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Physiological effects of an oil-adjuvanted vaccine on out-of-season Atlantic salmon (Salmo salar L.) smolt
Aquaculture 214 (2002) 397 – 409 www.elsevier.com/locate/aqua-online
Physiological effects of an oil-adjuvanted vaccine on out-of-season Atlantic salmon (Salmo salar L.) smolt Geir Olav Melingen *, Heidrun I. Wergeland Department of Fisheries and Marine Biology, University of Bergen, Bergen High Technology Centre, P.O. Box 7800, N-5020 Bergen, Norway Received 28 May 2001; received in revised form 16 October 2001; accepted 31 October 2001
Abstract Three groups of out-of-season smolts (0+) were studied; untreated smolt (UT), smolt injected with a multivalent commercial oil-adjuvanted vaccine (VA), and smolt injected with the same oiladjuvanted formulation without the vaccine components (AD). The 0+ smolts were induced by photoperiod and temperature manipulation. Growth, serum concentrations of proteins and IgM, and seawater tolerance were measured. Growth of VA-injected smolt was significantly reduced from three weeks post vaccination until two weeks after seawater transfer when compared to UT and AD. This indicates that vaccine antigen or the antigens in combination with adjuvant can cause reduced growth rates as previously reported using oil adjuvanted vaccines. One week after vaccination, the serum protein levels were significantly higher for UT fish compared to both VA and AD groups, but no significant differences were seen thereafter until seawater transfer. After seawater transfer, both VA and AD groups had significantly higher serum protein levels than the UT group. The serum IgM level increased after vaccination in the VA group and after seawater transfer was more than four times higher than in the UT group. At the same time, the AD group had an approximately 50% higher serum IgM level than UT fish, indicating that the oil-adjuvant alone stimulates IgM production. Using 96-h seawater challenge tests, there were no differences in seawater adaptability between UT, AD and VA treatments. The results show that the oil-adjuvanted vaccine had a negative effect
* Corresponding author. Tel.: +47-5558-46-16; fax: +47-5558-40-48. E-mail address: [email protected] (G.O. Melingen). 0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 1 ) 0 0 8 6 7 - 5
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on growth, and elevated IgM levels prior to seawater introduction, but had no negative effect on smoltification. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Salmon; Serum proteins; IgM; Smolting; Vaccination; Out-of-season smolt; Growth; Side-effects
1. Introduction Smolting in Atlantic salmon includes changes in morphology, behaviour and physiology, which pre-adapt the fish to life in seawater (McCormick and Saunders, 1987; Hoar, 1988). Central amongst these changes is the development of hypo-osmoregulatory mechanisms that allow smolts to move from freshwater to seawater with only minor disturbance in osmotic balance (Handeland et al., 2000). Photoperiod has been shown to be the major cue for the parr – smolt transformation, and the use of artificial photoperiods to alter timing of smoltification has been documented (Clarke, 1989; Duston and Saunders, 1990). To produce out-of-season (0 +) smolt, growth must be accelerated using heated water, and the fish must receive appropriate photoperiod cues (Saunders et al., 1990; Gaignon and Quemener, 1992; Stefansson et al., 1992). In commercial farming of Atlantic salmon (Salmo salar L.), vaccination against infectious diseases is required to avoid major losses (Press and Lillehaug, 1995). After the introduction of oil-adjuvanted, injectable bacterins against furunculosis (Midtlyng et al., 1996), polyvalent vaccine formulations have been widely used in salmon aquaculture (Lillehaug, 1997). Experience indicates that oil-adjuvanted vaccines are highly effective (Midtlyng, 1996). Besides providing protection against disease, various fish vaccines have been reported to increase growth rate (Sawyer and Strout, 1977) and improve feed conversion efficiency (Thorburn et al., 1988). However, oil-adjuvanted vaccines have also been reported to cause post-vaccination mortality, reduction in growth rate, internal abdominal adhesions, and injection-site lesions (Lillehaug et al., 1992; Midtlyng et al., 1996; Midtlyng and Lillehaug, 1998; Rønsholdt and McLean, 1999). In Norway, all farmed Atlantic salmon smolts are vaccinated prior to seawater transfer. In recent years, smolts are also being produced for out-of-season (0 +) seawater transfer in autumn (August – October), using artificial photoperiod and temperature regimes. These smolts are usually vaccinated only a few weeks prior to seawater transfer (Eggset et al., 1999). Thus, vaccination is often performed after the parr – smolt transformation is initiated. This accelerated production used for 0 + smolt might induce physiological, endocrine or immunological changes during parr – smolt transformation, which might differ from traditional smolt both in timing and strength of responses. The complex oil-adjuvant vaccines, might also affect the parr – smolt transformation. Use of oil-adjuvanted vaccine close to the start of smoltification has been shown to disturb the smoltification process and cause a delay of approximately two weeks (Eggset et al., 1999). A post-vaccination reduction both in serum immunoglobulin (IgM) and total serum protein has been reported during smolting for normal yearling smolt (1 +) (Melingen et al., 1995a). In addition, specific antibody levels were found to be reduced in fish vaccinated during smoltification, and vaccination with a non-adjuvanted vaccine had no negative
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impact on smoltification (Melingen et al., 1995b). However, for induced 0 + smolt, serum IgM levels remained low throughout the ‘‘winter’’ photoperiod, and were found to increase during the ‘‘summer’’ continuous photoperiod (Melingen and Wergeland, 2000). A synchronous pattern was not found for serum proteins, indicating that 1 + and 0 + smolts are not always comparable. The objective of this study was to examine the impact of oil-adjuvant and antigens in injectable vaccines on the growth, serum protein and IgM profiles, and seawater adaptability in out-of-season (0 +) Atlantic salmon smolt.
