Characterization and Pathogenicity of Vibrio Bacteria Isolated from Freshwater Prawn (Macrobrachium rosenbergii) Hatcheries:

Part 1: Isolation and Identification of Vibrio spp from Larval Stages

Tran Thi Tuyet Hoa, Dang Thi Hoang Oanh and Nguyen Thanh Phuong
Institute for Marine Aquaculture, College of Agriculture, Can Tho University, Can Tho, Viet Nam

Abstract
A total of 50 Vibrio spp were examined for classification by biochemical test. All of these bacteria were isolated from healthy and diseased larval prawn as well as larval rearing water of prawn hatcheries. According to the diagnostic scheme for Vibrio species described by Larsen and Pedersen (1999), four types of Vibrio spp were identified such as Vibrio cholerae, Vibrio alginolyticus,Vibrio carchariae and Vibrio mimicus with a percentage of 62% (31 strains), 20% (10 strains), 10% (5 strains) and 8% (4 strains), respectively. Some characteristics of these bacteria are given in this report.

1. Introduction
Two groups of bacteria cause serious disease in different phases of shrimp culture including Leucothrix sp. and several species of Vibrio (Lavilla-Pitogo, 1995). Vibrio bacteria are one of the pathogenic factors, which cause high mortality among economically important species of farmed marine fish and shrimp in Thailand (Ruangpan and Kitao, 1991). Vibriosis, especially luminous disease has caused serious loss in prawn hatcheries. Larval prawns are particularly susceptible to Vibrio harveyi succumbing to what has been termed luminescent bacterial disease (Lavilla-Pitogo et al. 1990). This disease has been identified as a major problem in the Phillipines, causing severe losses of juvenile prawns in several hatcheries (LavillaPitogo et al. 1992). In addition, Austin and Austin (1987) (cited by Hanna, 1991) have categorized the seven main Vibrio fish pathogen as being V. alginolitycus, Vibrio anguillarum, V. carchariae, V. cholerae, V. damsela, V. ordalii and V. vulnificus. In recent years the shrimp diseases encountered in Vietnam have included vibriosis in larvae and broodstock (Tho and Khang, 1990). In general, bacterial disease of shrimp has not been studied well in Vietnam. There is a need to carry out studies on the environment, pathogens and the preventive measures to support the shrimp farming industry in Vietnam (Tho and Khang, 1990). Considering the importance and immediate need for the country research works on the bacterial disease of shrimp, especially Vibriosis, we have been commenced systematically and developed step by step. In the first attempt, Vibriosis was taken for the study.

The present paper will describe some of characteristics and study the status of the composition of Vibrio bacteria in larvae and rearing water of prawn hatcheries in Can Tho,Vietnam.

2. Materials and Methods

Source of bacterial strains A total of 50 isolated strains, which are expected to be Vibrio spp are gram negative, oxidase-positive, glucose-fermenting and grows on Vibrio medium like TCBS agar. The Vibrios were isolated from the shell of artemia cyst, rearing water, healthy and diseased freshwater prawn (M. rosenbergii) larvae. Samples were collected from prawn larval rearing tanks from 1998 up to now, in which a part of them were collected in a complete cycle of larval rearing (from larval stage 1-11) (Table 1). There are 41 strains isolated from Can Tho prawn hatchery, while the remains of these strains are from Long My prawn hatchery. Nineteen reference strains were also used in this study. Isolated and reference strains were sub-cultured on nutrient agar, TCBS agar and stock cultures were maintained at 35oC in 10% glycerol/brain heart infusion broth medium with 1.5% sodium chloride. Table 1 : Isolated strains of Vibrio species presented in this study Isolated strains Larvae of prawn 1b 11aY 2b 11e 2g 163 2d 5b 1e TCX NTSH 3c 990605 IIB3 990607 8c 990608 11b 990609 4b 990610 3b 990611 At4a 990612 1fY 990613 C2a14.6 990614 2Ctom H 990615 7bG Labeled (TCX) Ld1 L1

