Growth and immune response of juvenile greasy groupers (Epinephelus tauvina) fed with herbal...

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Aquaculture 237 (2004) 9–20

Growth and immune response of juvenile greasy

groupers (Epinephelus tauvina) fed with herbal

antibacterial active principle supplemented diets

against Vibrio harveyi infections

V. Sivarama,*, M.M. Babua, G. Immanuela, S. Murugadassa,T. Citarasub, M.P. Mariana

aCenter for Marine Science and Technology, Manonmaniam Sundaranar University, Mount View Campus,

Rajakkamangalam-629 502, Tamil Nadu, IndiabSpic Bioprocess Laboratory, Centre for Biotechnology, Anna University, Chennai 600113, India

Received 18 August 2003; received in revised form 10 February 2004; accepted 15 March 2004

Abstract

Methanolic extracts of 10 antibacterial herbs were screened against Vibrio harveyi, the

causative agent of heavy mortalities in cultured greasy grouper Epinephelus tauvina. Ocimum

sanctum (OS), Withania somnifera (WS) and Myristica fragrans (MF) showed wider zones of

inhibition against this pathogen. The active principles from methanolic extracts were purified

through a preparative silica column and four different concentrations (100, 200, 400 and 800 mg/

kg diet) of purified active principles of OS, WS and MF were individually incorporated with the

basal diet and fed to greasy groupers (body weight of 30.0F 0.5 g) for a 12-week period to

evaluate the immunomodulatory and growth promoting characteristics of herbal active principles

in vivo. Significant improvements in immune parameters such as phagocytic activity, serum

bactericidal activity, Albumin–globulin (A/G) ratio and leukocrit were observed with the groups

fed on OS and WS supplemented diets at the concentrations of 100 and 200 mg/kg diet compared

to the control group. Supplementation of MF in the diet at all concentrations negatively affected

the immune parameters and immunosuppressive state was exhibited in E. tauvina. Growth and

feed conversion ratios were significantly increased in the WS fed group as well as the OS fed

group at the concentration of 100 and 200 mg/kg diet ( p < 0.05). Challenges were made with live

V. harveyi in both control and experimental groups (OS, WS and MF) and 100% mortality was

observed in the control group and fish fed a 100 mg/kg dietary concentration of MF. OS and WS

0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquaculture.2004.03.014

* Corresponding author. Tel.: +91-4652-253078; fax: +91-4652-221214.

E-mail address: [email protected] (V. Sivaram).

V. Sivaram et al. / Aquaculture 237 (2004) 9–2010

fed groups at the concentrations of 100, 200 reduced the mortalities by up to 5%. The application

of herbal principles in aquaculture is also discussed.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Epinephelus tauvina; Vibrio harveyi; Antibacterial herbal diet; Immune response

1. Introduction

Aquaculture production and trade for the grouper fishery is being limited by disease

outbreaks, nutrition and poor larval survival (Boonyaratpalin, 1993). Mariculture oper-

ations of grouper mainly depend upon fry and fingerlings caught from the wild, that

stresses the need to develop and standardize the culture of juveniles caught for grow-out in

an effective way. Vibriosis is a common problem in the intensive culture of grouper in

various stages of culture (Rasheed, 1989). Saeed (1995) reported the association of Vibrio

harveyi in mass mortalities of greasy groupers. Detrimental side effects such as toxicity,

resistance, residual impacts on public health and environmental hazard were major risk

factors while using the commercial antibiotics in disease treatment of fish (Harper, 2002;

Karunasager et al., 1994). Studies made by Ervik et al. (1994) demonstrated the increase in

resistant bacteria in the intestine of the fish receiving antibiotic drugs and also a total of

74–100% of wild fish in close proximity to treated ponds contained quinolone residues, a

group of antibiotics important in human therapy. To develop alternative practices for

disease management in aquaculture, attention should be diverted to find novel drugs,

especially from plant sources. Citarasu et al. (2002) describes the antimicrobial and

antistress effects of herbal product that significantly increased the survival rate of tiger

shrimp (Penaeus monodon) larvae. Babu and Marian (2001) demonstrated the effective

spent-spawner utilization, increasing fecundity and disease resistant larval production in P.

monodon by using herbal supplemented diets. These findings suggest that phytochemicals

could be an alternative to the chemotherapeutic molecules in aquaculture. An attempt to

develop multifunctional phytochemical based on grow-out diets for greasy grouper

juveniles was examined.

