Fish Physiology and Biochemistry vol. 12 no. 1 pp 61-73 (1993) Kugler Publications, Amsterdam/New York

Dietary vitamin C, immunity and disease resistance in Atlantic salmon (Salmo salar) Rune Waagbo 1 , Johan Glette 2 , Espen Raa-Nilsen 3 and Kjartan Sandnesl 2 I Institute of Nutrition, Directorateof Fisheries, P.O. Box 1900, N-5024 Bergen, Norway; Institute of Marine Research, Division of Aquaculture, P.O. Box 1870, N-5024 Bergen, Norway; 3University of Bergen, Centre of Biotechnology, N-5020 Bergen, Norway Accepted: January 11, 1993 Keywords: Atlantic salmon, fish nutrition, immunity, disease resistance, vitamin C, megadose ascorbic acid, ascorbate-2-monophosphate, incorporation Abstract Presmolt Atlantic salmon were fed a fish meal based experimental diet supplemented with graded levels of ascorbate-2-monophosphate (AP), equivalent to 40, 400, 2000 and 4000 mg ascorbic acid (AA)/kg for 6 months prior to a bacterial challenge experiment. The liver AA concentration reflected the dietary intake of AP, but not linearly. Growth, hematology and acid phosphatase activity in zymosan stimulated macrophages were not affected by dietary AP. Serum hemolytic complement activity was higher in fish fed the highest AP level, but the variation was not significant. Production of specific antibodies was significantly higher in fish fed the highest AP level 11 and 17 weeks after vaccination. Bacterial challenge with Aeromonas salmonicida showed increased survival in the 4000 AP group. Lysozyme activity in headkidney and serum complement activity and serum iron in fish surviving the challenge were higher in the 4000 AP group, indicating important roles of vitamin C on lysozyme, complement and iron in non-specific disease resistance. The results indicate that high dietary levels of AP favourably affect health in Atlantic salmon. Disease resistance was, however, not correlated with the AA status in the liver.

Introduction Vitamin C is essential for fish, and as recently reviewed by Sandnes (1991) a deficiency of this vitamin affects several metabolic functions. Among others, these include collagen formation and wound healing, hematopoiesis, detoxification reactions and immune factors (Sandnes 1991). As in landliving animals, vitamin C deficiency has been shown to reduce resistance against infections (Beisel 1982; Durve and Lovell 1982; Li and Lovell 1985; Wahli et al. 1986; Navarre and Halver 1989; Anggawati-Satyabudhy et al. 1989; Sandnes

et al. 1990; Hardie et al. 1991). Further, immune stimulating effects and increased disease resistance in fish fed high doses of vitamin C have been reported in channel catfish, Ictalurus punctatus (Durve and Lovell 1982; Li and Lovell 1985), rainbow trout, Oncoryhnchus mykiss (Blazer 1982; Wahli et al. 1986; Navarre and Halver 1989; Anggawati-Satyabudhy et al. 1989; Verlhac et al. 1993) and Atlantic salmon, Salmo salar (Hardie et al. 1991). The mode of action of vitamin C on immune functions is not, however, fully understood. On the other hand, immunological and physiological mechanisms participating in the host resistance

Correspondence to: Rune Waagbo, Institute of Nutrition, Directorate of Fisheries, P.O. Box 1900, N-5024 Bergen, Norway.

62 against infections have been successfully used in evaluation of nutritional status (Chandra 1988). In fish, vitamin C has been shown to stimulate serum hemolytic complement activity, proliferation of immune cells, phagocytosis, the release of signal substances and antibody production (Durve and Lovell 1982; Li and Lovell 1985; Navarre and Halver 1989; Hardie et al. 1991). However, some divergent results have been reported, which could probably be attributed to experimental design and fish species, the dietary level and form of vitamin C used and interacting nutrients (Bell et al. 1984; Lall et al. 1989; Leith et al. 1989; Erdal et al. 1991). The phosphate derivatives of ascorbic acid have been found to be bioactive as a source of vitamin C in salmonids (Waagbo et al. 1991; Sandnes and Waagbo 1991; Sandnes et al. 1992) and the minimum dietary requirement for growth in Atlantic salmon has been estimated to 10-20 mg AA equivalents per kg of feed, using ascorbate-2monophosphate (AP) as vitamin C source (Sandnes et al. 1992). In the present experiment the objective was to study the effect of increased levels of dietary vitamin C, in the form of AP, on immunity and resistance against infection in Atlantic salmon. Analyses of hematological parameters, serum hemolytic complement activity, total serum protein, albumin and iron concentrations, headkidney lysozyme activity and antibody production were carried out. Finally, the fish were challenged against Aeromonas salmonicida.