2. Materials and methods 2.1. Fish stock and rearing conditions Atlantic salmon (Salmo salar L.) fingerlings were obtained from AquaGen, Bolaks (Norway). The fish were healthy and had no prior history of disease and were inspected. The stock was certified free of specific pathogens. The experiment was carried out at Ewos Research Station Lønningdal, Bergen, Norway. The salmon were hatched December 30, 1998, and weighed 18.1 F 1.9 g (mean F s.d.) on June 8, 1999 at the start of the experiment. From hatching until June average temperature was maintained at 14 jC. In June average temperature was 11.5 jC, July 13.3 jC, and in August (until seawater transfer) 14.8 jC. After seawater transfer average temperature was 8.6jC. The fish were reared under continuous photoperiod (LD 24:0) at 14 jC until June 10, followed by a ‘‘winter’’ photoperiod (LD 12:12) for six weeks, and then continuous photoperiod for another four weeks before seawater transfer. After seawater transfer, the fish were reared under continuous photoperiod. The fish were reared at a density of 40– 45 kg/m3 until vaccination, using circular 6-m grey fibreglass tanks with a water depth of 170 cm. The tank had a lightproof cover (Helly Hansen-products), and a cycle timer adjusted the light provided by one metal-halogen headlight (8 w/m2). The oxygen level was adjusted by a process-control system (MCp18R, Marine Control, Norway), which supplied the particle filtered and UV-treated inlet water (0.5 ppt seawater added for buffering capacity) with supplemented oxygen to maintain at least 80% oxygen saturation in the outlet water. From the time of vaccination and the remaining experimental period, the fish groups were reared in separate 1 m grey square fibreglass tanks. The flow through inlet water was regulated to maintain above 70% oxygen saturation in the outlet water. The fish were fed from automatic feeders in excess of satiation with a commercial dry diet (Vextra, Ewos) with the manufacturers’ recommended pellet size. 2.2. Vaccination A commercial oil-adjuvanted vaccine containing Vibrio salmonicida, V. anguillarum, Aeromonas salmonicida and infectious pancreas necrosis (IPN) virus—Lipogen Quattro (Aqua Health, Canada)—was used. The fish were starved for 24 h and anaesthetised using metacaine (50 mg/l) prior to vaccination. At the time of vaccination on July 14, 1999, the
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fish were divided into three groups (n = 380 per group), and transferred to 1-m tanks. Temperature at time of vaccination was 15.4 jC. Fish were vaccinated according to label directions. 0.2 ml vaccine/fish was injected intraperitoneally (i.p.) to the vaccinated group (VA). This group was compared with a matching untreated group (UT), which was anaesthetized but not injected, and a group receiving 0.2 ml (i.p. injected) of a saline/oiladjuvant (Lipogen adjuvant equivalent to the Quattro vaccine, but no antigens. Prepared by Aqua Health) (AD). 2.3. Sampling procedures All fish were starved for 24 h prior to sampling. Thirty-five fish were sampled from the group prior to vaccination, and from each of the three treatment groups following vaccination according to regimes shown in Fig. 1, a total of 10 samplings. Five fish were used for flow cytometry analysis (Melingen et al., 2002), while the remaining 30 were used for serum protein and IgM analysis, for seawater tolerance test, as well as specific antibody values (Melingen et al., 2002). All fish were killed by a blow to the head before collecting the blood samples from the caudal vein using a syringe. The total body weight (BWT, g) and fork length (FL, cm) for each fish were measured at every sampling throughout the experiment. Condition coefficient was calculated as CF=(BWT/ FL3) 100. The blood coagulated at 4 jC over night, before centrifugation at 1000 g for 5 min. The serum was transferred to 1.5 ml Eppendorf tubes, and stored in aliquots at 20 jC for 24 h before transfer to 80 jC.
Fig. 1. The fish were reared under continuous photoperiod (LD 24:0) from hatching until start of the ‘‘winter’’ photoperiod (LD 12:12) on June 10. Six weeks later, a new continuous photoperiod was introduced, and the fish were transferred to seawater another four weeks later. During the freshwater period, sampling was performed at nine different times, and one sampling was performed after seawater transfer, all as indicated (*).
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2.4. Total serum protein and IgM determination Total serum protein was quantified using the Biuret Protein Assay with a bovine serum albumin (BSA, Sigma, USA) standard as described in Melingen and Wergeland (2000). Serum IgM was determined using a single radial immunodiffusion assay (SRID), as described by Melingen and Wergeland (2000). Rabbit antiserum to Atlantic salmon IgM was prepared as described by Ha˚varstein et al. (1988). 2.5. Seawater tolerance From vaccination and until seawater transfer, fish from all groups (UT, AD and VA, n = 12) were removed and transferred to seawater, and their hypoosmoregulatory ability assessed by measuring serum chloride levels after 96-h seawater tolerance test (33 – 34x ) at 10 jC. Their corresponding control groups (UT-C, AD-C and VA-C, n = 30) were measured using the isolated serum described in Section 2.3. Serum chloride levels were measured using 20 Al samples in a Radiometer CMT 10 titrator (Radiometer, Denmark). 2.6. Data processing and statistics Results are given as arithmetic mean values. Significant differences within and between groups were determined by one-way and two-way ANOVA, respectively, followed by Newman –Keuls post hoc test. Differences were considered significant when P < 0.05.
3. Results 3.1. Growth and condition coefficient The body weight and growth rate of the three treatment groups is summarized in Fig. 2. No significant differences (n.s.) in body weight were observed until three weeks post vaccination, when VA was significantly smaller than both AD and UT ( P < 0.05). VA remained smaller than UT and AD for the remainder of the experimental period ( P < 0.05). The CF increased from June 8 (1.10 F 0.07) to June 22 (1.16 F 0.25) ( P < 0.05), but remained stable for the rest of the experimental period. There were no significant differences between UT, AD and VA at any time. 3.2. Total serum protein Total serum protein levels are summarized in Fig. 3. The total serum protein tended to increase (n.s.) in all groups from introduction of the ‘‘winter’’ photoperiod (52.1 F 7.2 mg/ml) until four weeks later (58.0 F 9.6 mg/ml), but declined significantly in all three treatment groups at the time of vaccination (44.3 F 10.2 mg/ml) ( P < 0.001). One week
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Fig. 2. Body weight (bar indicates s.d., n = 35) of fish in the UT, AD and VA groups during the parr – smolt transformation period.
Fig. 3. Total serum protein concentration was measured by the Biuret Protein Assay (bar indicates s.d., n = 30) in UT, AD and VA throughout the experimental period.
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after vaccination, the protein level began to increase back to normal levels for UT, while both AD and VA remained significantly lower for two weeks ( P < 0.05). Two weeks after vaccination and until seawater transfer, there were no significant differences in total serum protein between VA, AD and UT. Two weeks after seawater transfer, a significant reduction in total protein was observed for UT ( P < 0.01), and at the same time a significant increase was observed for AD ( P < 0.05), while the values for VA remained stable. 3.3. Total serum IgM The serum IgM levels remained stable (0.30 F 0.15 mg/ml) until time of vaccination, when a significant increase was observed (0.71 F 0.10 mg/ml) ( P < 0.001) in all treatment groups (Fig. 4). One week after vaccination, a significant decrease in serum IgM was observed for all three groups ( P < 0.01), with UT having a significantly higher (0.59 F 0.12 mg/ml)( P < 0.001) and VA a significantly lower (0.25 F 0.15 mg/ ml) IgM level P < 0.001). Two weeks after vaccination, VA had a significantly higher (0.97 F 0.11 mg/ml) serum IgM level than both AD (0.79 F 0.09 mg/ml) and UT (0.84 F 0.09 mg/ml) ( P < 0.01). This situation remained throughout the experimental period ( P < 0.05). UT had a significantly higher serum IgM level than AD until four weeks after vaccination ( P < 0.01), when AD was similar to UT ( P>0.05). After seawater transfer, both UT (0.23 F 0.10 mg/ml) and AD (0.34 F 0.16 mg/ml) had a significant decrease in serum IgM levels ( P < 0.01), while VA (0.91 F 0.10 mg/ ml) remained stable. At this time AD had a significantly higher serum IgM level than UT ( P < 0.01).
Fig. 4. Total serum IgM concentration measured by single radial immuno-diffusion assay (SRID) (bar indicates s.d., n = 30) in UT, AD and VA throughout the experiment.
