Indian Journal of Experimental Biology Vol. 42, November 2004, pp. 1073-1077

Immunomodulation by 'ImmuPlus (Aqualmmu)' in giant freshwater prawn, Macrobrachium rosenbergii (De Man)

Jaya Kumari, P K Sahoo*, S S Giri & Bindu R Pillai Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, India

Received 24 November 2003; revised 12 August 2004

ImmuPius, a polyherbal commercial formulation was used to modulate the immune system of commercially important giant freshwater prawn M. rosenbergii. The prawns were fed with basal diet supplemented with ImmuPius@ lg/kg feed for 4 weeks. Results showed that the phenoloxidase activity (PO), haemagglutination and lysozyme activities were significantly elevated in lmmuPius-fed prawn up to 3 weeks of feeding and declined after 4 weeks of feeding. The total protein level in lmmuPius-fed prawn raised up to 2"d week of feeding. Incorporation of ImmuPlus at the rate of lg/kg feed in the diet of prawn for 3 weeks may be beneficial in raising the immune status of prawn.

Keywords: Macrobrachiwn rosenbergii, Immunomodulation, ImmuPius

The giant freshwater prawn, Macrobrachium rosenbergii (De Man), is commercially important worldwide as a primary inland cultured species 1•

However, the disease outbreaks caused by the pathogens and environmental stress result in declined production. A major constraint in the commercial scale aquaculture production of this species is the inadequate availability of seed2

. The serious effect of infections in wild stock are most probably underestimated as the diseased animals rapidly vanish in nature, and only in aquacultural farms mortality are reported causing economic Joss3

. Although the freshwater prawns are comparatively resistant than brackishwater shrimps, they are not free from diseases. Shell diseases such as Brown and Black spot caused by bacteria and vibriosis are commonly found in adult prawns4

. Further, high stocking density in culture system aggravates the infections. Studies towards a better understanding of prawn defense mechanisms constitute one approach towards overcoming di sease problems. Many workers have tried to study how shrimp defence factors, including possible induction mechanisms, influence shrimp health and resistance to disease, but the information gained has not yet Jed to any general conclusions. One problem may be the lack of reliable and effective

*Correspondent author: Phone: +91-674-2465421 ; Fax: +91-674-2465407 E-mail: pksahoo I @hotmail.com

indicators for measuring defence status5. To this end,

many cellular and humoral factors of shrimp and a few factors in prawns have been studied with the hope that they may be developed as indicators or as measures for the effectiveness of potential 'immunostimulants' 5

-8

. In this study, phenoloxidase (PO) enzyme activity, agglutinin, lysozyme and protein levels have been examined to determine whether these could be used as indicators for the stimulatory activity for one of the commercial polyherbal immunopotentiators, viz., ImmuPius.

Few of the immunostimulants viz., ~-1, 3 glucan5·6

,

peptidoglycan7, lipopolysaccharide (LPS) and

commercial stimulants, PENSTlM, BRM-01 and BRM-025 have been successfully tried to modulate non-specific immunity in shrimps and to enhance resistance against various bacterial, viral and fungal infections. Similarly, benzalkonium chloride8

, having immunostimulant property has been evaluated in freshwater prawn.

Medicinal plants, used in various traditional systems, have immune potential against various diseases9

. More than 13,000 plants have been studied during the last five years for various pharmacological properties in animals and human beings9

·10

. ImmuPlus (Aqualmmu), a polyherbal formulation (Indian Herbs Supply and Research Company Limited, Saharanpur, India) contains the extracts of selected Indian medicinal plants viz., Ocimum sanctum (Tul si), Withania somnifera (Ashwagandha), Tinospora

1074 INDI AN J EXP BIOL. NOVEMBER 2004

cordifolia (Guduchi) and Embfica officina/is (Amlaki ) as major constitutents in optimum concentration. Antioxidant and immunomodulatory activities of these plants in human being and animals have been well documented 11

• Therefore lmmuPiu was used in thi s investigation to modulate the immune system of freshwater prawn .

Materials and Methods Two diets were compared in the present study. The

control diet was a basal diet (Tab le I) containing 36.75% crude protein , 6% crude lipid and 22.0% ash . In the experimental diet, the basal diet was supplemented with ImmuPius (Aquaimmu, I g/kg) . Trial studies with postlarvae of M. rosenbergii showed that I g/kg supplementation of ImmuPlus (Aqualmmu) increased g rowth and resistance to stress. The feeding ingredients were thoroughly mixed, manually pelletized, air-dried and stored at room temperature. The pelleted feed was prepared at weekly interval s.