Materials and methods

cium L-ascorbate-2-monophosphate salt (F. Hoffmann La-Roche, Switzerland). The feed composition was given by Sandnes et al. (1992). The fish were fed according to standard feeding tables for Atlantic salmon (Austreng et al. 1987) corrected for biomass and water temperature during the trial which lasted 6 months prior to a final bacterial challenge experiment. The salinity was kept at 3 ppt by addition of filtered seawater to improve water quality. Water temperature was measured regularly and the mean monthly temperature during the experimental period from July 1990 to medio January 1991 were 10.4, 12.1, 11.4, 10.4, 8.4, 5.6 and 4.6°C, respectively.

Growth and mortality Body weight, length and condition factor (= weight x 100 x length - 3) and total mortality (sampled fish excluded) were recorded at the end of the experiment.

Liver ascorbate incorporation Ten fish from each tank were sacrificed after one week and then every second week and the AA status in pooled liver samples was analysed. At each subsequent sampling for immunological analyses, liver AA determinations were carried out as well. Liver AA was analysed fluorimetrically according to the method described by Roy et al. (1976). Analyses of AP in the diets were performed according to a reverse phase HPLC method (Ogata, Showa Denko K.K., Japan, pers. comm.).

Fish, feeds and experimental conditions Atlantic salmon parr which had been fed a fish meal based diet supplemented with ascorbate-2-monophosphate (AP) equivalent to 40 mg ascorbic acid (AA)/kg for 6 months were used in this study. In the feeding study 210 fish (mean weight 20 g) were distributed into 1 m3 fibreglass tanks and fed in duplicate the experimental diets supplemented with 40, 400, 2000 and 4000 mg AA equivalents of cal-

Vaccination study After 10 weeks of feeding the experimental diets, 30 fish (54-60 g) in each tank were vaccinated against cold water vibriosis with formalin killed Vibrio salmonicida(Norbio A/S, Norway). Twenty fish in each tank were sham-injected with the vaccine medium only. Four weeks thereafter the fish were

63 boosted with the vaccine or vaccine medium. At weeks 0, 7, 11 and 17 post-vaccination, blood was drawn from the caudal vein. Specific antibodies, total protein and albumin in serum samples (obtained after centrifugation at 3000 rpm for 10 min) from vaccinated (7 weeks, n = 4; 11 weeks, n = 10; 17 weeks, n = 5), sham-injected and untreated fish (7 weeks, n = 3; 11 weeks, n = 5) from each tank were quantified. Specific antibodies against Vibrio salmonicidawere measured using an ELISA assay described by Havardstein et al. (1990). Total protein and albumin were analysed according to the methods described by Sandnes et al. (1988). Hematological analyses including hemoglobin (Hb), hematocrit (Hct) and red blood cell count (Rbc) were performed on heparinized blood according to Sandnes et al. (1988). Hb was analysed at time of vaccination and 7 and 11 weeks after, in vaccinated, sham-injected and untreated control fish. Hct was analysed initially and after 11 weeks and Rbc after 11 weeks.

Immunological studies Serum samples obtained from untreated fish before and after bacterial challenge were analysed for serum total hemolytic complement activity assayed according to conventional methods using sheep red blood cells (SRBC) coated with A. salmon antiSRBC antibodies obtained according to the procedure described by Sakai (1981). The fish sera were diluted 1:5 prior to titration, and spontaneous hemolysis and total hemolytic complement activity were measured in microtitre plates (Ingram 1990). Headkidney macrophages were isolated by Percoll centrifugation, counted and cultured according to Secombes (1990). The ability to secrete acid phosphatase was recorded after stimulation with opsonized zymosan, prepared according to Scott and Klesius (1981) using Atlantic salmon serum. One ml of isolated macrophages (106 /ml) was stimulated with 0.2 ml opsonized zymosan (5 mg/ml). 0.2 ml Hanks' balanced salt solution was added to controls. After 24h incubation at 200 C the tubes were centrifuged (3000 rpm, 10 min) and the cells and supernatants were frozen at -80 0 C until

analysis. Prior to acid phosphatase determination the cells were diluted to equal volumes, acidified with 10 1 of 10% acetic acid, and analysed according to a conventional Technicon method (Technicon method no. SA4-0334J89). The results were calculated as the ratio supernatant acid phosphatase/cell acid phosphatase, from both zymosan stimulated (Z) and non-stimulated cells (C). Further, the stimulation index was calculated as the ratio (Z/C).

