Influence of dietary glucan and vitamin C on non-specific and specific immune responses of rainbow trout (Oncorhynchus mykiss)

Aquaculture 143 (1996) 123-I 33

Influence of dietary glucan and vitamin C on non-specific and specific immune responses of rainbow trout ( Oncorhynchus mykiss) Viviane Verlhac a7*, Jacques Gabaudan a, Alex Obach ‘, Willy Schiiep b, Reid Hole ’ a CRNA, Socihe’ Chimique Roche, BP 170, 68305 Saint-Louis Cedex, France b Analytical Department, F. Hoj7inann-La Roche Ltd., CH-4002 Basle, Switzerland ’ Nutreco Aquaculture Research Centre (ARC), P.O. Box 353, N-4033 Forus, Norway Accepted 3 December 1995

Abstract A trial was conducted to determine the effect of a combination of dietary glucan and vitamin C on the immune response of rainbow trout. After 3 weeks of adaptation on a control diet (without glucan but containing 150 ppm vitamin C), rainbow trout were fed the experimental diets containing yeast glucan and vitamin C at 150, 1000 and 4000 ppm for 2 weeks and then switched back to the control diet for the following 4 weeks. Macrophage activity (chemiluminescence response), complement activation and lysozyme levels were monitored just after feeding the experimental diets (week 0) and 2 and 4 weeks later. The kinetics of antibody response after vaccination against enteric redmouth disease were determined. The fish were immunised at week 0, at the end of the experimental feeding. Tissue ascorbic acid contents were monitored every 2 weeks and reflected the dietary treatments. No differences were observed in complement levels activated via the classical pathway. Two weeks after feeding the experimental diets, alternative pathway of complement activation and chemiluminescence response were enhanced by high doses of vitamin C. Significant enhancement of macrophage activity was still evident at week 2. No significant differences were observed in lysozyme levels. The specific immune antibody response was enhanced following vaccination, when glucan was also present in the diet. Keywords:

Immunomodulation; Nutrition; Glucan; Vitamin C; Rainbow trout

* Corresponding author. Tel.: 33 89 69 69 76; fax: 33 89 69 66 05. 00448486/96/$15.00 Copyright 0 1996 Elsevier Science B.V. AI1 rights reserved. SSDI 0044-8486(95)01238-9

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1. Introduction In mammals, glucans, insoluble polysaccharides derived from yeast cell walls, have been shown to potentiate non-specific immunity and to be effective in anti-bacterial defence (Biigwald et al., 1982; Di Luzio, 1985; Sherwood et al., 1987). Glucan injection to fish led to an enhancement of the immune response such as macrophage activities (Jeney and Anderson, 1993; Jorgensen et al., 1993a; Jorgensen et al., 1993b), complement activation and lysozyme level (Engstad et al., 1992). Furthermore, several studies have shown the influence of injected glucan on anti-bacterial defence and vaccination efficiency (Yano and Mangindaan, 1989; Robertsen et al., 1990; Nikl et al., 1991; Yano et al., 1991; Rprrstad et al., 1993; Aakre et al., 1994). When given orally, glucans were able to stimulate the immune response and disease resistance of fish (Raa et al., 1992; Nikl et al., 1993; Siwicki et al., 1994). Besides its crucial role in preventing deficiency symptoms at the minimum requirement level for fish, dietary vitamin C has been shown to stimulate immune responses such as macrophage activities, cell proliferation, natural killer cell activity, complement and lysozyme levels (Blazer, 1982; Li and Lovell, 1985; Verlhac et al., 1993; Waagbo et al., 1993). Several authors have demonstrated an improvement of the specific antibody response to vaccination by dietary vitamin C (Li and Lovell, 1985; Navarre and Halver, 1989; Waagbe, et al., 1993; Dunier et al., 1995). The role of vitamin C as an immunomodulator was emphasised by the presence of intracellular ascorbic acid (AA) in leukocytes at concentrations related to dietary levels (Verlhac and Gabaudan, 1994). The improvement of disease resistance by dietary vitamin C was reviewed by La11 and Olivier (1993) and Waagba (1994). The aim of this study was to determine the role of a dietary combination of yeast glucan and vitamin C (as ascorbate polyphosphate) in modulating the non-specific and specific immune. responses of rainbow trout.

