3. PNSB AS BIO-CONTROL AGENTS

3.1 INTRODUCTION

Farming of marine shrimps has spread rapidly in South-east and

South Asia, with exception of a few countries, including Myanmar.

Myanmar's neighbors Bangladesh to the north and Thailand to the

south are both major producers of cultured marine shrimps. Culture of

marine shrimp is now spreading rapidly in India. In all these countries

export of cultured marine shrimp is a major earner of foreign exchange

(http://www.fao.org/docrep/field/376565.html). Disease outbreaks and

mass mortalities of cultured shrimp are common now and the industry

is thus crippled economically (Ramasamy, 1995).

Shrimp disease is a major constraint to shrimp aquaculture

production around the world. Since 1994, average annual production

loss due to diseases in this sector in India amounts to about Rupees 350

– 400 crores (MPEDA/NACA. 2003). Pollution/deterioration of the

pond/nutritional imbalance/predisposing lesions cause stress leading to

the secondary invasion of bacteria and result in heavy mortalities.

Bacterial diseases of shrimp are on the body surface (erosion of the

appendages, cuticle), and shell disease (black discoloration/black

spot/brown spot) and tail rot are external infection while the internal

infection occur mainly in the hepatopancreas, gills, muscles, gut etc.

The external infections not only cause mortalities of infected hosts but

also reduce the market value of the shrimp (Ramasamy, 1995). Long

term exposure to even sublethal concentration of pollutants can make

shrimps more susceptible to bacterial diseases. Organisms like Vibrio

spp, Pseudomonas spp, Aeromonas spp, and Flavobacterium spp are

the most common pathogens associated with the disease of naturally

occurring shrimp in sea.

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Diagnosis of vibrio infection is based on clinical signs and the

histological demonstration of rod-shaped Vibrio bacteria in lesions,

nodules or haemolymph, excised organs and haemolymph may be

streaked on Robert bailey and scotts agar. (Ramasamy, 1995) followed

by TCBS agar.

3.1.1 Vibriosis in shrimps

Vibriosis is a serious problem in shrimp aquaculture, as they are

responsible for higher incidence of shrimp mortality . Species of Vibrio

viz., V. harveyi , V. fischeri, V.parahaemolyticus, V. alginolyticus,

V.anguillarum V. vulnificus and V.splendidus are associated with the

diseased shrimp (Lightner and Lewis, 1975; Lightner, 1993; Huervana

et al., 2006) . Shrimps suffering with vibriosis display localised

lesions of the cuticle typical of bacterial shell disease, localised

infections from puncture wounds, loss of limbs, cloudy musculature,

localised infection of the gut or hepatopancreas and/or general

septicemia, (Takahashi et al., 1985; Lightner, 1993). Mass mortalities

in hatcheries and grow-out ponds of shrimp attributed to outbreaks of

vibriosis, have been recorded from many regions of the world where

shrimp cultivation is done (Couch, 1978; Lightner, 1983; 1985; 1988;

1996; Jiravanichpaisal et al., 1994; Mohney et al., 1994).

Vibriosis is also rampant in the Indian region where brackish

water shrimp farming is the main aquaculture activity. The disease

problem is particularly severe in hatcheries, and in the past few years

many units were shut down due to invasion by luminous vibrios

(Karunasagar et al., 1994; Hameed et al., 1996; Shome et al., 1999).

Vibriosis is also having its impact in grow-out ponds and is frequently

responsible for the morbidity and mortalities of shrimp (Hameed,

1994; Abraham and Manley 1995; Jayasree et al., 2000). Surveys

undertaken on diseases caused by Vibrio spp. in Penaeus monodon

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from culture ponds of coastal Andhra Pradesh, India, recorded the

occurrence of five types of diseases: tail necrosis, shell disease, red

disease, loose shell syndrome (LSS), and white gut disease (WGD).

Among these, LSS, WGD, and red disease caused mass mortalities in

shrimp culture ponds. Six species of Vibrio viz., V. harveyi, V.

parahaemolyticus, V. alginolyticus, V. anguillarum, V. vulnificus, and

V. splendidus are associated with the diseased shrimp. The number of

Vibrio spp., associated with each disease ranged from two to five.

Additionally, shrimp with red disease had concurrent infections with

white spot syndrome virus. Vibrio harveyi in the case of LSS and

WGD, V. parahaemolyticus for red disease, and V. alginolyticus for

shell disease are the major etiological agents (Jayasree et al., 2000).

The situation is aggravated by the emergence of antibiotic-resistant

strains (Karunasagar et al., 1994) and the ability of Vibrio spp. to form

biofilms (Karunasagar et al., 1996).

Damage to shrimp stocks is frequently associated with bacterial

diseases that are mostly caused by luminous bacteria (Ruangpan et al.,

1997). Luminous vibriosis causes mortalities and termination of grow-

out activities especially in the first 45 days of culture (Lavilla-Pitogo et

al., 1998). Among the luminous Vibrio spp., prevalent in shrimp

ponds, Vibrio harveyi is more pathogenic to shrimps and causes mass

mortalities, and they are oppourtunistic pathogens, when the shrimps

are in a stressed state (Lavilla-Pitogo et al., 1990; 1998; Abraham et al,

2007).

3.1.2 Chemotherapeutic agents against the control of vibriosis in

shrimp ponds

Antibiotics are applied to shrimp pond to reduce the severity of

vibriosis. The normally used antibacterial agents in aquaculture are

Benzylpenicillin, Nitrofurans (furazolidone), Chloramphenicol, and

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Rifampicin. (Hernandez, 2005) The massive (mis)use of antibiotics to

control infections in aquaculture has resulted in the development of

resistant strains (Defoirdt et al., 2007).

Repeated usage of the antibacterial and other therapeutics in

coastal aquaculture has the potentiality to result in chemical residues

appearing in wild fauna of the local environment. The horizontal

transfer of resistance determinants to human pathogens and the

presence of antibiotic residues in aquaculture products for human

consumption constitute important threats to public health. In recent

years the use of nitrofurans in shrimp culture has been banned in USA,

UK, and other European countries (Hernandez, 2005; Defoirdt et al

2007). Consequently, antibiotics are no longer effective in treating

luminescent vibriosis (Karunasagar et al., 1994). In recent years, there

has been growing interest in biocontrol of microbial pathogens in

aquaculture to make the industry more sustainable.