Shell of artemia cyst A1 A3 A6

Rearing water A3IIT(a) A3IIT(b) P1 P2 P5 B3a B3T B3aT D1 D3IIT(a) D3IIT(b) A1aT P4G

Morphological and physiological study Colony morphology was observed on thiosulfate citrate bilesalts sucrose agar (TCBS, Himedia) after 24 hrs. of incubation at 300C. Cell morphology was determined from light microscope observations of Gramstained smear preparations. Motility was tested under light microscope of 100 magnification by using slide with a drop of young bacteria. Tolerance to NaCl was determined by the addition of NaCl to 1 %

pepton medium with percentage of 0, 3, 6, 8 and 10 % and cultures were examined for growth after 3 days at 30oC. Biochemical characteristics Selection of biochemical characters is followed the diagnostic scheme for Vibrio species associated with fish diseases described by Larsen & Pedersen (1999). Examination of characters was performed according to the principles of Cowan and Steels Manual (Barrow and Feltham, 1993) and methods of West and Colwell (1984). Test cultures were grown on Trypticase soy agar for approximately 24 hrs. before inoculation into test media. All test media were supplemented with 1.5 % NaCl and incubated at 30 0C. All the experimental isolates and the results were read after 24h at 30oC unless otherwise indicated. Statictical methods of data The phenotypic characters were coded in a binary format by scoring positive and negative character as 1 and 0 respectively. The data were examined using Euclidean distance with unweighted average linkage (UPGMA) clustering (Priest and Austin 1993) using NCSS 97 computer program.

3. Results and Discussion

Vibrio spp have great phenotypic diversity, therefore it is really complex to identify them. The genus Vibrio is gram negative, oxidase reaction positive, grow on TCBS agar, oxidative-fementative test positive. Besides these characteristic, they also give reaction with arginine, lysine, orthinine, amylase, indole, citrate, Voges-Proskauer, urease, gelatine, growth on 0, 6 and 8 % NaCl, growth at temperature 4, 35 and 40 OC, resistance with O/129 10g, produce acid from some of sugars, etc. According to the diagnostic scheme for Vibrio species described by Larsen and Pedersen (1999) and NCSS 97 computer program, it can be drawn a relative diagram between these isolated bacteria and typestrains that are shown in figure 1. In addition, four major groups were classified depending on main of biochemical and physiological characteristics, which are presented in table 2. The result of identified test demonstrated that the characteristics of 31 isolated bacteria are associated with Vibrio cholerae, 10 isolated strains with Vibrio alginolyticus, 5 isolated strains with Vibrio carchariae and 4 isolated strains with Vibrio mimicus. Table 2 show that four groups of isolated bacteria belong to group A/L/O = -/+/+ (arginine dihydrolase negative, Lysine decarboxylase positive and Orthinine decarboxylase positive). Vibrio cholerae 1With group of Vibrio cholerae, they were positive in lysine, ornithine, citrate utilisation, nitrate reduction, lipase, gelatinase, oxidase-fermentation test but were negative in arginine, urease and luminescence. They grew in 1% peptone medium containing 0, 3, 6% sodium chloride whereas there were about 30%, 62% strains not growing on 8% and 10% NaCl, respectively. These strains produced acid from glucose, galactose, glycerol, mannitol, sucrose but not from arabinose, salicin, xylose. All of strains do not produced gas from glucose. In the case of degraded starch, there were only 9.67% positive reaction.

