Fish & Shellfish Immunology 18 (2005) 327e334

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Stimulation of immunity in Indian major carp Catla catlawith herbal feed ingredients

Vasudeva Rao Y., Rina Chakrabarti*

Department of Zoology, University of Delhi, Delhi 110 007, India

Received 16 March 2004; accepted 5 August 2004

Available online 3 November 2004

Abstract

Catla catla, catla (150G 20 g) were fed a diet containing seed of Achyranthes aspera (0.5%) and control diet withoutA. aspera for four weeks prior to and after ip injection with chicken erythrocytes. Fish were sampled for four

consecutive weeks after immunization. Hemagglutination antibody titers were significantly higher in the test group offishes compared with the control group. Serum globulin levels were significantly (Pt-test! 0.05) higher in the test groupthan control group on days 14 and 21. Anti-trypsin activity due to total serum protease inhibitors and a1-antiproteasewas also significantly (Pt-test! 0.05) higher in the test group of fishes than the control. RNA/DNA ratio of spleen andkidney was also significantly (Pt-test! 0.05) higher in test group than the control group. All these results confirm thatA. aspera enhances the immunity of catla.� 2004 Elsevier Ltd. All rights reserved.

Keywords: Catla (Catla catla); Achyranthes aspera; Immunostimulation; Antigen-specific antibody response; Anti-proteases

1. Introduction

Immunostimulants increase resistance to infectious diseases by enhancing both specific and non-specificdefense mechanisms. Enhanced non-specific immunity against various bacterial fish pathogens (Aeromonashydrophila, Vibrio anguillarum, Vibrio salmonicida, Yersinia ruckeri) with glucans derived fromSchizophyllum commune and Saccharomyces cerevisiae has been reported [1,2]. Oral administration ofpeptidoglycan increased resistance to V. anguillarum in rainbow trout Oncorhyncus mykiss [3] and oraltreatment with glucans increased resistance to V. anguillarum and V. salmonicida in Atlantic salmon Salmosalar [4]. Injection of chitin increased the resistance in brook trout [5] and in rainbow trout [6] and injection

* Corresponding author. Tel.: C91 11 27666496.

E-mail address: rina@ndf.vsnl.net.in (R. Chakrabarti).

1050-4648/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.fsi.2004.08.005

328 V. Rao Y., R. Chakrabarti / Fish & Shellfish Immunology 18 (2005) 327e334

of glucans increased phagocytic activity and resistance to Streptococcus sp. in yellowtail Seriolaquinqueradiata [7]. Oral administration of quillaja saponin enhanced the immunity of yellowtail [8] andfeeding with Catharanthus roseus plant extract enhanced the immune response of Labeo rohita [9].

Achyranthes aspera Linn., a herb belonging to Amaranthaceae, is widely available and distributedthroughout India. The properties of this plant are thermogenic, expectorant, revulsive, carminative,digestive, stomachic, laxative, anodyne, depurative, anthelminthic, diuretic, linthontriptic, sudorific,demulcent, hematinic and anti-inflammatory; it is useful in treating cough, asthma, bronchitis, dyspepsia,flatulence, colic, painful inflammations, dropsy, ophthalmopathy, vomiting, leprosy, skin diseases, pruritus,helminthiasis, strangury, renal and vesical calculi, cardiac disorders, anaemia, vitiated conditions of kaphaand vata and general debility [10]. This plant has been shown to be immunostimulatory in mammals, as theaqueous extract of A. aspera enhanced the antigen-specific antibody response in mice of different geneticbackground, namely H-2b, H-2d and H-2q [11]. Among the different parts of the plant, the seed and rootpossess greater stimulatory activity [11]. Also A. aspera enhanced antigen-specific antibody levels, serumglobulin levels and RNA/DNA ratios of spleen [12], and anti-trypsin activity [13] in L. rohita. The presentinvestigation aims to study the immunomodulatory effect of A. aspera in Indian major carp Catla catla, themost important commercial fish in India.

2. Materials and methods

2.1. Experimental diet

Seed of A. aspera (grown at the Department of Zoology, University of Delhi) was collected, washed withtap water and air-dried. Seed (0.5 g) was ground in a mortar and pestle with water and the whole extractwas added to 99.5 g of experimental diet (30% protein) along with other ingredients (Table 1).

2.2. Experimental conditions

Indian major carp, C. catla, (150G 20 g) were obtained from the local fish market, Delhi, India and wereacclimatized for three weeks in outdoor cement tanks. Fish were divided into 8 groups (4 fish/group) undertwo feeding regimes, control: 4 groups, test: 4 groups. Fish were then fed either the experimental diet or thecontrol diet. Feeding was started four weeks prior to the immunization at 1% of body weight per day, onceat 9:00 AM and continued till the end of the experiment. The temperature ranged from 24 to 29 (C anddissolved oxygen was maintained above 5 mg/L throughout the experiment.