3.1.3 Biological control of Vibrio spp.,

Sea weed, sea sponges and marine algal extracts have been used

to control luminescent Vibrio spp., shrimp ponds (Selvin and Lipton

2004; Kanagasabhapathy et al., 2005; Huervana et al., 2006; Isnasetyo

et al., 2009). Vibriostatic activity has been reported in several bacterial

members such as, Bacillus spp., (Watchariya and Nontawith, 2007),

Flavobacterium spp., (Qi et al., 2009), Lactobacillus spp., (Ajitha et

al., 2004 and Vieira et al., 2007), Pseudomonas spp., (Bushra et al.,

2009), Phaeobacter spp., (Prado et al., 2009). Some of these bacteria

were reported to be effective in controlling vibriosis, where they were

also used as probiotics. Moriarty (1998), reported that probiotic strains

of Bacillus spp., were used in shrimp culture ponds, which were

effective in controlling luminous Vibrio spp. Apart from Bacillus spp.,

other probiotic bacterial strains, such as Lactobacillus spp., and

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Pseudomonas spp., are also commonly employed in shrimp culture

ponds to protect shrimp from vibriosis.

Abraham et al. (2001) suggested the use of co-existing bacteria

in the shrimp pond as potential biocontrol agents. Likewise, the

utilization of photosynthetic bacteria as probiotics is a common

practice in many fish or shellfish hatcheries and aquaculture farms in

China and are claimed to have multifunctional effects such as

improvement of water quality, enhancement of growth rate and

prevention of disease (Qi et al., 2009).

3.1.4 Antagonistic potentials of purple non sulfur bacteria

Some facultative anaerobic purple non sulfur bacteria such as

Rhodobacter sphaeroides, Rhodobacter capsulatus and

Rhodopseudomonas palustris have been found to produce bacteriocins

and exhibit inhibitory activity among the closely related species of

purple non sulfur bacteria (Guest, 1974; Wall et al., 1975; Willison et

al., 1987). Kaspari and Klemme (1977) reported about the antibiotic

effects of Rhodobacter sphaeroides against Bacillus subtilis, and found

that the antagonistic activity is not caused by bacteriocins, but by the

metabolites extractable with organic solvents, and further suggested

that the inhibitory substance from the PNSB may be a high molecular

(or) lipophillic substance.

The production of antibiotics by marine bacteria is well

documented (Rosenfeld, and Zobell, 1947; Krassilnikova, 1961;

Burkholder et al., 1966; Andersen et al., 1974; Gauthier, 1976;

Isnansetyo and Kamei, 2003), although, until now, no studies on the

antagonistic potentials of marine photosynthetic purple non sulfur

bacteria, against pathogenic Vibrio spp., have been performed, despite

their wide spread occurrence in shrimp ponds.

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Keeping this in mind, the present work aims in screening the

shrimp pond havest discharges from brackish and direct sea water

shrimp ponds and screening diseased shrimps in each shrimp farm for

pathogenic Vibrio spp., and studying the antagonistic potentials of

native Purple non sulfur bacterial strains against the pathogenic Vibrio

spp., responsible for vibriosis in Penaeus monodon.

In the light of the above, the present work was carried out with the

following objectives.

1. To isolate and characterize pathogenic Vibrio spp., from harvest

discharges and diseased shrimps.

2. To study the antagonistic potential of native PNSB strains

isolated from shrimp ponds, against pathogenic strains of Vibrio

spp.

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3.2 MATERIALS AND METHODS

3.2.1 Microbiological screening of shrimps and harvest discharges

with reference to heterotrophic bacteria

3.2.1.1 Selection of diseased shrimps

The tissues of Tiger prawn (Penaeus monodon), from shrimp

ponds exhibiting characters like, erratic / lethargic swimming, eroded

black or brown coloured cuticle, cloudy muscular tissues with necrosis

(Fig. 14a-14f) were chosen for the isolation of heterotrophic bacteria.

3.2.1.2 Selection of healthy shrimps

The tissues from healthy shrimps (Fig.15) that were growing in

other ponds in the same shrimp farm without any sign of infection

were also collected in order to compare them with regard to the

heterotrophic bacterial population with that of the infected shrimps.

3.2.1.3 Isolation and enumeration of heterotrophic bacteria from

infected and healthy tissues of Penaeus monodon. (Ramasamy

1995)

The surfaces of the infected tissues as well as the healthy tissues

were sterilized with a cotton swab soaked with rectified spirit. The

infected as well as healthy tissues were washed with (autoclaved)

0.85% physiological saline solution. One gram of the tissue was

homogenized using a glass tissue homogenizer. The tissue

homogenates were serially diluted and 100µl of homogenized serially

diluted tissue extracts was spread plated on Robert Bailey & Scott‟s

(1966) media (Appendix) and incubated overnight at 37°C. The

resulting colonies were enumerated.

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3.2.1.4 Isolation and enumeration of heterotrophic bacteria from

shrimp pond harvest discharge

One ml of harvest discharge was serially diluted in 9 ml of

(0.7% NaCl w/v) saline and was spread plated on modified Anderson‟s

marine agar media (Appendix) and incubated overnight at 37°C. The

resulting colonies were enumerated.

3.2.2 Screening of Vibrio spp., using TCBS agar

The colonies arising from Robert bailey and Scott‟s media

(1966) (shrimp tissue homogenate/extract) and modified Andersons

marine agar plates (harvest discharge), with different colony characters

were individually picked and streaked onto the plates containing

Thiosulphate citrate bile salts sucrose (TCBS) agar (Hi-Media,

India)(Appendix) and incubated overnight at 37°C. The presence of

green/greenish-yellow coloured colones indicated the presence of

Vibrio spp.

3.2.3 Purification of test strains

The green/greenish-yellow coloured colonies from TCBS agar

plates were picked and the purification of test strains was done by

repeatedly streaking onto modified Anderson‟s marine agar.

3.2.4 Characterization and identification of Vibrio spp.,

The purified test strains were identified based on simple

taxonomic key (Fig.) developed by Abraham et al. (1999). The key

was designed using the following tests on the basis of their

discrimination power and ease of application. These include growth

on modified Anderson‟s marine agar with and without 3% sodium

chloride (Nacl), gelatinase, arginine dehydrolase (ADH), lysine

decarboxylase (LDC), ornithine decarboxylase (ODC), yellow orange

pigmentation and luminescence on modified Anderson‟s marine agar,

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utilization of lactose, maltose and Voges-Proskauer reaction.

