MATERIALS AND METHODS

Location of study and rearing condition: This study was conducted in the Hatchery complex, Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia. Healthy shrimp larvae, L. vannamei, were provided from local hatchery and reared in aquaria measuring 0.85x0.38x0.24 m³ for one month to eliminate the weaker ones. The larvae were fed with Artemia and commercial pellet feed, from Vietnam containing 38% crude protein at daily rate of 8% b.wt., three times daily. During rearing period, pH, salinity and temperature was monitored daily using a pH meter (YSI, USA) and a hand refractometer (Atago, 8808).

Artemia preparation: Artemia cysts (supreme Plus, Golden Mark Brand) were hatched in conical fiberglass tanks with continues strong aeration under the optimal hatching condition (2 g L^-1; 20-30 ppt; 25-30°C) a day before feeding. Artemia cysts hatched after 18-24 h of incubation. Newly hatched Artemia nauplii were harvested by covering the top portion of the tank with a black material to let nauplii settle at the bottom. Accumulated Artemia nauplii were then siphoned out from the bottom of hatching tanks thoroughly. Artemia nauplii were then filtered with plankton net (80-120 μm) and washed thoroughly before being fed to the larvae.

Isolation of bacterial strain as pathogen: Vibrio parahaemolyticus, V. alginolyticus and V. cholerae which were isolated from digestive tract of moribund shrimp were used for antagonism test. Briefly digestive tract of moribund shrimp were dissected aseptically and homogenized in sterile distilled water. Samples were then serially 10-fold diluted in Normal Saline Solution (NSS) and subsequently 0.1 mL of each dilution was plated in Thiosulfate-citrate-bile salts-sucrose agar (TCBS agar, Difco). After 24 h incubation in 30°C single colonies were picked and preserved in 15% glycerol at -80°C for further investigation and identification.

Shrimp challenge test with isolated pathogen bacteria: Shrimp, L. vannamei was challenged with isolated Vibrio to confirm the bacterial pathogenicity using injection method. Healthy juveniles weighing approximately 4 g were acclimatized for one week in 20 ppt seawater. Shrimps were then distributed into 70 L glass aquaria of 15 animals each with a constant aeration. Three groups of shrimp in triplicates were separately injected with 0.1 mL of 10⁷ CFU mL^-1 of V. parahaemolyticus, V. alginolyticus and V. cholera. The third group was shrimp injected with 0.1 mL of sterile NSS as control. All shrimps were injected at the third abdominal segment using 1 mL sterile insulin syringe (29 G). Cumulated mortality was monitored daily for up to 15 days.

Isolation and screening for bacteria with antibacterial activity: Shrimps were washed gently in sterile distilled water to remove loosely adhering particles. Samples were dissected aseptically to remove digestive tract. All samples were homogenized in sterile distilled water and diluted up to 10^-16. Aliquot of 0.1 mL from each dilution were spread plated in two replicate. To ensure maximum recovery of cultivable bacteria, samples were cultured in marine blood agar, Tryptic Soya Agar, TSA (Difco, US). After incubation single colonies were replica plated onto plates containing 10 mL Muller Hinton Agar (MHA) supplemented with 1% NaCl and V. parahaemolyticus which was previously grown overnight in LB broth (Difco, USA). Plates were then incubated for 24 h at 30°C. Clear zones around replica-plated bacteria were interpreted as antagonistic activity against the pathogen and they were picked from the original plates. Antagonistic strains were stored in glycerol at -80°C for further investigation.

In vitro antagonism assay by cross streak method: For the study of inhibition by cross-streak method, an 18 h cultured of candidates bacteria in LB (Difco, US) were streaked as 2 cm thick band, across the diameter of the Muller-Hinton agar plate, MH (Difco, US). After incubation for 24 h at 30°C, the growth bacteria were scraped with a sterile slide. The remaining bacteria were killed by exposure to 5 mL chloroform (CHCl₃) poured on the glass lid and left for 15 min by keeping the medium inverted over the lid. The plates were then air dried for about 10 min to remove any residual chloroform. Then three different pathogens (V. parahaemolyticus, V. alginolyticus and V. cholerae) were used to evaluate the antagonist ability of the candidate bacteria and the same bacteria was streaked as positive control and the LB broth medium as negative control, perpendicular to candidates band using sterile swab dipped in an 18 h old culture of pathogen bacteria regulated by spectrophotometer to McFarland concentration (625 nm, OD: 0.08-0.1). Final concentration by this regulation was around 10⁶ CFU mL^-1. The resulted plates were incubated for 24 h at 30°C. The linear zone of inhibition was recorded in each case (Chythanya et al., 2002).

