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.     

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.     

三种免疫添加剂对草鱼非特异性免疫功能的影响

在饲料中添加不同剂量的Vc、酵母多糖和壳聚糖,连续投喂42d,并在试验的第14、28、35和42天取样检测,研究其对草鱼血清溶菌酶活性、血清超氧化物歧化酶活性（SOD）活性、血清碱性磷酸酶（AKP）活性、补体C3、含量丙二醛（MDA）含量和血液白细胞吞噬活性共6种指标的影响。结果显示：实验组与对照组相比,饲料中添加Vc可以提高血清溶菌酶活性、SOD活性、AKP活性、补体C3含量和血液白细胞吞噬活性高,并降低MDA含量。添加酵母多糖可以提高血清溶菌酶活性、AKP活性、补体C3含量和血液白细胞吞噬活性,并降低MDA含量,但不能提高SOD活性。添加壳聚糖可以提高血清溶菌酶活性、SOD活性、AKP活性、补体C3含量和血液白细胞吞噬活性,但不能降低MDA含量。饲料中添加Vc、酵母多糖和壳聚糖均对草鱼的非特异性免疫功能具有一定的促进作用。     

Dietary vitamin C,immunity and disease resistance in Atlantic salmon (Salmo salar)

Presmolt Atlantic salmon were fed a fish meal based experimental diet supplemented with graded levels of ascorbate-2-monophosphate (AP), equivalent to 40, 400, 2000 and 4000 mg ascorbic acid (AA)/kg for 6 months prior to a bacterial challenge experiment. The liver AA concentration reflected the dietary intake of AP, but not linearly. Growth, hematology and acid phosphatase activity in zymosan stimulated macrophages were not affected by dietary AP. Serum hemolytic complement activity was higher in fish fed the highest AP level, but the variation was not significant. Production of specific antibodies was significantly higher in fish fed the highest AP level 11 and 17 weeks after vaccination. Bacterial challenge with Aeromonas salmonicida showed increased survival in the 4000 AP group. Lysozyme activity in headkidney and serum complement activity and serum iron in fish surviving the challenge were higher in the 4000 AP group, indicating important roles of vitamin C on lysozyme, complement and iron in non-specific disease resistance. The results indicate that high dietary levels of AP favourably affect health in Atlantic salmon. Disease resistance was, however, not correlated with the AA status in the liver.     

投喂阿维拉霉素对鲤免疫应答的影响

将阿维拉霉素按照一定的浓度添加到饲料中,投喂经注射接种福尔马林灭活的嗜水气单胞菌(Aerom onashydrophila)菌苗的鲤,于免疫后的第7 d、14 d、21 d、28 d和35 d检测供试鲤血液中的白细胞吞噬活性、血清中溶菌酶活性、抗体效价、补体C3含量及免疫保护率(RPS),探讨了阿维拉霉素对受免鲤免疫应答的影响。结果表明,饲料中添加阿维拉霉素(4 mg/kg和8 mg/kg),投喂较长时间(≥28 d)会降低受免鲤的血液白细胞吞噬活性、血清中的溶菌酶活性和抗体效价,减少受免鲤血清中补体C3的含量,减弱鲤抵抗嗜水气单胞菌人工感染的能力,每公斤饲料中添加8 mg的阿维拉霉素其免疫抑制作用更强。     

Immunostimulant effects of nucleotide isolated from yeast RNA on carp, Cyprinus carpio L.

Nucleotides from yeast RNA were evaluated for their ability to enhance the non-specific immune responses in carp, Cyprinus carpio . Oral administration of nucleotides to fish daily for 3 days resulted in enhanced responses of phagocytic and nitroblue tetrazolium (NBT) activities in kidney phagocytic cells. This activation of kidney cells was observed for at least 10 days post-treatment. The serum complement and lysozyme activities also increased in fish treated with nucleotides. Furthermore, the number of Aeromonas hydrophila in nucleotide-treated fish significantly decreased in the blood, kidney and liver after intraperitoneal injection. Thus yeast nucleotides appear to enhance non-specific immune responses in fish.     