Additional 5 tests viz. D- glucosamine, growth at 8% Nacl, indole,

arabinose and ONPG (o-nitrophenyl-β-galactopyranoside) were done

to differentiate the stray luminous strains of Vibrio spp. All tests

proposed in the key were performed as per standard methods outlined

in Bergeys manual of systematic bacteriology (Bauman and Schubert,

1984).

3.2.4.1 Morphological, physiological and biochemical

characterization

3.2.4.1.1 Colony characteristics

The colonies of the purified test strains grown on modified

Andersons marine agar was observed for luminescence and colony

characteristics like colony shape, colony colour, texture, pigmentation

(yellow orange) and presence of swarming colonies.

3.2.4.1.2 Physiological and biochemical characters

3.2.4.1.2.1 Growth at various NaCl concentrations

The isolated pure cultures were grown at various NaCl

concentration (0% to 10%) at 37°C in modified Anderson‟s Marine

broth (Appendix) in 10 ml test tubes and growth was observed after 24

hours by analyzing the optical density (OD 600) using a colorimeter at

600 nm. The uninoculated medium with varying NaCl concentrations

served as the blank.

3.2.4.1.2.2 Decarboxylases and arginine dihydrolase test (ODC,

LDC, ADH)

Moeller Decarboxylase broth base (Hi-Media, India)

(Appendix) was prepared and separately added 1.0 gram each of, L-

Ornithine mono-hydrochloride, L-Lysine hydrochloride and L-

Arginine hydrochloride and the medium was heated at 60°C to

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solubilize all the contents. Dispensed 5 ml of the broth into 10 ml

tubes and 0.5ml of 20% NaCl was added to each test tube, with pH

adjusted to 6.8 and sterilized by autoclaving the tubes at 15 lbs

pressure (121°C) for 15 min. Two loops full of purified test strains

were inoculated into the medium and overlaid the inoculated broth

with sterilized paraffin oil up to 2mm thickness and incubated at 35 ±

2°C for up to 4 days. A tube of basal medium without amino acid was

inoculated in parallel with the test media. A tube containing basal

medium with respective amino acids served as the control. Incubated

tubes were observed daily after 24 hours of incubation and observed

for color change from purple to yellow and then from yellow to purple,

for up to 4 days.

3.2.4.1.2.3 Gelatin Liquefaction

Gelatin salt agar (Hi-Media, India) (Appendix) was prepared

and poured into sterile petri plates. A loop full of culture was streaked

on to the media and incubated at 37°C for 24 – 48 hours. Production of

gelatinase was seen as either a cloudy or clear zone around the area of

bacterial growth. The plates were flooded with ammonium sulphate

which assisted with clear view of zones of clearing. The plate was held

up to the light and read against a darkish background.

3.2.4.1.2.4 Voges-Proskauer Test

MRVP broth (Hi-media, India) was prepared and 5ml of the

broth was evenly distributed into 10 ml test tubes and each tube

containing MRPV broth (Appendix) was supplemented with an

additional 0.5 ml of 20% NaCl, and autoclaved. A loop full of culture

was inoculated and the tubes were incubated aerobically at 35 ± 2°C

for 72 hours. Six drops of Barrit‟s reagent –A (Appendix), and 2 drops

of Barrit‟s reagent –B (Appendix), were added and mixed gently and

69

allowed for colour development up to 1 hour. The appearance of red

colour indicated a positive result.

3.2.4.1.2.5 Indole test

Tryptone broth (Appendix) (Hi-Media, India) was prepared

and 5 ml of the broth was evenly distributed into 10 ml test tubes and

each of the test tubes containing the tryptone broth was supplemented

with an additional 0.5 ml of 20% NaCl and autoclaved. Two loop full

of pure culture was inoculated into the test tubes containing 5ml of

sterile tryptone broth, and incubated aerobically for 48 h at 37°C. After

48 hours, 6–7 drops of Kovac‟s reagent (Appendix) was added to the

test tubes and shaken. Development of a cherry red colour in the upper

reagent layer on top of the broth medium indicates positive result while

no colour development indicates a negative result.

3.2.4.1.2.6 Utilization of organic substrate as carbon sources

The test for sole carbon source utilization was done by

preparing the basal medium according to Bauman et al. (1984)

(Appendix). About, 5ml of basal medium was dispensed into 10 ml test

tubes and autoclaved at 15 lbs pressure (121°C) for 15 minutes. L-

arabinose, lactose, maltose and D-glucosamine were used as test

carbon sources. The test carbon source solutions were prepared at the

conc. of 2% using sterile deionized water and was filter sterilized using

a 0.22 mm membrane filter. Each test carbon source was added in 0.5

ml volumes into the individual test tubes containing sterile basal

medium. The purified test strains measuring 2µL with a McFarland

cell density of 0.5 was aseptically inoculated into tubes containing the

sterile basal medium along with individual test carbon source and

incubated aerobically at 35±2°C for 48 hours. After the incubation

period, the optical density was measured at 600 nm (OD600),

uninoculated tubes served as blank.

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3.2.4.1.2.7 ONPG (o-nitrophenyl-β-galactopyranoside) Test

Phosphate-ONPG solution was aseptically added to the peptone

water, and dispensed 2.5 ml of ONPG broth (Appendix) into sterile test

tubes. Inoculated a tube of ONPG broth with a loop of culture and

incubated at 35 ± 2 °C for 24–48 hours. The presence of yellow colour

was indicative of a positive result and indicated the presence of the

enzyme, β-galactosidase.

3.2.5 BIOCONTROL OF VIBRIO SPP., USING PNSB STRAINS

3.2.5.1 Screening of PNSB strains for antagonistic activity

3.2.5.1.1 Preparation of crude aqueous extracts from PNSB

strains (Burgees et al., 1991)

The purified PNSB strains, were cultured en-masse, using

modified Biebl and Pfennig‟s (1981) broth filled in 6 litre high grade

glass jars and sealed with noncorrosive stainless steel lid –with

scilicone rubber lining. The medium was poured up to the brim,

leaving no head space, bubbled with argon for 5 minutes and kept

under constant illumination at 2400lux at 30 ± 2°C, for 7 days until the

colour changed to red - brick red/ brown. Mass cultured photosynthetic

bacterial strains were harvested by centrifugation at 4000 rpm for 6

minutes and re-suspended in deionized water and placed in a boiling

water bath for 90 minutes. The cleared supernatant was recovered at

different time intervals, after cooling and used for the bioassay against

Vibrio spp., by the agar well diffusion method.