The ex perimental prawns (M. rosenbergii, 35-40 g) were artificially propagated and raised from a single stock at the Institute for six months prior to the experiment. The inter- moult prawns were selected and divided into two groups of 72 each. Each group was further divided into four subgroups (for each week of 4 weeks study). Each subgroup had 3 replicates of six prawns. Each replicate was held in a 300 I plastic tank with continous aeration. The prawns were acc limated to laboratory condition for 2 weeks before starting the experiment. The prawns were fed twice a day at the rate of 5% of their body weight at 1000 and 1700 hrs. Unconsumed food and excreta were removed every

Table 1--(:ompositi on of the basal di et of M. rosenbergii

Ingred ient

Groundnut cake Fi sh meal Ri ce bran Soybean meal Vitamin and mineral mixture* Vegetable oi l Carboxymethyl cellulose

Percentage

so 25 13 5 3 3

*Each 1000 g vitamin and mineral mixture provides vitamin A-2,000,000 i.u. , Vitamin D,-100,000 i.u. , vitam in Br 0.8 g. vitamin E-1 00 mg. vi tamin C-0.3 mg, vit amin K-0.4 g. calcium pantothenate-1.00 g, ni cotinamide-0.4 g, vitamin 8 12-2.4 mg, choline chloride-60 g, calcium -300 g, manganeese- 11 g. iodine-0.4 g, iron-3.00 g, zinc-6.00 g. copper-0 .8 g and cobalt-0. 18 g.

morning before feeding with replacement of 15% freshwater. During the experiment the mean physico­chemical parameters of water were a follows: water

te mperature 27°± 1.0°C, pH 7.2±0.04. dissolved oxygen 8.2±0.2 mg/1, hardness as CaCO, 92±0.5 mg/1 , N03-N 0.007±0.003 mg/1 and HJ 0.021±0.005 mg/1. The prawns were fed with the basal/experimental diet for 4 weeks .

Haemolymph was collected at the e nd of each week from the ventral-sin us cavity of three prawns from each tank using a 26-gauge needle and I ml syringe containing anticoagu lant solution ( l :9) (tri-sodium citrate: 0.114 M , sodium chloride: 0 .10 M, p H 7.45) 12

to measure phenoloxidase (PO) activity.

From the collected haemo lymph, haemocyte lysate supernatant (HLS) was prepared 13

• Briefly, PBS­washed haemocyte suspension was homogenized with a sonicator (Artek) equipped with a microtip (output 50, duty cycle 50%) and centrifuged ( 12,000 g) to collect HLS. PO acttvity was assayed spectrophotometrically by recording the formation of dopachrome from L-dihydroxyphenylalanine by usi ng L-3, 4 dihydroxyphenylalanine (L-DOPA, Hi-Media, Mumbai) as substrate in U-bottom microweli plates. HLS (50 ~-tl) was preincubated for 15 min at 37°C, after which 100 ~-tl of L-DOPA (1.6 mg/ml in tri s buffer saline, TBS: 50 mM Tris , 100 mM NaCI, pH 7.3) was added and allowed to react for I min . Each reaction mixture was further diluted with 100 j.tl TBS, and then the absorbance at 490 nm was measured . The control soluti on, which consi ' ted of 50 ~-tl PBS (to replace HLS), 100 ~-tl L-DOPA, and 100 ~-tl TBS, was used for the background PO activity in al l test conditions. The background PO activity (optical d nsity) values were in the range of 0.01-0.02. The PO activity in terms of optical density was expressed as dopachrome formation/50 j.tl HLS 1 ~ .

From the remaining three prawns in each replicate, haemolymph was collected as described above without anticoagu lant, a llowed to clot at room temperature for 30 min and left at 4°C for I hr. The tubes were then centrifuged at 4524 g at 4°C for 30 min to collect the supernatant serum. The serum samples were stored at -30°C till further analysis.

The haemagglutinin assay was carried out in U-bottom microwell plates using 1.5 % sheep red blood cell s (SRBC) 15

• Two-fold serial dilutions of serum (50 j.tl) samples were made in TBS . An equal vo lume of 1.5% (v/v) SRBC was added to each dilution of serum. The plates were incubated at 25°C

JAY A KUMAR! e1. a/.: IMMUNOMODULATION BY TMMUPLUS IN FRESHWATER PRAWN 1075

for 45 min. Haemagglutinin titre was recorded as the reciprocal of the last dilution, resulting in agglutination after 45 min incubation . Negative controls comprised mixed equal volumes of RBC and TBS.

Lysozyme levels in serum were measured using the turbidimetric assay 16 with slight modification. Briefly, a solution of 20 mg of Micrococcus lysodeikticus suspension in 100 ml acetate buffer (0.02 M, pH 5.5) was used. To 125 Jll of serum, 125 Jll of acetate buffer was added and then 1.25 ml of substrate suspension added to each tube. The reaction mixture was incubated for I hr at 25°C 17

• Difference between the initial and final turbidity ( ... OD) was taken as the measure of enzyme activity. The difference of 0.001 in OD observed at one hour is taken as one unit of enzyme activity. The protein content of serum was measured following Bradford method 18

, using bovine serum albumin as a standard protein. The mean±S E fo r each parameter was calculated for each group . Data were anal ysed using two-way analysis of variance. Means were compared using Duncan's multiple range test 19

. Difference was considered significant when P< 0 .05 .