2. Material and methods 2.1. Experimental

design

The experimental design was based on the feeding program established for prophylaxis with glucan, by Nutreco ARC whose application has led to an improvement of disease resistance in salmonids (Onarheim, 1992). After 3 weeks of adaptation on a control diet (no glucan and 150 ppm vitamin C), fish were fed the experimental diets (glucan and vitamin C in different combinations) for 2 weeks and then switched back to the control diet for the following 4 weeks. The immune response was investigated at the end of the experimental feeding period (week O), and at weeks 2 and 4 following the end of the experimental feeding period. 2.2. Animals and rearing conditions One-year-old rainbow trout (Oncorhynchus 72.5 k 1.4 g were obtained from the Institute

mykiss) with an initial body weight of for Fish Pathology (Berne, Switzerland).

V. Verlhnc et al./Aquaculture Table 1 Declared and measured

143 (1996) 123-133

vitamin C content of the experimental

Treatment

Yeast glucan

Control Glu+Vit C 150 Glu+Vit C 1000 Glu + Vit C 4000

+ + +

125

diets Vitamin C (mg ascorbic acid equivalent Declared Measured 150 150 1000 4000

kg-

’ feed)

145 152 1073 4311

Fish (n = 540) were divided between 12 205-l tanks which are part of a recirculating system supplied with ground water. Three tanks were randomly assigned to each dietary treatment. Water temperature was recorded daily and varied from 13°C to 15°C during the experiment. 2.3. Experimental

diets and feeding

conditions

The experimental diets (Table 1) were produced by Nutreco ARC (Stavanger, Norway). Vitamin C was added as ascorbate-2-polyphosphate (ROVIMIX @ STAY-C @, Hoffmann-La Roche, Basle, Switzerland) before extrusion. All vitamin C levels are given in milligrams ascorbic acid (AA) equivalent per kilogram of feed. Glucan (MacroGard@, Biotec-Mackzymal, Tromso, Norway) derived from Succharomyces cereuisiae cell wall was included in feed before extrusion. The amount added was according to the recommendations given by the manufacturer. Diet composition is given in Table 2. Fish were fed twice a day at a rate of 1.5% body weight per day, 7 days a week throughout the experiment. 2.4. Growth Fish from each individual tank were bulk-weighed every 2 weeks starting beginning of the trial. Specific growth rate (SGR) was calculated as SGR = ln( final body weight (g))

- In( initial body weight (g))

time interval (days)

Table 2 Composition

of the experimental

diets: proximate

analysis (% dry matter)

Ingredient

% dry matter

Protein Fat Carbohydrates Ash

47.7 f 0.3 25.2kO.l 11.0+0.2 16.2kO.2

a NFE, nitrogen

(NFE) a

free extract.

x 100

at the

126

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2.5. Immune parameters Two series of tanks were allocated to the study of non-specific immune response (macrophage activity, complement and lysozyme levels) and tissue vitamin C contents. Each parameter of non-specific immune response was determined on 12 fish per treatment at each sampling time. To test 48 individual fish for each parameter at exactly 2-week intervals, the start of experimental feeding was delayed by 3 days in the two series of tanks and the same parameter was evaluated on fish from the same series throughout the experiment. The third series of tanks was allocated to studies of antibody response after vaccination against enteric redmouth disease on 30 individual fish per treatment. 2.6. Chemiluminescence

response

Headkidney neutrophil and macrophage activity isolated on a Percoll gradient was measured by chemiluminescence assays using chicken serum-opsonised zymosan as the inducer of oxidative burst and luminol as the substrate (Verlhac and Gabaudan, 1994). Light emission was measured on an automated luminometer (Bioorbit 1251, Wallac). 2.7. Classical and alternative

pathways

of complement

activation

Rainbow trout blood was collected by caudal vein puncture in heparinised syringes and centrifuged at 3000 rev min-’ for 5 min at 4°C. Plasma samples were analysed for determination of complement activation. The 50%-haemolysis assays were conducted in microplates with goldfish and rabbit red blood cells. Classical pathway determination was carried out according to Verlhac and Gabaudan (1994). The alternative pathway was determined using the method of Yano (1992) with the following modifications: the tests were performed using rabbit red blood cells as target cells in the presence of ethylene glycol tetraacetic acid (EGTA) and Mg2+. 2.8. Lysozyme

level

Lysozyme level in plasma was determined by the turbidimetric assay in microplates according to the method of Ellis (1990). Results were expressed in units of lysozyme per millilitre plasma. One unit is defined as the amount of sample causing a decrease in absorbance of 0.001 min- ’ at 450 nm. 2.9. Antibody

response

At the end of the 2 week experimental feeding, groups of 30 fish from each dietary treatment were vaccinated against enteric redmouth disease by intraperitoneal injection of lo9 formalin-killed Yersinia ruckeri per fish in 0.2 ml of saline (Yersivax, RhaneMe’rieux Laboratories, Lyon, France). Every 2 weeks after immunisation, serum from all the fish was collected and antibody levels determined by enzyme-linked immunosorbent assay (ELISA) (Vergnet and Dunier, 19931, for up to 10 weeks post-treatment. Antibody

V. Yerlhuc et ul./Aquaculrure

levels were determined by measuring with a 540 nm reference filter.