Challenge with Aeromonas salmonicida After 27 weeks, 50 fish from each feeding regime were challenged with Aeromonas salmonicida.The administration route of the pathogen was by cohabitant challenge as described by Erdal and Reitan (1991). The fish were acclimatized in tanks (1 m3 ) for two weeks prior to the challenge. Water temperature was 12°C and salinity, 2 ppt. A stock suspension of A. salmonicida (109 bacteria/ml) was kept frozen (- 700C) in 20% glycerol. One ml of the suspension in 100 ml of Brain Heart Infusion Medium (Difco, Michigan, USA) were incubated for 24h at 22°C. After adjustment to a working suspension of 105 viable bacteria/ml, eight external cohabitants of similar size as the experimental fish were intraperitoneally injected with 0.1 ml of the working suspension and added to each tank. Dead fish were collected daily and Tryptic Soya Agar plates were smeared with kidney samples to confirm infection with A. salmonicida. As the cohabitant challenge resulted in an average loss of only 10% in the course of four weeks, the remaining fish were challenged with an intraperitoneal injection of 0.1 ml of the working suspension of A. salmonicida33 days after the start of the cohabitant challenge. The surviving fish from the challenge experiment were weighed. The liver and spleen were dissected and weighed and AA was analysed in the liver. Lysozyme activity was analysed in headkidney according to a turbidimetric assay (Parry et al. 1965) described by Ellis (1990). Serum samples were analysed for serum total complement activity. Serum

64 Table 1. Mean final (SEM) weight, length, condition factor and mortality (%) in Atlantic salmon fed increasing levels of dietary ascorbate-2-monophosphate (AP) for 27 weeks Feed AP (mg/kg)

(n)

Weight (g)

Length (cm)

Condition factor

Mortality (%0)

40 400 2000 4000

200 212 219 178

92 87 94 98

19.3 18.9 19.5 19.9

1.16 1.18 1.15 1.13

1.2 0.7 0.5 0.0

(3) (3) (3) (3)

(0.3) (0.3) (0.3) (0.3)

(0.01)al (0.01)a (0.01)b (0.01)b

IMeans in one column sharing a common superscript letter are not statistically different (p > 0.05).

iron was analysed according to a micro sample method by use of graphite furnace atomic absorption spectrophotometer, Perkin Elmer 5000 (Andersen, pers. comm.).

Statistics The data were statistically evaluated by means of a one way ANOVA analysis. Differences between two groups were evaluated using a t-test and difference in antibody response and complement activity were tested using a non-parametric MannWhitney U-test, all tests within a Statgraphics statistical program (Plus* WareIM Product, STSC).

Results Growth and mortality Growth and mortality during the feeding period of 27 weeks did not differ according to dietary AP (Table 1).

AA liver content The AP content analysed in the diets confirmed the feed supplementations showing recoveries between 94 to 102%o. The liver AA retention during the first 10 weeks of the trial is presented in Figure 1. The liver levels increased in all dietary groups and reflected the dietary contents of AP, but the differences were most pronounced after one week of feeding.

Vaccination study At vaccination, 10 weeks after the start of the experiment, the liver AA status was significantly different (p < 0.05), except for the two lowest AP levels (Fig. 1). The liver AA status of fish sampled 7 and 11 weeks after vaccination was significantly different (p < 0.05) between all groups at each sampling, with a systematic elevation from week 7 to 11 (Table 2). After week 17 there were again lower levels with no difference in AA status between the two highest dietary groups. Specific antibodies against Vibrio salmonicida analysed 7, 11 and 17 weeks after vaccination are presented in Table 3. No differences were observed between vaccinated fish in the dietary groups after 7 weeks. However, fish in the 4000 AP group increased antibody titre significantly during the next 4 weeks compared to the other groups (p < 0.05). A significant elevated antibody level in vaccinated fish from the 4000 AP group was also observed in serum collected after 17 weeks. Sham-injected fish and untreated control fish from all dietary groups showed antibody concentrations below 0.1 absorbance units (Table 3). Table 4 shows total protein and albumin in sera from vaccinated, sham-injected and control fish after vaccination. After 7 weeks the concentration of protein and albumin in pooled serum samples was highest in the 4000 AP group. Serum protein and albumin from vaccinated fish after 11 weeks showed significantly higher levels in 40 and 400 AP groups compared to the other groups. Regardless of treatment, the serum concentrations of protein and albumin were higher in the 40 AP group. Except for the 400 AP group, no significant differences were

