Pakistan Journal of Marine Sciences, Vol. 17 (Special Issue), 161-167, 2008

ISOLATION AND IDENTIFICATION OF VIBRIO SPP. AND

AEROMONAS SPP. IN WILD AND HATCHERY REARED PENAEUS

MONODON POSTLARVAE FROM COX’S BAZAR, BANGLADESH

S. Aftab Uddin, M. Zafar, A. S. M. Noman and A. Sharmin

Institute of Marine Sciences and Fisheries, University of Chittagong, Chittagong-4331,

Bangladesh (SAU, MZ, AS); Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong-4331, Bangladesh (ASMN).

E-mail: aftabims@yahoo.com

ABSTRACT: The present study was undertaken to determine the pathogenic bacteria of wild and hatchery reared Penaeus monodon postlarvae collected from the Cox’s Bazar area during December 2005 to March 2006. A total of thirty five isolated strains of bacteria were selected for identification through physiological and bio chemical tests from five P. monodon hatcheries in Bangladesh. The wild postlarvae were collected from the adjacent seawater of Cox’s Bazar beach. Among the isolated strains, two groups of bacteria Vibrio and Aeromonas bacteria were detected. The Vibrio group was comprised four species; V. alginolyticus, V.

harveyi, V. parahaemolyticus and V. vulnificus. Among them V. harveyi was the dominantly isolated species followed by V. alginolyticus, V. vulnificus and V. parahaemolyticus. All the identified vibrio spp. and Aeromonas hydrophila were found to be associated with both in wild and hatchery reared post larvae of P.

monodon. Vibrio species were recorded both in diseased and healthy post larvae. It was observed that recorded water parameters i.e. temperature, salinity and pH were favourable for the growth of identified pathogenic bacteria. The present investigation indicated that Vibrio was more frequent than other bacteria and it was found both hatchery and wild postlarvae of P. monodon.

KEYWORDS: Vibrio sp., Aeromonas sp., Penaeus monodon, Bangladesh.

INTRODUCTION

Shrimp culture plays an important role in the fisheries economy of Bangladesh. The tiger shrimp Penaeus monodon is one of the largest shrimp among the penaeid shrimps and it is a most suitable for aquaculture in Bangladesh. With the rapid expansion of shrimp production, the Bangladeshi shrimp hatchery and its related activities industry has been progressed rapidly during the last five years. The shrimp hatchery industry was developed on different levels of economic and management scale. Bangladesh stood seventeenth by volume (23,000 MT) and eighth by value (US$ 246.6 million) of shrimp (wild and cultured) exports in the year 2002 (WB, NACA, WWF and FAO, 2002.). However, failures in shrimp production occurred due to different diseases and caused by bacteria, viruses and fungi. Bacteria are the most common biological agents in the mericulture and it is also known that marine crustaceans can be infected by one or more type of bacteria. In many shrimp producing countries, Vibrio and Aeromonas are considered as the most common and significant infectious pathogens (Lightner, 1996; Moriarty, 1997; Vaseeharan et al., 2005). Vibrio is one of the most important pathogen for reared aquatic organisms such as Penaeid shrimps (Lightner, 1993). Some Vibrio species or strains of certain species have been identified as primary pathogens (Lavilla-Pitogo, et al., 1990; de la Pena et al., 1995). Only a few Vibrio species have been proven to be pathogen for shrimp, the closely related species Vibrio harveyi and V.

campbelii have caused disease in shrimp postlarvae (Abraham, et al., 1999, Karunasagar, et.al., 1994; Sahul-Hameed et al., 1996; Prayitno and Latchford, 1995). Vibriosis is caused by a number of Vibrio species of bacteria including Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V.

alginolyticus, V. penaeicida and Vibrio sp. (Brock and Lightner, 1990; Ishimaru, et al., 1995).

162 Pakistan Journal of Marine Sciences, Vol. 17 (Special Issue), 2008.

There have been occasional reports on Vibriosis caused by Vibrio damsela, V. fluvialis and other unidentified Vibrio species (Lightner, 1996). Low quality of postlarvae is one of the major technical constraints currently faced in this sector in Bangladesh. The high incidence of disease outbreak in recent years has been blamed for the poor quality of hatchery reared postlarvae. Farmers doubt the quality of seed who buy seed from hatcheries as well as from the natural seed collectors. This doubt poses a serious threat to shrimp culture industry of Bangladesh. Therefore this study was taken for the first time to isolate and identify the pathogenic Vibrio bacteria of P. monodon postlarvae from different hatcheries and wild sources.