?43 (19996) 123-133

the absorbance

127

of sera diluted at 1:400 at 450 nm

2.10. Vitamh C analyses Ascorbic acid (AA) contents of liver, headkidney, plasma and leukocytes of five fish per treatment were measured by the fluorimetric assay (Vuilleumier and Keck, 1989) prior to the experimental feeding and at each sampling time. Ascorbate polyphosphate content of the experimental diets was also determined according to Wang et al. (1988). 2. Ii _ Statistical analysis Analysis of variance and Duncan’s multiple range test were run to compare the dietary treatment values at any one sampling time using the SAS@ program. Statistical analysis was run in the same way for all parameters tested.

3. Results

and discussion

Growth performance of fish was represented by a specific growth rate ranging from 1.90 to 1.96% per day among treatments throughout the experiment. Feed ascorbic acid (AA) contents (Table 1) reflected the different amounts added to the feed before extrusion. Liver and plasma AA confents (Fig. 1) responded to the dietary doses of vitamin C at the end of feeding the experimental diets (week 0). Two

PLASMA

LIVER

~ Gl” + Yit C ,50 mGtu +

Vit C

1000

I Glu + Vit C 4000 week -2 week 0 week 2 week 4 HEADKIDNEY 2501

week

-2 week

week -2 week0 week 2 week 4 I

0 week 2 week 4

Fig. I. Tissue ascorbic acid content of rainbow trout fed the experimental diets at the different sampling times. Results represent the mean of 5 fish per treatment. Statislics: when na significant differences were detected between the dietary treatments at any one sampling time, no letters were assigned to bars. Significant differences at p < 0.05 are indicated by different letters above bars.

V. Verlhac et aL/Ayuaculture

128

H

143 (1996) 123-133

6

x 2 x

mO

4

.E 0 2

2 ES3Glu +

Vit C 1.50

MGlu + Vit C 1000 0 week 0

week 2

week 4

Fig. 2. Effect of glucan and vitamin C on phagocytosis measured by chemiluminescence in rainbow trout at weeks 0, 2, 4 after the end of the experimental feeding. Results are under the curve of chemiluminescence (AUC in lo5 x mV x set) and represent the treatment. Statistics: when no significant differences were detected between the dietary sampling time, no letters were assigned to bars. Significant differences at p < 0.05 are letters above bars.

induced by zymosan, expressed as the area mean of 12 fish per treatments at any one indicated by different

weeks after being switched back to the control diet (week 21, both liver and plasma AA levels were strongly reduced. These results confirmed the close relationship between liver and plasma AA levels and dietary intake of vitamin C. Headkidney, in which AA content had strongly increased at week 0, still presented a significantly higher AA level at week 2 in the group fed glucan and vitamin C at 4000 mg AA equivalent kg-’ feed (Fig. 1). No significant differences were observed after 2 weeks between the groups fed 150 and 1000 ppm vitamin C. At week 4, AA concentration reached similar levels in all treatments. Differences in AA storage patterns between liver and headkidney stimulated interest in the study of AA levels in headkidney, a major lymphoid organ in fish, when investigating the relationship between vitamin nutrition and immunity. Considering peripheral blood leukocytes (Fig. 11, AA concentration was significantly higher in fish fed vitamin C at 4000 ppm compared with those fed 1000 and 150 ppm at all sampling times. These results showed that leukocytes are able to store elevated amounts of ascorbic acid in their cytosol and therefore require a longer period of time to become depleted. This could explain the slower AA depletion in the headkidney. The capacity of leukocytes to store and keep AA in their cytosol may be related to the requirement for antioxidant substances to maintain the integrity of membranes and the optimal functioning of immune cells. These results corroborated findings in humans (Evans et al., 1982), demonstrating that human mononuclear leukocytes could maintain high AA levels despite variations in plasma. Chemiluminescence (CL) response at week 0 (Fig. 2) was significantly increased in fish fed glucan and vitamin C at 4000 ppm compared with those fed vitamin C at 150 ppm with or without glucan. At this sampling time, no significant differences were