Presence of anti-Neoparamoeba sp. antibodies in Tasmanian cultured Atlantic salmon, Salmo salar L

Previous studies have indicated that when Atlantic salmon, Salmo salar L., are exposed to Neoparamoeba sp. the fish produce anti-Neoparamoeba sp. antibodies. It appears unlikely that these antibodies elicit any specific protection against amoebic gill disease (AGD) as fish with demonstrable activities have been affected by AGD. Experiments were conducted on Atlantic salmon cultured throughout Tasmania to assess the natural production of antibodies towards Neoparamoeba sp. Fish were sampled from areas where AGD was prevalent and from areas where there had been no reported cases. An enzyme-linked immunosorbent assay (ELISA) was used to measure anti-Neoparamoeba sp. antibody activities in serum. All fish from sea water had antibody activities greater than the negative control fish, including fish from areas with no reported cases of AGD. Time trial samples indicated that time after transfer to sea water did not appear to be a significant (P > 0.05) factor in antibody activity, however location was (P < 0.05). There was no agreement (corrected kappa value, 0.16) between the ELISA result and the isolation of Neoparamoeba sp. from the gills of the same fish. The results suggest that Atlantic salmon in seawater culture in Tasmania produce anti-Neoparamoeba sp. antibodies regardless of infection history, suggesting the presence of Neoparamoeba sp. in the environment.     

Changes in the innate immune response of Atlantic salmon, Salmo salar L., exposed to experimental infection with Neoparamoeba sp

An experiment was conducted to determine the effect of Neoparamoeba sp. infection on the innate immune responses of Atlantic salmon. Atlantic salmon were experimentally infected with Neoparamoeba sp. and serially sampled 0, 1, 4, 6, 8 and 11 days post-exposure (dpe). Histological analysis of infected fish gill arches identified the presence of characteristic amoebic gill disease lesions as early as 1 dpe with a steady increase in the number of affected gill filaments over time. Immune parameters investigated were anterior kidney phagocyte function (respiratory burst, chemotaxis and phagocytosis) and total plasma protein and lysozyme. In comparison with non-exposed control fish basal respiratory burst responses were suppressed at 8 and 11 dpe, while phorbol myristate acetate-stimulated activity was significantly suppressed at 11 dpe. Variable differences in phagocytic activity and phagocytic rate following infection were identified. There was an increase in the chemotactic response of anterior kidney macrophages isolated from exposed fish relative to control fish at 8 dpe. Total protein and lysozyme levels were not affected by Neoparamoeba sp. exposure.     

Salmonid macrophages: separation, in vitro culture and characterization

Abstract. A method is described for the separation of fish leucocytes and the establishment of pure monolayers of fish macrophages in vitro. The method makes it possible to study the important role of cellular immunity in fish. Fish leucocytes were obtained from the pronephros of rainbow trout, Salmo gairdneri Richardson, and compared to those obtained from the pronephros of Atlantic salmon, Salmo salar L. Single cell suspensions were separated by density gradient centrifugation and seeded on glass cover slips for maintenance in culture. After 20 h in culture a subpopulation of the cells had adhered and spread out on the cover slips and were macrophage-like by morphological criteria. About 90–99% of these cells had the ability to phagocytose a variety of particles, including fixed sheep erythrocytes, latex, carbon particles, yeast and Vibrio anguillarum. Opsonization of particles with mammalian immunoglobulins and mammalian complement did not enhance the phagocytic activity.     

Cryptobia salmositica: an in vitro and in vivo study on the mechanism of anaemia in infected rainbow trout, Salmo gairdneri Richardson

Abstract. There are two basic antigenic components in Cryptobia that are responsible for the anaemia in infected rainbow trout. A 'lytic component', which is dosage-dependent, causes lysis of red blood cells independent of antibody or complement. The second, an 'immune-complex-forming component', attaches to red blood cells, forms immune complexes with specific antibody and activates complement resulting in haemolysis. These two antigenic components, from both live and lysed Cryptobia , were present in the serum of infected fish. When sonicated antigen or heat-inactivated antiserum (from infected fish) was incubated with red cells from uninfected fish, a portion of the red cells was lysed and a positive Coombs' reaction was observed with the remaining intact red cells. The positive Coombs' reaction was due to immune complexes adsorbed onto the red cells and these lysed when incubated with complement. Antibody by itself did not adsorb onto the red cells. From the fourth week post-infection, a positive Coombs' reaction was observed in all infected fish and haemolysis occurred with complement. The authors suggest that, in infected fish, one or more components of the complement cascade is depleted continually during infection and that the anaemia is due to the lytic action of the antigen and immune complex formation on red cells. These lead to intra-vascular haemolysis as well as erythrophagocytosis. In general, the mechanism of anaemia in cryptobiosis appears similar to that in African trypanosomiasis.     

