Abstract

The bacterial flora in hepatopancreas and hemolymph of 110 male and 9 female broodstock of normal Giant Black Tiger Shrimp (Penaeus monodon) was isolated by using Trypticase Soy Agar with 1% Nacl and Thiosulfate Citrate Bile Salt Agar. Identification of bacteria was done by using API 20E and NFT kit. The result shows that majority of the bacteria found belong to genus Vibrio ( 93.9% ) which consist of V. alginolyticus 36.3%, V. parahaemolyticus 27.3% and V. vulnificus 9.1% . Other gram negative bacteria found were 6.1%.

Introduction

Giant Black Tiger Shrimp has been an important aqua-cultural product of Thailand during the past few years. Broodstocks have been brought from nature, mostly from the Andaman Sea, for breeding and the off-springs will be raised in the pond until reaching market size (approximately 4 months).

In natural seawater, there are many types of bacteria including Vibrio sp. in every region all year round (Raeungpan et.al, 1985). In penaeid shrimp, the intestinal bacterial flora found were in Genus Vibrio, Aeromonas, Pseudomonas (Letochawalit and Chaichanasirividdhaya, 1988; Dempsey and Kitting, 1987 ), Photobacteria , Xanthomonas, Alteromonas, and Chromobacterium ( Dempsey and Kitting, 1987 ). The pathogenic bacteria that cause diseases in Penaeid shrimp are mostly Vibrio sp., such as V. alginolyticus, V. parahaemolyticus, V. anguillarum (Lhimsuwan et al., 1987; Johnson, 1978 ), V. metschnikovii and V. fluvialis (Kaper et al., 1983 ) Also from genus Flavobacterium, Pseudomonas ( Brock, 1986 ). These bacterias can be found in the internal organs and hemolymph of the diseased shrimps.

This study intends to identify the normal flora in hepatopancreas and hemolymph of the male and female broodstock in order to manipulate the selection for healthy and higher productivity of shrimp hatchery farm management which will result in faster growing and higher survival rate of shrimp larvae.

Material and Methods

Male and female brood stock of Black Tiger shrimps were collected from Andaman Sea and kept in a shrimp hatchery for breeding purposes. One hundred and ten females and nine males weight and length were measured.

Hemolymph was drawn from cardiac puncture, then streak on Trypticase soy agar (TSA) with 1% NaCl and Thiosulfate Citrate Bile Sucrose (TCBS) agar. Then incubate at 35°C for 24 hours. The colonies of bacteria from hemolymph will be isolated and identified.

By using aseptic technique, the carapace of each shrimp was dissected into hepatopancreas. A tissue portion of anterior hepatopancreas, approximately 1 gram, was spread on TSA and TCBS agar for bacteria culture. The plates were incubated at 35°c for 24 hours.

After incubation, bacterial colonies were isolated and identified by motility test, gram stain. Oxidase test (spot test), catalase test (3% H₂ 0₂ method), and using API 20 E, NFT test kits to characterize each colony and identify them.

Result

From the 110 female broodstock, the average weight was 198-86 ± 37.56 gram and the average length was 28.37±1.59 centimeter.

From the 9 male broodstock, the average weight was 104.00±14.87 gram and the average length was 23.06±0.93 centimeter.

There were 33 samples that showed evidence of bacterial flora. 29 were in female and 4 were in male broodstock. There were 26 samples that carried bacteria in both hepatopancreas and hemolymph. But only 7 samples that the bacteria was found in hepatopancreas alone.

Table 1. Number of Samples with Bacteria

Identification of bacteria found showed the presence of Vibrio alginolyticus 36.3%, V. parahaemolyticus 27.3%, V. cholerae 18.2%, Vibria vulnificus 9.1% and unidentified bacteria 6.1%.

Table 2. Type and Number of Bacteria in Samples

Discussion

From the result of the study of bacterial flora in hepatopancreas and hemolymph of male and female Black Tiger shrimp broodstock caught from the Andaman Sea for breeding purposes, only gram, negative bacteria were found and most of them were in genus Vibrio. The characteristics of this genus are that they are gram negative, motile, rod shape, catalase positive, oxidase positive, (Cowan and Steel, 1970 The incidence of Vibrio alginolyticus was the most evidenced in the study. The less evidenced were V. parahaemolyticus, V. cholerae and V. vulnificus respectively. Using API 20E and NFT system generally gives 92% confidence level in the laboratories. The difference in bacterial identification (genus and specie categories) between API and R/B Tube (Corning diagnosis system) and the conventional tube media are not significantly different at 95% confidence level. (Rutherford et al., 1977).

Vibrios are commonly found in seawater in this region (Raeungpan et al., 1985). And they can be found in Penaeid shrimp gastrointestinal tract (Dempsey et al., 1989; Chandrasekaram et al., 1984). Vibrios can be found at, low level in hemolymph when there is stress from intensive farming, improper management, molting, or harvesting. These stress can induce invasion of bacteria into hemolymph (Sinderman and Lightner, 1988 ). The similarity of species of bacteria found in hemolymph corresponded with those in the hepatopancreas. But some of the samples showed that bacteria can occur in hepatopancreas alone without hemolymph invasion. Generally bacteria can be both primary and secondary cause of diseases. Most of the bacterial flora in animal would be secondary infection from other primary cause such as, over stressed. injuries, wounds, or malnutrition (Sinderman and Lightner, 1988 ). In this study, all the animal used did not have any clinical abnormalities which could be seen in shrimp with Vibriosis or Vibrio disease of Penaeid shrimp. The lesions are abnormal swimming, anorexia, delayed clotting-of haemo-lymph, an opaque body. (Johnson, 1978; Brock, 1986). This showed that Shrimp can have Vibrios as normal bacteria flora without any recognizable disease signs and lesions.

When comparing between male and female broodstock, the percentage of bacteria found in male was higher than in female (44.4% in male: 26.4% in female). This may due to the limitation in number of male (9 samples) used was much less than female (110 samples). But in commercial shrimp hatcheries, the ratio of male to female can be up to 1:10. In many cases, the broodstock that carry bacteria can cause infection in shrimp larvae and egg especially by Vibrios (Schnick, 1987)

In conclusion, bacteria found in Black Tiger Shrimp broodstock were Vibrio species. The presence of these Vibrios did not cause any external abnormalities. But there is a possibility that in case of improper environment, the infection may cause disease in egg, larvae and even the broodstock themselves.

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