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Aquaculture 252 (

Seasonal variation in the innate immune parameters

of the Asian catfish Clarias batrachus

Jaya Kumari, P.K. Sahoo *, T. Swain, S.K. Sahoo, A.K. Sahu, B.R. Mohanty

Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, India

Received 7 March 2005; received in revised form 5 July 2005; accepted 25 July 2005

Abstract

To determine if the innate immunity of Asian catfish (Clarias batrachus) raised in captivity are affected by the rearing

temperature or seasons, various indices of the humoral and cellular innate immune functions were measured in fish reared at a

wide range of water temperatures over a period of 2 years. Measures of humoral immunity included the estimation of serum

myeloperoxidase, lysozyme, haemagglutinin and alternative complement activities. Cellular assay quantified the ability of

blood phagocytes to mount superoxide production. Fish were sampled during different periods of two consecutive years,

maintained at similar prevailing ranges of water temperature. Experiments were performed at mean temperatures of 19, 24, 28,

31, 32.5 8C during the same time of 2 years. The kinetics of the temperature or season mediated immunomodulation in the

innate immune parameters was remarkably fluctuated among the individuals and at different periods or temperatures. Although

a clear seasonal variation was marked in the innate immune parameters of this species, the fluctuations of all the parameters are

not consistent to any of the temperatures except for lysozyme levels that remained significantly lower during summer compared

to other seasons. The probable compensatory mechanism among the innate defence molecules might be playing role to protect

from infections during different water temperatures or seasons.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Innate immunity; Annual variation; Temperature; Season; Clarias batrachus

1. Introduction

Clarias batrachus is a tropical, highly nutritive,

medium-sized catfish of Asian region, which is very

hardy in nature and tolerant to adverse ecosystems. In

0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquaculture.2005.07.025

* Corresponding author. Tel.: +91 674 2465421; fax: +91 674

2465407.

E-mail addresses: cifa@ori.nic.in, pksahoo1@hotmail.com

(P.K. Sahoo).

intensive culture systems, productions of 100 tonnes/

ha have been reported (Thakur and Das, 1986; Are-

erat, 1987; Zheng et al., 1988). This species experi-

ences large differences in the ambient water

temperature during growout like most aquaculture

species. For instance, during summer, water tempera-

tures rise to around 29–35 8C, whereas in winter the

fish experience temperatures as low as 17–19 8C. Theclose relationship that exists between teleost and their

environment is the basis of a wide variety of studies.

2006) 121–127

J. Kumari et al. / Aquaculture 252 (2006) 121–127122

The fish have a body temperature that is essentially

the temperature of the surrounding water (Fry, 1967),

so that their entire physiology, including immune

functions, is influenced by environmental temperature

(Le Morvan et al., 1998). During different periods of

the seasons or year, C. batrachus show increased

susceptibility to various pathogens. At the onset of

the rainy season during June–July, the early stages of

this fish become highly susceptible to bacterial septi-

caemia (caused by Aeromonas hydrophila, Edward-

siella tarda, etc.) (Sahoo et al., 1998; Swain et al.,

2001) and epizootic ulcerative syndrome (Callinan et

al., 1995). The winter period is also associated with

decreased growth rates and increased susceptibility to

opportunistic pathogens (P.K. Sahoo, unpublished

observations). Little has been explored about the

immune status of this species (Sinha and Chakravarty,

1997; Kumari et al., 2003; Kumari and Sahoo, 2005).

The pathological condition in fish depends both on

temperature-dependent immune system regulation and

pathogenicity of the organism. Numerous studies have

reported the thermosensitivity of specific immune

responses (Avtalion et al., 1973; Stolen et al., 1984;

Bly and Clem, 1992; Le Morvan et al., 1998), whereas

few data are available on the effects of temperature or

season on innate immunity of fish. Lower environ-

mental temperatures adversely affect both cellular and

humoral immune responses in various fish species.

The immunologically dpermissiveT and dnon-permissiveT temperatures depend on fish species,

and the non-permissive temperature has been estab-

lished as 4 8C in salmonids, 14 8C in carp and 22 8Cin channel catfish (Bly and Clem, 1992). The non-

specific immune parameters are useful to determine

the health status of fish, evaluate immunomodulatory

substances, and act as markers of pollution and dis-

ease resistance. Many cells (mostly leucocytes and

macrophages) and their products [myeloperoxidase

(MPO), superoxides, lysozyme, complement, acute-

phase proteins, interferons, agglutinins, properdins,

lysins] contribute to the general immunological

defence mechanism (Sahoo et al., 2005).