壳聚糖对花鲈生长和非特异性免疫力的影响

在基础饲料中分别添加0.5%、1%和2%的壳聚糖,连续投喂花鲈(Lateolabrax japonicus)60d,探讨壳聚糖对花鲈生长和非特异性免疫力的影响。结果表明,添加0.5%和1%壳聚糖可以显著促进花鲈的生长,但是不同添加浓度对成活率均无影响。在30d时添加0.5%或1%壳聚糖能有效提高花鲈的补体活性、溶菌酶活性和吞噬活性;在60d时仅明显提高补体活性。添加壳聚糖对白细胞的数量没有影响。     

Immune response and disease resistance in Atlantic salmon (Salmo salar L.) fed three levels of dietary vitamin E and the effect of vaccination on the liver status of antioxidant vitamins

Atlantic salmon were fed 40, 300 or 1100 mgall-rac-α-tocopheryl acetate kg feed^?1 for 12 weeks.After 6 weeks half of the fish in each group were marked and vaccinated (i.p.injection) against furunculosis and vibriosis. Liver α-tocopherol levelsreflected the dietary input after 6 and 12 weeks of feeding. Noimmunomodulatoryeffects of dietary vitamin E on baseline levels of the immune parameters beforevaccination were detected in this study, as evaluated by antibody dependent andspontaneous plasma complement activities. In general, vaccination increasedplasma complement activities and the number of antigen specific antibodyproducing cells as compared with unvaccinated control fish, but with nodifferences with respect to the vitamin E regimes. Also the ability ofunvaccinated fish to withstand experimental furunculosis was unaffected bydietary vitamin E. The concentrations of α-tocopherol and ascorbic acidinthe liver were, however, negatively affected 6 weeks post vaccination comparedto respective unvaccinated fish.     

Immune response, growth and survival of Labeo rohita fingerlings fed with levamisole supplemented diets for longer duration

The study was to determine the effect of long-term administration of different dosages of levamisole on growth, immune response and disease resistance against Aeromonas hydrophila & Edwardsiella tarda in Labeo rohita fingerlings. Fish were fed with four different dosages of levamisole (0, 125, 250 and 500 mg kg⁻¹ diet) for 56 days. Different serum biochemical and haematological parameters such as serum total protein content, albumin content, globulin content, albumin/globulin ratio, glucose content, leucocytes count; cellular immune parameters including superoxide anion production, phagocytic activities, lymphokine production index; humoural immune parameters including lysozyme, complement and serum bactericidal activities were evaluated after 14 days interval. After 56 days, fish were divided into two subgroups under each treatment group for challenge with pathogens A. hydrophila and E. tarda . The cumulative mortality (%) and agglutinating antibody titre was recorded on 28th day postchallenge. WBC count, phagocytic ratio, lymphokine production index, lysozyme activity and serum bactericidal activity were increased upon administration of levamisole dosages for long term. However, the growth performance and survival against pathogens was not significantly changed over 56 days administration of levamisole. But incorporation of moderate dosage of levamisole for 42 days results better immune response without effect on growth and survival of L. rohita fingerlings.     

Protective effect and antibody response of DNA vaccine against salmonid alphavirus 3 (SAV3) in Atlantic salmon

This work reports the effect of two DNA vaccines against salmonid alphavirus 3 (SAV3) in Atlantic salmon. Presmolts were vaccinated by intramuscular injection of plasmids encoding the SAV3 structural polyprotein C‐E3‐E2‐6K‐E2 (pCSP), E2 only (pE2), or plasmid without insert (pcDNA3.3). E2 is expressed at the surface of cells transfected with pCSP and internally in cells transfected with pE2. A commercial vaccine based on inactivated SAV (NCPD) was used for comparison. At 10 weeks post‐vaccination, only fish vaccinated with pCSP showed antibody against E2 and virus‐neutralizing activity. Vaccinated fish were infected with SAV3 to determine protection by virus quantitation in serum after 7 days and scoring of pathological changes after 21 days. Fish vaccinated with both pCSP and NCPD vaccines showed significant virus reduction in serum, while fish vaccinated with pE2 did not. All fish vaccinated with pcDNA3.3 and pE2 showed pathological changes in organs typical of PD, 60% of fish vaccinated with NCPD showed PD pathology, while fish vaccinated with pCSP did not show PD pathology. Taken together, DNA vaccination with pCSP provided strong protection for salmon against SAV3 infection, which in part may be due to production of virus‐neutralizing antibodies.     