Identification of bacterial strains: Conventional biochemical methods followed by Biolog GN microplates (Biolog, Hayward, CA, USA) (Olsson et al., 2004) were applied for all identifications. After the Gram-staining and morphological characteristics, strains were subjected to phenotypic tests for conventional identification, including catalase, oxidase, motility, urease, citrate, indole, Voges-Proskauer, H₂S production, glucose, inositol, manitol and reduction ability of nitrate to nitrite.

RESULTS

Water quality parameters: The temperature was maintained at 26-28°C and the range of water salinity of the water was 25-28 g L^-1. Total ammonium (0-0.1 mg L^-1), nitrite (0-0.05 mg L^-1) and pH (7.0-7.6) were stable and within acceptable ranges (Boyd and Tucker, 1998).

Identification of bacterial isolates: All bacterial isolates including pathogens and candidate antagonistic isolates were identified using conventional biochemical test followed by Biolog GN microplates and Microlog Software. Identification of pathogenic bacteria using biochemical tests showed the assessment of three species of Vibrionaceae including V. parahaemolyticus, V. alginolyticus and V. cholera (Table 1). On the other hand, candidate antagonistic strains were identified as Pseudomonas sp. using biochemical tests. Bacterial identification using biochemical tests were confirmed with identification using Biolog Microlog Software where the same identifications were resulted.

Isolation and assessment of pathogenic bacterial strains: Based on the morphological observations in TCBS agar, three different species including V. parahaemolyticus, V. alginolyticus and V. cholera were successfully isolated and identified. Subsequently they were subjected for their virulent effects and pathogenicity on white shrimp L. vannamei using the injection method. Cumulated mortality of shrimp infected with V. parahaemolyticus was recorded 43% after 10 days of injection, however no mortality was observed for those shrimp injected with V. alginolyticus and V. cholera. These results clearly revealed the pathogenicity effect of V. parahaemolyticus for white shrimp L. vannamei.

Isolation and assessment of antagonistic bacterial strains: A total of 43 bacteria were isolated from digestive tract of shrimp, L. vannamei. They were tested for their possibility of antibacterial activity against V. parahaemolyticus using replica-plating method. Only six bacterial isolates produced clearing zones of different sizes. To confirm the antagonistic activity of isolates, cross streak assay was carried out. Out of six antagonistic isolates, four isolates (isolates: 5, 7, 15 and 30) displayed strong inhibitory activity against V. parahaemolyticus, V. alginolyticus and V. cholera (Fig. 2). Results of antagonistic test of four Pseudomonas sp. isolated bacteria showed great antagonistic activity against three pathogens (Fig. 2) while the other two Pseudomonas sp. (isolates: 9 and 12) did not exhibit strong antagonistic ability against three pathogens (Fig. 1).

Table 1:	Biochemical characteristics of pathogens and candidate antagonistic bacteria
+: Positive reaction, -: Negative reaction

Fig. 1(a-b):	Low inhibitory effect of two Pseudomonas sp. isolates No., (a) 9 and (b) 12 against three pathogens V. parahaemolyticus (V. par), V. alginolyticus (V. al) and V. cholerae (V. ch) in cross-streak method, Co+: Positive control, Co¯: Negative control, Arrows show the linear zone of inhibition

Fig. 2(a-d):	Inhibition zone of four isolated Pseudomonas sp. isolates No., (a) 5, (b) 7, (c) 15 and (d) 30 against V. parahaemolyticus (V. par), V. alginolyticus (V. al) and V. cholerae (V. ch) in cross-streak method, Co+: Positive control, Co¯: Negative control, Arrows show the linear zone of inhibition