Enzymes such as myeloperoxidase, lysins and

lysozyme are microbicidal. Activation of comple-

ment leads to the destruction and elimination of

invading pathogens such as bacteria, viruses and

fungi (Yano, 1992). Out of the three pathways of

the complement system, the alternative complement

pathway (ACP) of fish is many-fold higher than that

of mammals indicating its importance in the innate

response compared to mammals (Holland and Lam-

bris, 2002). The nitroblue tetrazolium (NBT) assay

is indicative of oxidative radical production from

neutrophils and monocytes for use in defence

against pathogens (Anderson and Siwicki, 1995).

Natural fish haemagglutinins/agglutinins bind to car-

bohydrate moieties of microbes causing agglutina-

tion for inactivation and easy immune clearance

(Dalmo et al., 1997).

This work investigated the seasonal fluctuations,

reflecting thermal influences in some of the para-

meters pertaining to non-specific immunity to estab-

lish a physiological normal range in Asian catfish

C. batrachus.

2. Materials and methods

2.1. Experimental design and fish maintenance

C. batrachus (42.23F10.06 g) utilized in this

study were collected from different culture systems

of the Central Institute of Freshwater Aquaculture,

Bhubaneswar farm. Ten to fifty-eight immature fish

were sampled at each time of the year based on the

availability. Apparently healthy fish were collected

and acclimatized in the laboratory for 15 days during

the above periods with the prevailing temperatures in

ferro-cement tanks each with a 500 l water volume.

Each tank was stocked with 10 fish and a similar

stocking density was maintained throughout the

experiment. The fish were fed with a formulated pellet

diet at the rate of 3% of their body weight once daily,

throughout the experimental period. About 10% of

water was removed daily along with the waste feed

and faecal material. The basic physicochemical water

parameters were measured systematically at 3-day

intervals to maintain optimal levels (dissolved oxy-

gen: 5.65F0.72 mg/l; pH: 8.2F0.82; nitrites:

0.015F0.009 mg/l; ammonia: 0.109F0.024 mg/l)

throughout the experiment.

Fish were tested five times a year over a period of

two consecutive years at various mean water tempera-

tures (winter, 19 8C; spring, 24 8C; summer, 32.5 8C;rainy, 31 8C; autumn, 28 8C). The water temperatures

mentioned here represent the mean values over 15

J. Kumari et al. / Aquaculture 252 (2006) 121–127 123

days of acclimatization period in the wet laboratory in

that particular season and that also simulates mean

water temperatures in the culture systems during the

corresponding seasons. The fish were sampled over 2

years in the last week of December, March, May,

August and October, representing winter, spring, sum-

mer, rainy and autumn seasons, respectively.

2.2. Sample collection

A previous health control in these fish indicated the

absence of any parasitic or bacterial infections. In order

to sample the blood, fish were quickly caught, placed in

anaesthetic (0.1 ml/l 2-phenoxyethanol) and sampled

through caudal puncture with a syringe. A part of fresh

blood was placed in heparinized tube for nitroblue

tetrazolium (NBT) test. The rest of the blood was left

to clot at 4 8C for 2 h, the clot removed after centrifuga-

tion, and the serum aliquoted and stored at �70 8C for

lysozyme, complement, haemagglutination and MPO

measurements. The complement activity was studied

on the day following bleeding.

2.3. Non-specific immunological parameters

The total myeloperoxidase content present in

serum was measured according to Quade and Roth

(1997) and a partial modified technique (Sahoo et al.,

2005). Respiratory burst activity was assayed by the

reduction of nitroblue tetrazolium (NBT) to formazan

as a measure of reactive oxygen radical production

(Anderson and Siwicki, 1995).

Hemolytic assays for the determination of the activ-

ity of the alternative complement pathway were per-

formed following the technique described by

Matsuyama et al. (1988) and Yano (1992) with partial

Table 1

Mean and standard errors for water temperature during a particular season,

corresponding immune parameters analysed over 5 different periods in tw

Variable (period of the year/season)

(mean temperature, 8C)No. of fish sampled/

season/year

Weight

December/Winter (19.00F0.15a) 15 42.80FMarch/Spring (24.00F0.16b) 25 39.65FMay/Summer (32.50F0.36e) 58 39.72FAugust/Rainy (31.00F0.11d) 42 44.18FOctober/Autumn (28.00F0.12c) 10 44.76F

Means bearing different superscript(s) column-wise are significantly ( P b

modifications (Kumari and Sahoo, 2005). Briefly, rab-

bit red blood cells (RaRBC) were washed thrice in

EGTA-Mg-GVB buffer (veronal-buffered saline con-

taining 10 mM EGTA, 10 mM MgCl2, and 0.1%

gelatin) and resuspended to 2�108 cells/ml in the

same buffer. To individual 125 Al aliquots of seriallydiluted test sera in EGTA-Mg-GVB buffer, 50 Al ofRaRBC suspension was added and each mixture was

incubated for 120 min at 20 8C. The hemolytic reaction

was stopped by adding 1.575 ml EDTA-GVB buffer

(10 mM EDTA and 0.1% gelatin) and each reaction

mixture was centrifuged at 1600�g for 5 min. The

supernatant was taken and the absorbance read at 414

nm. Control consists of complete haemolysis (1.7 ml

distilled water and 50 Al RaRBC) and a blank without

serum sample (containing only buffer and RaRBC).