3.2.5.1.1.1 Agar well diffusion method

The petriplates containing Muller Hinton agar (Appendix) were

seeded with 24hr broth cultures of purified Vibrio spp by swabbing

them on the surface of medium. Agar wells measuring 6 mm in

diameter were cut with the help of sterilized cork borer. Using a

micropipette, 500µL of aqueous PNSB extracts were added into the

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wells. The plates were incubated in an upright position at 37° for 24

hours. The diameter of inhibition zones was measured in mm and the

results were recorded. The aqueous extracts of PNSB strains showing

inhibitory action against Vibrio spp., were further subjected to disc

diffusion assay and broth tube dilution method to determine their

antagonistic efficiency and MIC (minimum inhibitory concentration)

to bring about vibriocidal activity, by preparing crude intracellular

solvent extracts.

3.2.5.1.2 Extraction of crude intracellular extracts from positive

PNSB strains using chemical solvents (Burgees et al., 1991)

Mass cultured positive PNSB strains (50ml) were evenly

distributed in sterile glass centrifuge tubes and centrifuged at 4000 rpm

for 6 minutes, the supernatant was discarded and the cell pellet was

washed twice by centrifugation, with double deionized distilled water.

The cell pellets was subjected to solvent extraction. Solvents like (E1)

Chloroform: methanol: water (1:2:0.8), (E2) Acetone: methanol (7:2)

and (E3) Toluene: methanol (3:1) were used in the extraction of

intracellular extracts. The intracellular extracts were obtained by

vortexing the cell pellets for 10 minutes suspended in solvents along

with presterilized 0.5mm glass beads and filtered using ultrafine nylon

filter. The solvent extracts were collected and then concentrated in

vacuum at 40°C using a rotary evaporator (Photonix, India), the dry

residues were collected and stored in dry eppendorf tubes. The dry

residues (0.5grams) were further dissolved in 10 ml of 2% dimethyl

sulfoxide (DMSO) (SRL chem. India) for studying the antibacterial

activity by disk diffusion and broth tube dilution method.

3.2.5.1.3 Bioassay of crude PNSB extracts

The sensitivity of purified strains of Vibrio spp., to solvent

extracts like (E1) chloroform: methanol: water (1:2:0.8), (E2) acetone:

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methanol (7:2) and (E3) toluene: methanol (3:1) of PNSB was tested

by measuring the zone of inhibition of a given concentration of the

extract by the disk-diffusion method (Bauer et al., 1966) and by

determining the minimal inhibitory concentration (MIC) by Broth tube

dilution method.

3.2.5.1.3.1 Bioassay of crude extracts by disc diffusion (Bauer et al.,

1966)

The broth cultures containing various Vibrio spp., isolated from

infected shrimps and harvest discharges were streaked on to Andersons

Marine agar plates and incubated aerobically at 35±2°C for 24 hours

and the individual colonies were picked and diluted to approximately

108 CFU/mL, according to the 0.5 McFarland standard (Roe-Carpenter,

2007) (Appendix). 0.1ml of the inoculum were spreaded on Mueller-

Hinton agar incorporated with 2 % Nacl and sterile paper disks (6 mm

in diameter) were laid on the inoculated substrate after being soaked

with 15 μL of PNSB extract at a concentration of 50 mg/ml. The plates

were incubated for 24 h at 37°C. Antimicrobial activity was

determined by measuring the diameter of the zone of inhibition around

the disk. As a positive control of growth inhibition, Chloramphenicol

30 μg/ml (Hi-media) antibiotic disc (6mm) was used.

3.2.5.1.3.2 Bioassay of crude extracts by broth tube dilution

(Qaiyumi, 2007 modified)

The broth tube dilution assay was performed to determine the

minimal inhibitory concentration (MIC) of the solvent extracts over

Vibrio spp.Twelve sterile screw capped test tubes were labeled from 1

to 12. One ml of solvent (DMSO) was taken in tube 1 (negative

control). One ml of sterile Mueller Hinton broth was taken in each of

the tubes from 3-12. About 1 ml of working test extract (dissolved in

2% DMSO with a conc. of 50mg. /ml) was taken in tube 2 and another

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Broth tube dilution test

1 m

L o

f M

&H

bro

th +

1m

L o

f t

est

extr

act

so

luti

on

1m

l o

f te

st e

xtr

act

fro

m P

NS

B i

sola

te

at t

he

con

c. (

50

mg

/mL

) +

1m

L i

no

culu

m

1m

l o

f D

MS

O +

1m

L i

no

culu

m

1ml 1ml 1ml 1ml 1ml 1ml 1ml Discard 1 ml

1 2 3 4 5 6 7 8 9 10 11 12

1mL Mueller Hinton broth

Final Conc. (mg/mL) 50 25 12.5 6.25 3.12 1.56 0.78 0.39

Note: Inoculum of the test pathogen was added in all the tubes except the tube 12.

Tube 11 serves as control and receives no test extract solution.

Tube 12 contains only 1 ml of M&H broth without inoculum, i.e broth control

The concentration of the working test extract solution due to the doubling dilutions from the tube 3 to tube 10, ranges from 50mg/ml to 0.39 mg/ml

74

1ml of the test extract solution was added to tube 3. One ml of the

solution (working test extract + Mueller Hinton broth) was transferred

from tube 3 to 4 and this process of transfer was continued through

tube 10. About 1ml from tube 10 was discarded. Tube 11 serves as

control and receives no working test extract solution.

Isolated colonies from an overnight culture of the test pathogen

growing in Andersons marine agar plates were diluted in the Mueller

Hinton broth to a turbidity comparable to that of a 0.5 Mc Farland

turbidity standard (approximately 1.0 × 108 CFU/mL). This suspension

was further diluted 1:100 (106) with Mueller Hinton broth. Within 15

minutes of the preparation of this suspension of test pathogen 1ml of

the bacterial broth suspension was added to each tube except the 12th

(last) tube which serves as broth control tube.

The tubes were incubated at 37°C for 24 hours. The presence of

turbidity in the tubes indicates growth. The boundary dilutions having

lowest concentration of test extract dilution containing tubes without

any visible growth was taken as the minimal inhibitory concentration

(MIC) against test Vibrio spp. Cfu/ml was determined by spreading

10µl of test pathogen suspension from tube 11, onto Andersons marine

agar plate and incubating overnight at 37°C.