Vibrio alginolitycus With group of Vibrio alginolitycus, they were positive in lysine, nitrate reduction, lipase, gelatinase, oxidase-fermentation test but were negative in arginine, urease and luminescence. All ten isolated strains grew in 1% peptone medium containing 3, 6, 8, 10% sodium chloride but not growing on 0% NaCl. These strain produced acid from glucose, glycerol, mannitol, sucrose but not from lactose, salicin. All of strains do not produced gas from glucose. In the case of degraded starch, there were only 10% positive reaction and VP reaction had 20% positive reaction. Vibrio carchariae With group of Vibrio carchariae, they were positive in lysine, ornithine, citrate utilisation, indole, nitrate reduction, lipase, gelatinase, oxidase-fermentation test but were negative in arginine, VP reaction and luminescence. They grew in 1 % peptone medium containing 3, 6 % sodium chloride whereas there were about 40%, 100%, 100% strains not growing on 0%, 8%, 10% NaCl, respectively. These strain produced acid from glucose, cellobiose, galactose, glycerol, lactose, mannitol, salicin, sucrose but not from arabinose, xylose. All of strains did not produce gas from glucose. In the case of degraded starch and urease, there were a 100% positive reaction. Vibrio mimicus They give positive reaction with lysine, citrate utilisation, indol, nitrate reduction, lipase, gelatinase, oxidase-fermentation test but were negative in arginine, VP reaction, urease and luminescence. They grew in 1 % pepton medium containing 3, 6 % sodium chloride but not 0 %, 8 %,10 % NaCl. There were only 25 % strains giving positive reaction with ornithine. These strain produced acid from glucose, galactose, lactose, mannitol, salicin but not from arabinose, cellobiose, sucrose, xylose. All of strains did not produce gas from glucose.

Table 2 : Biochemical characteristics between four groups of isolated bacteria and typestrains
Char act er i s t i c H em ol ys i s M ot i l i t y Cat al as e O xi das e Ar gi ni ne Lys i ne Or ni t hi ne Gr ow t hi n 0% N aCl 3% 6% 8% 10% O/ Ft es t Ur eas e TCBS Lum i nes cence Ci t r at e I ndol e VP Ni t r at er educt i on A m yl as e( St ar ch) G el at i nas e Tw een 20 ( Li pas e) Gl ucos e G as f r om gl ucos e Ar abi nos e Cel l obi os e G al act os e Gl ycer ol Lact os e M anni t ol Sal i ci n Sucr os e X yl os e

Vibrio cholerae Vibrio alginolyticus Vibrio carchariae Vibrio mimicus Typestrain Isolated strains Typestrain Isolated strains Typestrain Isolated strains Typestrain Isolated strains
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + (+) (+) (+) + (+) v v + (-) + (+) v v + + (+) + (-) v (+) (+) v + (-) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + v + + + + + + v v (-) + + (+) + v v v + + (+) v + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + (+) (+) + + v + + (+) (+) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + (-) + + + + + + + v + + + (-) (-) + v + + + -

Notes:

+ (+) v (-) -

the test is positive with a percentage higher than 90% the test is positive with percentage between 75 and 89% the test is positive with percentage between 26 and 74% the test is positive with percentage between 25 and 11% the test is positive with percentage lower than 10%

Relative diagram between these isolated bacteria and typestrain

100 120 20 40 60 80 0

Figure 1 : Relative diagram between these isolated bacteria and typestrains

V.fluvialis V. furnissii V. anguillarum V. pelagius I V. tubiashii V. splendidus II V. splendidus I V. nigripulchritud V. pen. V. natriegens V. ordalii V. ich. V. holllisae V. pelagius II V. harveyi A1 990614 Ld1 A2 990612 990615 990610 990613 990609 990607 A3 P2 2CtomH 2a14.6 A3IIT(a) D3IIT(b) B3T B3a L1 2d 2g 1e TCX NTSH 11e 11b 4b 8c 990607 at4a 3c 3b V. cholerae label(TCX) 7bG 5b 11aY V.mimicus 990605 P5 A1aT 990611 1b V.carchariae P1 D3IIT(a) P4G 2b A6 163 B3aT D1 1fY IIB3 V.alginolyticus

