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Fish & Shellsh Immunology (2001) 11, 347355 doi:10.1006/fsim.2000.0322 Available online at http://www.idealibrary.

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Effect of ascorbic acid on the immune response of the catsh, Mystus gulio (Hamilton), to different bacterins of Aeromonas hydrophila


Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India (Received 10 January 2000, accepted after revision 29 October 2000)
Ascorbic acid is one of the essential vitamins for normal physiological activities of any organism. The present study demonstrates an immunostimulatory e#ect of vitamin C on the humoral and cell mediated immunity of the bagrid catsh, Mystus gulio, determined using di#erent bacterins of Aeromonas hydrophila. Humoral as well as cell mediated immune responses were elucidated in the vitamin supplemented, vaccinated and unvaccinated groups. The vitamin supplemented lipopolysaccharide (LPS) vaccinated group exhibited greater immune (both humoral and cell mediated) responses than its formalin killed (FK) and heat killed (HK) bacterin vaccinated counterparts. Nevertheless, in the challenge study, the relative percent survival (RPS) was found to be the same for both FK and LPS immunised vitamin treated groups while lower for the HK immunised vitamin treated group.
 2001 Academic Press

Key words:

ascorbic acid, Mystus gulio.




I. Introduction Dietary deciencies of various micronutrients inuence the resistance of animals to infectious diseases. Ascorbic acid is one of the essential vitamins for the normal physiological functioning of organisms, including sh (Wilson & Poe, 1973; Andrews & Murai, 1975; Lim & Lovell, 1978), and is a well known immunostimulator in mammals. Hardie et al. (1990, 1991) while assessing the e#ects of vitamin C and E depletion on the immune response of Atlantic salmon, Salmo salar, found a signicantly increased mortality rate of vitamin decient sh following challenge with Aeromonas salmonicida. However, Floyd (1987) and Li et al. (1993) reported that high concentrations of dietary vitamin C were not benecial in improving disease resistance of channel catsh. During challenge of unvaccinated sh, the non-specic immune response may be relatively more important for disease resistance than the specic immune response, since the latter requires a longer time for antibody
*Corresponding author. E-mail: Present address: Department of Microbiology, Ayya Nadar Janaki Ammal College (autonomous), Sivakasi-626124, Tamil Nadu, India. 10504648/01/040347+09 $35.00/0 347  2001 Academic Press



build-up and specic cellular activations (Anderson, 1992). The non-specic response e#ected by phagocytes and non-specic cytotoxic cells is quickly activated by immunostimulants, and may be manipulated to protect sh against pathogens. Incorporating an optimum level of vitamin C with feed seems to be a simple and e#ective way of improving the general health status of sh and consequently resistance to microbial diseases. The present study is an attempt to evaluate the requirement of vitamin C by the immune system of the catsh, Mystus gulio, as assessed by vaccination with di#erent bacterins of Aeromonas hydrophila. II. Materials and Methods

Bagrid catsh, Mystus gulio (Hamilton), weighing 21 12 g were obtained from Muthupet Saline Swamp (10 25 N and 79 39 E) and acclimatised to laboratory conditions in freshwater. During acclimation the sh were maintained in large cement cisterns (1000 l capacity) for a month: the water temperature was 275 18 C, salinity c. 3, normal day/night exposure (12 h dark/light). During experimentation acclimatised sh of the same size were grouped and maintained in separate glass tanks of 100 l capacity. The composition of the pelletised feed used was as follows: feed for the control group consisted of 90 g Tapioca our, 270 g rice-bran, 230 g sh meal, 140 g groundnut oil cake, 270 g silk worm pupae and 200 mg vitamin (thiamine 10 mg, riboavin 10 mg, pyridozine 40 mg, pantothenic acid 30 mg, vitamin C 10 mg, vitamin A 20 mg, vitamin E 80 mg). For the experimental groups 90 mg of ascorbic acid was added to the above feed.

Aeromonas hydrophila used in the present study was obtained from a diseased sh during an outbreak in the Tiruchirappalli region of Central Tamil Nadu, identied following standard biochemical tests (Cowan & Steel, 1965) using the criteria of Austin & Austin (1993).

Formalin and heat killed bacterins of A. hydrophila were prepared as described by Anbarasu et al. (1998). Crude LPS was obtained by the phenol hot water extraction method as described by Westphal & Jann (1965).