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3.3 RESULTS

3.3.1 Microbiological screening of shrimps and harvest discharges

with reference to heterotrophic bacteria

The diseased shrimps and harvest discharges, when subjected to

microbiological screening using Robert Bailey & Scott‟s (1966)

medium and modified Anderson‟s marine agar plates yielded bacterial

colonies with different colony colour (pale white, greyish white,

orange, pale orange-yellow), size and colonial morphology . The

tissue extracts from the healthy, diseased shrimp samples and harvest

discharges from various stations that were subjected to serial dilution

and spread plating onto Robert Bailey & Scott‟s (1966) and modified

Anderson‟s marine agar plates respectively (Fig.16a-17), yielded

microbial colonies ranging from 8.90 ×105 to 2.07 × 10

6(tissue

extracts) and 1.0 × 106 to 2.13 × 10

6 (harvest discharges) (Tables 21

and 22).

Heterotrophic bacterial count was more in diseased shrimp

samples than from healthy shrimp samples in all four stations.

However in the Karankadu station, shrimps showing erratic swimming

(indicating diseased nature) had significant heterotrophic bacterial

counts. Harvest discharge samples also contained heterotrophic

bacterial counts comparable to diseased shrimp samples. Types of

shrimp diseases prevalent in various stations are as follows. Red

disease was found in 3 stations viz., Vadakkupoygainallur, Pappakovil

and Karankadu. Shell disease and necrotic lesions in the abdomen was

found in 2 stations viz., Vadakkupoygainallur and Pappakovil. Loose

shell disease and erratic swimming was found in 2 stations viz.,

Sethubavachatram and Karankadu. White spot disease was found only

in one station, Karankadu.

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3.3.2 Screening for Vibrio spp., using TCBS agar

All the colonies representing samples from shrimp tissues and

harvest discharge were either green/yellow or yellowish green and thus

indicated the presence of Vibrio spp., (Tables 23 – 26; Fig.18).

3.3.3 Purification of test strains

Green/yellow or yellowish green colonies from TCBS agar

plates were purified by streaking onto plates containing modified

Andersons marine agar. A total number of 66 strains were reported

after purification. The details of the purified strains with reference to

their growth in modified Anderson‟s marine agar are given in tables 23

to 26.

3.3.4 Characterization and identification of strains

The characterization details of the 66 strains are given in tables

27 to 30. The identification of the strains based on the simple

taxonomic key developed by Abraham et al. (1999) revealed that

majority of the strains (60) belonged to Vibrio spp., and the remaining

(6) strains belonged to Photobacterium spp. Details regarding the

species identification of the 66 strains are given in table 31, and their

prevalence across the stations are given in table 32. List of shrimp

pathogens in each station is given in the table 33. For the biological

control of shrimp pathogens using indigenous PNSB strains three

species of shrimp pathogens namely Vibrio harveyi, Vibrio fischeri and

Vibrio alginolyticus were used as test pathogens.

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3.3.5 Antagonistic activity of purple non sulfur bacterial strains

against Vibrio spp.,

3.3.5.1 Screening of PNSB strains for Antagonistic activity by agar

well diffusion method

Screening of antagonistic activity of PNSB strains using their

hot water extracts (aqueous) by agar well diffusion (Fig.19) revealed

the following. BRP1, BRP2, BRP3, BRP4, BRP6, BRP10 and BRP11

did not show antagonistic activity against Vibrio harveyi and Vibrio

fischeri. Besides the above mentioned strains BRP5 and BRP7 also

did not show antagonistic activity against Vibrio alginolyticus. Among

the strains showing antagonistic activity, the aqueous extracts of BRP9

require a minimum boiling time of 30 minutes, BRP8 requires 40

minutes, BRP5 requires 50 minutes and BRP12 requires an hour of

heating to show antagonistic activity. However prolonged heating up to

90 minutes did not have any negative effect on the antagonistic

activity. Among the strains showing antagonistic activity against

Vibrio harveyi, BRP9 (26.03±0.33) ranks first followed by BRP12

(20.60±0.65), BRP8 (15.87±0.62), BRP5 (12.10±0.62) and BRP7

(11.83±0.5). Against Vibrio fischeri, again BRP9 showed maximum

antagonistic activity (22.50±0.17) followed by BRP12 (16.20±0.41),

BRP7 (11.40±0.24), BRP8 (9.40±0.22) and BRP5 (9.07±0.26). In the

case of Vibrio alginolyticus again BRP9 registered maximum

antagonistic activity (23.40±0.24) followed by BRP8 (9.23±0.05) and

BRP12 (9.13±0.4). The PNSB strains exhibiting antagonistic activity

against 3 strains of Vibrio spp., is summarized in the tables 34 to 36.

As only 5 strains of PNSB (BRP5, BRP7, BRP8 and BRP12) showed

antagonistic activity against the test Vibrio strains, they alone were

considered for further bioassay against test Vibrio strains using disk

diffusion method and broth tube dilution method.

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3.3.5.2 Bioassay of crude PNSB extracts by disk diffusion

From the disk diffusion assay (Fig.20) for assessing antagonistic

activity of different solvent extracts of PNSB strains, the following

observations were made. Among the PNSB stains BRP9 showed

maximum antagonistic activity (27.50±0.34), followed by BRP12

(21.23±0.17), BRP8 (21.17±0.12), BRP5 (12.20±0.08), and BRP7

(12.17±0.05) against Vibrio harveyi. Against Vibrio fischeri, BRP9

(23.83±0.26) was found to be more effective, followed by BRP12

(18.07±0.74), BRP5 (12.20±0.22), BRP7 (11.20±0.22), and BRP8

(10.17±0.17). BRP9 showed maximum inhibition (23.17±0.17) against

Vibrio alginolyticus followed by BRP8 (11.07±0.09) and BRP12

(9.00±0.22). Of the different solvents used for extracting PNSB

extracts E1 (Chloroform:methanol:water (1:2:0.8)) was found to be

more effective, since it showed maximum antagonistic activity against

all the three Vibrio spp. Against Vibrio harveyi and Vibrio fischeri, the

E1 extracts of BRP9 registered significant antagonistic activity and it is

almost on par with the antibiotic standard viz. Chloramphenicol

(30µg). However E2 (acetone:methanol (7:2)) and E3

(Toluene:methanol (3:1)) extracts also showed inhibitory potentials.