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3.4. Serum chloride levels One week post vaccination all groups being held in freshwater experienced a significant increase in serum chloride levels ( P < 0.05) (Fig. 5). For UT (129.4 F 4.8 mmol/l) the levels remained stable until seawater transfer, while AD (127.8 F 3.8 mmol/l) and VA (129.9 F 4.1 mmol/l) decreased significantly two weeks after vaccination ( P < 0.05). VA (124.2 F 3.3 mmol/l) continued its decrease the following week ( P < 0.05). The levels then remained stable for AD and VA until seawater transfer. Two weeks after seawater transfer, UT had the largest increase in serum chloride levels ( P < 0.001), AD had a small increase ( P < 0.01), while VA remained stable ( P>0.05). At this time, UT (139.4 F 12.2 mmol/l) had a significantly higher serum chloride level than AD (126.3 F 11.7 mmol/l) and VA (126.3 F 11.7 mmol/l) ( P < 0.05). The results from the comparable seawater challenge test groups showed that one week post vaccination, all three seawater challenged groups (UT, AD and VA) exhibited significant increases in serum chloride levels ( P < 0.01). These increases continued to a maximum two weeks after vaccination ( P < 0.01), then the values decreased for all seawater challenged groups until seawater transfer. For UT the reduction in the last week was not significant. The only times there were differences between the seawater challenged groups were one week post vaccination, when AD (148.3 F 12.0 mmol/l) had a significantly lower serum chloride level than VA (165.3 F 13.1 mmol/l) and UT (166.5 F 18.7 mmol/l) ( P < 0.05), and two weeks prior to seawater transfer when VA (177.2 F 14.9 mmol/l) had a significantly higher serum chloride level than UT (162.2 F 13.0 mmol/l) and AD (159.1 F 8.9 mmol/l) ( P < 0.05).
Fig. 5. Serum chloride levels (bars indicate s.d.) for control fish groups (UT-C, AD-C and VA-C, n = 30 for each group), and for seawater challenge groups (UT, AD and VA, n = 12 for each group).
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4. Discussion Vaccination of Atlantic salmon using oil-adjuvanted vaccines may affect growth of the fish due to loss in appetite or side-effects. A better understanding of what causes this reduction is needed. Three weeks post vaccination, the vaccinated group was significantly smaller than either the untreated group or the group receiving the oil-adjuvant only. These observations are in accordance with the findings of others (Midtlyng and Lillehaug, 1998; Rønsholdt and McLean, 1999), who reported a growth reduction in fish receiving oil-based adjuvant vaccines. Based on our results, it appears that the antigens and/or interactions between the oil-adjuvant and antigens rather than the adjuvant itself may cause the growth reduction. The handling and injection process seems to have little or no effect upon growth, as AD and UT had similar growth patterns. The conclusion is consistent with Rønsholdt and McLean (1999) who determined that the effect upon growth was caused by the antigenic component (A. salmonicida) in the vaccine, rather than the adjuvant and/or interactions between the two. Colquhoun et al. (1998) also concluded that the oil-adjuvant, although an important factor, may not be completely responsible for some serious side-effects associated with vaccination such as abdominal adhesions. These side-effects affect physiological function (Poppe and Breck, 1997) and growth performance of fish (Midtlyng, 1997). Our experiment cannot demonstrate whether it is one or more of the antigens or the combination of antigens and adjuvant that cause the growth reduction observed. It has been reported from the field that fish vaccinated shortly after an IPNV outbreak have shown more side-effects than vaccinated fish without previous infection (Kaurstad, personal communication). However, Colquhoun et al. (1998) showed that pre-injection with IPNV had no effect on the degree of vaccine adhesions, while co-injection with Pseudomonas fluorescens and additional environmental stress resulted in a significant increase in adhesion level. If side-effects like abdominal adhesions are caused by a strong immune response from the antigens, or the combination of antigens and adjuvant, and not by the adjuvant itself, one could consider vaccinating at lower temperatures when the immune activity is lower. In the present experiment, temperature at time of vaccination was 15.4 jC, which is much higher than the temperatures at which 1 + smolt are typically vaccinated. Photoperiod gives the 0 + smolt a signal that it is winter, while at the same time the temperature indicates that it is summer. The higher temperature is expected to contribute to a higher immune response for the 0 + smolt than if vaccinated at lower temperatures as for the 1 + smolt. Perhaps a lowered, but increasing temperature during ‘‘winter’’ photoperiod would be more favourable for the 0 + smolt immune response? A low and increasing stimulation over a long period may be more favourable for the fish with respect to physiological and immunological development. Total serum protein and IgM were significantly lower for the vaccinated group (VA) and adjuvant group (AD) compared to the untreated group (UT) one week after vaccination, but two weeks after vaccination and until seawater transfer the serum proteins were similar for VA, AD and UT, while in the same period VA showed higher serum IgM levels than the others. The mechanisms behind the observed changes in IgM might be due to cortisol acting on B-cells directly, or other mediators (Espelid et al., 1996). The increase in serum IgM might also be due to antigen stimulation,
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indicating stimulation by the antigen specific antibodies or non-specific IgM production caused by the combination of antigens and oil adjuvant. A similar increase in serum IgM concentration was reported for vaccinated fish one week post vaccination (Melingen and Wergeland, 2000), with increasing serum protein levels after vaccination, except for one week post vaccination when a reduction was observed for vaccinated fish. The serum IgM and protein levels were both higher after vaccination in that experiment compared to present experiment. These differences may have been caused by the different experimental protocol, as fish in Melingen and Wergeland (2000) were kept in 6-m tanks, while in the present experiment the fish were transferred to 1-m tanks at time of vaccination. This may have caused differences in water quality that may affect the immune status of the fish. The reduction in serum IgM and protein observed for VA and AD one week post vaccination may indicate stress caused by the vaccination, resulting in an increase in cortisol, which is known to inhibit B-cell activity (Maule et al., 1988, Maule and Schreck, 1991). It has been shown that B-lymphocytes are directly affected by cortisol, and reduced plasma IgM levels were found in rainbow trout after cortisol administration (Hou et al., 1999). A possible redistribution of cells may also cause a lower B-cell activity for blood leucocytes. Cortisol is known to inhibit some components of the immune response and enhance other parts that may be functional in stressful situations. Effects of cortisol on leucocyte viability are also cell type-specific. For example, B-cells from carp are especially sensitive to cortisol, whereas thrombocytes