MATERIALS AND METHODS

Sample collection: The present study was conducted from December 2005 – March 2006. Samples were collected from shore water (wild source) and five commercial hatcheries of Cox’s Bazar namely: Golden Hatchery, Prime Hatchery, ADB Hatchery, Niribili Hatchery and Quality Hatchery. Concurrently water parameters were recorded from the hatcheries and wild sources, physical parameters of the water were also observed. The isolation method was done accordingly by Vaseeharan et al., (2005). Identification tests were carried out both in Institute of Marine Sciences and Fisheries, University of Chittagong and the leading shrimp processing plant named M/s. SAR & Co. Ltd., Chittagong.

Homogenization and Dilution: The samples were surface sterilized with 25 ppm of sodium hypochlorite for 2 minutes before rinsed in filtered seawater. 1gm sample of postlarvae from each hatchery were taken separately and placed in individual sterile test tube and homogenized in 1ml filtered seawater using a sterile glass rod. This homogenate sample was made to be 1:10 dilution. The homogenate was then serially diluted up to 10-3 in filtered seawater. Plating: 1 ml from each dilution was plated by pipette onto thiosulphate citrate bile salts sucrose (TCBS) agar media and nutrient agar supplemented with NaCl. After solidifying of the media the plates were inverted and placed in incubator to incubate at room temperature (30º C) for 48 h. Identification and biochemical characterization: The bacteria were examined to standard morphological, physiological and biochemical tube and plate tests according to the procedures of Colwell et al., (1984), and Bergey’s Manual of Determinative Bacteriology (1994). Aeromonas spp. were isolated and identified on the basis of the biochemical characterization of Kaper et al., (1979) and Esteve, (1995). All samples were incubated for 24 h at 30° C and representative colonies were purified on TCBS agar and tryptic soy agar (TSA).

RESULTS AND DISCUSSION

Bacteria were identified in respect to their morphological and biochemical characteristics. Thirty

five gram negative bacterial strains were isolated from the larvae of P. monodon in Cox’s Bazar, Bangladesh. Based on morphological and biochemical identification, two groups of bacteria were eminent. Among the isolated strains, 27 strains were Vibrio bacteria and 8 strains were Aeromonas bacteria. The species of Vibrio were separated into two groups on the basis of colour on TCBS agar; species that usually give yellow colonies on TCBS include Vibrio alginolyticus, V. cholerae, V.

anguillarum and V. fluvials. Those that usually give green colour include Vibrio parahaemolyticus,

V. vulnificus, V. splendidus, V. damsela, V. mimicus, V. harveyi, V. fischeri (Lightner, 1996). In the experiment the yellow and green colours were observed in the isolated strains on TCBS agar.

Uddin et al.: Isolation and identification of Vibrio spp. and Aeromonas spp. 163

Distribution of the bacterial strains of the present investigation is presented in Table 1. Table 2 shows the morphological characteristics of all the isolated Vibrio strains. The biochemical characteristics of all the isolated Vibrio strains showed arginine dihydrolase (A) negative, lysine decarboxylase (L) positive and ornithine decarboxylase (O) positive (Table 3).

Table 1. List of Vibrio and Aeromonas species and their strains number isolated during the present investigation.

Species name No. of strains Source

Hatchery reared PL Wild PL

Vibrio harveyi 12 9 3

V. parahaemolyticus 4 3 1

V. alginolyticus 6 4 2

V. vulnificus 5 4 1

Aeromonas hydrophila 6 4 2

A. sorbia 2 2 -

Vibrio harveyi: Twelve bacterial strains, which were apparently equated with the V. harveyi. These strains were A- , L+, O+, positive growth on citrate, growth in 8 % NaCl was positive or negative, Voges-Proskauer reaction was negative. All strains reduce nitrate to nitrite.

V. parahaemolyticus: Four bacterial strains which were A- , L+, O+, positive growth on citrate, positive growth in 8 % NaCl, negative Voges-Proskauer reaction.

V. alginolyticus: Six bacterial strains, all were A- , L+, O+, positive growth on citrate, positive growth in 8 % NaCl, and positive Voges-Proskauer reaction. V. vulnificus: Five bacterial strains, all were A- , L+, O+, positive growth on citrate, negative growth in 8 % NaCl, and negative Voges-Proskauer reaction. There were morphological and biochemical evidence for separation of motile aeromonads into two species, namely A. hydrophila and A. sorbia (Table 4).

Aeromonas hydrohpila: Six strains seem to characterize A. hyrophila. These strains were straight rods, positive growth on nutrient agar, colonies were white to buff. These strains could use L-arginine and L-arabinose, grown in KCN medium, produced gas from glucose.

A. sorbia: Two strains were isolated from hatchery reared postlarvae. These strains were not used L-arginine and L-arabinose. They were not grown in KCN medium. The physical parameters of the investigated hatcheries were shown in Table 5.