65 Liver AA (mg/kg)

nen

ZOu

20C 150 100 50 n 0

0

1

2

3

4

5

6

7

8

9

10

Weeks AA equivalents of AP (mg/kg): -40

-400 x 2000

4000

1

Fig. 1. Mean liver ascorbic acid concentration (AA) during the first 10 weeks of the study with Atlantic salmon fed different levels of ascorbate-2-monophosphate (AP) in the diet (at last sampling n = 10, range of SEM 7-9). Table 2. Mean (SEM) liver ascorbic acid (AA), 7, 11 and 17 weeks after vaccination against cold water vibriosis (Vibrio salmonicida) Liver AA (g/g) Feed AP (mg/kg)

7 weeks I

11 weeks 2

40 400 2000 4000

52 108 131 148

82 167 184 221

(2)a4 (4 )b (6)c (4)d

(2) a (4)b (4)C (7)d

17 weeks 3 55 116 137 132

(3) a (5)b (7)C (4)C

In = 20. 2n = 8 pooled liver samples, each of 5 fish. 3n = 10. 4 Means in one column sharing a common superscript letter are not statistically different (p > 0.05).

seen between vaccinated, sham-injected and untreated fish after 11 weeks. Blood Hb measured prior to vaccination showed increasing Hb with increasing dietary AP (Table 5). Seven and 11 weeks later, no similar trend according to dietary regime was observed in untreated fish, showing mean Hb values in the range of 10.0-11.0 g/100 ml. Blood Hct in untreated control fish was statistically higher (p < 0.05) in the 40 AP group 11 weeks after vaccination. As for serum

protein and albumin, Hb and Hct varied somewhat according to treatment. Mean Rbc in untreated fish was highest in the 40 AP group, but only significant different from the 2000 AP group.

Nonspecific immune response Serum total complement activity presented as CH50 units/ml was higher in fish fed the two

66 Table 3. Mean (SEM) serum specific antibody level (OD4 9 2 , serum dilution 1:500) in vaccinated (V), sham-injected (S) and untreated (C) Atlantic salmon in the dietary groups, 7, 11 and 17 weeks after vaccination against cold water vibriosis (Vibrio salmonicida) Antibody response (OD4 92 ) Feed AP (mg/kg)

Treatment

7 weeks

11 weeks 2

17 weeks 3

40

V S C

0.38 (0.09)a4 0.04 (0.01) 0.02 (0.00)

0.63 (0.07)a 0.08 (0.01) 0.07 (0.00)

0.28 (0.04)a

400

V S C

0.43 (0.08)a 0.07 (0.04) 0.08 (0.04)

0.65 (0.06)a 0.08 (0.01) 0.06 (0.03)

0.33 (0.06)a

2000

V S C

0.43 (0.10)a 0.07 (0.02) 0.06 (0.03)

0.64 (0.06)a 0.09 (0.02) 0.06 (0.00)

0.30 (0.05)a

4000

V S C

0.37 (0.05)a 0.10 (0.05) 0.05 (0.01)

0.78 (0.0 6 )b 0.10 (0.02) 0.06 (0.00)

0.37 (0.06)b

IV: n = 8; S and C: n = 6. 2 V: n = 20; S and C: n = 10. 3 n = 10. 4 Means in one column sharing a common superscript letter are not statistically different (p > 0.05). Table 4. Mean (SEM) serum total protein and albumin concentration in vaccinated (V), sham-injected (S) and untreated (C) Atlantic salmon fed different levels of ascorbate-2-monophosphate (AP) in the diet, 7 and 11 weeks after the first vaccination against cold water vibriosis (Vibrio salmonicida) 11 weeks 2

7 weeks Feed AP (mg/kg)

Protein (g/l)

Albumin (g/l)

Protein (g/l)

Albumin (g/l)