and cells in the T-cell fraction are insensitive (Weyts et al., 1998). A loss of sensitivity to cortisol in leucocytes from juvenile salmonids during winter has been previously documented (Slater and Schreck, 1998), and it has been indicated that the cortisol levels during smolting probably are controlled more by photoperiod than by endogenous rhythm or temperature (Olsen et al., 1993). Time of vaccination for 0 + smolt in the present experiment was during the ‘‘winter’’ photoperiod, which probably will not correlate to a normal winter, as the temperature in this experiment was much higher. With the high temperatures used in present study, a stronger immune response is expected than if vaccinated at lower temperatures. A reduction in serum protein levels after seawater transfer for UT may indicate that the physiological status, measured by concentration of serum IgM and proteins, of UT differ from AD and VA. The vaccine and/or adjuvant seem to cause a major physiological impact on the fish as indicated by variations in serum IgM and protein levels. The consequences of or reasons for these fluctuations are unknown. This may be caused by endocrine interactions with the immune system (Weyts et al., 1999), or other physiological factors may be involved. The adjuvant seems to have an impact during seawater transfer, as the serum IgM levels for AD does not have a similar reduction as UT. This may be due to non-specific stimulation by the adjuvant at time of seawater transfer, while these cells have been down regulated or inhibited during parr – smolt transformation. This observation for serum IgM correlates with the observation for serum proteins. There are no indications of a negative effect on seawater capability caused by vaccination. However, after seawater transfer, the serum chloride levels increased more for UT than for VA and AD. This may indicate a positive effect caused by the vaccine or
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adjuvant, where physiological or endocrine impact caused by the vaccine/adjuvant result in increased seawater adaptability as measured by serum chloride levels. Using the 96-h seawater challenge test, a totally different profile was found compared to using a 24-h seawater challenge test for similar smolt as described by Melingen and Wergeland (2000). It is shown that the acute hyperosmotic stress and duration of the stabilization period during seawater acclimation of Atlantic salmon is temperaturedependent (Handeland et al., 2000). However, Handeland et al. (2000) were not able to detect any differences in plasma chloride levels between groups acclimated to temperatures of 4.6 –18.9 jC. The present experiment indicates that the increased serum chloride levels found by Melingen and Wergeland (2000) after vaccination using a 24-h seawater challenge test, is just a short-term effect which is not present after 96-h in seawater, and the 96-h seawater challenge test was concluded to be more appropriate than the 24-h test. The oil-adjuvanted vaccine may affect the physiological status of the fish resulting in a different serum chloride profile during the first 24-h in seawater, but what triggers this physiological effect is not known. Adjuvants are foreign to the fish, and if appropriately administered they lead to sterile inflammations that attract the various cells of the aseptic defence system. Most adjuvants directly or indirectly stimulate the generation of IL-11, but also other factors that support growth and differentiation (Leenaars et al., 1997). Weyts et al. (1999) have reviewed the complex interactions between the immune- and endocrine system, but there are still potential interactions between these systems that are not understood and can affect the physiological status of the fish. The results indicate that the vaccine used had a negative effect on growth in the observation period. The changes in serum protein and IgM also indicate changes after vaccination, and resulted in elevated IgM levels for VA. Vaccination was carried out at a time when levels of serum IgM were low, which might indicate a favourable time to vaccinate Atlantic salmon at high temperatures. If some of the reported side-effects from using oil-adjuvanted vaccines are caused by a strong immune response, higher IgM levels indicating higher immune activity, may result in more side-effects. The physiological and immunological status of a 30 – 40 g 0 + parr/smolt will probably be different compared to a 1 + parr/smolt of similar size due to different photoperiods and temperatures. These possible differences may give different responses to oil-adjuvanted vaccines at the time of immunization.
References Clarke, W.C., 1989. Photoperiod control of smolting: a review. Physiol. Ecol. Jpn. 1, 497 – 502. Colquhoun, D.J., Skjerve, E., Poppe, T.T., 1998. Pseudomonas fluorescens, infectious pancreatic necrosis virus and environmental stress as potential factors in the development of vaccine related adhesions in Atlantic salmon, Salmo salar L. J. Fish Dis. 21, 355 – 364. Duston, J., Saunders, R.L., 1990. Control of the timing of smoltification in Atlantic salmon: endogenous rhythms and environmental factors. In: Saunders, R.L. (Ed.), Proceedings of Can.-Nor. Finfish Aqua. Workshop. Can. Tech. Rep. Fish. Aquat. Sci., vol. 1761, St. Andrews, N.B., pp. 99 – 105. Eggset, G., Mortensen, A., Lo¨ken, S., 1999. Vaccination of Atlantic salmon (Salmo salar L.) before and during smoltification; effects on smoltification and immunological protection. Aquaculture 170, 101 – 112.
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Espelid, S., Løkken, G.B., Steiro, K., Bøgwald, J., 1996. Effects of cortisol and stress on the immune system in Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol. 6, 95 – 110. Gaignon, J.L., Quemener, L., 1992. Influence of early thermic and photoperiodic control on growth and smoltification in Atlantic salmon (Salmo salar). Aquat. Living. Resour. 5, 185 – 195. ˚ ., Bjo¨rnsson, B.Th., Lie, Ø., Stefansson, S.O., 2000. Seawater adaptation by Handeland, S.O., Berge, A out-of-season Atlantic salmon (Salmo salar L.) smolts at different temperatures. Aquaculture 181, 377 – 396. Ha˚varstein, L.S., Aasjord, P.M., Ness, S., Endresen, C., 1988. Purification and partial characterization of an IgM-like serum immunoglobulin from Atlantic salmon (Salmo salar). Dev. Comp. Immunol. 12, 773 – 785. Hoar, W.S., 1988. The physiology of smolting salmonids. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, vol. XIB. Academic Press, New York, NY, pp. 275 – 343. Hou, Y.Y., Suzuki, Y., Aida, K., 1999. Effects of steroid hormones on immunoglobulin M (IgM) in rainbow trout, Oncorhynchus mykiss. Fish Physiol. Biochem. 20, 155 – 162. Leenaars, M., Claassen, E., Boersma, W.J.A., 1997. Antigens and antigen presentation. In: Lefkovits, I. (Ed.), Immunology Methods Manual, vol. 2. Academic Press, USA, pp. 991 – 1013. Lillehaug, A., 1997. Vaccination strategies in seawater cage culture of salmonids. In: Gudding, R., Lillehaug, R., Midtlyng, P.J., Brown, F. (Eds.), Fish Vaccinology. Dev. Biol. Stand., vol. 90. Karger, Basel, pp. 401 – 408. Lillehaug, A., Lunder, T., Poppe, T.T., 1992. Field testing of adjuvanted furunculosis vaccines in Atlantic salmon, Salmo salar L. J. Fish Dis. 15, 485 – 496. Maule, A.G., Schreck, C.B., 1991. Stress and cortisol treatment changed affinity and number of glucocorticoid receptors in leukocytes and gill of coho salmon. Gen. Comp. Endocrinol. 