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.     

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.     

Isolation, cultivation and characterization of head kidney macrophages from Atlantic cod, Gadus morhua L.

Head kidney macrophages from Atlantic cod, Gadus morhua L., were isolated by density sedimentation and maintained under serum-free conditions for up to one week. The cells adhered and spread well on glass and plastic, were highly phagocytic, and had typical macrophage morphology as shown by phase contrast and scanning and transmission electron microscopy. Histochemical studies with the light microscope showed that the macrophages were acid phosphatase and non-specific esterase positive, and alkaline phosphatase and peroxidase negative. Compared with unstimulated control cells, LPS-treated cells showed enhanced superoxide anion formation, as measured by the reduction of nitroblue tetrazolium, and increased levels of acid phosphatase activity.     

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.     

Immunomodulatory effect of prolactin on Atlantic salmon (Salmo salar) macrophage function

The in vitro and in vivo effect of prolactin (PRL) on kidney macrophages from Atlantic salmon (Salmo salar) was investigated under the assumption that PRL stimulates immune innate response in mammals. Kidney macrophages were treated two ways: first, cultured in RPMI 1640 medium containing 10, 25, 50 and 100 ng/mL of PRL and second, isolated from a fish with a PRL-injected dose of 100 ng/Kg. Reduced nitro blue tetrazolium (formazan) was used to produce intracellular superoxide anion. Phagocytic activity of PRL was determined in treated cells by optical microscopy observation of phagocytized Congo red-stained yeast. Kidney lysozyme activity was measured in PRL-injected fish. In vitro and in vivo macrophages treated with PRL presented an enhanced superoxide anion production, elevated phagocytic index and increased phagocytic activity. Treated fish showed higher levels of lysozyme activity in the head kidney compared to the control. These results indicate that PRL-stimulated innate immune response in Atlantic salmon and future studies will allow us to assess the possibility of using PRL as an immunostimulant in the Chilean salmon industry.     

Accumulation of immunomodulatory laminaran [β(1,3)-D-glucan] in the spleen and kidney of Atlantic salmon, Salmo solar L.

Abstract. The tissue distribution of ³H- and FITC-laminaran in Atlantic salmon, Salmo salar L., was investigated after intravenous administration. Liquid scintillation counting and whole-body autoradiography revealed that the concentration of ³H-labelled laminaran in the spleen and anterior kidney was higher than in blood throughout the experimental period (1 h to 12 days). Renal excretion and intestinal exsorption were the main elimination pathways for laminaran. Microscopic examination revealed accumulation of FITC-labelled laminaran in macrophages in kidney and spleen. In addition, endothelial cells in the kidney, spleen, liver and intestine contained the immunomodulator. Thus, these experiments show that laminaran accumulates and is retained in immunologically relevant cells in the kidney and spleen.     

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.     

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.     

A biologically active IL-1β derived peptide stimulates phagocytosis and bactericidal activity in rainbow trout, Oncorhynchus mykiss (Walbaum), head kidney leucocytes in vitro

The present work provides the first information concerning the ability of a rainbow trout interleukin-1β (IL-1β) derived peptide, P3, to enhance phagocytosis and bactericidal activity. P3 was synthesized to complex with the IL-1 receptor and corresponds to fragment 197–206 (YRRNTGVDIS) of the trout sequence. A dose-dependent effect of P3 on phagocytosis in head kidney leucocytes was documented, with an optimal response occurring at 0.25 m M . With respect to the temporal kinetics of P3 induced activation, an incubation period of 2 h resulted in enhanced phagocytic activity, although incubation times of between 4 and 24 h were optimal. An additive effect was observed when P3 (0.1 m M ) was combined with another IL-1 derived peptide, P1 (0.1 m M ). The latter peptide corresponds to fragment 146–157 (YVTPVPIETEAR) and is equivalent to a region in the mammalian protein that is known to be part of the receptor binding domain, but does not overlap with P3. With respect to bactericidal activity, macrophages incubated for 24 h with 0.1 m M P3 demonstrated an enhanced ability to kill an avirulent strain (MT004) of the Gram-negative salmonid pathogen Aeromonas salmonicida . Further investigation revealed that extracellular production of superoxide anions was not responsible for the enhanced bactericidal activity of macrophages exposed to P3.     