The value y/(1�y) and the reciprocal of the serum

dilution were plotted on log–log graph paper and the

ACH50 (units/ml), the reciprocal dilution giving 50%

haemolysis [ y/(1�y)=1], was read from the graph.

The turbidimetric assay for lysozyme was carried

out according to Sankaran and Shanto (1972) with

partial modifications. A suspension of 150 Al ofMicro-

coccus lysodeikticus (0.2 mg/ml in 0.02 M sodium

acetate buffer, pH 5.5) was added to previously dis-

pensed 15 Al of test serum in a 96-well U-bottom

microtitre plates (Tarson, India). Initial O.D was

taken at 450 nm immediately after adding the substrate

and final O.D was taken after 1 h incubation at 24 8C.Lyophilized hen egg white lysozyme, HEWL (Sigma),

was used to develop a standard curve. Serum lysozyme

values were expressed as Ag/ml equivalent to hen egg

white lysozyme activity.

Haemagglutination assays were performed as in

Kumari and Sahoo (2005). Double serial dilution of

the inactivated sera (56 8C for 20 min) were made in

weight of the fish sampled, number of fish sampled/season/year and

o consecutive years in C. batrachus

(g) Lysozyme

(Ag/ml)

ACH50

(units/ml)

Haemagglutination

titre

0.75 16.79bF0.67 44.84bcF7.43 74.67aF21.43

1.70 17.67bF0.62 57.41cF7.47 145.07abF41.41

1.09 11.21aF0.68 46.54bcF6.39 195.23bF27.95

1.59 15.17bF0.59 28.27abF2.14 45.64aF6.81

1.18 16.97bF0.87 18.19aF7.38 204.8bF96.58

0.05) different.

Fig. 1. Nitroblue tetrazolium (NBT) and myeloperoxidase (MPO)

activities measured at 540 nm and 450 nm, respectively, in Asian

catfish C. batrachus at various seasons of the year. Data represent

the meanFS. E. Statistical differences ( P b0.05) among various

seasons for a particular parameter are indicated by different letters

(a, b, c) over the bars.

J. Kumari et al. / Aquaculture 252 (2006) 121–127124

PBS (with Ca2+ and Mg2+), and then 50 Al of 1%

RaRBC was added to each well of the microtitre plate

and incubated for 1 h at 37 8C. The HA titre was

defined as the last dilution of serum showing minimal

positive agglutinin. Values are expressed as reciprocal

of HA titre.

2.4. Statistical analysis

The experimental assays for each parameter of the

individual fish were run in triplicate, and the meanFS.E. for each parameter calculated from the 2 years

Table 2

Range of variation and annual average of several immunological param

occasions considered to represent the normal physiological non-specific

ranges of 19–32.5 8C

Parameter Range

NBT activity (OD at 540 nm) 0.22–0

MPO activity (OD at 450 nm) 0.09–2

Haemagglutination titre 8–1

Lysozyme activity (Ag/ml) 2.10–2

Alternative complement (ACH50) activity (units/ml) 2.02–1

data. Differences among various time periods were

assessed using one-way ANOVA. Similarly, the dif-

ferences among various temperatures prevailing over

five seasons as well as weight of the fish sampled

during each time period were assessed using one-

way ANOVA. Mean values were compared using

Duncan’s (1955) multiple range test. In all cases,

0.05 was used as the level for accepted significance.

3. Results

A significant (P b0.05) difference in the mean

water temperatures observed in the aquaria over 15

days of acclimatization (temperature recorded twice a

day for 15 days) among the five sampling periods was

recorded (Table 1). There was no significant differ-

ence in the weight of the fish sampled during various

seasons (Table 1).