2. Materials and methods

2.1. Bacterial culture

V. harveyi (CMB57) was isolated (Sheeja et al., 2003) on Thiosulphate Citrate

Bilesalt Sucrose (TCBS) agar (HIMEDIA, India) from mortalities among cultured

grouper farms from India, and identified in our laboratory using biochemical

reactions (Farmer and Hickman-Brenner, 1992) as well as based on the characteristics

described in Bergey’s Manual of systematic bacteriology (Holt et al., 1994). The

isolates were oxidase positive, sensitive to vibriostatic agents, grown in nutrient broth

(HIMEDIA, India) with 3% and 6% NaCl and TCBS agar. Serological tests were

also done with standard anti V. harveyi sera for species confirmation. The pure

V. Sivaram et al. / Aquaculture 237 (2004) 9–20 11

cultures were stocked in TCBS slants at 4 jC. The virulence of stock cultures (CMB57)

were evaluated at their lethal dose (Saeed, 1995; Sheeja et al., 2003) in sterile 1% saline

through intramuscular injection in five healthy fish selected from aquaria and main-

tained in separate 250 l flow through sea water aquaria for observations of pathological

signs.

2.2. Screening and purification of active principles from antimicrobial herbs

Ten herbal plants possessing antibacterial activity against Vibrio species (Thomson and

William, 1978; Cowan, 1999) were selected for the present study. The name of the herbal

plants and the parts used are given in Table 1. Fresh leaves of herbal plants were collected

based on their availability from various geographical locations of South India and seeds

were purchased from local market. The plant parts were shadow dried, ground to a fine

texture and the sieved powder was then soaked with equal part of methanol (1:1) for 48

h (Eloff, 1998). The slurry was then filtered, washed to remove non-soluble fractions and

filtrate was centrifuged (20,000� g for 30 min) for clarification. The clarified extracts

were condensed at 35 jC, until the solvent residue had evaporated. Each 500 Ag of

condensate was impregnated in 5 mm diameter sterile paper discs (HIMEDIA, India);

screened against V. harveyi through replicates of disc diffusion test as described by Bauer

et al. (1966) and zone of inhibition of each herbal extract was measured and recorded.

Based on the in vitro inhibitory activity, Ocimum sanctum (OS), Withania somnifera (WS)

and Myristica fragrans (MF) were selected for further characterization. Purification of

active principles from crude methanolic extracts of OS, MF and WS was done through

preparative silica column chromatography (mesh size 50–80 Am, 30 cm length, 0.5 ml

flow rate, 3 bed volume elution) with Chloroform/Methanol (90:10) as mobile phase,

fractions were condensed in a rotary evaporator and again screened against V. harveyi to

the determine active fraction. One positive fraction with inhibitory effect against the

pathogen from each extract was selected for growth and immune studies in greasy grouper

juveniles.

Table 1

Name of the plants and their inhibitory activity against V. harveyi

Herb Name Family Parts used for

antibacterial

screening

Zone of inhibition

against V. harveyi

(mm)a

Azadirachta indica Meliaceae Leaf 13.2F 0.1

Murraye Koenigii Myristicaceae Leaf 11.4F 0.4

Andrographis paniculata Acanthaceae Leaf 16.3F 0.3

Daemia extensa Asclapidaceae Leaf 10.1F 0.7

Ocimum sanctum Lamiaceae Leaf 29.4F 0.5

Solanum surattense Solanaceae Leaf 14.3F 0.1

Tinospora cardifolia Menispermaceae Leaf 7.3F 0.6

Withania somnifera Solanaceae Root 24.3F 0.1

Terminalia bellirica Combretaceae Seed 7.1F 0.3

Myristica fragrans Myristicaceae Seed 21.4F 0.9

a Disc diffusion test.

2.3. Preparation of herbal diets

Ingredients and formulation of the basal ration (Table 2) was followed as described by

Boonyaratpalin (1993). The proximate composition of the basal diet was 45.1% protein;

7.2% lipid; 14.6% ash; 7.1% moisture; 3% fibre. Twelve test diets (4� 3) were prepared

by incorporating three herbal principles separately at the concentration of 100, 200, 400

and 800 mg/kg diet. First, dry ingredients were mixed thoroughly and 4% gelatin solution

containing active principles with appropriate concentration were added along with oil

ingredients. Water was added and pH of the diets were adjusted to 7.0F 0.1 with saturated

sodium hydroxide, and mixed thoroughly in mixer for 20 min. The resulting dough was

extruded through a pelletizer with a die into small strands of pellets. The diets were dried

at room temperature for 48 h, and then stored in air tight containers at room temperature

until fed.