V. Verlhac

et ul./Aquaculture

143 (1996) 123-133

129

observed between the two high vitamin C supplementation levels (1000 and 4000 ppm) and no effect of glucan was observed. Two weeks after being switched back to the control diet (week 2) macrophages from fish fed glucan and vitamin C at 1000 and 4000 ppm gave a significantly higher response in CL than those from fish fed vitamin C at 150 ppm without glucan. At week 4, differences among the treatments were less evident. CL response seemed to be the most sensitive parameter because the enhancement lasted for at least 2 weeks after the end of the experimental feeding. The increase in CL response seemed to be related to vitamin C rather than to glucan. The effect of glucan on phagocytic and bactericidal capacities of macrophages was only demonstrated after injection (Chen and Ainsworth, 1992; Jorgensen et al., 1993a; Jorgensen et al., 1993b). Enhancement could be related to an increase in the production of superoxide anions (02-) and not hydroperoxides. In our experiment, the CL response was investigated using luminol as a substrate, which allowed the detection of major reactive oxygen species (ROS): hydrogen peroxide (H,O,), hydroxyl radicals (OH-) and singlet oxygen (‘0, . ). The specific production of 02-, which occurs during the initial step of the oxidative burst and can be measured using lucigenin as a substrate in the CL test was not determined. Regarding the effect of dietary vitamin C on CL response, previous experiments have shown an enhancement in the production of either O*- or the other ROS in salmonids (Verlhac and Gabaudan, 1992; Verlhac and Gabaudan, 1994) while Hardie et al. (1991) did not find any effect on 02- production in Atlantic salmon. Blazer (1982) and Verlhac et al. (1993) demonstrated a stimulatory effect of vitamin C on the ingestion of latex beads by trout macrophages. Results from the present study confirmed the modulatory role of dietary vitamin C on macrophage activity and also highlighted the need to determine which of the different phagocytic processes are affected by dietary vitamin C. With respect to another aspect of non-specific immune response, complement activation, the results of this trial (Fig. 3) showed that, at week 0, the two groups of fish fed glucan and vitamin C at 1000 and 4000 ppm presented higher complement levels when activated through the alternative pathway than those fed 150 ppm with or without glucan, which reached similar complement activity. In contrast to the CL response, the enhancement was short-lived and observed only at week 0. Our results showed that the increase in the alternative pathway of complement activation was related to vitamin C rather than to glucan but this remains to be confirmed. An effect of glucan on the alternative pathway of complement activation has been demonstrated in mammals (Glovsky et al., 1983) and also in fish (Engstad et al., 1992) in the case of intraperitoneal injection of glucan. The results from this trial did not show such an influence of glucan on this parameter but the administration route was different. Regarding the effect of glucan on complement levels activated through the classical pathway, no data seemed to be available in fish and this study did not show any effect of dietary glucan. Concerning the effect of vitamin C on complement activation via the classical pathway, Verlhac and Gabaudan (1994) did not observe any effect of dietary vitamin C on this parameter in rainbow trout and Atlantic salmon fed 1000 ppm for at least 2 months. On the other hand, Li and Love11 (1985), Hardie et al. (1991) and Waagbo et al. (1993) showed an increase in complement activity in fish fed vitamin C at doses exceeding 2000 ppm, following long-term feeding. It may, therefore, be possible that activation of

130

V. Verlhac et al./Ayuaculture

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CLASSICAL PATHWAY

ALTERNATIVE PATHWAY

50

E 2 I B

501

40

30

E ‘3i g

20

5: 5

10 &IPGIu + Vii C lOOil 0

week 0

week 2

week 4

week 0

week 2

week 4

Fig. 3. Effect of glucan and vitamin C on classical and alternative pathways of complement activation in rainbow trout at weeks 0, 2, 4 after the end of the experimental feeding. Results are expressed in CH50 and APSO units per ml of serum and represent the mean of 12 fish per treatment. Statistics: when no significant differences were detected between the dietary treatments at any one sampling time, no letters were assigned to bars. Significant differences at p < 0.05 arc indicated by different letters above bars.