Results and Discussion The ImmuPlus-fed prawns showed a significant

(?<0.05) ri se in PO activity than the control group throughout the experimental period and enhanced activ ity was observed ti ll 3 weeks of lmmuPlus feeding which declined after 4 weeks of feeding (Fig. I a). A fluctuation in PO activity was noticed in control group prawns throughout the four weeks of experiment. A similar trend of rise in haemagglutinin levels in immunostimulant-fed prawns in comparison to control group was observed for 3 weeks of feed ing wh ich became comparable with the control value after 4 weeks (Fig. l b). Lysozy me acti vity diffe red considerably amongst individual animals and a fl uctuation in lysozy me level in control prawns was no ticed during different peri ods. T he ImmuPiu s-fed

rawns showed significantl y (?<0.05 ) nhanced serum lysozy me level till 3 weeks o f feed ing (Fig. i c). A rise in serum tota l protein concentration was marked in ImmuPlus-fed prawns until 2 weeks of feeding compared to contro l which subsequentl y decl ined and beca me on par with level of control group (Fig. l d).

!n any culture sys tem special care must be taken to control opportun istic pathogens such as vibrios,

chitinoclastic bacteria, filamentous bacteria, fungi and protozonas. Although the immunostimulants do not eradicate all pathogens, they can increase non-specific disease resistance, leading to reduction in mortality caused by opportunistic pathogens or stressors. The present results showed that oral administration of one of the commercial herbal immunomodulators (ImmuPlus) could enhance non-specific immunity of

0.25

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-~ 0.2 -.;: 0 .. ., 0 .15 1/) .. "0

·~ 0.1 0 r:: ~ 0 .05 c.

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0 16

14

-.;: 12 r:: .g 10 .. r:: "5 8

~ 6 .. ~ 4 .. :I: 2

0 250

i:' 200

:~ ~ 150 ., E ~ 100 0 1/) >.

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"0 :§ 12 o; 10 > ~ 8 r:: ·a; 6 0 a. 4 ;;;

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2 3 4

V.feeks after feeding

Fig. 1- Phcnoloxidase ac ti vity (a) in haemoc yte lysate supernatan t. haemagglutinatio n titre (b). lysozy me ac ti vity (c). and tOta l prote in leve l (d) in the serum o f M. rose11 bergii afte r I. 2. 3 and 4 weeks o f feedi ng basal (D) or lmmuPius-mixed (• ) d iet. [Each ba r represents the mean ± SE va lue fro m 9 dete rminati ons. Bars with aste ri sk are s ignificamly (P<0.05 ) d iffe rent among the prawns at d iffe rent feeding pe ri ods]

1076 INDIAN J EXP BIOL. NOVEMBER 2004

the adult prawns. Some of the plant extracts/products have been ev:.Juated for their immunostimulatory and antioxidant properties in fishes 20

'21

. ImmuPlus is one of the above herbai products which has a lso been evaluated for its efficacy to stimulate the non-specific immunity in vertebrates21

. The herbs included in ' ImmuPlus' have been studied to stimulate immune system of human beings and animals through enhanced phagocytosis , production of reactive oxygen and nitrogen species 11

. The herbal product contains Amlaki (Emblica officina/is), as one of its ingre­dients, is rich in vitamin C which is a potent immunostimulant and antioxidane-25

. The ascorbic acid present in this fruit is conjugated to gallic acid and reducing sugars, forming a tannoid complex, which is more stable in nature and enhances the bioavailability of ascorbic acid 11

. Similarly, another constituent, Guduchi (Tinospora cord(folia) , is well known to augment phagocytic cell functions and enhance protection against infections in animals and human beings26

. The other constituents, Aswagandha (Withania somnifera) and Tulsi (Ocimum sanctum) are also well known for their immunomodu latory

I I I ry 7 Th . . d h . ' J h . ro es ·- . us , It IS expecte t at su111 ar en ancmg effects may be reflected on the immune parameters in treated prawns.

In the present study, elevated PO activity , haemagglutinin, lysozyme and total protein levels were recorded in ImmuPius-fed M. rosenbergii. The preliminary assay of lysozyme activity in prawns as an index of immunostimulation was made and the activ ity was well COITelated with the other immune indices viz., PO activity and haemagglutinin titre. The enhanced immunity indices were noticed to be there up to three weeks of feeding, which declined after 4 weeks of feeding. That is why it is essenti a l to know the optimum dose and period of feeding of each of the substances in any species.

Much more to be studied to understand the mechanisms behind these immunostimulatory effects as well as to employ these herbal substances as possible therapeutants . This concept of using herbal products for health, also ga ins a little more credibility, when we realize that herbal antioxidants concurrently exhibit significant immunomodulatory activit ies.

Acknowledgement This work, in part, was supported by a grant from

the Indi an Herbs Research & Supply Co. Ltd., Saharanpur, India.

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