Respective bacterins adjusted to 5 108 cells ml 1 and 02 ml sh 1 were injected intraperitoneally on two occasions 13 days apart, as shown schematically in Fig. 1. Control sh were injected with an equal volume of sterile phosphate bu#ered saline. After 7, 14 (after primary immunisation), 15, 30 and 60 (after secondary immunisation) days, blood samples were taken from ve sh of each group by cardiac puncture. After collection, the blood was allowed to clot at room temperature (30 C) for an hour and then placed in a refrigerator overnight at 4 C. It was then centrifuged for 10 min at 400 g,



Primary immunisation

Secondary immunisation

Monitoring of immune responses and challenge study 15 30 60




sample bleeding

Fig. 1. Schematic diagram of the experimental design.

serum was separated and inactivated by placing it in a water bath at 56 C for 30 min, and then stored at 20 C prior to use.

Antibody titres from vaccinated and control sh were determined by a bacterial agglutination test, as described by Anbarasu (2000). The results were expressed as the geometric mean value.

The inammatory reaction was measured following Anbarasu et al. (1998). Briey, respective bacterins and sterile PBS were injected in the caudal region of the sh of the respective groups. After 24, 48, 72 and 96 h the diameter of inammatory zones was measured using a vernier caliper.

Sixty days after immunisation, sh were killed and a single cell suspension of spleen prepared. The cells were used to assess the adherence, phagocyte and respiratory burst (NBT test) activity. For the NBT test, each cell suspension (100 cells) was mixed with an equal volume of 05% NBT and incubated for 20 min in a 96 well microtitre plate following the manufacturers instructions (Sigma, MO, U.S.A.). Into each well of another microtitre plate an equal volume of spleen single cell suspension (100 cells) and A. hydrophila formalin inactivated whole cells were added, to measure phagocytic and adherence activities following the method of Van Oss et al. (1973). The experiments were performed at room temperature (30 C).

After 60 days of immunisation, all groups were challenged with 01 ml of the homologous virulent A. hydrophila (108 cells ml 1) injected intraperitoneally, and mortality was noted daily for 4 days. The maintained water temperature was 270 10 C. The relative percent survival (RPS) was calculated using the formula



Table 1. Determination of antibody titres in catsh, Mystus gulio, immunised with Aeromonas hydrophila (n =5) Agglutinating antibody titres Groups Days FK HK LPS FK+Vit HK+Vit LPS+Vit Vit-Control Control Before immunisation 0 Nil Nil Nil Nil Nil Nil Nil Nil 2 2 2 2 2 4 After primary immunisation 7 052 073 000 000 073 089b Nil Nil 4 2 2 4 4 8 14 103 073 000 103 103 179b Nil Nil 8 4 8 8 4 8 2 After secondary immunisation 15 179a 103 179a 000a 137a 179ab 052 Nil 32 4 32 32 4 32 2 30 60

000a 32 716ac 103 8 273c 716a 64 000ac 716a 32 716ac 273a 16 000ac ab 1073 128 000abc 073 2 052 Nil Nil

Superscripts a, b and c denote signicant values from vitamin control, heat killed groups and at day 60, respectively. FK=formalin killed, HK=heat killed, LPS=lipopolysaccharide, FK+Vit=formalin killed with vitamin, HK+Vit=heat killed with vitamin, LPS+Vit=lipopolysaccharide with vitamin, Vit-Control=vitamin control.


All experiments were performed in triplicate and the mean S.E. calculated for each group are presented in the tables. The bacterial agglutination test and the delayed type hypersensitivity were treated with single/multiple ANOVA and Tukey test. The respiratory burst activity, phagocytosis and adherence activity were treated with Students t-test. Di#erences were considered statistically signicant when P< 005. III. Results This study has revealed a requirement for ascorbic acid for optimal immune responses in the catsh, Mystus gulio, vaccinated against A. hydrophila. The titre of agglutinating antibody (Table 1) was nil for both the experimental and control groups prior to immunisation. The control group did not show any visible agglutination throughout the period of study. Among the groups given unsupplemented diets, those vaccinated with formalin killed whole cells showed a higher antibody titre after primary immunisation than groups given heat killed whole cells or LPS (P< 005). In groups given dietary vitamin administration, the LPS vaccinated group demonstrated the highest initial antibody titre (4 090) after primary vaccination. A similar titre was observed in the groups vaccinated with the formalin killed bacterin with or without vitamin administration on day 14. After secondary immunisation higher antibody titres were generally seen in the groups given vitamin C supplementation, especially on day 60, compared with the groups given unsupplemented diets. In addition, after secondary immunisation the antibody



