The Effect of Levamisole as an Adjuvant on the Humoral Immune Response of Atlantic Salmon (Salmo salar L.)

Pre and post-smolt Atlantic salmon (Salmo salar L.) were administered a levamisole adjuvanted Vibrio anguillarum vaccine by bath or intraperitoneal (IP) injection. A significant serum anti-V. anguillarum antibody response was elicited in groups of fish administered an IP injection of bacterium only. Fish (pre and post-smolt) treated with the levamisole adjuvanted vaccine (IP and bath) showed a suppressed response relative to the respective positive (vaccinated) control groups. No detectable antibody response was elicited in fish not treated or treated with a placebo. Results from this trial suggest that levamisole as an adjuvant has a narrow range of efficacy. Introduction Vaccination of fish is an elegant means of enhancing the protective capabilities of fish to specific pathogens. While many vaccines are effective when used insularly, the efficacy of others may benefit from the inclusion of adjuvants. Recent studies have demonstrated that levamisole (used as an adjuvant) increased resistance to pathogenic challenge through the elevation of antibody response and non-specific immune parameters (Siwicki et al., 1990; Anderson and Jeney, 1992; Jeney and Anderson, 1993; Midtlyng et al., 1996). Evidence has suggested that the efficacy of levamisole as an adjuvant is dependent on the antigen with which it is administered. The aim of this study was to determine the effect of levamisole as an adjuvant with a commercial Vibrio anguillarum vaccine on the humoral immune response of Atlantic salmon (Salmo salar L.). Materials and Methods Fish Atlantic salmon (S. salar L.) pre-smolt (10 g) or post-smolt (585 g) were held in fresh water (0 ‰) or sea water (35 ‰) respectively. Presmolts (n = 378) were held in eighteen 50 L tanks in a flow through system, while postsmolts (n = 32) were individually tagged and held in a single 4000 L rathburn tank in a recirculation system. Fish were fed commercial salmon diets (Gibson’s). Both groups of fish were kept at a constant temperature using temperature control apparatus. The presmolts were held at 14 ± 0.7OC, whilst the post-smolts were held at 11 ± 0.5OC. Vaccination Fish were divided into six (pre-smolt) or four (post-smolt) treatment groups (Table 1). Bath vaccine was prepared by diluting a Vibrio anguillarum (serovar O1) whole cell vaccine Bull. Eur. Ass. Fish Pathol., 20(3) 2000, 102 (Anguillivac-C; DPIWE, Launceston, Tasmania) containing a minimum of 1x1010 cells mL1 of formalin killed whole cells. Levamisole (Levamisole gold, Virbac, Peakhurst, NSW) in the form of levamisole hydrochloride (5 μgmL-1) was added to the bath. Injectable vaccine was prepared by centrifuging 10 mL of Anguillivac-C for 3 minutes at 2130 g. After removal of the supernatant, cells were washed twice with sterile 0.1 M PBS (pH = 7.2) then resuspended in the same volume of PBS. Injectable levamisole in the form of levamisole phosphate (Nilverm®, Coopers, Sydney, NSW) was diluted in PBS and vaccine to create the required volume and concentration (table 1). Fish were acclimated to the tanks over fourteen days prior to treatment. Fish in all groups were anaesthetised with benzocaine (ethyl-paminobenzoate; 50 mgL-1) and weighed prior to administration of treatments. Bathing of fish occurred over a one hour period in an adjuvanted or non-adjuvanted vaccine bath. For the injected treatments, fish were intraperitoneally (IP) injected Sampling When sampling, fish were euthanased with an overdose of benzocaine (100 mgL-1). At 4 weeks post-treatment approximately half the pre-smolts (n = 10 from each triplicate tank) and all the post-smolts (n = 8) were euthanased. Blood was removed from the caudal vein and allowed to clot on ice. Blood was centrifuged at 1890 g for 3 minutes and serum removed and frozen (-80°C) until required. At 8 weeks post-treatment, the remaining pre-smolts were sampled as described. Enzyme linked immunosorbent assay (ELISA) The serum anti-V anguillarum antibody responses were monitored after an indirect ELISA had been developed (Crowther, 1995). Briefly, plates were coated with sonicated V. anguillarum diluted in carbonate buffer and incubated overnight at 4°C. Wells were blocked with 3% casein sodium in PBS (pH = 7.2) for 30 minutes at 37°C. Serum samples were added to each well and incubated for 90 minutes at room temperature. Mouse antiAtlantic salmon IgM monoclonal antibody (MAb; 4610) or rabbit anti-Atlantic salmon IgM were used to probe the pre-smolt and o N t n e m t a e r T t n e m t a e r t t l o m s e r P c n o C ( e m u l o V a) t n e m t a e r t t l o m s t s o P c n o C ( e m u l o V a) 1 t n e m t a e r t o N a n b a n b 2 ) P I ( S B P ) h s i f g / g μ 0 ( l m 2 . 0 ) h s i f g / g μ 0 ( l m 5 . 0 3 ) P I ( e n i c c a V ) h s i f g / g μ 0 ( l m 2 . 0 ) h s i f g / g μ 0 ( l m 5 . 0 4 ) P I ( e l o s i m a v e L + e n i c c a V ) h s i f g / g μ 5 . 0 ( l m 2 . 0 ) h s i f g / g μ 5 . 0 ( l m 5 . 0 5 ) h t a b ( e n i c c a V ) h s i f g / g μ 0 ( l / l m 2 i n c 6 ) h t a b ( e l o s i m a v e L + e n i c c a V ) h s i f g / g μ 5 ( l / l m 2 i n c Table 1. Vaccination treatments alevamisole concentration (μg.g fish-1) bna = not applicable cni = not included Bull. Eur. Ass. Fish Pathol., 20(3) 2000, 103 post smolt antibody titres respectively (60 minutes at 37°C). Rabbit anti-mouse HRP (DAKO P161) or goat anti-rabbit HRP (DAKO P0448) was added to each well for 60 minutes at 37°C and colour developed using ophenylene diamine in sodium citrate phosphate buffer. Plates were washed 4 times with PBS in between each step. Plates were read using a Titertek Plus MS212 plate reader at 492 nm. Positive (vaccinated) control as well as negative (non-vaccinated) control serum were included on each plate to enable data comparison. Statistical analysis of treatment groups was by one way ANOVA after the assumptions of the test had been satisfied. Differences between individual treatment groups were detected using Tukey’s posteriori test. Results Pre-smolt administered an IP injected vaccine alone exhibited a significant elevation of antibody level (Figure 1). In addition, the fish bathed in vaccine displayed a detectable antibody response at week 4. Antibody levels of fish treated with the vaccine and levamisole (bath and IP) were above the negative control groups. However, when compared to the respective positive control groups the antibody levels of fish treated with the levamisole adjuvanted vaccine were suppressed. Statistically, the means of treatment groups were significantly different (P = 0.003) however, at week 8 the response had tapered and means were not significantly different (P = 0.107). A similar trend in antibody response amongst treatment groups was observed in post-smolt salmon. Little or no antibody was observed in post-smolt from the control (null and PBS treatment) groups (Figure 2). Fish in groups administered the vaccine showed a significant elevation of antibody titre (P = 0.000) while the antibody level of fish administered the levamisole adjuvanted vaccine was suppressed relative to the positive control group. However the difference between the positive control and treatment groups was not significant. 0 0.25 0.50 0.75 1.00 Control PBS-IP Vaccine-Bath Vaccine + Lev.-Bath Vaccine-IP Vaccine + Lev.-IP Week 4 Week 8 O pt ic al D en sit y (4 92 nm ) Weeks Post Vaccination a a a a a b