84, 83 – 93. Maule, A.G., Tripp, R.A., Kaattari, S.L., Schreck, C.B., 1988. Stress alters immune function and disease resistance in chinook salmon (Oncorhynchus tshawytscha). J. Endocrinol. 120, 135 – 142. McCormick, S.D., Saunders, R.L., 1987. Preparatory physiological adaptations for marine life of salmonids: osmoregulation, growth and metabolism. Am. Fish. Soc. Symp. 1, 211 – 229. Melingen, G.O., Wergeland, H.I., 2000. Serum protein and IgM profiles in connection with the smolting and vaccination of out-of-season Atlantic salmon (Salmo salar L.). Aquaculture 188, 189 – 201. Melingen, G.O., Stefansson, S.O., Berg, A., Wergeland, H.I., 1995a. Changes in serum protein and IgM concentration during smolting and early post-smolt period in vaccinated and unvaccinated Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol. 5, 211 – 221. Melingen, G.O., Nilsen, F., Wergeland, H.I., 1995b. The serum antibody levels in Atlantic salmon (Salmo salar L.) after vaccination with Vibrio salmonicida at different times during smolting and early post-smolt period. Fish Shellfish Immunol. 5, 223 – 235. Melingen, G.O., Pettersen, E.F., Wergeland, H.I., 2002. Leucocyte populations and responses to immunization and photoperiod manipulation in Atlantic salmon (Salmo salar L.) 0 + smolt. Accompanying paper, Aquaculture. Midtlyng, P.J., 1996. A field study on intraperitoneal vaccination of Atlantic salmon (Salmo salar L.) against furunculosis. Fish Shellfish Immunol. 6, 553 – 565. Midtlyng, P.J., 1997. Vaccinated fish welfare: protection versus side-effects. In: Gudding, R., Lillehaug, A., Midtlyng, P.J., Brown, F. (Eds.), Fish Vaccinology. Developments in Biological Standardization, vol. 90. Karger, Basel, pp. 371 – 379. Midtlyng, P.J., Lillehaug, A., 1998. Growth of Atlantic salmon Salmo salar after intraperitoneal administration of vaccines containing adjuvants. Dis. Aquat. Org. 32, 91 – 97. Midtlyng, P.J., Reitan, L.J., Speilberg, L., 1996. Experimental studies on the efficacy and side-effects of intraperitoneal vaccination of Atlantic salmon (Salmo salar L.) against furunculosis. Fish Shellfish Immunol. 6, 335 – 350. Olsen, Y.A., Reitan, L.J., Røed, K.H., 1993. Gill Na+,K+-ATPase activity, plasma cortisol level, and non-specific immune response in Atlantic salmon (Salmo salar) during parr – smolt transformation. J. Fish Biol. 43, 559 – 573. Poppe, T.T., Breck, O., 1997. Pathology of Atlantic salmon Salmo salar intraperitoneally immunised with oil-adjuvanted vaccine. A case report. Dis. Aquat. Org. 29, 219 – 226. Press, C.M., Lillehaug, A., 1995. Vaccination in European salmonid aquaculture: a review of practice and prospects. Br. Vet. J. 151, 45 – 69.
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Rønsholdt, B., McLean, E., 1999. The effect of vaccination and vaccine components upon short-term growth and feed conversion efficiency in rainbow trout. Aquaculture 174, 213 – 221. Saunders, R.L., Duston, J., Harmon, P.R., Knox, D.E., Stewart, M.W., 1990. Production of underyearling Atlantic salmon smolts. Bull. Aquacult. Assoc. Can. 90 (4), 61 – 63. Sawyer, E.S., Strout, R.G., 1977. Survival and growth of vaccinated, medicated and untreated coho salmon (O. kisutch) exposed to V. anguilarum. Aquaculture 10, 311 – 315. Slater, C.H., Schreck, C.B., 1998. Season and physiological parameters modulate salmonids leucocyte androgen receptor affinity and abundance. Fish Shellfish Immunol. 8, 379 – 391. Stefansson, S.O., Berg, A.E., Hansen, T., Saunders, R.L., 1992. The potential for development of salinity tolerance in underyearling Atlantic salmon (Salmo salar). World Aquacult. 23 (2), 52 – 55. Thorburn, M.A., Carpenter, T.E., Ljunberg, O., 1988. Vibriosis vaccination of rainbow trout Salmo gairdneri at varying temperatures and seasons: I. Effects of mortality and feed conversion in four Swedish field trials. Dis. Aquat. Org. 5, 179 – 183. Weyts, F.A.A., Flik, G., Rombout, J.H.W.M., Verburg-van Kemenade, B.M.L., 1998. Cortisol induces apoptosis in activated B cells, not in other lymphoid cells of common carp, Cyprinus carpio L. Dev. Comp. Immunol. Weyts, F.A.A., Cohen, N., Flik, G., Verburg-van Kemenade, B.M.L., 1999. Interactions between the immune system and the hypothalamo – pituitary – interrenal axis in fish. Fish Shellfish Immunol. 9, 1 – 20.
Physiological effects of an oil-adjuvanted vaccine on out-of-season Atlantic salmon (Salmo salar L.) smolt Geir Olav Melingen *, Heidrun I. Wergeland Department of Fisheries and Marine Biology, University of Bergen, Bergen High Technology Centre, P.O. Box 7800, N-5020 Bergen, Norway Received 28 May 2001; received in revised form 16 October 2001; accepted 31 October 2001
Abstract Three groups of out-of-season smolts (0+) were studied; untreated smolt (UT), smolt injected with a multivalent commercial oil-adjuvanted vaccine (VA), and smolt injected with the same oiladjuvanted formulation without the vaccine components (AD). The 0+ smolts were induced by photoperiod and temperature manipulation. Growth, serum concentrations of proteins and IgM, and seawater tolerance were measured. Growth of VA-injected smolt was significantly reduced from three weeks post vaccination until two weeks after seawater transfer when compared to UT and AD. This indicates that vaccine antigen or the antigens in combination with adjuvant can cause reduced growth rates as previously reported using oil adjuvanted vaccines. One week after vaccination, the serum protein levels were significantly higher for UT fish compared to both VA and AD groups, but no significant differences were seen thereafter until seawater transfer. After seawater transfer, both VA and AD groups had significantly higher serum protein levels than the UT group. The serum IgM level increased after vaccination in the VA group and after seawater transfer was more than four times higher than in the UT group. At the same time, the AD group had an approximately 50% higher serum IgM level than UT fish, indicating that the oil-adjuvant alone stimulates IgM production. Using 96-h seawater challenge tests, there were no differences in seawater adaptability between UT, AD and VA treatments. The results show that the oil-adjuvanted vaccine had a negative effect
* Corresponding author. Tel.: +47-5558-46-16; fax: +47-5558-40-48. E-mail address: [email protected] (G.O. Melingen). 0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 1 ) 0 0 8 6 7 - 5
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on growth, and elevated IgM levels prior to seawater introduction, but had no negative effect on smoltification. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Salmon; Serum proteins; IgM; Smolting; Vaccination; Out-of-season smolt; Growth; Side-effects