Beneficial effects of Bacillus subtilis on water quality,growth, immune responses,endotoxemia and protection against lipopolysaccharide‐induced damages in Oreochromis niloticus under biofloc technology system

The present research explored the effects of Bacillus subtilis on water quality, growth, immune responses, endotoxemia and protection against lipopolysaccharide (LPS) damages in Nile tilapia Oreochromis niloticus under biofloc system. B. subtilis was added at 0, 1, 2, 3 and 4 grams (1.19 × 10⁸ CFU/g) per kg of basal diet, named T1 (control), T2, T3, T4 and T5, respectively, and fed to fish (14.82 ± 0.42 g) for 50 days. The concentrations of TAN, NO₂ and NO₃ were significantly reduced, and fish fed probiotics displayed significantly better growth performances versus the control, concomitantly with significantly enhanced activities of digestive enzymes. They also showed significantly declined serum glucose and cholesterol vice versa significantly improved immune responses (total protein, albumin, globulin, lysozyme, alternative complement, protease, immunoglobulins, alkaline phosphatase and respiratory burst), antioxidant capacity (superoxide dismutase, total antioxidant capacity, malondialdehyde) and skin mucus parameters (total protein, lysozyme, alternative complement, protease, immunoglobulins). Meanwhile, significantly lower endotoxin (LPS) concentrations were detected in the intestines and serum of fish fed probiotics. LPS challenge induced profound oxidative stress and impaired immune responses. Interestingly, probiotic alleviated LPS‐induced damages and restored mentioned parameters. In conclusion, B. subtilis effectively enhanced fish production, immunity and protection against LPS‐induced damages in tilapia under biofloc system.     

Dietary modulation of digestive enzymes by the administration of feed additives to rainbow trout,Oncorhynchus mykiss Walbaum

The aim of this study was to assess the metabolic and digestive enzyme activities in rainbow trout, Oncorhynchus mykiss Walbaum fed dietary supplements. The biometric indices and the following enzymes activities were measured: acid protease and pepsin from the stomach homogenates, alkaline phosphatase (AP) from the small intestine and the brush border membrane, total proteolytic enzymes and trypsin from the small intestine and liver/pancreas. Dietary garlic induced a higher pepsin activity in the stomach than lipopolysaccharide (LPS) and ginger. Conversely, dietary ginger and LPS showed a significant difference (P > 0.05) in acid protease activity compared to the controls. There was not any significant difference observed between treated groups and the controls for hepato‐pancreas proteolytic enzyme activity, except for trypsin. AP activity increased significantly with dietary garlic, ginger and LPS compared to the controls.     

Effect of dietary supplementation with Bacillus subtilis on the growth,performance, immune response and antioxidant activities of the shrimp (Litopenaeus vannamei)

The objective of this study was to evaluate the effect of dietary supplementation of a probiotic bacterium, Bacillus subtilis, on the growth, immune response and antioxidant activities of shrimp (Litopenaeus vannamei). Shrimps with an average initial weight of 2.11±0.17 g were randomly assigned to four groups with three replicates. The control group was fed a basal diet, and three treated groups were fed diets supplemented with B. subtilis at doses of 1 × 10⁴, 5 × 10⁴ and 10 × 10⁴ colony‐forming unit (CFU) g^?1 feed respectively. After 40 days of culture, 10 shrimps from each replicate were taken randomly for the determination of immune response and oxidization resistance indices. The results showed that the shrimps fed with B. subtilis at a dose of 1 × 10⁴, 5 × 10⁴ CFU g^?1 feed showed significantly better growth than that of the control diet. The phenoloxidase activities showed a tendency to increase with an increased dose of B. subtilis in diets but there was no significant difference among the three treated groups. In addition, phenoloxidase activities were found to be significantly higher (P<0.05) in the groups treated with B. subtilis than that of the control group. Shrimps treated with 5 × 10⁴ CFU g^?1 feed probiotic bacterium showed the highest lysozyme activity and it was significantly higher (P<0.05) than the other groups. However, there was no significant difference in acid phosphatase and alkaline phosphatase activity across all the groups. The total antioxidant capacity, superoxide dismutase and glutathione peroxidase activities in the probiotic‐treated groups were significantly increased (P<0.05) as compared with the control groups. Both maleic dialdehyde concentration and superoxide anion activities in the probiotic‐treated groups were significantly lower (P<0.05) than those of the control. The probiotic did not affect the nitric oxide synthase and the catalase activity in any of the control and treated groups. These results indicated that the probiotic B. subtilis could significantly promote the growth rate of shrimp by increasing the immune function and antioxidant capacity. The most effective dose of B. subtilis in the diet was 5 × 10⁴ CFU g^?1 feed.     

