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Summary Previous studies have shown that leucine metabolite β‐hydroxy‐β‐methylbutyrate (HMB) increases the immune function in animals as measured by cellular and humoral immune responses. In the present study the influence of feeding HMB on the nonspecific cellular and humoral defence mechanisms and protection against furunculosis in rainbow trout (Oncorhynchus mykiss) was examined. HMB was fed in a pelleted ration at either 0 (control), 10, 25 or 50 mg  kg−1 bw day−1 for 8 weeks. Blood and pronephros cells were taken at random from 10 fish in each group for the analyses. The respiratory burst activity (RBA) and potential killing activity (PKA) of phagocytes, lymphocyte proliferation stimulated by either concanavalin (ConA) or lipopolysaccharide (LPS), lysozyme activities and total immunoglobulin (Ig) levels in plasma were analysed at 0, 1, 2, 3, 4, 6 and 8 weeks. After 4 weeks of feeding HMB, a challenge test was performed by injection of Aeromonas salmonicida into the fish. HMB approximately doubled the respiratory burst activity and potential killing activity ability of polymorphonuclear (PMN) and morphonuclear (MN) cells (P < 0.01) and increased the mitogen‐stimulated lymphocyte proliferation (P < 0.01) when compared with the control group. HMB feeding also increased (P < 0.01) the lysozyme activity in plasma and total Ig levels in serum. During the 14‐day challenge test, mortality was decreased (P < 0.01) by up to 62% in HMB‐fed fish compared with the control group mortality. In conclusion, the non‐specific immune enhancement by HMB resulted in protection against furunculosis in the rainbow trout.

IntroductionImmune-potentiating functions of Lactobacillus plantarum strains in the common carp were evaluated.Material and MethodsFourteen days of feeding fish dry diet supplemented with the bacteria provided parameters of nonspecific humoral immunity (lysozyme, ceruloplasmin, γ-globulin, total protein levels, and serum bactericidal activity) and cellular immunity (pinocytosis, respiratory burst activity, and potential killing activity of organ phagocytes), as well as the proliferative response of organ lymphocytes stimulated with mitogens. The resistance of fish to infection with Aeromonas hydrophila was also determined.ResultsDietary supplementation with L. plantarum had a substantial influence on the activity of organ phagocytes, especially the potential killing activity of head kidney cells. A significant increase in the proliferative activity of LPS-stimulated B lymphocytes and in the levels of γ-globulins and total protein was observed. The supplemented diet conveyed higher resistance than the control diet as the cumulative fish mortalities after infection with A. hydrophila were 65% and 85%, respectively.ConclusionThe results indicate that dietary supplementation with L. plantarum stimulates the antibacterial resistance of common carp and may reinforce defence against bacterial infections, but further studies need to be conducted.

Immunostimulants activate nonspecific defence mechanisms, cell-mediated immunity and specific immune responses. Immunostimulants can be administered before, with, or after vaccines to amplify the specific immune response in generating elevations of humoral antibody levels and the number of antibody-secreting cells (ASC). Special applications of immunostimulants include assisting spray or other regimens to increase the topical uptake of vaccines. In the present study, the influence of dimerized lysozyme (KLP-602) and inosine pranobex (groprinosin) on the cellular and humoral immune response after in vitro and in vivo immunization of rainbow trout with Yersrnia ruckeri vaccine (Yersivax) was determined. Immunostimulants were administered by injection before, with, or after the vaccine applied by immersion. ELISPOT assays were used for the quantification of total and specific ASC after in vitro and in vivo immunization. Also, the specific immune response was determined 7, 14, 18,2 1,28,35 and 45 days after in vivo immunization. Immunostimulants administered before, with, or after the vaccine increased cell-mediated immunity and the specific immune response, compared with the control groups (vaccine only). The higher responses (adjuvant effect) after the application of dimerized lysozyme (KLP-602) were observed in each experimental study.