1. Introduction Smolting in Atlantic salmon includes changes in morphology, behaviour and physiology, which pre-adapt the fish to life in seawater (McCormick and Saunders, 1987; Hoar, 1988). Central amongst these changes is the development of hypo-osmoregulatory mechanisms that allow smolts to move from freshwater to seawater with only minor disturbance in osmotic balance (Handeland et al., 2000). Photoperiod has been shown to be the major cue for the parr – smolt transformation, and the use of artificial photoperiods to alter timing of smoltification has been documented (Clarke, 1989; Duston and Saunders, 1990). To produce out-of-season (0 +) smolt, growth must be accelerated using heated water, and the fish must receive appropriate photoperiod cues (Saunders et al., 1990; Gaignon and Quemener, 1992; Stefansson et al., 1992). In commercial farming of Atlantic salmon (Salmo salar L.), vaccination against infectious diseases is required to avoid major losses (Press and Lillehaug, 1995). After the introduction of oil-adjuvanted, injectable bacterins against furunculosis (Midtlyng et al., 1996), polyvalent vaccine formulations have been widely used in salmon aquaculture (Lillehaug, 1997). Experience indicates that oil-adjuvanted vaccines are highly effective (Midtlyng, 1996). Besides providing protection against disease, various fish vaccines have been reported to increase growth rate (Sawyer and Strout, 1977) and improve feed conversion efficiency (Thorburn et al., 1988). However, oil-adjuvanted vaccines have also been reported to cause post-vaccination mortality, reduction in growth rate, internal abdominal adhesions, and injection-site lesions (Lillehaug et al., 1992; Midtlyng et al., 1996; Midtlyng and Lillehaug, 1998; Rønsholdt and McLean, 1999). In Norway, all farmed Atlantic salmon smolts are vaccinated prior to seawater transfer. In recent years, smolts are also being produced for out-of-season (0 +) seawater transfer in autumn (August – October), using artificial photoperiod and temperature regimes. These smolts are usually vaccinated only a few weeks prior to seawater transfer (Eggset et al., 1999). Thus, vaccination is often performed after the parr – smolt transformation is initiated. This accelerated production used for 0 + smolt might induce physiological, endocrine or immunological changes during parr – smolt transformation, which might differ from traditional smolt both in timing and strength of responses. The complex oil-adjuvant vaccines, might also affect the parr – smolt transformation. Use of oil-adjuvanted vaccine close to the start of smoltification has been shown to disturb the smoltification process and cause a delay of approximately two weeks (Eggset et al., 1999). A post-vaccination reduction both in serum immunoglobulin (IgM) and total serum protein has been reported during smolting for normal yearling smolt (1 +) (Melingen et al., 1995a). In addition, specific antibody levels were found to be reduced in fish vaccinated during smoltification, and vaccination with a non-adjuvanted vaccine had no negative
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impact on smoltification (Melingen et al., 1995b). However, for induced 0 + smolt, serum IgM levels remained low throughout the ‘‘winter’’ photoperiod, and were found to increase during the ‘‘summer’’ continuous photoperiod (Melingen and Wergeland, 2000). A synchronous pattern was not found for serum proteins, indicating that 1 + and 0 + smolts are not always comparable. The objective of this study was to examine the impact of oil-adjuvant and antigens in injectable vaccines on the growth, serum protein and IgM profiles, and seawater adaptability in out-of-season (0 +) Atlantic salmon smolt.
2. Materials and methods 2.1. Fish stock and rearing conditions Atlantic salmon (Salmo salar L.) fingerlings were obtained from AquaGen, Bolaks (Norway). The fish were healthy and had no prior history of disease and were inspected. The stock was certified free of specific pathogens. The experiment was carried out at Ewos Research Station Lønningdal, Bergen, Norway. The salmon were hatched December 30, 1998, and weighed 18.1 F 1.9 g (mean F s.d.) on June 8, 1999 at the start of the experiment. From hatching until June average temperature was maintained at 14 jC. In June average temperature was 11.5 jC, July 13.3 jC, and in August (until seawater transfer) 14.8 jC. After seawater transfer average temperature was 8.6jC. The fish were reared under continuous photoperiod (LD 24:0) at 14 jC until June 10, followed by a ‘‘winter’’ photoperiod (LD 12:12) for six weeks, and then continuous photoperiod for another four weeks before seawater transfer. After seawater transfer, the fish were reared under continuous photoperiod. The fish were reared at a density of 40– 45 kg/m3 until vaccination, using circular 6-m grey fibreglass tanks with a water depth of 170 cm. The tank had a lightproof cover (Helly Hansen-products), and a cycle timer adjusted the light provided by one metal-halogen headlight (8 w/m2). The oxygen level was adjusted by a process-control system (MCp18R, Marine Control, Norway), which supplied the particle filtered and UV-treated inlet water (0.5 ppt seawater added for buffering capacity) with supplemented oxygen to maintain at least 80% oxygen saturation in the outlet water. From the time of vaccination and the remaining experimental period, the fish groups were reared in separate 1 m grey square fibreglass tanks. The flow through inlet water was regulated to maintain above 70% oxygen saturation in the outlet water. The fish were fed from automatic feeders in excess of satiation with a commercial dry diet (Vextra, Ewos) with the manufacturers’ recommended pellet size. 2.2. Vaccination A commercial oil-adjuvanted vaccine containing Vibrio salmonicida, V. anguillarum, Aeromonas salmonicida and infectious pancreas necrosis (IPN) virus—Lipogen Quattro (Aqua Health, Canada)—was used. The fish were starved for 24 h and anaesthetised using metacaine (50 mg/l) prior to vaccination. At the time of vaccination on July 14, 1999, the
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fish were divided into three groups (n = 380 per group), and transferred to 1-m tanks. Temperature at time of vaccination was 15.4 jC. Fish were vaccinated according to label directions. 0.2 ml vaccine/fish was injected intraperitoneally (i.p.) to the vaccinated group (VA). This group was compared with a matching untreated group (UT), which was anaesthetized but not injected, and a group receiving 0.2 ml (i.p. injected) of a saline/oiladjuvant (Lipogen adjuvant equivalent to the Quattro vaccine, but no antigens. Prepared by Aqua Health) (AD). 2.3. Sampling procedures All fish were starved for 24 h prior to sampling. Thirty-five fish were sampled from the group prior to vaccination, and from each of the three treatment groups following vaccination according to regimes shown in Fig. 1, a total of 10 samplings. Five fish were used for flow cytometry analysis (Melingen et al., 2002), while the remaining 30 were used for serum protein and IgM analysis, for seawater tolerance test, as well as specific antibody values (Melingen et al., 2002). All fish were killed by a blow to the head before collecting the blood samples from the caudal vein using a syringe. The total body weight (BWT, g) and fork length (FL, cm) for each fish were measured at every sampling throughout the experiment. Condition coefficient was calculated as CF=(BWT/ FL3) 100. The blood coagulated at 4 jC over night, before centrifugation at 1000 g for 5 min. The serum was transferred to 1.5 ml Eppendorf tubes, and stored in aliquots at 20 jC for 24 h before transfer to 80 jC.
Fig. 1. The fish were reared under continuous photoperiod (LD 24:0) from hatching until start of the ‘‘winter’’ photoperiod (LD 12:12) on June 10. Six weeks later, a new continuous photoperiod was introduced, and the fish were transferred to seawater another four weeks later. During the freshwater period, sampling was performed at nine different times, and one sampling was performed after seawater transfer, all as indicated (*).