The influence of haemocyte number on the resistance of the freshwater crayfish, Pacifastacus leniusculus Dana, to the parasitic fungus Aphanomyces astaci

Abstract. The number of circulating haemocytes in crayfish, Pacifastacus leniusculus Dana, was reduced after injection of yeast cell walls (Zymosan) into the animals. Crayfish harbouring the parasitic fungus A. astaci as a 'latent' infection in melanized areas in the cuticle died due to unrestricted growth of unmelanized hyphae when they were injected with Zymosan. The β–1,3–gIucan laminaran, introduced into the haemocoel of P. leniusculus harbouring A. astaci hyphae in the cuticle, also caused a decrease in the total haemocyte counts (THC), but these animals survived the treatment and no unmelanized hyphae of A, astaci were found. The recovery of THC was found to be more rapid after laminaran injection than after Zymosan injection, which could explain why laminaran treated crayfish retained their resistance and could combat the A. astaci hyphae in the cuticle. The importance of the number of haemocytes in defence and well-being of crayfish is discussed.     

盐度突变对凡纳滨对虾非特异性免疫因子的影响

研究了盐度对凡纳滨对虾非特异性免疫因子的影响。结果表明,盐度突变对凡纳滨对虾非特异性免疫因子的影响是显著的。研究结果表明,盐度突变组(30～27、30～24、30～21、30～18和30～15)血细胞内O2-产量呈先升后降的趋势;酚氧化酶(PO)活力升高,盐度突变组显著高于对照组(P<0.05);盐度突变组SOD活性显著低于对照组(P<0.05);血清蛋白含量逐渐降低,对照组显著高于盐度突变组(P<0.05)。     

Immunohistochemical changes in production of pituitary hormones during artificial maturation of female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

ABSTRACT:   Specific antibodies against follicle-stimulating hormone β subunit (FSHβ), prolactin (PRL), and somatolactin (SL) of the Japanese eel Anguilla japonica were produced. These antibodies, as well as antibodies against luteinizing hormone β subunit (LHβ) and growth hormone (GH) produced previously, were used to examine changes in the production of pituitary hormones in female eels during maturation induced by salmon pituitary homogenate (SPH) injection. Immunohistochemical observations showed a decrease in FSH production after SPH injection, suggesting that SPH inhibits FSH production. In contrast, LH production increased markedly with maturation. The number of GH producing cells decreased gradually during maturation, possibly because of inhibition by exogenous GH present in the SPH and/or endogenous insulin-like growth factor-I produced by the stimulation of salmon GH. Although changes in the number of PRL producing cells with maturation were not evident, the number of SL producing cells showed a peak at the late vitellogenic stages, and thereafter decreased to the migratory nucleus stage. These results suggest that GH and SL are involved in sexual maturation in SPH injected eels, as in other fishes.     

Polysaccharide-induced protection of carp, Cyprinus carpio L., against bacterial infection

Abstract. The efficacy of 10 polysaccharides (curdlan, inulin, krestin, laminaran, lentinan, levan, schizophylian, selerogiucan, yeast glucan and zymosan) to enhance protection of carp, Cyprinus carpio L., against bacterial infection was investigated. Carp were intraperitoneally injected with the polysaceharides (2–l0 mgkg^-1) on days 1 and 4, and challenged with Edwardsiella tarda on day 7. Among the polysaccharides tested, lentinan, schizophyllan and scleroglucan, which are l,6-branchcd-β-l,3-glucans, significantly increased the survival rate. They also induced a protective effect against Aeromonas hydrophila at a dose of 5 mg kg^-1. The ability of the polysaccharides to activate the alternative complement pathway (ACP) was examined by incubating the polysaccharides with carp serum and measuring the residual ACP activity. At a final concentration of 0.l mgml^-1, l,6-branched-β-1,3-glucans greatly reduced (76–77%) the ACP activity. Therefore, it is suggested that the protective effect of the l,6-branched-β-1,3-glucans may be associated with the activation of ACP.     