Influence of beta-1.3/1.6-glucan (Macrogard) on the innate immunity and protection against Aeromonas hydrophila in tench (Tinca tinca (L.)) was assessed. Macrogard was fed at doses of 0, 0.5, 1 and 2 g kg -1 of pellets for 1 month. The blood, spleen and head kidney from 10 fish of each group were separated and analysed for immunity parameters. Twenty tench from each group were infected with A. hydrophila. Macrogard at doses 1 and 2 g kg -1 of feed significantly (P \ 0.05) increased the phagocytic activity of macrophages and proliferative response of mitogens stimulated lymphocytes. The same doses significantly (P \ 0.05) increased lysozyme activity and Ig level in serum, compared to the control and dose 0.5 g kg -1 of feed. The challenge test showed that Macrogard reduced mortality of tench after experimental infection (5-35%).

Studies have shown that in both in vitro and in vivo tests, b-hydroxy-b-methylbutyrate (HMB) increases the nonspeci¢c cellular and humoral immune response and protection against diseases in animals. The present study examines the in£uence of HMB on nonspeci¢c humoral defense mechanisms and protection against furunculosis in pikeperch (Sander lucioperca). b-hydroxy-b-methylbutyrate was fed in a pelleted ration of 50 mg kg À1 feed day À1 for 4 weeks. Blood was drawn from 12 HMB-fed and control-fed pikeperch. The lysozyme and ceruloplasmin activities in the plasma, total immunoglobulin (Ig) levels, and total serum protein were analysed prior to and then after 2 and 4 weeks of HMB ingestion. After 4 weeks of HMB ingestion, a challenge test was performed by injecting the ¢sh with live pathogenic Aeromonas salmonicida bacteria. b-hydroxy-bmethylbutyrate at a dose of 50 mg kg À1 feed resulted in a statistically signi¢cant (Po0.05) increase in the lysozyme activity of the plasma, total Ig, and serum protein levels. Additionally, reduced mortality (40%) after the in vivo challenge with pathogenic A. salmonicida suggested that HMB-activated nonspeci¢c protection against furunculosis in pikeperch.

b-Hydroxy-b-methyl butyrate (HMB) has been shown to counteract many of the negative effects of intensive fish production methods and results in increased growth and protection against diseases. In the present study, the in vitro and in vivo effect of HMB on the immunocompetence cell activity in tench Tinca tinca (L.) was examined. In the in vitro study pronephric phagocytes and lymphocytes were isolated from the fish and grown in culture medium (RPMI-1640) containing 0, 0.1, 1, 5, 10, 25, 50 or 100 lg HMB cm )3 of medium. The effects of HMB on the respiratory burst activity (RBA), the potential killing activity (PKA) and lymphocyte proliferation stimulated by concanavalin A (ConA) or lipopolysaccharide (LPS) were examined. The in vitro study showed that HMB increased the RBA and PKA of the phagocytes, compared to the medium over that of cells grown without HMB. Lymphocyte proliferation stimulated by ConA and LPS was also increased approximately when HMB was added to the culture medium at concentrations between 10 and 100 lg HMB cm )3 . In the in vivo study fish were fed daily with pellets containing HMB at doses 0, 10, 25 and 50 mg kg )1 body weight day )1 . The study showed that HMB statistically increased the RBA and PKA and highest activity at 50 mg kg )1 body weight day )1 were observed. Also lymphocyte proliferation stimulated by ConA and LPS were maximally stimulated at dose 50 mg kg )1 body weight day )1 . In conclusion, the current study shows that HMB could potentially improve immunocompetence cell activity in tench through increased cell proliferation and functionality.

The influence of deltamethrin on the innate immunity in rainbow trout was examined. Fish were immersed in deltamethrin at doses of 1, 2, and 4 microg/L for 30 min. The results showed that deltamethrin at doses of 2 and 4 microg/L decreased phagocytic activity of spleen macrophages and proliferative response of pronephros lymphocytes at days 1, 2, and 5 after immersion. Deltamethrin at these doses decreased the lysozyme activity, total protein, and immunoglobulin levels in serum. The greatest immunosuppressive influence of deltamethrin at dose 4 microg/L was observed at the end of the study.