The linear zones of inhibition for isolate number 5 (Fig. 2a) were 5, 10 and 14 mm for V. parahaemolyticus, V. alginolyticus and V. cholerae, respectively. The linear zones of inhibition activity for isolate number 7 (Fig. 2b) were 15, 14 and 12 mm for V. parahaemolyticus, V. alginolyticus and V. cholera, respectively. The linear zones of inhibition for isolate number 15 (Fig. 2c) were 15, 16 and 13 mm for V. parahaemolyticus, V. alginolyticus and V. cholera, respectively. The linear zones of inhibition for isolate number 30 (Fig. 2d) were 16, 17 mm for V. parahaemolyticus, V. alginolyticus, respectively, without any zone for V. cholera.

DISCUSSION

In selecting a potential probiotic strain for beneficial health effects on the host, many criteria must be examined. Considering the infectious problems in shrimp aquaculture, antagonism properties against pathogen could be a key factor of candidate strains. Although, isolation and screening of potential probiotics is usually a long process and time-consuming, the replica-plating method could be used successfully for the isolation of bacterial strains with antibacterial properties (Hjelm et al., 2004). In the present study candidate probiotic bacteria were isolated during a course of one week, using replica plating method, based on the antibacterial activity against shrimp pathogens.

Before the experiment was conducted we isolated and identified the pathogen bacteria from moribund shrimp and subsequently their pathogenicity to white shrimp, L. vannamei was examined. Among the isolated pathogens only V. parahaemolyticus showed highly virulent to shrimp, however, all pathogen isolates were subjected for cross streak antagonism assay.

Six out of 43 isolated strains exhibited inhibitory activity in replica plating method; however, using the cross streak method to confirm the antagonism properties, four isolates showed strong inhibitory activity against V. parahaemolyticus, V. alginolyticus and V. cholera. Although the other two isolates did not show strong antagonistic activity (Fig. 1) against pathogens, but the reasonable inhibitory activity of these two isolates might suggest these bacteria as candidate probiotics. All candidate isolates were identified as Pseudomonas sp. using two methods of conventional biochemical tests and Biolog GN microplates using Microlog Software.

Isolation and antagonistic activity of Pseudomonas sp. against pathogens has been reported by literatures. For instance, Alavandi et al. (2004) isolated Pseudomonas sp. PM11 as candidate probiotics from the gut of farm-reared sub adult shrimp and tested for their effect on the immunity indicators of black tiger shrimp. This study reported promising in vitro results for shrimp culture. In another study, Irianto and Austin (2002) reported the inhibitory roles of Pseudomonas I-2 against V. harveyi and V. fluvialis in shrimp culture. Additionally, Gram et al. (1999) reported that a Pseudomonas fluorescens AH2 reduced the mortality of rainbow trout (Oncorhynchus mykiss) infected with a pathogenic V. anguillarum strain.

In vitro promising achievements were resulted from the present study where strong anti-vibrio activities of four Pseudomonas sp. isolates were recorded. Vijayan et al. (2006) reported the production of siderophores by Pseudomonas PS-102, responsible for inhibition of pathogenic vibrios and Aeromonas spp. Therefore it can be concluded that the inhibition of pathogens in the present study might be due the siderophore production of Pseudomonas sp. isolated from digestive tract of white shrimp, L. vannamei. Gatesoupe (1997) already stated that harmless bacteria with the ability to produce siderophores have been used as probiotics to compete and overgrow pathogenic microbes.

CONCLUSION

We report on isolation and identification of six candidate probiotics, Pseudomonas sp. which showed probiotic properties in terms of inhibitory activity against pathogenic Vibrio species. This could be a promising result for shrimp aquaculture that has been affected by vibriosis during the recent years.

Further experiments, including the application of candidate probiotics and shrimp challenged with pathogens, could provide valuable information of probiotic potential abilities of these candidates for shrimp aquaculture facilities.

ACKNOWLEDGMENTS

This study was funded by the Malaysian government E-Science, grant No. 05-01-04SF0103. We would like to thank the staff of the Marine Science Research Station and Biology Field Station, UPM, Port Dickson, Malaysia, for their assistance during this study.