2.4. Experimental fish and husbandry

Greasy grouper juveniles (Epinephelus tauvina) caught from wild (Gulf of Mannar,

India) were stocked in quarantine tanks to assess their disease free health status

(through disease symptoms, microbiological examinations of Vibrios load as well as

the visual signs) for the period of 15 days and healthy fish were used for the present

study. All healthy fish were acclimatized in ambient laboratory condition for 30 days

in 250 l seawater aquaria and fed with commercial diet. The proximate composition

(%) of the commercial diet was moisture 13, Crude protein 33; Ether extract 9; ash 3;

crude fibre 6. After acclimatization, 12 experimental groups and control group in

triplicate were stocked (50 individuals/group at the body weight range of 30.5F 0.5 g/

individual) in 39 individual 250 l capacity flow through aquaria at the water exchange

rate of 2 l/min. High pressure sand filtered sea water used with acceptable limits of

water quality throughout the experiments. Temperature was 28F 1 jC, dissolved

oxygen concentration was >6.0 mg/l, ammonia–nitrogen concentration was not


Table 2

Composition of basal diet (Boonyaratpalin, 1993)

Materials g/kg diet

Brown fish meal 280

Shrimp head meal 100

Squid meal 20

Squid liver powder 30

Wheat gluten 60

Wheat flour 200

Soy bean meal 100

Broken rice 102

Fish oil 20

Vitamin premix 7.8

Mineral mix 40

Other 40.2


exceeded >0.22 mg/l and nitrite–nitrogen did not exceed 0.07 mg/l at any point during

the study. 12:12 L/D photoperiod was maintained in aquaria through out the study

period. Fish were fed with their respective diet at the rate of 5% of their body weight/

day for the period of experiment. The daily ration was subdivided into two and fed at

0900 and 1700 h for each day. Faecal matter was collected at 1500 h daily and

uneaten feed was collected after each feeding and the dry matter content was

determined (dried at 105 jC). Specific growth rate and feed conversion ratio (FCR)

was estimated for both control and experimental groups using the procedures described

elsewhere (Ricker, 1979).

2.5. Collection and immunological analysis of blood samples

Every four weeks, five number of randomly chosen fishes from each experimental

and control groups were anaesthetized with 50 mg/dm3 MS-222, taken after 24 h of

final feeding for blood collection. Blood was collected from caudal vein, with 1 ml

plastic syringe rinsed with anticoagulant, and part of the blood was transferred

immediately in to 2 ml micro centrifuge tubes containing equal volume of anticoag-

ulant (12.5% sodium citrate) and stored at 4 jC for leukocrit study and remaining

blood was kept in room temperature for 1 h without anticoagulant to collect serum for

immunological assessment and stored at � 40 jC until further analysis. Part of the

blood in triplicate were analysed for leukocrit value (Blaxhall and Daisley, 1973). Rate

of phagocytosis of the blood samples was estimated (Siwicki et al., 1994) by mixing a

drop of freshly collected blood from both control and experimental groups mixed with

formalin killed V. harveyi (1�108 cells/ml) on a glass cover slip and placed in a

humidified chamber for 30 min at 20 jC. After incubation, the cover slips were

washed with PBS and cells were fixed with methanol for 5 minutes and stained with

May Grunwald’s Giemsa (MERCK, Germany) and washed three times with distilled

water and dried at 40 jC. The slides were observed at 1000� in oil immersion

microscope for phagocytes engulfing more than a bacterium. Bactericidal activity was

determined in vivo i.e., fish were injected with 0.1 ml/kg body weight with live V.

harveyi suspension (containing 105 cell/ml) and blood samples were collected from the

animals after 10 and 90 min intervals of the experiment. One hundred microliters of

blood was serially diluted and spread plated in triplicate on TCBS agar plates (HI

MEDIA, India) and incubated at 34 jC for 24 h. From the number of V. harveyi

colonies in each plate, total Colony Forming Units (CFU) in each blood sample was

estimated. Albumin–Globulin ratio (A/G) of experimental and control sera was

determined as described by Sahoo et al. (1999).

2.6. Challenge experiments

Once in four weeks, 10 fish from each group were challenged with the lethal

dose of V. harveyi was injected intramuscularly, transferred to 100 l aquaria and

observed for a period of 4 days for mortality and pathological signs. From the

number of survival in control and experimental group, percentage of mortality was

calculated.