Effects of dietary vitamin C on immunity, growth and survival of Indian major carp Labeo rohita, fingerlings

An 8‐week feeding trial was conducted to evaluate the efficacy of dietary l ‐ascorbic acid (AA) dosages on immunity, growth and survival of the fingerlings of Labeo rohita. AA was supplemented at 0, 100, 200 and 500 mg kg⁻¹ basal diets. Each diet was fed for 8 weeks to duplicate groups of fingerlings of rohu with initial body weight of 35 ± 5 g. Selected biochemical and haematological analyses were evaluated every second week. At the end of the feeding trial of 56 days, fish were divided into two subgroups under each major treatment group for challenge with two pathogens, Aeromonas hydrophila and Edwardsiella tarda. The mortality (%) and agglutinating antibody titre were recorded on 28th day postchallenge. Non‐specific immune parameters like superoxide anion production, lysozyme activity and complement activity increased independently of the levels of AA in the diet. Whereas phagocytic ratio, phagocytic indices, lymphokine production indices and serum bactericidal activity were better in the group of fish fed with 200 mg AA kg⁻¹ feed for 42 days. However, growth parameters such as specific growth rate, feed conversion ratio and survival were better in the group of fish fed with the highest level of AA through feed for 56 days. These data indicated that elevated levels of dietary AA could be recommended for optimum immunity, growth and survival of fingerlings of L. rohita.     

Effects of dietary supplementation of A3α-peptidoglycan on innate immune responses and defense activity of Japanese flounder (Paralichthys olivaceus)

To determine the effect of dietary supplementation of A3α-peptidoglycan (A3α-PG), extracted from Bifidobacterium sp. cell wall, on the immune responses of Japanese flounder (Paralichthys olivaceus) and explore a possible influence of doses and administration length, a 40-day feeding trial was conducted to evaluate graded levels (0, 0.5, 1, 2, 4, 8 and 16 g A3α-PG/kg diet) in the diet of juvenile Japanese flounder, followed by a challenge with Vibrio anguillarum by intraperitoneal injection. Growth, serum lysozyme, hemolytic complement activity, blood leucocyte phagocytic activity, respiratory burst, and total serum protein were monitored at intervals of 10, 20, 30 and 40 days.Growth, serum lysozyme, hemolytic complement activity, blood leucocyte phagocytic activity of juvenile Japanese flounder were generally enhanced by dietary supplementation of 1, 2, 4 and 8 g A3α-PG/kg diet and also enhanced immune responses, but not growth. The beneficial influence of A3α-PG on most of the tested immune responses were dose- and/or administration length-dependent except serum lysozyme, which was only stimulated by higher doses (more than 2 g A3α-PG/kg) after 40 days feeding. No significant influence of dietary supplementation of 0.5 g A3α-PG/kg on innate immunity was observed on juvenile fish. The highest dose explored in the present study, 16 g A3α-PG/kg, also failed to show any significant influences, although no obvious pathology was observed. Dietary supplementation of A3α-PG showed protection against V. anguillarum. The present study showed an immunomodulatory effect of dietary supplementation of A3α-PG and the potential use in health management of cultured economic fish species such as Japanese flounder.     