Dose-dependent influences of dietary β-1,3-glucan on innate immunity and disease resistance of hybrid striped bass Morone chrysops×Morone saxatilis

To investigate potential use of dietary β-1,3-glucan for health management of hybrid striped bass, juvenile fish were fed diets supplemented with yeast glucan (MacroGuard^®) at 0.05%, 0.1% or 0.2% of diet for 4 weeks, followed by immune response assays and a bath challenge with Streptococcus iniae . Dietary glucan significantly ( P <0.05) enhanced neutrophil oxidative radical production, and fish fed 0.1% glucan had a significant ( P <0.05) reduction in mortality (10%) after bacterial challenge compared with fish fed the control diet (46.7%). However, accumulative mortality of fish fed 0.2% glucan was not significantly different from that of fish fed the control diet. To further elucidate this observation, macrophages from sub-adult hybrid striped bass were isolated and cultured in L-15 medium with 10% foetal calf serum and penicillin/streptomycin supplemented with 0.2, 0.5, 1, 2, 5, 20 and 100 μg soluble glucan (MacroGuard^®) mL⁻¹ for 24 and 48 h. Intracellular superoxide anion production was significantly ( P <0.001) increased by 0.5 μg glucan mL⁻¹, but significantly ( P <0.001) suppressed by doses >5 μg glucan mL⁻¹. It is concluded that dietary yeast glucan has potential for use in diet formulations of hybrid striped bass to limit the adverse effects of S. iniae , but dosage should be an important consideration in administration.     

The in vitro effect of CpG-ODNs on the innate immune response of common carp, Cyprinus carpio L.

Unmethylated CpG dinucleotides within bacterial DNA or synthetic oligodeoxynucleotides (ODNs) can activate immune cells. A panel of synthetic oligodeoxynucleotides was evaluated in vitro for their ability to enhance the innate immune response of common carp (Cyprinus carpio L.). In vitro addition of CpG-ODNs enhanced the phagocytic activity and superoxide anion production of stimulated kidney phagocytes. The CpG-ODNs also induced lymphocyte proliferation in the fish kidney cells. The ODNs containing multiple CpGs generally resulted in greater stimulatory capacity, although CpGs located at the terminus of an ODN were ineffective. These results indicated that CpG-ODNs enhance the innate immune response of common carp.     

Changes of enzyme activity and hematopoiesis in Chinese prawn Fenneropenaeus chinensis (Osbeck) induced by white spot syndrome virus and zymosan A

White spot syndrome virus and zymosan A were used to stimulate the Chinese prawn Fenneropenaeus chinensis (Osbeck), and enzyme activities in organs involved in immune defence e.g., lymphoid organ, hepatopancreas and gill, total haemocyte count and mitotic index of haematopoietic tissue (HPT) were measured. The results showed that activities of phenoloxidase (PO), superoxide dismutase (SOD), alkaline phosphatase (ALP) and acid phosphatase (ACP) were higher in lymphoid organ than in gill. The hepatopancreas had no PO activity, its SOD activity was similar to the lymphoid organ, and its ALP and ACP activities were higher than lymphoid organ and gill. Phenoloxidase activity in lymphoid organ was higher than that of the control group at 2 h after injection and lower than control after 24 h. Superoxide dismutase activity in all organs showed an increase. Alkaline phosphatase activity in lymphoid organ and gill was higher than that of the control group at 2 and 24 h after injection respectively. The difference in ACP activity was not significant. After injection with zymosan A, the mitotic index of HPT increased by 1.5‐fold compared with the control, and returned to control level after 48 h.     

副溶血弧菌感染日本蟳后对几种免疫活性酶的影响

用副溶血弧菌悬液和生理盐水(对照)注射感染日本蟳,研究其血清超氧化物歧化酶(SOD)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、过氧化氢酶(CAT)活性的变化,并进行统计学分析。试验结果表明,注射副溶血性弧菌悬液组12、24、48 h后SOD活性均高于对照组,48 h达到最高;122、4 h CAT活性增加明显,24 h达最高;AKP活性5、12、24 h均有增加,高于对照组,24 h最高;ACP活性12 h开始上升,24 h达最高。在人工感染副溶血性弧菌48 h内,日本蟳体内的免疫因子发生明显的变化,血清中SOD、ACP、AKP、CAT活性在不同时间段均有显著升高趋势,高于对照组。