Fish & Shellfish Immunology (2001) 11, 347–355

doi:10.1006/fsim.2000.0322
Available online at http://www.idealibrary.com on

Effect of ascorbic acid on the immune response of the

catfish, Mystus gulio (Hamilton), to different bacterins of
Aeromonas hydrophila

K. ANBARASU† AND M. R. CHANDRAN*

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 immuno-
stimulatory e#ect of vitamin C on the humoral and cell mediated immunity of
the bagrid catfish, 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, immunomodulation, Aeromonas hydrophila,

Mystus gulio.

I. Introduction
Dietary deficiencies of various micronutrients influence the resistance of
animals to infectious diseases. Ascorbic acid is one of the essential vitamins
for the normal physiological functioning of organisms, including fish (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 significantly increased mortality rate of vitamin
deficient fish following challenge with Aeromonas salmonicida. However,
Floyd (1987) and Li et al. (1993) reported that high concentrations of dietary
vitamin C were not beneficial in improving disease resistance of channel
catfish. During challenge of unvaccinated fish, the non-specific immune
response may be relatively more important for disease resistance than the
specific immune response, since the latter requires a longer time for antibody
*Corresponding author. E-mail: mrc@bdu.ernet.in
†Present address: Department of Microbiology, Ayya Nadar Janaki Ammal College
(autonomous), Sivakasi-626124, Tamil Nadu, India.

347
1050–4648/01/040347+09 $35.00/0  2001 Academic Press
348 K. ANBARASU AND M. R. CHANDRAN

build-up and specific cellular activations (Anderson, 1992). The non-specific

response e#ected by phagocytes and non-specific cytotoxic cells is quickly
activated by immunostimulants, and may be manipulated to protect fish
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 fish and conse-
quently resistance to microbial diseases. The present study is an attempt to
evaluate the requirement of vitamin C by the immune system of the catfish,
Mystus gulio, as assessed by vaccination with di#erent bacterins of Aeromonas
hydrophila.

II. Materials and Methods

FISH

Bagrid catfish, Mystus gulio (Hamilton), weighing 211·2 g were obtained

from Muthupet Saline Swamp (1025 N and 7939 E) and acclimatised to
laboratory conditions in freshwater. During acclimation the fish were main-
tained in large cement cisterns (1000 l capacity) for a month: the water
temperature was 27·51·8 C, salinity c. 3‰, normal day/night exposure (12 h
dark/light). During experimentation acclimatised fish 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 flour, 270 g rice-bran, 230 g fish meal, 140 g
groundnut oil cake, 270 g silk worm pupae and 200 mg vitamin (thiamine
10 mg, riboflavin 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.

BACTERIAL STRAIN

Aeromonas hydrophila used in the present study was obtained from a

diseased fish during an outbreak in the Tiruchirappalli region of Central
Tamil Nadu, identified following standard biochemical tests (Cowan & Steel,
1965) using the criteria of Austin & Austin (1993).

PREPARATION OF VACCINES

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).

EXPERIMENTAL DESIGN

Respective bacterins adjusted to 5108 cells ml
1 and 0·2 ml fish
1 were

injected intraperitoneally on two occasions 13 days apart, as shown schemati-
cally in Fig. 1. Control fish 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 five
fish 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 400g,
 EFFECT OF ASCORBIC ACID ON THE IMMUNE RESPONSE OF THE CATFISH 349

Primary Secondary Monitoring of immune

immunisation immunisation responses and
challenge study

Day 0 1 7 14 15 30 60

prebleeding 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.

BACTERIAL AGGLUTINATION TEST

Antibody titres from vaccinated and control fish were determined by a

bacterial agglutination test, as described by Anbarasu (2000). The results were
expressed as the geometric mean value.

DELAYED HYPERSENSITIVITY TEST

The inflammatory reaction was measured following Anbarasu et al. (1998).

Briefly, respective bacterins and sterile PBS were injected in the caudal
region of the fish of the respective groups. After 24, 48, 72 and 96 h the
diameter of inflammatory zones was measured using a vernier caliper.

IMMUNOLOGICAL ASSAYS

Sixty days after immunisation, fish 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 0·5% NBT and incubated for
20 min in a 96 well microtitre plate following the manufacturer’s 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).

CHALLENGE STUDY

After 60 days of immunisation, all groups were challenged with 0·1 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 27·01·0 C. The relative percent survival (RPS) was calculated using the
formula
350 K. ANBARASU AND M. R. CHANDRAN

Table 1. Determination of antibody titres in catfish, Mystus gulio, immunised with

Aeromonas hydrophila (n=5)

Agglutinating antibody titres

Groups Before After primary
After secondary immunisation
immunisation immunisation
Days 0 7 14 15 30 60

FK Nil 20·52 41·03 81·79a 320·00a 327·16ac

HK Nil 20·73 20·73 41·03 41·03 82·73c
LPS Nil 20·00 20·00 81·79a 327·16a 640·00ac
FK+Vit Nil 20·00 41·03 80·00a 327·16a 327·16ac
HK+Vit Nil 20·73 41·03 41·37a 42·73a 160·00ac
LPS+Vit Nil 40·89b 81·79b 81·79ab 3210·73 ab
1280·00abc
Vit-Control Nil Nil Nil 20·52 20·73 20·52
Control Nil Nil Nil Nil Nil Nil

Superscripts a, b and c denote significant 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.

STATISTICAL ANALYSIS

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 Student’s t-test. Di#erences were
considered statistically significant when P<0·05.