40

V S C

55.5 53.0 56.1

21.7 19.4 23.3

68.0 (2.7)a3 67.7 (2.7) 72.0 (5.5)

28.3 (1.2)a 27.7 (1.4) 30.4 (2.8)

400

V S C

52.6 52.4 54.6

22.1 18.0 20.0

66.4 (1.7)a 56.6 (2.2) 55.7 (2.9)

25.9 (0.8)b 20.8 (1.0) 20.7 (1.7)

2000

V S C

52.4 50.3 58.0

21.3 17.8 24.0

60.8 (1.9)b 56.7 (2.1) 55.1 (3.2)

23.6 (1. )b 20.8 (0.7) 22.7 (1.7)

4000

V S C

58.5 57.4 59.8

23.7 23.5 26.4

60.6 (2.2)b 58.3 (3.6) 61.1 (3.6)

23.4 (1. )b 21.8 (1.8) 26.0 (1.9)

IV: n = 8, S and C: n = 6 of pooled serum samples each of 3 to 4 fish. 2V: n = 20, S and C: n = 10. 3 Means in one column sharing a common superscript letter are not statistically different (p > 0.05).

highest AP levels at the end of the experiment, but not statistically different (Table 6). The ability of stimulated isolated macrophages to produce or secrete acid phosphatase was not cor-

related to dietary AP showing ratio values of 1.29, 1.34, 1.22 and 1.25 (pooled SEM = 0.03) for the 40, 400, 2000 and 4000 AP groups, respectively.

67 Table 5. Mean (SEM) hematological analyses in vaccinated (V), sham-injected (S) and untreated (C) Atlantic salmon fed different levels of ascorbate-2-monophosphate (AP) in the diet, initially and 7 and 11 weeks after vaccination against cold water vibriosis (Vibrio salmonicida) 7 weeks 2

Initially Feed AP (mg/kg) 40

400

V S C V S C

2000 V S C 4000 V S C

Hb (g/100 ml)

7.7 (0.2)a4

8.0 (0.1)a

8.3 (0.3)ab

8.8 (0. 3 )b

11 weeks 3

Hct (°70)

Hb (g/100 ml)

Hb (g/100 ml)

Hct (0)

Rbc (*1012/1)

40 ()a

9.7 (0.3) 11.1 (0.4) 11.0 (0.3)a

9.6 (0.2) 10.6 (0.2) 10.2 (0.2)a

51 (1) 52 (1) 53 () a

1.35 (0.03)a

38 (2)

10.3 (0.3) 11.1 (0.2) 10.3 (0.4)a

9.9 (0.2) 9.5 (0.4) 10.0 (0.4)a

52 (1) 40 (3) 47 (2)

1.28 (0.04)ab

42 (I) a

10.7 (0.2) 11.0 (0.5) 10.5 (0.4)a

9.6 (0.2) 9.8 (0.4) 10.5 (0.5)a

49 (1) 44 (1) 46 (2 )b

1.22 (0.02)b

41 (2) a

10.2 (0.1) 11.1 (0.3) 11.0 (.)a

9.9 (0.2) 10.4 (0.6) 10.5 (0.6)a

49 (1) 49 (2) 48 (2)b

1.29 (0.05)ab

a

3 4 In = 10. 2 V: n = 8, S and C: n = 6. V: n = 20, S and C: n = 10. Means in one column sharing a common superscript letter are not statistically different (p > 0.05).

Table 6. Mean (SEM) serum complement hemolytic activity (CH 50 U/ml) in Atlantic salmon from the dietary groups before (n = 6) and after bacterial challenge (n = 13) Feed AP (mg/kg)

40 400 2000 4000

Complement hemolytic activity (CH 50 U/ml) After 27 weeks

Surviving fish

4.8 4.4 6.2 5.8

5.1 5.7 4.8 7.5

(0.9) (0.3) (1.8) (0.7)

(1.0) (0.6) (0.4) (1.7)

Challenge againstAeromonas salmonicida Figure 2 shows cumulative mortality during the challenge experiment. All infected cohabitants died during the first 10 days. Only minor mortality (< 10%70) occurred in fish from the 40, 400 and 2000 AP groups, and no mortality occurred in the 4000 AP group until day 30. The surviving fish were rechallenged by i.p. injection at day 33, which gave a mortality ranging from 12 to 16% in all groups. The total mortality in the 40, 400 and 2000 AP groups ranged from 20 to 24%, while the 4000 AP group showed 12°70 mortality.

