine fishes, which have radiated into fresh water through using river beds as protected spawning grounds. It also takes the view that the maturation process has priority over somatic growth in fish, and that it has already been initiated by the time of first feeding. Its completion is environmentally dependent, and can be arrested annually. Whether or not it will be arrested depends on the status of the energy stores of the individual at particular critical times of year. This mechanism has adaptive value both for immediate reproductive success — adequate energy to provision the next generation — and for later overwinter survival, ensuring that if energy stores are inadequate for reproduction they are spared. Atlantic salmon show variation in their reproductive patterns, and examples are given from laboratory and aquaculture experiments to demonstrate some environmental controls which result in these variations. A hypothetical model is presented to account for the operation of these controls.     

Nocardiosis in tank-reared Atlantic salmon, Salmo salar L.

Nocardiosis is an infection caused by aerobic Gram-positive, branching, filamentous rods of the genus Nocardia . The organism is resident in both soil and plants ( Austin & Austin 1993 ; Frerichs 1993 ), and is closely related to Mycobacterium spp. Nocardiosis caused by Nocardia asteroides and N. seriolae (previously N. kampachi ) has been reported in several finfish species, both freshwater and marine. The first reported incidence was recorded by Valdez & Conroy (1963) in neon tetras, Hyphessobrycon innesi (Myers), and subsequently in other fish species, some of commercial significance including rainbow trout, Oncorhynchus mykiss (Walbaum) ( Snieszko, Bullock, Dunbar & Pettijohn 1964 ), brook trout, Salvelinus fontinalis (Mitchill), ( Campbell & MacKelvie 1968 ) and yellowtail, Seriola quinqueradiata (Temminck & Schlegel; Kubota, Kariya, Nakamura & Kira 1968 ). Despite two of these incidences occurring in salmonids, infection by Nocardia spp. in Atlantic salmon, Salmo salar L., has not previously been reported.     

Absorption of immunomodulating β(1,3)-glucan in yolk sac larvae of Atlantic halibut, Hippoglossus hippoglossus (L.)

A soluble immunomodulating β(1,3)-glucan, laminaran, was given in drinking water to yolk-sac larvae of Atlantic halibut, Hippoglossus hippoglossus L. The larvae absorbed laminaran when the concentration was 25 mg l^–1. After 5 days exposure to laminaran, each larva contained 2.3 ng and 46 ng of ³H- and ¹²⁵I-labelled laminaran, respectively. After 10 days exposure, each larva contained 4.5 ng and 47 ng of ³H- and ¹²⁵I-laminaran, respectively. The absorption was confirmed by fluorescence microscopy of larvae exposed to fluorescein-labelled laminaran (FITC-laminaran). The fluorescence was localized to cells in the intestinal epithelial layer and the skin epithelial layer.     

Retained levels of dietary α-, γ- and δ-tocopherol in tissues and body fluids of Atlantic salmon, Salmo salar, L.

The purpose of this study was to describe the relationship between dietary supplementation and concentration of γ- and δ-tocopherol (TOH) in tissues of Atlantic salmon, Salmo salar , L., when α-TOH was fed at a constant level. Post-smolt Atlantic salmon (230 g) in duplicate tanks were fed a fish-meal-based diet with 250 g kg⁻¹ lipid. Different combinations of RRR -tocopherylacetate preparations were added to give the following levels of TOH (mg kg⁻¹) in diets 1–4, respectively: 150α; 150α+ 100δ; 150α+ 50γ+ 50δ; 150α+ 100γ+ 100δ. The concentrations of γ- and δ-TOH in most tissues were directly proportional to the feed concentrations. Exceptions were γ-TOH in erythrocytes, spleen and kidney and δ-TOH in heart muscle, where the intercept with the y -axis was significantly different from zero. The relative retained levels of α-, γ- and δ-TOH in most tissues were similar to their biological activities in mammals. An exception was muscle and adipose tissue, where γ- and δ-TOH were retained at rates of 70–90% and 20–35% compared with α-TOH, respectively. α-TOH was retained to a greater extent when α-TOH was fed alone than when γ- and δ-TOH were added to the diet. Fish growth was significantly correlated to total dietary TOH concentration. The data suggest that turnover of tocopherols in Atlantic salmon is similar to that in mammals, i.e. that discrimination between the tocopherols is mediated through competition for a tocopherol-binding protein in the liver, and consequently that α-TOH is preferentially secreted in very low density lipoprotein. Muscle and adipose tissue appear to be supplied with tocopherols from chylomicrons that have not yet been exposed to the hepatic discrimination mechanism.     

Haematological and biochemical analyses of Atlantic salmon, Salmo solar L., suffering from coldwater vibriosis ('Hitra disease')

Abstract. Juvenile Atlantic salmon, Salmo salar L. were sampled from a commercial Norwegian fish farm during an outbreak of 'Hitra-disease'. One group of fish subjectively judged as healthy and another as diseased were defined on the basis of the classical apathetic behaviour seen in'Hitra-disease ď salmon. Haematological and biochemical analyses were carried out from blood and organs in 10 fish from each group. The diseased fish were severely anaemic. The blood indices MVC, MCH and MCHC indicated active erythropoiesis to compensate for the loss. Alkaline phosphatase (AP) activity, total protein, albumin, creatinine, triglycerides and total cholesterol were significantly reduced in the serum of diseased fish, while the activities of aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) showed normal and significantly increased values respectively. Liver and spleen weight relative to the body weight and the content of water and lipid in the liver were significantly elevated in diseased fish. Furthermore, the iron content of the spleen was increased, whereas the zinc content showed no changes. The branched-chain amino acids valine, leucine and isoleucine were significantly higher and serine significantly lower in muscle extracts of diseased fish.     

Seasonally changing metabolism in Atlantic salmon (Salmo salar L.) II –β-oxidation capacity and fatty acid composition in muscle tissues and plasma lipoproteins

With the aim of elucidating seasonally changing lipid metabolism in immature Atlantic salmon (Salmo salar L.) in sea water, one group was reared under simulated natural light, while one group was reared under continuous light. Fatty acid profile in plasma lipoproteins did not vary during the experiment, while β‐oxidation capacity increased during spring, concurrent with decreasing temperatures. Simultaneously, the relative level of monounsaturated fatty acids (MUFA) in muscle tissue decreased (42–36%). Muscle levels of saturated fatty acids were low during early spring (19%), but then increased slowly, and muscle levels of polyunsaturated fatty acids increased during spring (from 36% to 39%). It is suggested that increased spring growth and the concomitant energy demand was met by increased lipid oxidation, where MUFA were preferred as energy substrate.