III. Results

This study has revealed a requirement for ascorbic acid for optimal immune
responses in the catfish, Mystus gulio, vaccinated against A. hydrophila.
The titre of agglutinating antibody (Table 1) was nil for both the experimen-
tal 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<0·05). In groups given dietary vitamin
administration, the LPS vaccinated group demonstrated the highest initial
antibody titre (40·90) 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
 EFFECT OF ASCORBIC ACID ON THE IMMUNE RESPONSE OF THE CATFISH 351

Table 2. Determination of delayed hypersensitivity in catfish, Mystus gulio, challenged

with Aeromonas hydrophila (n=4)

Groups Diameter of zone of inflammation (mm)

Hours 24 48 72 96

FK 0·200·04 0·280·05 0·300·03 0·300·04

HK 0·200·07 0·200·00 0·200·07 0·200·07
LPS 0·300·04a 0·400·00a 0·500·04a 0·400·00a
FK+Vit 0·300·00a 0·300·04a 0·400·00a 0·500·00a
HK+Vit 0·280·05a 0·300·00a 0·300·04a 0·180·03a
LPS+Vit 0·280·03a 0·500·04ab 0·700·06ab 0·700·00ab
Vit-Control 0·180·03 0·200·07 0·200·00 0·100·00
Control 0·100·00 0·100·00 0·000·00 0·000·00

Superscripts a and b denote significant 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 significantly higher as days progressed for both vitamin supple-
mented 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 inflam-
mation varied for the di#erent groups. As with the agglutinating antibody
titres, groups given vitamin supplementation showed a wider inflammation
zone than the non-supplemented groups. The vitamin supplemented, LPS
immunised group had the widest zone of inflammation (P<0·05).
The nitroblue tetrazolium (NBT) test revealed (Table 3) significant di#er-
ences between vitamin C supplemented and unsupplemented groups. The
vitamin supplemented fish immunised with LPS had the highest values. The
phagocytic and adherence activity observations reflected a similar pattern as
that of the NBT test. For all the above tests, the statistical significance was
calculated relative to the vitamin control. However, the vitamin supple-
mented control value was significant at the 5% level (P<0·05) 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 findings indicate that
ascorbic acid supplemented diets fed to bagrid catfish give better protection
352 K. ANBARASU AND M. R. CHANDRAN

Table 3. Determination of nitroblue tetrazolium test, phagocytic and adherence

activities in catfish, Mystus gulio, immunised with Aeromonas hydrophila (Mean S.E.)
(n=6)

Nitroblue Phagocytic Adherence

Groups
tetrazolium test index index

FK 40·20·20a 12·00·37 9·20·17

HK 38·20·20a 11·30·21 8·70·33
LPS 41·60·25a 12·70·21a 10·20·17a
FK+Vit 41·00·45a 12·20·17a 9·20·17
HK+Vit 40·00·32ab 12·20·17ab 9·00·80
LPS+Vit 45·20·20ab 14·30·21ab 13·20·17ab
Vit-Control 35·20·37 11·00·26 9·00·37
Control 30·60·25 10·20·17 7·50·22

Superscripts a and b denote significant values (P<0·05) from vitamin control and unsupple-
mented 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 catfish, Mystus gulio,

challenged with Aeromonas hydrophila (n=10)

Groups Number of fish mortalities after Total number

Hours 24 48 72 96 of mortalities RPS

FK 2 NIL 2 NIL 4/10 59

HK 2 1 2 1 6/10 39
LPS 1 1 1 NIL 3/10 69
FK+Vit 1 1 NIL NIL 2/10 79
HK+Vit 2 2 1 NIL 5/10 49
LPS+Vit 1 NIL NIL 1 2/10 79
Vit-Control 6 2 1 NIL 9/10 9
Control 6 4 — — 10/10 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 catfish, 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 first line defence against the
entry of pathogens is vitamin C sensitive. Optimal functioning of non-specific
defence mechanisms has an important role in defence (Anderson, 1992) since
nonspecific defences have a wide spectrum of activity and are stimulated
immediately when a pathogen enters an organism. Specific immunity has a
narrow spectrum, takes a longer time to develop, but is highly persistent.
 EFFECT OF ASCORBIC ACID ON THE IMMUNE RESPONSE OF THE CATFISH 353

Several previous reports indicate that it is not easy to confirm the amount of
ascorbic acid required for the normal functioning of lymphoid as well as other
physiological functions of fish. 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 catfish, 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 prep-
arations, 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 fish 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 fish, 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 bacteri-
cidal and phagocytic activity. The present study has also shown that spleno-
cytes from fish 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 inflammation
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 inflammatory reaction.
The challenge study confirmed the overall protective e#ect and showed that
ascorbic acid fed fish had a higher relative percent survival than their
counterparts fed with unsupplemented diets. Nevertheless, the LPS immu-
nised 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 deficiency
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 0·4 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 fishes, 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.
354 K. ANBARASU AND M. R. CHANDRAN

Based on the above investigation it is evident that an optimal level of

vitamin C (100 mg kg
1) certainly enhances both the specific and non-specific
immunity of the catfish, M. gulio. Thus, Vitamin C at optimal level acts as an
immunostimulator which will improve the immune status of fish. It is hoped
that this base line information will be of immense use in fish farming.

The authors are grateful to the Indian Council of Agricultural Research (New Delhi),
for financial assistance, and to Prof. C. J. Secombes (University of Aberdeen, U.K.) for
critically reading the manuscript.

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