Body weight, hepatosomatic index (HSI) and spleen somatic index (SSI) and liver AA from surviving fish are presented in Table 7. Body weight showed no significant differences according to dietary regime. The HSI and SSI were not influenced by the dietary regime, showing mean values of 0.88% and 0.92 ppt, respectively. Lysozyme activity in head kidney tissue (Fig. 3) from surviving fish was significantly higher in the 4000 AP group compared to the other groups (p < 0.05). Fish from the 40 AP group showed significantly lower lysozyme activity compared to the 400 and 2000 AP groups. Total serum complement activity was moderately higher in the 4000 AP group compared to the other groups (Table 6). Serum iron (Fig. 4) was significantly higher (p < 0.01) in surviving fish from the 4000 AP (1.75 mg/l) compared to the 40 AP group (0.93 mg/l).

Discussion Phosphate derivatives of AA have been shown to be good vitamin C sources in fish feeds due to their

68 Cumulative mortality % 25

_..-

20

15 _. 10

.

5

..

_ II

I

0

5

O

10

I

20

15

I

25

I

30

I

I

45

50

I

35

40

I

55

60

Days AA equivalents of AP (mg/kg): -' 40 -400

'- 2000 - 4000

Fig. 2. Accumulated mortality in Atlantic salmon fed different levels of ascorbate-2-monophosphate (AP) in the diet for 27 weeks prior to cohabitant challenge with Aeromonas salmonicida. The fish were re-challenged with i.p. injection at day 33. Table 7. Mean (SEM) weights, hepatosomatic index (HSI), spleen somatic index (SSI) and liver ascorbic acid (AA) in Atlantic salmon from the dietary groups after bacterial challenge (surviving fish) Feed AP (g/kg)

(n)

Weight (g)

HSI (g/100 g)

SSI (g/kg)

AA (lg/g)

40 400 2000 4000

40 34 37 43

119 97 113 100

0.91 0.83 0.93 0.84

0.91 0.95 0.92 0.90

43 94 109 119

(7)al (5)b (6 )ab (5)b

(0.05) (0.05) (0.05) (0.05)

(0.05) (0.07) (0.06) (0.06)

(2) a (3)b (4)C (7)C

1

Means in one column sharing a common superscript letter are not statistically different (p > 0.05).

stability during processing and storage, and to their superior bioactivity compared to other AA derivatives (Sandnes and Waagbo 1991; Waagbo et al. 1991; El Naggar and Lovell 1991). The minimum dietary requirement of AA in Atlantic salmon, using AP as vitamin C source, was determined to be in the range of 10-20 mg AA equivalents/kg feed (Sandnes et al. 1992). Growth and mortality were not negatively affected by the high dietary levels fed during 6 months in the present experiment. Thus the results indicate that

there are no harmful effects of dietary doses of AP up to 4000 mg AA equivalents/kg in this species. Generally, high levels of water soluble vitamins are regarded as harmless to fishes compared to fat soluble vitamins which accumulate when the fish is fed in excess (Tacon 1985). The salmon used in this experiment was prefed 40 mg AP/kg from startfeeding (Sandnes et al. 1993). During this period liver AA gradually increased to 21 ag/g, which is the initial AA status in the present experiment (Sandnes et al. 1992). The dietary sup-

69

* 1000 U/g tissue 18 c

16 14 12

b

b

10 a

8 6 4 2 0

-

_

40 AP

I

I

400 AP

-

2000 AP

4000 AP

Diets Fig. 3. Head kidney lysozyme activity in surviving Atlantic salmon fed different levels of ascorbate-2-monophosphate (AP) in the diet for 27 weeks prior to bacterial challenge (n = 7 pooled samples of 5 fish).

mg iron/I 2,5 b I

ab

ab

1,5

.

_L

[

-r-1

a

1

0,5 0

40 AP

400 AP

2000 AP

4000 AP

Diets Fig. 4. Serum iron concentration in surviving Atlantic salmon fed different levels of ascorbate-2-monophosphate (AP) in the diet for 27 weeks prior to bacterial challenge (n = 10).