The inhibitory potentials of the various PNSB strains varied depending

on the solvents used (Table 37).

3.3.5.3 Bioassay of crude extracts by broth tube dilution

Broth tube dilution test performed to find out the minimum

inhibitory concentration (MIC) of the different solvent extracts of

PNSB strains revealed the following (Table 38). Among the strains

BRP9 was the most effective, confirming the results of earlier two

assays. Solvent E1 could bring out the inhibitory potential of the PNSB

strains better compared to E2 and E3. The various tests undertaken for

the study of antagonistic activity of PNSB strains brought to light that

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Rhodobacter sphaeroides (BRP9) could be considered as a potential

antagonistic agent for the control of Vibrio spp., that are pathogenic to

shrimps.

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3.4 DISCUSSION

In shrimp culture ecosystem, most of the bacteria play a

negative role as they compete with shrimps for food and oxygen,

causing stress and disease (Moriarty, 1997). Due to the influence of

turbidity, heavy organic matter plays a major role in the distribution of

bacterial population in pond water (Sharmila et al., 1996).

In the present study the enumeration of total heterotrophic

bacteria from the harvest discharge and shrimps (healthy and diseased)

was done in order to quantify the total heterotrophic bacterial counts

using modified Andersons marine agar for harvest discharge and

Robert Bailey & scott‟s (1966) media for shrimp tissue extracts. The

shrimp tissue extracts and harvest discharge yielded bacterial cells

ranging from 8.9 × 105 to 1.05 × 10

6 cell/mg(healthy shrimps), 1.04 ×

106 to 9.30 × 10

6 cells /mg (Diseased), likewise the total heterotrophic

bacteria from harvest discharges from the four stations yielded

bacterial cells ranging from 1.00 × 106

to 2.13 × 106 cells /ml of

sample. From the results obtained one can ascertain that the

heterotrophic bacterial count is lesser in the healthy shrimps than that

of the diseased shrimp and the level of the heterotrophic bacteria in the

harvest discharge is also higher. This may be due to the fact that the

population of heterotrophic bacteria tends to increase, as the days of

culture progressed (Abraham et al., 2004; Ganesh et al., 2010). The

higher heterotrophic bacterial counts in the harvest discharges may due

to the abundance and availability of nutrients derived from excess feed,

shrimp excreta and other dead and decaying organic matter, which is

conducive for bacterial growth, as observed by Abraham et al., (2004).

Among the total heterotrophic bacteria (THB), the members of

the Genus Vibrio spp., play a vital role in shrimp culture ecosystem

(Ruangpan and Kitao, 1991) as they damage water quality causing

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diseases and mortality to the shrimp as primary and secondary

pathogens. Vibrio spp., is part of the autochthonous flora of the coastal

pond ecosystem (Aiyamperumal, 1992; Ruangpan and Tabkaew,

1994).

Bacteria of the genus Vibrio are ubiquitous in marine and

estuarine aquatic ecosystems in which shrimp occur naturally and are

farmed. Several Vibrio spp. form part of the natural biota of fish and

shellfish (Vanderzant et al., 1971; Colwell, 1984; Ruangpan and Kitao,

1991; Otta et al., 1999). Some of the Vibrio species such as V. harveyi

and Vibrio parahaemolyticus are also associated with bacterial

infections in shrimp (Lightner, 1993; Jiravanichpaisal and Miyazaki,

1995; Lavilla-Pitogo, 1995) and are generally considered to be

opportunistic pathogens causing disease when shrimps are stressed

(Gopal et al., 2005). In shrimp farms from India, Otta et al., (1999) and

Vaseeharan and Ramasamy (2003) noted that Vibrio spp., accounted

for 38–81% of the bacterial biota.

In the present study, the shrimps showing symptoms of various

types of shrimp diseases could be observed. For example, Shell disease

was observed in shrimps collected from the stations

Vadakkupoygainallur and Pappakovil, Red disease could be observed

in shrimps in the station Vadakkupoygainallur , Pappakovil and

Karankadu, necrosis in the abdomen could be observed in the stations

Vadakkupoygainallur and Pappakovil, loose shell in

Sethuvbavachatram and Karankadu, White spot disease could be found

in only one station viz., Sethubavachatram and erratic swimming in

diseased shrimps could be observed in two stations viz.,

Sethubavachatram and Karankadu. Shrimp diseases, like shell disease,

red disease, necrosis, loose shell and white spot disease are commonly

found in many regions of the world where shrimp cultivation is done.

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(Lightner, 1988; Sindermann, 1990; Nash et al., 1992; Yang et al.,

1992; Hameed, 1994; Abraham and Manley, 1995; Hameed et al.,

1996). In India too the above mentioned diseases are common and

prevalent in shrimps as observed by Jayasree et al. (2006), in the

shrimp ponds of Andhrapradesh. Different species of Vibrio are

associated with shrimp diseases.

3.4.1 Purification, Isolation and characterization of pathogenic

Vibrio spp.,

The colonies arising in Robert Bailey and Scotts media and

modified Andersons marine agar were streaked onto TCBS agar and all

the colonies representing samples from shrimp tissue and harvest

discharge from all stations were either green/yellow or yellowish green

and thus indicated the presence of Vibrio spp. The occurrence of

various Vibrio spp., in water, sediment and shrimp samples from

multiple shrimp farm environments was also reported from the east and

west coast of India by Gopal et al. (2005). Further purification of

green/yellow /yellowish green colour colonies on modified Andersons

marine agar, yielded a total of 66 strains.

These purified (66) strains of Vibrio spp., were characterized

and identified using the simple taxonomic key developed by Abraham

et al. (1999), as they had better discriminatory power and ease of

application. The species identified include, Vibrio fischeri, V.

mediterranei, V.harveyi, V.orientalis, V.splendidus, V. logei, V.

alginolyticus, V.damsela and V. marinus. Among them the

nomenclature of last two species has been revised and the new names

are Photobacterium damselae for V. damsela (Osorio et al., 1999) and

Moritella marina for V. marinus (Urakawa et al., 1998) respectively.