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2.4. Total serum protein and IgM determination Total serum protein was quantified using the Biuret Protein Assay with a bovine serum albumin (BSA, Sigma, USA) standard as described in Melingen and Wergeland (2000). Serum IgM was determined using a single radial immunodiffusion assay (SRID), as described by Melingen and Wergeland (2000). Rabbit antiserum to Atlantic salmon IgM was prepared as described by Ha˚varstein et al. (1988). 2.5. Seawater tolerance From vaccination and until seawater transfer, fish from all groups (UT, AD and VA, n = 12) were removed and transferred to seawater, and their hypoosmoregulatory ability assessed by measuring serum chloride levels after 96-h seawater tolerance test (33 – 34x ) at 10 jC. Their corresponding control groups (UT-C, AD-C and VA-C, n = 30) were measured using the isolated serum described in Section 2.3. Serum chloride levels were measured using 20 Al samples in a Radiometer CMT 10 titrator (Radiometer, Denmark). 2.6. Data processing and statistics Results are given as arithmetic mean values. Significant differences within and between groups were determined by one-way and two-way ANOVA, respectively, followed by Newman –Keuls post hoc test. Differences were considered significant when P < 0.05.
3. Results 3.1. Growth and condition coefficient The body weight and growth rate of the three treatment groups is summarized in Fig. 2. No significant differences (n.s.) in body weight were observed until three weeks post vaccination, when VA was significantly smaller than both AD and UT ( P < 0.05). VA remained smaller than UT and AD for the remainder of the experimental period ( P < 0.05). The CF increased from June 8 (1.10 F 0.07) to June 22 (1.16 F 0.25) ( P < 0.05), but remained stable for the rest of the experimental period. There were no significant differences between UT, AD and VA at any time. 3.2. Total serum protein Total serum protein levels are summarized in Fig. 3. The total serum protein tended to increase (n.s.) in all groups from introduction of the ‘‘winter’’ photoperiod (52.1 F 7.2 mg/ml) until four weeks later (58.0 F 9.6 mg/ml), but declined significantly in all three treatment groups at the time of vaccination (44.3 F 10.2 mg/ml) ( P < 0.001). One week
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Fig. 2. Body weight (bar indicates s.d., n = 35) of fish in the UT, AD and VA groups during the parr – smolt transformation period.
Fig. 3. Total serum protein concentration was measured by the Biuret Protein Assay (bar indicates s.d., n = 30) in UT, AD and VA throughout the experimental period.
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after vaccination, the protein level began to increase back to normal levels for UT, while both AD and VA remained significantly lower for two weeks ( P < 0.05). Two weeks after vaccination and until seawater transfer, there were no significant differences in total serum protein between VA, AD and UT. Two weeks after seawater transfer, a significant reduction in total protein was observed for UT ( P < 0.01), and at the same time a significant increase was observed for AD ( P < 0.05), while the values for VA remained stable. 3.3. Total serum IgM The serum IgM levels remained stable (0.30 F 0.15 mg/ml) until time of vaccination, when a significant increase was observed (0.71 F 0.10 mg/ml) ( P < 0.001) in all treatment groups (Fig. 4). One week after vaccination, a significant decrease in serum IgM was observed for all three groups ( P < 0.01), with UT having a significantly higher (0.59 F 0.12 mg/ml)( P < 0.001) and VA a significantly lower (0.25 F 0.15 mg/ ml) IgM level P < 0.001). Two weeks after vaccination, VA had a significantly higher (0.97 F 0.11 mg/ml) serum IgM level than both AD (0.79 F 0.09 mg/ml) and UT (0.84 F 0.09 mg/ml) ( P < 0.01). This situation remained throughout the experimental period ( P < 0.05). UT had a significantly higher serum IgM level than AD until four weeks after vaccination ( P < 0.01), when AD was similar to UT ( P>0.05). After seawater transfer, both UT (0.23 F 0.10 mg/ml) and AD (0.34 F 0.16 mg/ml) had a significant decrease in serum IgM levels ( P < 0.01), while VA (0.91 F 0.10 mg/ ml) remained stable. At this time AD had a significantly higher serum IgM level than UT ( P < 0.01).
Fig. 4. Total serum IgM concentration measured by single radial immuno-diffusion assay (SRID) (bar indicates s.d., n = 30) in UT, AD and VA throughout the experiment.
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3.4. Serum chloride levels One week post vaccination all groups being held in freshwater experienced a significant increase in serum chloride levels ( P < 0.05) (Fig. 5). For UT (129.4 F 4.8 mmol/l) the levels remained stable until seawater transfer, while AD (127.8 F 3.8 mmol/l) and VA (129.9 F 4.1 mmol/l) decreased significantly two weeks after vaccination ( P < 0.05). VA (124.2 F 3.3 mmol/l) continued its decrease the following week ( P < 0.05). The levels then remained stable for AD and VA until seawater transfer. Two weeks after seawater transfer, UT had the largest increase in serum chloride levels ( P < 0.001), AD had a small increase ( P < 0.01), while VA remained stable ( P>0.05). At this time, UT (139.4 F 12.2 mmol/l) had a significantly higher serum chloride level than AD (126.3 F 11.7 mmol/l) and VA (126.3 F 11.7 mmol/l) ( P < 0.05). The results from the comparable seawater challenge test groups showed that one week post vaccination, all three seawater challenged groups (UT, AD and VA) exhibited significant increases in serum chloride levels ( P < 0.01). These increases continued to a maximum two weeks after vaccination ( P < 0.01), then the values decreased for all seawater challenged groups until seawater transfer. For UT the reduction in the last week was not significant. The only times there were differences between the seawater challenged groups were one week post vaccination, when AD (148.3 F 12.0 mmol/l) had a significantly lower serum chloride level than VA (165.3 F 13.1 mmol/l) and UT (166.5 F 18.7 mmol/l) ( P < 0.05), and two weeks prior to seawater transfer when VA (177.2 F 14.9 mmol/l) had a significantly higher serum chloride level than UT (162.2 F 13.0 mmol/l) and AD (159.1 F 8.9 mmol/l) ( P < 0.05).
Fig. 5. Serum chloride levels (bars indicate s.d.) for control fish groups (UT-C, AD-C and VA-C, n = 30 for each group), and for seawater challenge groups (UT, AD and VA, n = 12 for each group).