70 plementations were reflected in the liver AA storage during the first 10 weeks (Fig. 1). However, different kinetics of AA incorporation were observed. Maximum liver concentrations of AA were reached after one week in fish fed the highest dietary level of AP, after 3 weeks in the 400 AP group and after 5 weeks in the 40 AP group. Fish fed the two highest levels of AP in the diet showed a decline in liver AA concentrations after the first week of feeding and seemed to adjust the hepatic contents to lower levels. This indicates an adaptation with regard to liver AA which may involve excretion of excess vitamin C. The liver AA status fluctuated somewhat during the experiment, as seen from the analyses carried out during the vaccination trial and at weeks 26 and in week 36, pre- and post-challenge, respectively (Tables 1, 2 and 6). The lower levels found after vaccination may be related to a reduced feed intake due to handling stress or lower water temperatures. At lower supplementations of vitamin C in the feed, the status of the fish can be evaluated by the liver AA concentration (Hilton et al. 1977; Sandnes 1982; Sandnes et al. 1992). The relatively narrow range in the steady state liver AA status found in the present study shows that liver AA does not reflect dietary intake linearly when fed very high levels. A significant negative correlation (p < 0.05) was seen between body weight and liver AA concentration in all feeding groups at the time of vaccination. This correlation increased with increasing dietary AP level, indicating limitations in storing excess vitamin C as well as changes in storage capacity with body weight. The AA level in lymphoid organs as head kidney, spleen or even isolated leucocytes may be a more suitable index of vitamin C status for immunological related studies. Reduced activity of immunoreactive cells has been shown in vitamin C deficient fish while supplementations 50 to 100 times above the minimal requirement have increased activity (Blazer 1982; Verlhac et al. 1993). However, megadoses of dietary vitamin C have not been shown to enhance the killing activity of isolated phagocytes measured as intracellular bactericidal index or respiratory burst in fish (Li and Lovell 1985; Hardie et al.

1991). In this experiment the activity of zymosanstimulated macrophages, measured as the ability to secrete acid phosphatase, was not influenced by dietary AP. In an experiment with a marine toad (Bufo marinus) Ghoneum et al. (1990) point to a possible role of ascorbic acid in suppressing natural killer cell activity by acting as a scavenger of oxidative radicals. Phagocytosis also involves release of radicals during respiratory burst, which may be influenced by AA. Injection with formalin killed Vibrio salmonicida induces production of specific antibodies in Atlantic salmon (Havardstein et al. 1990). This is also shown by the high antibody levels compared to sham-injected and untreated control fish in the present study. The only significant differences in specific antibody production were found between the 4000 mg AP group and the other groups 11 and 17 weeks after immunization. These results confirm previous findings by Navarre and Halver (1989) and Li and Lovell (1985) who reported that high dietary levels of vitamin C stimulated the antibody production. However, Liu et al. (1989), Sandnes et al. (1990) and Hardie et al. (1991) did not find effects of high dietary vitamin C on antibody production in channel catfish (Ictalurus punctatus) and Atlantic salmon. As suggested by Navarre and Halver (1989), the increased production of antibodies may be related to an increased activity of lymphoid cells. Verlhac et al. (1993) found higher concentrations of serum antibodies after vaccination against Yersinia ruckeri in rainbow trout fed 1000 mg AA equivalents of AP/kg compared to 100 mg/kg and vitamin C deprived fish. However, the differences between the two AP supplemented groups levelled out rapidly and the area under the antibody production curve during 16 weeks was not statistically different. The present data support the view that the increase reported in antibody levels when feeding high doses of vitamin C occurs rapidly, and may be related to functions controlling the early phase of the production. Serum total protein, albumin, Hct and Rbc showed highest concentrations in the 40 AP group. These results are indicative of some homeostatic disturbance in this group. The reason for this is