the classical pathway requires high doses of vitamin C during longer periods. In our experiment, feeding fish with vitamin C at 4000 ppm for 2 weeks, did not lead to any enhancement of the classical pathway of complement activation. Further investigation is required in order to determine whether the levels of vitamin C fed prior to trials and the feeding duration could influence this parameter. Results on lysozyme levels from the plasma of rainbow trout ranged from 840 to among the different groups and sampling times (Table 3). No 1140 units ml-’ significant differences between the dietary treatments appeared at any sampling times and individual variations were very high in all of the treatment groups. Engstad et al. (1992) observed an increase in plasma lysozyme in Atlantic salmon injected with glucan. In their experiment, lysozyme activity started to increase 1 week after intraperitoneal injection of 10 mg glucan and reached a peak after 2 weeks. Waagbpr et al. (1993) showed an increase in lysozyme levels in fish fed vitamin C at 4000 ppm. The kinetics of antibody formation determined after vaccination at week 0 led to higher antibody levels in fish fed diets supplemented with both glucan and vitamin C

Table 3 Effect of glucan and vitamin C on plasma lysozyme

level (units ml-

’ ) in rainbow trout

Treatment

Week 0

Week 2

Week 4

Control GlufVitC 150 Glu + Vit C 1000 Glu + Vit C 4000

842.2 * 278.4 840.0 + 276.4 902.2 ?c 413.4 974.4+ 547.5

614.4+ 186.5 710.9f202.1 770.3 + 302.7 678.8 * 396.1

189.1 k269.9 1081.7+ 363.6 1030.0f410.0 780.7* 196.1

Results are the mean of 12 fish per treatment. means at any one sampling time.

No significant

differences

were observed between the treatment

V. Verlhuc et al./Aquaculrure Table 4 Effect of glucan rainbow trout

and vitamin

C on antibody

response

143 (19961 123-133

after vaccination

131

against

enteric redmoutb

Treatment

Week 0

Week 2

Week 4

Week 6

Week 8

Control Glu+VitC 150 Glu + Vit C 1000 Glu+VitC4000

0

0.10 + 0.06 0.12+0.10 0.11 kO.06 0.14+0.06

0.31 kO.lSb 0.53+0.31a 0.54 * 0.32a 0.56 + 0.37a

0.21 0.35 0.39 0.37

0.18*0.1Sb 0.23 Jo 0.17a 0.24+0.18a 0.3 1 f 0.22a

0 0 0

t0.14b *0.25a + 0.28a *0.23a

disease

in

‘Week10 0.13*0.10 0.17f0.13 0.20+0.14 0.17+0.16

Results are expressed as tire optical density and represent the means of 30 fish per treatment. Significant differences at P < 0.05 were indicated by different letters. When no significant differences were detected between the dietary treatments at any one sampling time, no letters were assigned to means.

compared with the control (Table 4). This difference was still observed 8 weeks after immunisation. The three groups of fish fed the different vitamin C levels had similar antibody levels. Ten weeks after immunisation, antibody levels had decreased in all groups and the effect of glucan had disappeared. In our experiment, fish were immunised at week 0 which means the fish had been fed glucan for 2 weeks when vaccination occurred. These results are in agreement with Chen and Ainsworth (1992) who demonstrated that glucan and vaccine injection should occur on the same day to improve vaccine efficiency. Since it has been demonstrated in mammals that glucans act at the cellular (macrophage) membrane level via a specific receptor (Czop and Austen, 19851, it may be possible that the linkage of glucan particles to specific receptors modifies the structure of the membrane in such a way that the macrophage becomes more efficient in the function of antigen processing and presentation. It is also possible that vitamin C could have helped in this matter because its strong influence on macrophage activities have been clearly demonstrated. Most of the trials on the modulating effect of vitamin C on antibody production after vaccination have shown an improvement of the response (Li and Lovell, 1985; Navarre and Halver, 1989; Waagbo et al., 1993; Dunier et al., 1995). In our experiment, drawing conclusions on the specific effect of glucan or vitamin C on antibody production remains difficult due to the absence of groups treated with the high vitamin C doses but without glucan.

4. Conclusion The results of this first trial showed that, under the experimental conditions used, the combination of glucan and high doses of vitamin C for a short feeding period had a stimulatory effect on the non-specific and specific immune parameters tested except for plasma lysozyme. The dose response in relation to the feeding period of glucan and vitamin C should be further investigated, as well as the immunomodulatory role of dietary glucan, which has been little studied. These results confirm the important role of micronutrients and feed additives in fish health status and suggest means for prophylaxis.

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Acknowledgements Denis Constant and Christophe Nutrition (CRNA, So&W Chimique

Zickler from the Research Centre for Animal Roche) have collaborated in this study.

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