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4. Discussion Vaccination of Atlantic salmon using oil-adjuvanted vaccines may affect growth of the fish due to loss in appetite or side-effects. A better understanding of what causes this reduction is needed. Three weeks post vaccination, the vaccinated group was significantly smaller than either the untreated group or the group receiving the oil-adjuvant only. These observations are in accordance with the findings of others (Midtlyng and Lillehaug, 1998; Rønsholdt and McLean, 1999), who reported a growth reduction in fish receiving oil-based adjuvant vaccines. Based on our results, it appears that the antigens and/or interactions between the oil-adjuvant and antigens rather than the adjuvant itself may cause the growth reduction. The handling and injection process seems to have little or no effect upon growth, as AD and UT had similar growth patterns. The conclusion is consistent with Rønsholdt and McLean (1999) who determined that the effect upon growth was caused by the antigenic component (A. salmonicida) in the vaccine, rather than the adjuvant and/or interactions between the two. Colquhoun et al. (1998) also concluded that the oil-adjuvant, although an important factor, may not be completely responsible for some serious side-effects associated with vaccination such as abdominal adhesions. These side-effects affect physiological function (Poppe and Breck, 1997) and growth performance of fish (Midtlyng, 1997). Our experiment cannot demonstrate whether it is one or more of the antigens or the combination of antigens and adjuvant that cause the growth reduction observed. It has been reported from the field that fish vaccinated shortly after an IPNV outbreak have shown more side-effects than vaccinated fish without previous infection (Kaurstad, personal communication). However, Colquhoun et al. (1998) showed that pre-injection with IPNV had no effect on the degree of vaccine adhesions, while co-injection with Pseudomonas fluorescens and additional environmental stress resulted in a significant increase in adhesion level. If side-effects like abdominal adhesions are caused by a strong immune response from the antigens, or the combination of antigens and adjuvant, and not by the adjuvant itself, one could consider vaccinating at lower temperatures when the immune activity is lower. In the present experiment, temperature at time of vaccination was 15.4 jC, which is much higher than the temperatures at which 1 + smolt are typically vaccinated. Photoperiod gives the 0 + smolt a signal that it is winter, while at the same time the temperature indicates that it is summer. The higher temperature is expected to contribute to a higher immune response for the 0 + smolt than if vaccinated at lower temperatures as for the 1 + smolt. Perhaps a lowered, but increasing temperature during ‘‘winter’’ photoperiod would be more favourable for the 0 + smolt immune response? A low and increasing stimulation over a long period may be more favourable for the fish with respect to physiological and immunological development. Total serum protein and IgM were significantly lower for the vaccinated group (VA) and adjuvant group (AD) compared to the untreated group (UT) one week after vaccination, but two weeks after vaccination and until seawater transfer the serum proteins were similar for VA, AD and UT, while in the same period VA showed higher serum IgM levels than the others. The mechanisms behind the observed changes in IgM might be due to cortisol acting on B-cells directly, or other mediators (Espelid et al., 1996). The increase in serum IgM might also be due to antigen stimulation,
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indicating stimulation by the antigen specific antibodies or non-specific IgM production caused by the combination of antigens and oil adjuvant. A similar increase in serum IgM concentration was reported for vaccinated fish one week post vaccination (Melingen and Wergeland, 2000), with increasing serum protein levels after vaccination, except for one week post vaccination when a reduction was observed for vaccinated fish. The serum IgM and protein levels were both higher after vaccination in that experiment compared to present experiment. These differences may have been caused by the different experimental protocol, as fish in Melingen and Wergeland (2000) were kept in 6-m tanks, while in the present experiment the fish were transferred to 1-m tanks at time of vaccination. This may have caused differences in water quality that may affect the immune status of the fish. The reduction in serum IgM and protein observed for VA and AD one week post vaccination may indicate stress caused by the vaccination, resulting in an increase in cortisol, which is known to inhibit B-cell activity (Maule et al., 1988, Maule and Schreck, 1991). It has been shown that B-lymphocytes are directly affected by cortisol, and reduced plasma IgM levels were found in rainbow trout after cortisol administration (Hou et al., 1999). A possible redistribution of cells may also cause a lower B-cell activity for blood leucocytes. Cortisol is known to inhibit some components of the immune response and enhance other parts that may be functional in stressful situations. Effects of cortisol on leucocyte viability are also cell type-specific. For example, B-cells from carp are especially sensitive to cortisol, whereas thrombocytes and cells in the T-cell fraction are insensitive (Weyts et al., 1998). A loss of sensitivity to cortisol in leucocytes from juvenile salmonids during winter has been previously documented (Slater and Schreck, 1998), and it has been indicated that the cortisol levels during smolting probably are controlled more by photoperiod than by endogenous rhythm or temperature (Olsen et al., 1993). Time of vaccination for 0 + smolt in the present experiment was during the ‘‘winter’’ photoperiod, which probably will not correlate to a normal winter, as the temperature in this experiment was much higher. With the high temperatures used in present study, a stronger immune response is expected than if vaccinated at lower temperatures. A reduction in serum protein levels after seawater transfer for UT may indicate that the physiological status, measured by concentration of serum IgM and proteins, of UT differ from AD and VA. The vaccine and/or adjuvant seem to cause a major physiological impact on the fish as indicated by variations in serum IgM and protein levels. The consequences of or reasons for these fluctuations are unknown. This may be caused by endocrine interactions with the immune system (Weyts et al., 1999), or other physiological factors may be involved. The adjuvant seems to have an impact during seawater transfer, as the serum IgM levels for AD does not have a similar reduction as UT. This may be due to non-specific stimulation by the adjuvant at time of seawater transfer, while these cells have been down regulated or inhibited during parr – smolt transformation. This observation for serum IgM correlates with the observation for serum proteins. There are no indications of a negative effect on seawater capability caused by vaccination. However, after seawater transfer, the serum chloride levels increased more for UT than for VA and AD. This may indicate a positive effect caused by the vaccine or
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adjuvant, where physiological or endocrine impact caused by the vaccine/adjuvant result in increased seawater adaptability as measured by serum chloride levels. Using the 96-h seawater challenge test, a totally different profile was found compared to using a 24-h seawater challenge test for similar smolt as described by Melingen and Wergeland (2000). It is shown that the acute hyperosmotic stress and duration of the stabilization period during seawater acclimation of Atlantic salmon is temperaturedependent (Handeland et al., 2000). However, Handeland et al. (2000) were not able to detect any differences in plasma chloride levels between groups acclimated to temperatures of 4.6 –18.9 jC. The present experiment indicates that the increased serum chloride levels found by Melingen and Wergeland (2000) after vaccination using a 24-h seawater challenge test, is just a short-term effect which is not present after 96-h in seawater, and the 96-h seawater challenge test was concluded to be more appropriate than the 24-h test. The oil-adjuvanted vaccine may affect the physiological status of the fish resulting in a different serum chloride profile during the first 24-h in seawater, but what triggers this physiological effect is not known. Adjuvants are foreign to the fish, and if appropriately administered they lead to sterile inflammations that attract the various cells of the aseptic defence system. Most adjuvants directly or indirectly stimulate the generation of IL-11, but also other factors that support growth and differentiation (Leenaars et al., 1997). Weyts et al. (1999) have reviewed the complex interactions between the immune- and endocrine system, but there are still potential interactions between these systems that are not understood and can affect the physiological status of the fish. The results indicate that the vaccine used had a negative effect on growth in the observation period. The changes in serum protein and IgM also indicate changes after vaccination, and resulted in elevated IgM levels for VA. Vaccination was carried out at a time when levels of serum IgM were low, which might indicate a favourable time to vaccinate Atlantic salmon at high temperatures. If some of the reported side-effects from using oil-adjuvanted vaccines are caused by a strong immune response, higher IgM levels indicating higher immune activity, may result in more side-effects. The physiological and immunological status of a 30 – 40 g 0 + parr/smolt will probably be different compared to a 1 + parr/smolt of similar size due to different photoperiods and temperatures. These possible differences may give different responses to oil-adjuvanted vaccines at the time of immunization.
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