71 unknown, but it is not likely that this was caused by a deficiency of vitamin C. Hardie et al. (1991) did not find any impact of dietary AA (50, 310 and 2750 mg AA/kg feed) on serum total protein in Atlantic salmon. In contrast, Komarov and Knyazeva (1984) found an increased level of total serum protein in rainbow trout (Oncorhynchus mykiss) fed a vitamin C-enriched diet. The cohabitant model of challenge in this experiment was chosen because of its similarity to natural conditions (Erdal and Reitan 1991). The initial cohabitant challenge showed low mortality, with less than 10% in the 40 to 2000 AP groups and no mortality in the 4000 mg AP group. The mortality was low compared to the cumulative mortality (60%) in a parallel running challenge experiment, confirming the pathogenicity of the bacteria (Albrektsen, Sandnes, Glette and Waagbo, unpublished). Even after an additional injection of bacteria at day 33, the mortality remained low, which confirmed the results of the initial cohabitant challenge. Low mortality rate in fish with high liver AA status (> 80 /g/g) is in accordance with a similar study in Atlantic salmon (Hardie et al. 1991). Serum hemolytic activity was somewhat enhanced in the 4000 AP group, which indicates an effect of vitamin C on the complement system. An AA dose-related increase in complement activity was found in channel catfish (Li and Lovell 1985), Atlantic salmon (Hardie et al. 1991) and rainbow trout (Verlhac et al. 1993). Vitamin C may influence the activity of the complement system by stimulating the synthesis of the first component in the classical way as discussed by Hardie et al. (1991). In contrast, Yano et al. (1988), Lui et al. (1989) and Lall et al. (1989) did not find differences in hemolytic activity related to dietary vitamin C in red sea bream (Pagrusmajor), channel catfish and Atlantic salmon, respectively. This inconsistency may be related to the methods used or to genetic differences. In the present experiment, a titration of the Atlantic salmon hemolysins used prior to the serum analyses showed that the reagent was relatively weak, causing a maximum hemolysis of 50% at 5% dilution of fish serum. Thus a non-optimal hemolysin may have overshadowed possible dose-

related differences between the dietary groups. Head kidney lysozyme activity analysed in surviving fish at the end of the challenge study showed significantly higher levels in the 4000 AP group, and a dose-dependent increase in activity was apparent. Monocytes, macrophages and neutrophils are believed to be the main sources of lysozyme in the tissues and high levels of this acute-phase protein are regarded as protective against infectious agents in fish (Grinde 1989; Lie et al. 1989). Although controversial, indirect protective effect of AA has earlier been proposed to be synergistic with H2 0 2 and lysozyme in immuno-active cells (Beisel 1982). The principle used for the lysozyme assay in the present study leaves the question open whether the rise in activity was due to an increased number of lysozyme containing immune cells, increased intracellular lysozyme concentration or lastly, increased proportion of a more potent high molecular weight variant of lysozyme (Lie et al. 1989). Ascorbate has been shown to influence the absorption and utilization of iron in fish (Sandnes et al. 1984; Maage et al. 1990), and anemia has been reported in AA deficient fish (Hilton 1984; Sandnes et al. 1990). The total serum iron concentration was significantly (p < 0.005) correlated to the liver AA concentration in the present study. Decreased ironbinding capacity in vitamin C-deficient rainbow trout was reported by Blazer (1982), and serum iron constituted only 3 -7% of the iron-binding capacity in all groups (Blazer 1982). The decreased ironbinding capacity in vitamin C-deficient fish may therefore be due to malfunctions in the iron transport as have been suggested by Maage et al. (1990), rather than a depression of a nonspecific resistance factor. Dabrowski et al. (1990) found only minor effects of AA and ascorbate-2-sulfate on iron metabolism in rainbow trout. The present increase in serum iron concentration may be related to enhanced bactericidal action during the previous infection. The role of iron in disease and immunity was recently reviewed by Weinberg (1992). The highest serum concentration in this experiment is in the same range as reported in other studies with Atlantic salmon (Waagbo and Maage 1992). From the present observations on serum iron concentra-

72 tion and head kidney lysozyme activity, these parameters seem to play roles in the nonspecific defence, either by continuously elevated levels in fish fed high dietary levels of AA or elevations induced by inflammation/infection. In summary, the results from this study suggest that high levels of AP in the diet favourably affect health in Atlantic salmon. According to results from vitamin C studies with several fish species under different rearing conditions there are strong indications that feeding high levels of this vitamin enhances immune functions and health in fish. Further studies have to be carried out to investigate the exact role and the effective dietary level of AA for optimal immune defence in fish.

Acknowledgements This study was kindly supported by F. Hoffman La Roche Ltd., Basle, Switzerland. The authors wish to thank Matre Aquaculture Research Station for wet laboratory facilities. The technical assistance given by Mrs. Hildegunn Fauskanger and Mr. Vidar Fauskanger in Matre, Mr. Hari Rudra and Mrs. Kari Andersen at the Institute of Marine Research in Bergen and the staff at the Institute of Nutrition in Bergen is highly appreciated.

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