Table 2. Determination of delayed hypersensitivity in catsh, Mystus gulio, challenged with Aeromonas hydrophila (n =4) Groups Hours FK HK LPS FK+Vit HK+Vit LPS+Vit Vit-Control Control 020 020 030 030 028 028 018 010 24 004 007 004a 000a 005a 003a 003 000 Diameter of zone of inammation (mm) 48 72 028 020 040 030 030 050 020 010 005 000 000a 004a 000a 004ab 007 000 030 020 050 040 030 070 020 000 003 007 004a 000a 004a 006ab 000 000 030 020 040 050 018 070 010 000 96 004 007 000a 000a 003a 000ab 000 000

Superscripts a and b denote signicant values from vitamin control, and all other groups, respectively. FK=formalin killed, HK=heat killed, LPS=lipopolysaccharide, FK+Vit=formalin killed with vitamin, HK+Vit=heat killed with vitamin, LPS+Vit=lipopolysaccharide with vitamin, Vit-Control=vitamin control.

titres were signicantly higher as days progressed for both vitamin supplemented and non-supplemented groups (based on statistical analysis) relative to the respective primary responses. The delayed hypersensitivity test (Table 2) showed that the zone of inammation varied for the di#erent groups. As with the agglutinating antibody titres, groups given vitamin supplementation showed a wider inammation zone than the non-supplemented groups. The vitamin supplemented, LPS immunised group had the widest zone of inammation (P< 005). The nitroblue tetrazolium (NBT) test revealed (Table 3) signicant di#erences between vitamin C supplemented and unsupplemented groups. The vitamin supplemented sh immunised with LPS had the highest values. The phagocytic and adherence activity observations reected a similar pattern as that of the NBT test. For all the above tests, the statistical signicance was calculated relative to the vitamin control. However, the vitamin supplemented control value was signicant at the 5% level (P< 005) for the NBT test and adherence activity but not for phagocytosis. Table 4 shows the relative percent survival (RPS) of the groups challenged with the homologous virulent A. hydrophila strain. Vitamin supplemented groups had high RPS values compared to the non-supplemented groups. IV. Discussion The use of dietary supplements in aquaculture for prevention of diseases is a promising recent trend (Lovell, 1973; Lim & Lovell, 1978; Li & Lovell, 1985; Wilson et al., 1989; Sakai et al., 1990; Hardie et al., 1990, 1991; Anderson, 1992; Onarheim, 1992; Raa et al., 1992; Duncan & Lovell, 1993; Siwicki et al., 1994; Thompson et al., 1995; Meydani & Beharka, 1996; Mulero et al., 1998; Ortuno et al., 1999; Sakai, 1999). Indeed, the present ndings indicate that ascorbic acid supplemented diets fed to bagrid catsh give better protection



Table 3. Determination of nitroblue tetrazolium test, phagocytic and adherence activities in catsh, Mystus gulio, immunised with Aeromonas hydrophila (Mean S.E.) (n =6) Groups FK HK LPS FK+Vit HK+Vit LPS+Vit Vit-Control Control Nitroblue tetrazolium test 402 382 416 410 400 452 352 306 020a 020a 025a 045a 032ab 020ab 037 025 Phagocytic index 120 113 127 122 122 143 110 102 037 021 021a 017a 017ab 021ab 026 017 Adherence index 92 87 102 92 90 132 90 75 017 033 017a 017 080 017ab 037 022

Superscripts a and b denote signicant values (P< 005) from vitamin control and unsupplemented vaccinated groups, respectively. FK=formalin killed, HK=heat killed, LPS=lipopolysaccharide, FK+Vit=formalin killed with vitamin, HK+Vit=heat killed with vitamin, LPS+Vit=lipopolysaccharide with vitamin, Vit-Control=vitamin control.