The percentage of prevalence of various strains of Vibrio spp., in all

the 4 stations are as follows, Vibrio fischeri (7.69%), Vibrio

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mediterrranei (28.84%),Vibrio harveyi (34.61%), Vibrio orientalis

(3.84%), Vibrio splendidus (7.69%),Vibrio logei (13.46%) and Vibrio

alginolyticus (3.84%). The prevalence of some of the Vibrio spp., viz.,

Vibrio harveyi (94.86%), V. splendidus (4.57%) and V. fischeri

(0.57%) reported from shrimp farms by Abraham et al. (2007), show a

pattern that has been replicated in the present study also.

Among the strains of Vibrio isolated in the present study, the

percentage of luminous Vibrio spp., was 37.87% and the non luminous

Vibrio spp., was 62.12%. The higher prevalence of non luminous

vibrios in shrimp ponds was also reported by Ruangpan (1998). With

regard to the prevalence of pathogenic Vibrio spp., in each station, V.

fischeri and V. splendidus could be isolated in the healthy shrimps

from two stations viz., Vadakkupoygainallur and Pappakovil (brackish)

respectively, but pathogenic Vibrio spp., could not be isolated from the

healthy shrimps collected from the stations Sethubavachatram and

Karankadu (direct sea water). Vibrio harveyi could be isolated from the

shrimps exhibiting shell disease from the stations

Vadakkupoygainallur and Pappakovil. Likewise Vibrio fischeri and

Vibrio splendidus, could be isolated from the shrimps showing shell

diseases from the station Vadakkupogainallur.

Vibrio fischeri, though considered to be pathogenic to shrimps,

were reported to be isolated from shrimp pond only. However isolation

of the same species from the tissues of diseased shrimp has been

reported by Chandrasekaran and Kumar (2011), which to the best of

our knowledge is the first report on the isolation of V. fischeri from

diseased shrimps in the natural environments though Manilal et al.

(2010) reported the pathogenic ability of the above species under

invitro conditions. Among the three stations showing red disease viz.,

Vadakkupoygainallur, Pappakovil, and Karankadu, only Vibrio harveyi

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could be isolated, and that too in one station (Karankadu) only. The

shrimps exhibiting necrosis were found only in two stations viz.,

Vadakkupoygainallur and Pappakovil. Vibrio harveyi and Vibrio

splendidus could be isolated from the necrotic sample collected from

Vadakkupoygainallur, but shrimp sample exhibiting necrosis collected

from Pappakovil yielded Vibrio alginolyticus.

Loose shell syndrome could be observed only in two stations

viz., Sethubavachatram and Karankadu and both the samples yielded

Vibrio harveyi. White spot disease could be observed only in one

station (Sethubavachatram) and Vibrio harveyi could be isolated. In the

shrimps showing erratic swimming obtained from two stations viz.,

Sethubavachatram and Karankadu, Vibrio harveyi could be isolated

only from Sethubavachatram. The number of pathogenic Vibrio spp.,

isolated from each type of shrimp disease ranged from 1 to 3, of which

Vibrio harveyi was prevalent in all the diseased shrimps, but non

prevalent in healthy shrimps. V. harveyi, could be isolated from the

harvest discharges in stations viz., Vadakkupoygainallur, Pappakovil,

and Sethubavachatram. Likewise Vibrio fischeri could be isolated in

the harvest discharge only from one station (Vadakkupoygainallur).

Vibrio alginolyticus and Vibrio splendidus could not be isolated from

harvest discharges from any of the stations.

The disease wise prevalence of pathogenic Vibrio spp., are as

follows, Vibrio harveyi was isolated from shrimps exhibiting shell

disease, red disease, necrosis of the abdominal region, loose shell,

white spot syndrome, and also in the shrimps exhibiting erratic

swimming. Vibrio alginolyticus was isolated in shrimps exhibiting

necrosis of the abdominal region, Vibrio fischeri was isolated from

shrimps exhibiting shell disease and Vibrio splendidus from the

shrimps exhibiting shell disease and necrosis of the abdominal region.

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In an earlier report on the isolation of pathogenic Vibrio spp., from

diseased shrimps in India by Jayasree et al. (2006), Vibrio harveyi and

Vibrio alginolylticus was isolated from shrimps showing loose shell

syndrome, and red disease, Vibrio splendidus was isolated from loose

shell syndrome alone. Likewise Vibrio fischeri, was isolated from

shrimp pond water, where there was incidence of shell disease by

Manilal et al. (2010) and established that Vibrio fischeri as moderate

opportunistic pathogen. In other words, none of the species of Vibrio

could be termed as specific to any particular type of vibriosis.

Among the pathogenic Vibrio spp., isolated from diseased

shrimps Vibrio harveyi was dominant and have been documented in

the previous studies of Abraham et al. (2003;2004;2007), possibly

because of its ability to grow well in shrimp pond environment with

wide ranging conditions of physicochemical parameters, which might

have favored the prevalence of Vibrio harveyi in cultured shrimps also.

The occurrence of luminous bacteria shows the inherent fertility of the

shrimp pond environment providing media, for bacterial growth and

favoring luminescent vibrios. The effect of pond soil on water quality

had been recognized by Chen et al. (1992) and its fertility was

apparently influenced by the accumulated excess feed, waste products,

and shrimp exuviae. Although it was shown that thorough drying of

soil eliminates Vibrio, populations that get established within ponds

always enter with the incoming water (Moriarty, 1997; Lavila-pitogo et

al., 1998).

Traditionally antibiotics have been used in attempts to control

bacterial disease in aquaculture (Defoirdt et al., 2007). But antibiotics

are no longer effective and the prevalence of antibiotic resistant strains

in the shrimp aquaculture has become an issue of concern

(Karunasagar et al., 1994; Alderman and Hastings, 1998). The massive

86

usage of commercially available antibiotics results in natural

emergence of antibiotic-resistance bacteria, which can transfer their

resistance genes to other bacteria that have never been exposed to

antibiotics (Austin et al., 1995; Moriarty, 1997).

This has led to the, suggestion on the usage of non pathogenic

bacteria as probiotic biocontrol agents instead of antibiotics (Fuller,

1978; Gatesoupe, 1999; Mishra et al., 2001). The use of probiotics is

prevalent in the aquaculture industry (particularly in shrimp culture) as

a means of controlling disease and in turn improving water quality by

balancing bacterial populations and nutrient (e.g., nitrogen and

phosphorus) concentrations. Probiotics that have been examined for

use in shrimp aquaculture include bacteria, yeasts, bacteriophage, and

microalgae (Park et al., 2000; Iriano and Austin, 2002; Ajitha et al.,

2004; Balcazar et al., 2006; Farzanfar, 2006). Vibriostatic activity has

been observed by Abraham (2004) in bacterial members belonging to

the Genera Alteromonas spp., and they were effective against Vibrio

harveyi. Prado et al. (2009) reported on the inhibitory activity of

Phaeobacter strain pp154 against shrimp pathogens like Vibrio

alginolyticus, Vibrio fischeri and Vibrio harveyi.