2.3. Antigen

Chicken red blood cells (c-RBC) were collected in Alsever’s solution one day before immunization, washedthrice with phosphate buffered saline (PBS, pH 7.4) and made to 20% (v/v) suspension in the same solution.

Table 1

Composition of experimental diets

Ingredients/100 g of feed Experimental diet Control diet

Fish meal (g) 32.88 32.88

Wheat flour (g) 54.62 55.12

Cod liver oil (g) 10 10

Vitamin and mineral premix (g) 2 2

Achyranthes aspera (g) 0.5 e

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2.4. Immunization of fish

Fish were anaesthetized with MS-222 (Sigma chemicals) and injected intraperitoneally with 500 ml of thec-RBC suspension in PBS.

2.5. Sampling

Fish were anaesthetized with MS-222 and bled by cutting the tail. Blood was collected from 4 fish of eachgroup on days 7, 14, 21 and 28 after immunization and allowed to clot at room temperature. Serum wasobtained by centrifugation. After blood collection, the spleen and kidney were removed from the fish forRNA/DNA estimation.

2.6. Hemagglutination assay

The antigen-specific antibody response was determined by hemagglutination assay [14]. Blood fromchicken was collected in Alsever’s solution and the cells were washed in PBS and resuspended to 2% (v/v) inPBS. Serum of control/test fish (50 ml) was serially diluted in PBS in 96 well round-bottomed microtiterplates. Equal volume of 2% c-RBC was added to all wells and kept for 1 h at room temperature and thenovernight at 4 (C. The reciprocal of the highest dilution that gave agglutination was taken as thehemagglutinating antibody titer.

2.7. Determination of total serum protein, serum albumin and globulin

Saturated ammonium sulfate (100 ml) was added to 100 ml of serum in a micro centrifuge tube, mixedwell and left to stand for 1 h at room temperature. The tubes were then centrifuged (Sigma-3K30) at10,000! g for 10 min and the supernatants (albumin fraction) were collected into separate tubes. Theprecipitate (globulin fraction) was dissolved in 500 ml of distilled water. The protein content of bothalbumin and globulin fractions was determined by the Lowry method [15]. Total serum protein wasdetermined at 280 nm in a spectrophotometer by using standard DNA/Protein program pack, Ver.No.2.00, UV-1600 Series of Shimadzu UVevisible spectrophotometer.

2.8. Determination of a1-antiprotease

The level of a1-antiprotease in fish serum was determined [16] by incubating 10 ml of serum with 20 mg oftrypsin dissolved in 100 ml of TriseHCl (50 mM, pH 8.2). In the serum blank, trypsin was replaced with100 ml of TriseHCl, and in the positive control no serum was added to trypsin. All tubes were made up to200 ml with TriseHCl and incubated at room temperature for 1 h. After incubation, 2 ml of 0.1 mMsubstrate, BAPNA (Na-benzoyl-DL-arginine-p-nitroanilide HCl, Sigma) dissolved in TriseHCl (containing20 mM calcium chloride), was added to all tubes and incubated for a further 15 min. Finally, the reactionwas stopped by adding 500 ml of 30% acetic acid. Optical density (OD) was measured at 410 nm ina UVevisible spectrophotometer, Shimadzu UV-1601.

The OD of the positive control, i.e. the activity of trypsin without the addition of any serum was taken as100% activity. The OD values of the serum blank were deducted from trypsin plus serum, and thepercentage trypsin inhibition by the serum was calculated.

330 V. Rao Y., R. Chakrabarti / Fish & Shellfish Immunology 18 (2005) 327e334

2.9. Determination of total serum anti-proteases

Total protease inhibitors in fish serum was performed [16] by incubating 10 ml of serum with 20 mg oftrypsin dissolved in 100 ml of PBS (pH 7.4). No serum was added to trypsin in the positive control but 10 mlPBS was added. All tubes were incubated at room temperature for 30 min. After incubation, 1 ml of caseindissolved in PBS (2.5 mg/ml), was added to all tubes and incubated for a further 15 min. The reaction wasstopped by adding 500 ml of 10% trichloroacetic acid. To remove the precipitate, all tubes were centrifugedat 10,000! g for 5 min. The optical density of the supernatants was measured at 280 nm in a UVevisiblespectrophotometer, Shimadzu UV-1601, and the percentage trypsin inhibition was calculated.

2.10. RNA/DNA ratio

Nucleic acids were isolated from spleen and kidney according to Schneider [17]. DNA and RNA weredetermined using diphenylamine and orcinol, respectively.