Table 4. Determination of relative percent survival (RPS) in catsh, Mystus gulio, challenged with Aeromonas hydrophila (n =10) Groups Hours FK HK LPS FK+Vit HK+Vit LPS+Vit Vit-Control Control 24 2 2 1 1 2 1 6 6 Number of sh mortalities after 48 72 96 NIL 1 1 1 2 NIL 2 4 2 2 1 NIL 1 NIL 1 NIL 1 NIL NIL NIL 1 NIL Total number of mortalities 4/10 6/10 3/10 2/10 5/10 2/10 9/10 10/10 RPS 59 39 69 79 49 79 9 0

FK=formalin killed, HK=heat killed, LPS=lipopolysaccharide, FK+Vit=formalin killed with vitamin, HK+Vit=heat killed with vitamin, LPS+Vit=lipopolysaccharide with vitamin, Vit-Control=vitamin control.

post-vaccination than in groups fed with unsupplemented diets. Thus it reveals that ascorbic acid acts as an immunostimulator in the catsh, Mystus gulio, under optimal concentration. It is clear that adequate amounts of ascorbic acid are essential for normal physiological functions, and probably a component of the immune system that act as a rst line defence against the entry of pathogens is vitamin C sensitive. Optimal functioning of non-specic defence mechanisms has an important role in defence (Anderson, 1992) since nonspecic defences have a wide spectrum of activity and are stimulated immediately when a pathogen enters an organism. Specic immunity has a narrow spectrum, takes a longer time to develop, but is highly persistent.



Several previous reports indicate that it is not easy to conrm the amount of ascorbic acid required for the normal functioning of lymphoid as well as other physiological functions of sh. It may vary from species to species and under di#erent climatic conditions. In the present study, 100 mg kg 1 has yielded good immune responses in mature catsh, M. gulio. The exact level required has been standardised by introducing di#erent concentrations of ascorbic acid and calculated (data not published). A slightly higher level of ascorbic acid (150 mg kg 1) incorporated with feed is advisable for commercial feed preparations, as the half-life is very low and it is easily oxidised to an inactive form (Wilson et al., 1989; Teskeredzic et al., 1989). Determination of the exact level of incorporation required for optimal immune responsiveness is very helpful for feed formulation because ascorbic acid is not cheap and the feed cost will increase as higher amounts are incorporated. Prior to immunisation, no antibody titre was present in all groups, as reported in previous studies (Baba et al., 1988; Velji et al., 1990; Sakai et al., 1990; Anbarasu et al., 1998; Anbarasu & Chandran, 1998). After primary and secondary immunisation with LPS the highest levels of antibody titre (64, 128) were induced. Interestingly LPS administered to sh fed with unsupplemented diets also gave high titres after secondary immunisation, next to the LPS+ascorbic acid treated groups, perhaps because LPS itself can act as an immunostimulator. These results are in accordance with many previous reports (Baba et al., 1988; Burrel, 1990; Wycko# et al., 1998). LPS is a known macrophage activator for both mammals and sh, under both in vitro and in vivo conditions. Jorgensen & Robertsen (1994) have shown that salmon macrophages stimulated by LPS were activated in terms of enhanced bactericidal and phagocytic activity. The present study has also shown that splenocytes from sh fed with ascorbic acid supplemented and unsupplemented diets had the highest phagocytic and adherence activity after LPS administration. The delayed hypersensitivity test demonstrated that the ascorbic acid fed LPS immunised group had the strongest and most prolonged inammation relative to other experimental groups. Secombes (1996) has pointed out that if any antigen enters into the organism, the macrophages and granulocytes found in the blood can act as mobile phagocytes, while Burrel (1990) points out that even LPS is an endotoxin and may itself create an inammatory reaction. The challenge study conrmed the overall protective e#ect and showed that ascorbic acid fed sh had a higher relative percent survival than their counterparts fed with unsupplemented diets. Nevertheless, the LPS immunised groups had the highest RPS, presumably due to both humoral as well as cell mediated immunity. Further, Duncan & Lovell (1993) have reported that folic acid is necessary for the proper formation of blood cells and a deciency has been connected to anaemia in various animals. Whilst 4 mg kg 1 of folic acid is required to reduce columnaris mortality, with high concentrations of ascorbic acid just 04 mg folic acid kg 1 is enough to prevent columnaris mortality. Hardie et al. (1991) have already reported that a high level of ascorbic acid is essential for reducing the e#ects of physiological stress as well as wound healing in shes, and in the feed can inhibit serum cortisol levels. Hence, during intensive aquaculture the addition of ascorbic acid can act at a number of levels to be highly e#ective.



Based on the above investigation it is evident that an optimal level of vitamin C (100 mg kg 1) certainly enhances both the specic and non-specic immunity of the catsh, M. gulio. Thus, Vitamin C at optimal level acts as an immunostimulator which will improve the immune status of sh. It is hoped that this base line information will be of immense use in sh farming.
The authors are grateful to the Indian Council of Agricultural Research (New Delhi), for nancial assistance, and to Prof. C. J. Secombes (University of Aberdeen, U.K.) for critically reading the manuscript.

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