2.7. Statistical analysis

All data obtained from experiments were analyzed using one-way ANOVA ( p < 0.05 as

significant level) in Statistica 6.0 computer package (Statsoft, UK). Means were compared

using Duncan’s multiple range test (Duncan, 1955) for multiple range of variations.


3. Results

3.1. Antibacterial screening and characterization of methanolic extracts from herbs

Methanolic extracts of all herbs showed different degree of inhibition against V.

harveyi in disc diffusion test. The maximum inhibitory zone was observed for O.

sanctum (OS) (29.4F 0.5 mm) followed by W. somnifera (WS) (24.3F 0.1 mm) and M.

fragrans (MF) (21.4F 0.9 mm). The results of zone of inhibition test of herbal extracts

are given in Table 1.

3.2. Growth

Variance in specific growth rate, FCR and weight gain were significant between control

and groups fed with OS or WS supplemented diets at 100 and 200 mg/kg diet

concentration ( p < 0.05). Specific growth rate was observed at maximum of 0.46F 0.01

% body weight/day for WS supplemented diet at the concentration of 100 mg/kg diet. M.

fragrans does not exhibit any significant improvement in growth at any concentrations

(Table 3).

Table 3

Growth parameters of greasy grouper juveniles fed with experimental diets during 12-week study

Diet Concentration of the

herbal principles

(mg/kg diet)

Final wt (g) Average

weight gain

(g/day)

Specific

growth rate*

(% body

weight/day)

Feed

Conversion

Ratio*

Control 0 56.7F 1.3 0.31F 0.01 0.32F 0.01a 1.86F 0.02a

WS 100 74.0F 2.1 0.52F 0.50 0.46F 0.01b 2.19F 0.02b

200 73.2F 3.2 0.51F 0.03 0.45F 0.02b 2.18F 0.03b

400 60.7F 2.3 0.40F 0.04 0.38F 0.03a 2.02F 0.06b

800 61.7F 1.0 0.37F 0.01 0.36F 0.01a 1.98F 0.01a

OS 100 66.7F 1.1 0.43F 0.01 0.40F 0.01b 2.08F 0.01b

200 71.5F 0.8 0.44F 0.07 0.41F 0.04b 2.08F 0.01b

400 59.3F 0.8 0.33F 0.02 0.33F 0.02a 1.89F 0.04a

800 55.4F 1.6 0.29F 0.02 0.31F 0.01a 1.82F 0.03a

MF 100 54.6F 2.3 0.31F 0.04 0.32F 0.03a 1.84F 0.08a

200 57.3F 2.4 0.30F 0.01 0.31F 0.01a 1.84F 0.02a

400 55.6F 1.1 0.29F 0.01 0.31F 0.01a 1.82F 0.03a

800 54.3F 2.8 0.28F 0.03 0.30F 0.02a 1.79F 0.06a

*Values receiving same superscript are statistically not significant ( p>0.05).

Fig. 1. Immune parameters of fish fed with herbal principles of O. sanctum (OS), W. somnifera (WS) and M.

fragrans (MF) supplemented diets at the concentrations of 100, 200, 400 and 800 mg/kg diet for 12-week study

period. ( —4th week; —8th week; —12th week) (groups receiving same superscript has no significance

( p>0.05)). (a) Serum Lysozyme activity. (b) Phagocytic activity (%) against V. harveyi. (c) Leukocrit values. (d)

Albumin–Globulin (A/G) ratio. (e) In vivo bactericidal activity (%).



3.3. Immune indices

No significance was observed in serum lysozyme activity between control and

experimental groups fed with herbal diets (Fig. 1a). Phagocytic activity was decreased

Fig. 1 (continued).

Fig. 1 (continued).


in groups fed with MF supplemented diets than control group at all concentrations. In

groups fed at the concentration of 100 and 200 mg/kg OS supplementation in diet,

significant increase of phagocytic activity was observed ( p < 0.05). Significant

difference in phagocytic activity between control and experimental groups was

obtained at the concentration of 400 mg/kg dietary supplementation of WS (Fig.

1b). Significant difference in leukocrit values was detected in the experimental groups

fed with OS as well as WS supplemented diets at the lower concentrations of 100

and 200 mg/kg diet (Fig. 1c) than control group. Increased value of A/G ratio

indicates immunosuppressive state and was observed in fishes fed on all concen-

trations of MF supplemented groups. O. sanctum as well as W. somnifera signifi-

cantly increased the serum globulin level in animals fed with 100 and 200 mg/kg

dietary concentrations (Fig. 1d). Variance in serum bactericidal activity was observed

in groups fed on OS as well as WS supplemented diets at the concentrations of 100

and 200 mg/kg. Feeding of M. fragrans also increased the serum bactericidal activity

at 800 mg/kg supplementation in diet (Fig. 1e).