It was observed that MPO content showed a

seasonal pattern with the lowest significant values

recorded in the coldest months (winter and spring) at

a mean temperature range of 19–24 8C and the

highest values during the rest of the period with

water temperature above 28 8C (Fig. 1). Whereas,

a reverse trend was marked with superoxide produc-

tion levels with the highest activity at lowest tem-

perature of 19 8C during winter and a lower activity

at higher temperature range of 28–31 8C during rainy

and autumn seasons. This was not the case for

ACH50 level, as no clear trend with regard to tem-

perature variations was recorded (Table 1). However,

the lowest ACH50 activity (18.19F7.38 units/ml)

was recorded during the autumn season and the

highest of 57.41F7.47 units/ml during spring sea-

eters determined in C. batrachus over a period of 2 years at 10

immune response under culture conditions at mean temperature

Annual average

Year I Year II

.89 0.54F0.05 0.52F0.05

.20 0.77F0.15 0.99F0.24

024 133.08F31.79 130.78F31.03

4.45 15.56F1.16 14.06F1.12

64.06 39.05F6.99 41.37F6.87

J. Kumari et al. / Aquaculture 252 (2006) 121–127 125

son. Significantly lower lysozyme activity was

noticed during the peak summer (mean temperature

of 32.5 8C) (Table 1) compared to other seasonal

temperatures. On the other hand, there was a

decrease in haemagglutination titre in the coldest

winter period at 19 8C and rainy season at a tem-

perature of 31 8C compared to high titre during

summer and autumn seasons (Table 1). Wide varia-

tions in all the parameters were noticed throughout

the year among the individuals (Table 2). The mean

values of each of the immune parameters obtained

over two consecutive years did not differ signifi-

cantly (Table 2).

4. Discussion

A number of reports have shown the changes in

non-specific immune parameters with relation to

infection, toxicity, diet, stressors, pollution or tem-

perature fluctuations (Ingram, 1980; Studnicka et al.,

1986; Anderson, 1990; Anderson et al., 1992; Dalmo

et al., 1997; Ellis, 2001). Nevertheless, just a few of

these studies are related to C. batrachus (Saha et al.,

1993; Sinha and Chakravarty, 1997; Kumari and

Sahoo, 2005). No report on the effect of temperature

fluctuations or normal range in the innate immune

parameters in C. batrachus is available.

Temperature has an effect on a number of phy-

siological and immune parameters (Sinha and Chak-

ravarty, 1997; Hernandez and Tort, 2003; Tort et al.,

2004). On the other hand, the temperature fluctua-

tions have shown variable effects in the immune

parameters among different species. For example,

alternative complement activity did not reveal any

change with relation to variable temperatures (12 or

24 8C) in snapper (Pagrus auratus) (Cook et al.,

2003); whereas a clear reduction in ACH50 activity

was marked in Sparus aurata during winter period

(temperature of b10 8C). In this study, although a

significant seasonal variation was noticed in ACH50

activity of C. batrachus, the variation was not

temperature-dependent. The lowest activity was

marked during autumn at a mean temperature of

28 8C. Collectively, our results are in agreement

with recent studies in cyprinid fish on the alternative

complement pathway activities which were found to

be predominant at lower temperatures (Yano et al.,

1984; Collazos et al., 1994; Le Morvan et al., 1998;

Alcorn et al., 2002).

Similarly, one study revealed that lysozyme level

in S. aurata was less sensitive to seasonal or tempera-

ture changes (Hernandez and Tort, 2003); whereas a

clear reduction in lysozyme level was marked in S.

aurata at lower temperature (Tort et al., 2004). In our

study, the lowest lysozyme level was noticed at the

highest temperature (32.5 8C) in C. batrachus. The

wide variations in all the immune parameters noticed

in this species were also marked in other tropical

Indian major carps (Sahoo et al., 2005). Earlier studies

have shown that the adaptation to lower temperature

did lead to an increase in the respiratory burst activity

in fish (Dexiang and Ainsworth, 1991; Hardie et al.,

1994; Le Morvan et al., 1998). Similarly, the highest

superoxide production level was marked in winter at

19 8C in C. batrachus. On the contrary, the lowest

MPO activities were evident during winter or spring at

temperature of 19–24 8C. Natural haemagglutinin

level appeared to be insensitive to low temperatures

although seasonal fluctuations in this parameter was

noticed in this study.

A clear seasonal variation in all the immune para-

meters is marked in this species. However, the fluctua-

tions noticed in all the parameters are not consistent in

any one of the temperatures. This indicated that C.

batrachus might be adaptable to this temperature

changes and the compensatory mechanism among

the defence molecules might be playing a role to

protect from major pathogen attacks at various periods

of the year. The specific pathogen problem at a parti-

cular period needs a thorough elucidation into the

pathogenic mechanism of the etiological agent to say

which part of the immune system is particularly

responsible for the failure of defence. The baseline

data obtained here might be of help to work as health

indicators in pollution, stress, nutrition or infection-

related studies.

Acknowledgements

The authors wish to thank the Director, Central

Institute of Freshwater Aquaculture, Kausalyaganga,

Bhubaneswar, India and Catfish Unit for providing

the necessary facilities and fish, respectively, during

this study.

J. Kumari et al. / Aquaculture 252 (2006) 121–127126

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