2.11. Statistical analysis

All the data were statistically analysed by Student t-test using Microsoft Excel. The level of significancewas P! 0.05.

3. Results and discussion

After immunization, the antigen-specific antibody response measured as hemagglutination antibody(HA) titers reached maximum levels on day 14 and then decreased. As shown in Fig. 1, thehemagglutinating antibody levels of the test group were significantly higher than for the control groupon days 7 and 21. On day 14, PZ 0.063 and on day 28, PZ 0.054. In the test group, the highest titersamongst the 4 sampled fish were 32, 2048, 128 and 32 on days 7, 14, 21 and 28, respectively; whereas in thecontrol group, the highest titers were 16, 32, 16 and 16 over the same period.

1

10

100

1000

10000

7 14 21 28Days after Immunization

Control

Test

*

*

Hem

aggl

utin

atio

n tit

re

Fig. 1. Effect of feeding A. aspera on antigen-specific antibody response in Catla catla. The values represent the meanG S.E. of four

fish (*P ! 0.05).

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In the test group, the total serum protein level was higher on days 14 and 21, and on day 28 it was lowerthan the control, but none was significantly (Pt-testO 0.05) different (Fig. 2a). The serum albumin andglobulin levels are shown in Fig. 2b and c, respectively. No significant differences were detected between thetest and control groups except for globulin levels on day 21 when the test group was significantly higherthan the control.

25

28

31

34

37

40Control

Test

10

12

14

16

18

20

5

7

9

11

13

15

7 14 21 28

Days after Immunization

*

b

a

c

Tot

al p

rote

in (

mg/

ml s

erum

)A

lbum

in (

mg/

ml s

erum

)G

lobu

lin (

mg/

ml s

erum

)

Fig. 2. Effect of feeding A. aspera on total serum protein (a), albumin (b), and globulin (c) levels in Catla catla. The values represent the

meanG S.E. of four fish (*P ! 0.05).

332 V. Rao Y., R. Chakrabarti / Fish & Shellfish Immunology 18 (2005) 327e334

Protease inhibitors principally a1-protease inhibitor and a2-macroglobulin play a role in restricting theability of bacteria to invade and to grow in fish [18]. The percentage trypsin inhibition by a1-antiproteasewas significantly (Pt-test! 0.05) higher in the test groups than the control groups (Fig. 3a). The percentagetrypsin inhibition due to total protease inhibitors in serum reached a maximum level on day 21 and decreasedon day 28. While the trend was similar in both groups (Fig. 3b), the percentage total trypsin inhibition inserum was significantly (Pt-test! 0.05) higher in the test groups than the control groups throughout the studyperiod, except day 14. These results indicate that feeding of C. catla with A. aspera incorporated diet alsoenhances certain non-specific factors of the immune system by enhancing the level of natural anti-proteasesin the serum, which may provide defense against invading bacterial pathogens.

The RNA/DNA ratio is used as an indicator of the growth of fish, the higher the RNA/DNA ratio thehigher the protein synthesis and the better the growth. The RNA/DNA ratio in spleen and kidney reflectsthe active protein synthesis (i.e. immunoglobulin and other molecules associated with the immuneresponse). The RNA/DNA ratio of spleen was significantly higher in the test group on days 7 and 14(Fig. 4a). The RNA/DNA ratio of kidney was significantly higher in the test group than the control groupon days 21 and 28 (Fig. 4b).

In conclusion, the present investigation indicates that both specific and non-specific immunity wereenhanced in catla when fed with the experimental diet containing A. aspera compared with the control fish

80

85

90

95

100Control

Test

**

* *

50

55

60

65

70

75

7 14 21 28

Days after Immunization

*

**

b

a

Try

psin

Inhi

bitio

n (%

)T

ryps

in In

hibi

tion

(%)

Fig. 3. Effect of feeding A. aspera on a1-antiprotease (a) and total protease inhibitors (b) levels in fish serum. The values represent the

meanG S.E. of four fish (*P! 0.05).

333V. Rao Y., R. Chakrabarti / Fish & Shellfish Immunology 18 (2005) 327e334

fed with the normal diet. This was revealed by higher serum antibody levels and higher serum anti-proteasesin the test group fish than control groups. Serum globulin level and RNA/DNA ratio of the spleen werealso significantly enhanced in the fish fed with the A. aspera containing diet. These results confirm thatA. aspera stimulates both specific immunity and non-specific immunity in Indian major carp, C. catla.

Acknowledgements

I am very much thankful to my colleague Mr. M. Romesh Singh for his help during the experiment.Authors express their sincere thanks to the unknown reviewers for their valuable suggestions.

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0.5

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