The antibacterial effect of probiotic bacteria results from factors

such as the production of antibiotics, bacteriocins, sideropheros,

lysozyme, protease, hydrogen peroxide, the alteration of pH values,

and the production of organic acids and ammonia (Verschuere et al.,

2000). Lactic acid bacteria and Bacillus produce several compounds

that may inhibit the growth of competing bacteria. Among these

compounds, the bacteriocins are the most important (Gildberg et al.,

1997; Ali, 2000). These are proteins, or protein complexes, produced

by certain strains of bacteria that can have an antagonistic action

against species that are closely related to the producer bacterium.

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Bacteriocins are divided into four classes: class-I antibiotics; class-II

small hydrophobic, heat-stable peptides; class-III large heat-stable

peptides; and class IV complex bacteriocins: probiotics with lipid

and/or carbohydrate (Fooks and Gibson, 2002).

Purple non sulfur bacteria do produce bacteriocins and

antibiotics (Guest, 1974; Wall et al., 1975; Willison et al., 1987;

Kaspari and Klemme, 1977). A study on the role of antagonistic

bacteria, especially the co-existing bacteria as biocontrol agents

appears worthwhile in lieu of the negative impacts of antibiotics

(Abraham et al., 2001). PNSB are found to co-exist in shrimp ponds

along with Vibrio spp., in the present study and this has prompted us to

investigate the antagonistic potentials of PNSB members against

pathogenic Vibrio spp. The present study focuses on the biocontrol of

three, pathogenic Vibrio spp., viz., V. harveyi, V. fischeri, and V.

alginolyticus, isolated from shrimps and harvest discharges, using

native purple non sulfur bacteria.

3.4.2 Antagonistic activity of purple non sulfur bacterial strains

against Vibrio spp.,

The antagonistic study was performed by subjecting pathogenic

Vibrio spp., to the aqueous extracts of 12 PNSB strains and the

influence of boiling time was also checked for the antagonistic effect

on the pathogenic Vibrio spp. The aqueous extracts of 12 PNSB strains

(BRP1 to BRP12), were tested for their antagonistic activity by agar

well diffusion method. The strains BRP1, BRP2, BRP3, BRP4, BRP6,

BRP10 and BRP11 could not elicit antagonistic activity against the 3

strains of pathogenic Vibrio spp., viz., V. harveyi, V. fischeri, and V.

alginolyticus. Likewise BRP5 and BRP7 did not show any antagonistic

activity against the pathogen Vibrio alginolyticus. Among the 12

PNSB strains screened for antagonistic activity against the 3 strains of

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pathogenic Vibrio spp., only 5 PNSB strains showed antagonistic

activity. The positive PNSB strains are as follows: Rhodobacter

spheroides (BRP9), Rhodobacter capsulatus (BRP12), Rhodobium

marinum (BRP8), Rhodovulum sulphidophilum (BRP5) and

Rhodobium orientis (BRP7). However the last two species were

negative in their antagonistic response to V.alginolyticus. Against

Vibrio harveyi, the PNSB strains BRP9 showed highest inhibitory

activity followed by BRP12, BRP8, and BRP5and BRP7. Against

Vibrio fischeri BRP9 showed maximum antagonistic activity followed

by BRP12, BRP7, BRP8, and BRP5. Against Vibrio alginolyticus the

only 3 PNSB strains viz., BRP9, BRP8 and BRP12 showed maximum

antagonistic activity. The inhibitory activity of the aqueous extracts of

the PNSB isolates was influenced by heating and found to be time

dependent in eliciting antagonistic activity against the pathogenic

Vibrio spp.

Thus the supernatants of BRP9 started to inhibit the V. harveyi

strains only after 30 min of boiling, BRP8 after 40 min., BRP5 and

BRP7 after 50 min. and BRP12 after 60 min. For the antagonistic

activity against Vibrio fischeri the aqueous extracts of BRP9 and BRP7

required a minimum heating time of 50 min to inhibit followed by

BRP12, BRP8 and BRP5, which required 60 minutes of heating to

produce antagonistic activity. In the case of antagonistic activity

against Vibrio alginolyticus the aqueous extract of BRP8 showed

inhibiton after 40 minutes of heating, BRP12 after 60 min and BRP9

after 70 min. of heating. In all the positive strains of PNSB, the zones

of inhibition increased with the time of heating up to 90 min.

This indicates that the antibacterial compounds are intracellular

as observed by Burgees et al. (1991) and the bioactive compounds are

thermostable, even during prolonged periods of boiling (upto 90 min.),

89

a characteristic feature observed in non protein compounds of

Pseudomonas aeruginosa CMG1066 by Bushra et al. (2009), thus

suggesting that the bioactive compound might be a non-protein

compound.

Among the PNSB strains that tested positive for antagonistic

interaction against pathogenic Vibrio spp., by disk diffusion method,

Rhodobacter sphaeroides (BRP9) was found to be most efficient

strain, in terms of its antagonistic activity against all the three species

of Vibrio tested using E1 solvent extracts (chloroform:methanol:water)

than others.

Vibriostatic activity has been observed in Bacillus spp.

(Watchariya and Nontawith, 2007), Flavobacterium spp. (Qi et al.,

2009), Lactobacillus spp. (Ajitha et al., 2004; Vieira et al., 2007),

Pseudomonas spp. (Bushra et al., 2009) and Phaeobacter spp. (Prado

et al., 2009). The reports available on the utilization of PNSB against

marine Vibrio sp., or any other shrimp pathogen are rather limited. The

effectiveness of utilizing these anoxygenic purple non-sulphur bacteria

depends on the outcome of extensive research in this perspective in the

coming future. Now with the outcome of this study it is possible to

consider using PNSB as candidates to control Vibriosis in Penaid

shrimps and the scope is wide open to harness the bio-pharmaceutical

potential of Rba. sphaeroides as well. However invivo studies are

required to confirm the above findings in the pond environment.