维生素E及脂肪源对大菱鲆非特异性免疫的影响

在大菱鲆饲料中分别添加鲤鱼鱼油及花生油作脂肪源,每种脂肪中添加两个水平的维生素E（8mg／kg和300mg／kg维生素E醋酸酯）,饲养大菱鲆84d,探讨其对大菱鲆非特异性免疫的影响。结果发现,（1）以鲤鱼鱼油为脂肪源的添加高剂量的维生素E能提高大菱鲆血清补体活性,而以花生油为脂肪源的饲料添加高剂量的维生素E不影响大菱鲆血清补体活性;（2）以鲤鱼鱼油和花生油为脂肪源的饲料添加高剂量的维生素E不影响大菱鲆中性粒细胞的吞噬率及白细胞总数;（3）以鲤鱼鱼油为脂肪源的饲料添加高剂量的维生素E能提高大菱鲆的溶茵酶活性,但随着时间推移其影响减弱;（4）饲喂n-3HUFA含量高的饲料,对大菱鲆腹水病的抵抗能力高于n-3HUFA含量低的饲料。     

The effect of dietary vitamin A on the immunocompetence of Atlantic salmon (Salmo salar L.)

Atlantic salmon, Salmo salar L. were maintained on diets containing low (0.37 mg kg^–1 diet), normal (1.95 mg kg^–1 diet) and high (15 mg kg^–1 diet) levels of vitamin A fed at 1.5% body weight per day. After 4 months, liver vitamin A levels reflected dietary intake and growth rates of all three groups were similar. Kidney leucocyte migration and serum bactericidal activity were found to be significantly reduced in fish fed low levels of vitamin A. On the other hand, high levels of vitamin A in the diet were found to augment serum antiprotease activity relative to the levels found in the other dietary groups. However, phagocyte respiratory burst activity, bactericidal activity and eicosanoid production were unaffected by the dietary vitamin A regime, as were lymphocyte functions (lymphokine and antibody production) and both serum lysozyme and classical complement activity. That the overall immunomodulatory effect of vitamin A was small was reflected in the resistance to Aeromonas salmonicida. No significant differences were found between the different vitamin A intake groups despite a trend to decreased resistance in the low vitamin A diet group.     

Effect of Dietary Lipids on Macrophage Function,Stress Susceptibility and Disease Esistance in Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

As the supply of marine fish oil is becoming a limiting factor in the production of Atlantic salmon (Salmo salar), new diets and alternative sources of energy are being tested. Plant oils are natural potential candidates to replace fish oil, but the different levels of essential polyunsaturated fatty acids may influence the health and growth of salmon. In this study, we have investigated the resistance to transport stress and bacterial infection, phagocytic activity in head kidney macrophages and eicosanoid metabolism in salmon fed three different diets. In high-energy fishmeal based diets, 50% and 100% of the supplementary fish oil (FO) was replaced with soybean oil (SO). The three dietary groups were fed for 950 day-degrees at 5 °C (27 weeks) and 12 °C (11 weeks) before challenging the fish with Aeromonas salmonicida, analyzing the lipid composition of head kidney and examining macrophage function in vivo and in vitro. Dietary fatty acids affected the lipid composition of the kidney. The level of eicosanoid precursor’s 20:4n-6 and 20:3n-6 were 3 and 7-fold higher in the 100% SO group compared with the FO group. The total fraction of n-3 lipids in kidney was 19% in the SO group, compared to 16% and 12% in the 50% or 100% SO groups, respectively. However, the production of leucotriene B₄ (LTB) and prostaglandin E₂ (PGE) immunoreactive materiel from exogenously added arachidonic acid in head kidney macrophages was only affected by the composite diet (increased) at 5 °C. In addition, the phagocytic activity of kidney macrophages in vivo and in vitro was not affected by diet. No effect of diet was observed on transport stress or susceptibility to a bacterial infection with Aeromonas salmonicida. Atlantic salmon therefore seems to tolerate a diet solely based on soybean oil as lipid source, without any detrimental effects on growth, health and immune functions.     

The immune response of Atlantic salmon, Salmo salar L., to the causative agent of bacterial kidney disease, Renibacterium salmoninarum

Abstract. Both under-yearling and post-yearling Atlantic salmon parr produced high agglutinating antibody titres in response to a single intraperitoneal injection of killed bacterial kidney disease (BKD) cells emulsified in. Freund's complete adjuvant (FCA), Low or no response was observed in animals injected with BKD cells in saline or in animals vaccinated by hyperosmotic immersion. Immunological duration was insufficient in fish vaccinated as under-yearling parr to provide protective immunity 2 years later when the fish had become smolts. Atlantic salmon post-yearling parr injected with BKD cells in FCA demonstrated a reduced prevalence of BKD lesions compared to control animals when both were observed as smolts 1 year after vaccination.