There are more than 30 currently recognized Vibrio species (Farmer, 1992); only four species were clearly identified in this investigation. All 27 stains showed the key phenotypical features of bacteria belonging to the genus Vibrio. These strains were gram negative, motile, oxydase positive and able to grow in TCBS media. We observed that V. vulnificus and V.

harveyi strains mostly were isolated from diseased P. monodon postlarvae. These strains were able to utilize cellobiose but were not able to utilize sucrose. Lightner, (1996) reported that V.

harveyi and V. vulnificus were able to utilize cellobiose, D-gluconate, D-glucuronate but negative on utilization of sucrose and L-serine.

164 Pakistan Journal of Marine Sciences, Vol. 17 (Special Issue), 2008.

Table 2. Morphological characteristics of different Vibrio species.

Species V. harveyi V. parahaemolyticus V. alginolyticus V. vulnificus

Shape Straight rod Straight rod Straight rod Curved rod

Gram staining - - - -

Swarming on solid complex

- - - -

Spore staining No Spore No Spore No Spore No Spore

Growth on TCBS Y/G G Y G

Motility test + + + +

Growth in 8% NaCl V + + -

Oxidase (Kovac’s) + + + +

Vibrio alginolyticus, the second most frequently isolated bacterial species here (6 strains) is

very common in the marine environment (Lightner, 1996). Karunasagar, (1994) reported that mass mortalities of P. monodon associated with Vibrio spp. infections have been observed in shrimp hatcheries in India. Lavilla-Pitogo et al., (1992) reported that larval prawns are particularly susceptible to V. harveyi. Nash et al., (1992) reported that pathogenic strains of V. harveyi, V.

vulnificus, V. parahaemolyticus have caused massive epidemics in Thailand.

Table 3. Biochemical characteristics of different Vibrio (Strain No. 1-5).

Tests V. harveyi V. parahaemolyticus V. alginolyticus V. vulnificus

Arginine dihydrolase - - - -

Ornithine decarboxylase + + + +

Lysine decarboxylase + + + +

Voges-Proskaur - - + -

Citrate + + + +

Urease test - V - -

NO3- to NO2

- + + + +

Growth at NaCl (%)

0 - - - -

3 + + + -

8 + + + -

Utilization of D- Glucose + + + +

Sucrose V - + -

Cellobiose + + - +

Mannitol + + + V

Maltose + + + +

Xylose - - - -

Symbols: +, positive; -, negative; V, strain instability (+ or -); G, green; Y, yellow.

Uddin et al.: Isolation and identification of Vibrio spp. and Aeromonas spp. 165

Table 4. Morphological and biochemical characteristics of Aeromonas hydrophila and Aeromonas sorbia.

Tests A. hydrophila A. sorbia

Shape Straight Straight, curved rod

Gram staining Gram Negative Gram Negative

Spore staining No Spore No Spore

Motility test + +

Growth in nutrient broth at 37° C + +

Growth in KCN broth + -

Voges-Proskauer + +/-

L-arginine utilization + -

L-arabinose utilization + -

Fermentation of sucrose + +

Fermentation of mannitol + +

Gas from glucose + +

Baumann and Baumann, (1981) reported that V. alginolyticus is very common in coastal waters of temperate and tropical regions. Nash et al., (1992) reported that V. alginolyticus is to be pathogen for P. monodon larvae. Six Vibrio species including V. harveyi and V. splendid cause luminescence which are readily visible at night and infected postlarvae, juveniles and adult (Ruby, et.al., 1980; Lightner, et.al., 1992). Vibriosis is a common problem in the worldwide hatchery industry. Information on Vibriosis and other bacteria in shrimp hatchery is therefore essential to support healthy seed supply activity.

Table 5. Major water parameters of different hatchery and natural sea water of Cox’s Bazar area.

Hatchery Salinity ‰ Temperature (° C) pH

Hatchery Natural Hatchery Natural Hatchery Natural

Golden Hatchery 20 30 26 25 8.0 7.8

Prime Hatchery 20 30 26.5 25 7.8 7.8

ADB Hatchery 22 30 27 25.5 8.0 7.8

Niribili Hatchery 22 30 27 25 8.4 7.8

Quality Hatchery 24 30 26.5 25 8.2 7.8

A number of pathogenic bacteria belonging to the genus Vibrio, Staphylococcus, E. coli and Salmonella have been isolated from cultured shrimp in India (Ahmed et al., 1995; Sugumar et al.,

2001). In the present study we observed that Aeromonas hydrophilla were found both in hatchery reared and wild postlarvae while A. sorbia was found in hatchery reared diseased postlarvae of P.

monodon. Aeromonas spp. Pseudomonas spp. are the normal microflora of wild and cultured crustaceans and are opportunistic pathogens (Lightner, 1993). They are associated with mortality less frequently than Vibrio spp. and are not considered primary pathogens. Aeromonas sp. is associated with soft shell syndrome of P. monodon reported from the Philippines (Baticados et al., 1986).

166 Pakistan Journal of Marine Sciences, Vol. 17 (Special Issue), 2008.

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(Received: 20 February, 2008)