3.4. Mortality

Control group showed 100% mortality after challenging with lethal dose of live V.

harveyi cell suspension. Feeding of OS as well as WS at the concentrations of 100 and

200 mg/kg diet significantly reduced the mortality than control at the final week of

experiment up to the 5% level. Reduction in mortality was also observed in fishes fed

Fig. 2. Percentage of Mortality in control and experimental groups of juvenile E. tauvina fed with herbal active

principles of O. sanctum (OS),W. somnifera (WS) andM. fragrans (MF) supplemented diets at the concentrations

of 100, 200, 400 and 800 mg/kg diet for 12-week study period. ( —4th week; —8th week; —12th week).


MF supplemented diets at the concentrations of 400 and 800 mg/kg but not statistically

significant (Fig. 2).

4. Discussion

Saeed (1995) suggested the treatment for V. harveyi outbreaks in E. tauvina with

oxytetracycline, but also pointed out the potential risk behind the treatment in fish. As

the alternative to chemotherapy, application of multifunctional phytochemicals in

aquaculture is new venture and few works were carried out in fish (Citarasu et al.,

2002; Babu and Marian, 2001; Jian and Wu, 2003). In the present study 10 herbs were

screened for their inhibitory activity against V. harveyi; methanolic extracts of all herbs

remarkably inhibited the growth of pathogen in agar plates. Among them, O. sanctum,

W. somnifera and M. fragrans exhibited wide zone of inhibition which are comparable

with commercial antibiotics used in aquaculture. As shown in results, the fish fed

antibacterial active principles of O. sanctum and W. somnifera at the concentrations of

100 and 200 mg/kg diet, significantly improved growth, immunity and survival rate.

Higher concentration of these herbal principles does not significantly influence the

experimental parameters (Jian and Wu, 2003). Immunosuppression state was observed in

group fed on M. fragrans extracts. Macrophages activity was inhibited by methanolic

extracts of M. fragrans (Tezuka et al., 2001). Also volatile oil from M. fragrans

exhibited antibacterial activity against 25 different genera of plant and animal pathogens

(Dorman and Deans, 2000). However myristicin, the principal aromatic constituent of


volatile oil of M. fragrans was reported for intoxication after ingestion of approximately

1–2 mg/kg body weight in humans (Hallstrom and Thuvander, 1997). O. sanctum and

W. somnifera were reported for their immunostimulant properties in experimental

animals (Davis and Kuttan, 2000). Mediratta et al. (2002) explained the possible

mechanism of immunostimulating effect of O. sanctum in experimental mice. In present

study, both O. sanctum and W. somnifera significantly increased the phagocytic activity,

serum immunoglobulin level and survival rate in E. tauvina against V. harveyi infections.

Reduced mortality against pathogenic challenges at lower dosage of herbal principles

was also reported by Kim et al. (1999) and Jian and Wu (2003). Citarasu et al. (2002)

developed an Artemia enriched herbal diet for Penaeus monodon with the combination

of five herbs, which significantly increased the growth and survival during stressed

conditions. Also, feeding with W. somnifera, Ferula asafetida and Mucuna pruriens

remarkably accelerated the spawning rate, fecundity and larval quality in spent spawners

of P. monodon (Babu and Marian, 2001). Several herbal principles were tested for their

growth promoting activity in aquatic animals (Citarasu et al., 2002; Jayaprakas and

Eupharsia, 1996). The present study also demonstrated the growth promoting activity of

O. sanctum and W. somnifera in E. tauvina. Increase in SGR and weight gain in groups

fed W. somnifera at lower concentrations in E. tauvina is comparable with the work

carried out by Citarasu et al. (2002) in shrimp P. monodon. In conclusion, 100 and 200

mg/kg supplementation of W. somnifera and O. sanctum significantly improves the

growth and non-specific immune system and also reduces the mortalities in E. tauvina

juveniles during V. harveyi infections.

Acknowledgements

The authors thanks Indian Council for Agricultural Research (ICAR), New Delhi for

the research grant (28(1)2000-NATP/CGP-II/223) through National Agricultural Tech-

nology Programme (NATP), Mr. R. Manoharan, Fish farm Manager (MSU) was kindly

acknowledged for his dedicated efforts in animal collection and maintenance.

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