A total of 50 identified strains of Vibrio spp. belong to 4 groups with percentage rate as following in Table 3. Distribution of these bacteria in different sample are shown in Table 4. Table 3 : Percentage of identified bacteria Identified bacteria Vibrio cholerae Vibrio alginolyticus Vibrio carchariae Vibrio mimicus Table 4 : Percentage of identified bacteria in distinct samples Sample Rearing water Larvae of prawn Identified bacteria Vibrio cholerae Vibrio alginolitycus Vibrio carchariae Vibrio mimicus Vibrio cholerae Vibrio alginolitycus Vibrio carchariae Vibrio alginolitycus Vibrio cholerae Number of samples 6 6 2 4 22 3 3 1 3 Percentage (%) 42.9 42.9 14.3 12.5 68.8 9.38 9.38 25 75 Percentage (%) 62 20 10 8

Egg of Artemia

As shown in above table 4, Vibrio cholerae is the dominant bacteria, accounting for 62 % of the total isolated strains in this study. In distinct case of samples, Vibrio cholerae also occupy a large number of bacterial composition in rearing water, larvae of prawn as well as egg of artemia with percentage rate 42.9 %, 68.8 % and 75 % respectively (Table 5). As a result of Austin & Austin (1987), Vibrio cholerae, Vibrio alginolyticus, Vibrio carchariae are main pathogen that are collected from prawn hatchery in Can Tho, Vietnam. Therefore, it is very important to pay attention that considerable on the environmental parameters in these hatcheries, which may promote susceptibility to infection of larvae of prawn. Furthermore, research on toxicity of identified bacteria should be carried out through experimental infection so that prawn hatcheries can avoid the effect of these pathogens.

Literature Cited
Ruangpan L, Kitao T, 1991. Vibrio bacteria isolated from black tiger shrimp, Penaeus monodon Fabricius. Journal of Fish diseases 14.383-388. Song YL, Cheng W, Shen CH, Ou1 YC and Sung HH, 1990. Occurrence of Vibrio vulnificus infection in cultured shrimp and eel in Taiwan. Pro. ROC-JAPAN Symp. Fish Disease: 172-179. Mercedes A and Anicet RB, 1994. Improvement and update of a set of keys for biochemical identification of Vibrio species. Journal of Applied Bacteriology 77.719-721. Mercedes Alsina and Anicet RB, 1994. A set of keys for biochemical identification of environmental Vibrio species. Journal of Applied Bacteriology 76.79-85. Morris P and Robert GH, 1995. Classification of isolates of Vibrio harveyi virulent to Penaeus monodon larvae by protein profile analysis and M13 DNA fingerprinting. Disease of aquatic organism 21. 61-68.

Hanna PJ, Altmann K, Chen D, Smith A, Cosic S and Moon P, 1991. Development of monoclonal antibodies for the rapid identification of epizootic Vibrio species. Journal of Fish Disease 15.63-69. Tho, N. H. and Khang LT, 1990. Report on Shrimp disease in Vietnam. Proceedings of the SEAADCP Shrimp health Management Workshop 103-106. Santos Y, Romalde JL, Bandin I, Magarinos B, Nunez S, Barja JL and Toranzo AE, 1993. Usefulness of the API-20E system for the identification of bacterial fish pathogens. Aquaculture, 116. 111-120. Lavilla-Pitogo CR, Albright LJ, Paner MG, Sunaz NA, 1992. Studies on the source of luminescent Vibrio harveyi in Penaeus monodon hatcheries. Disease in Asian aquaculture 157-164. Lavilla-Pitogo CR, Baticados MCL, Cruz-Larcierda ER, de la Pena LD, 1990. Occurrence of luminous bacterial disease of Penaeus monodon larvae in the Philippines. Aquaculture 91: 1-13. Lavilla-Pitogo CR, 1995. Bacterial disease of Penaeid shrimp. Disease in Asian Aquaculture II 107121.