INTRODUCTION
Groupers are high priced and popular for seafood fishes among the major maricultured fish species in Indonesia and Southeast Asia. However, with the rapid development of grouper aquaculture in the recent years, the marine environment is becoming more and more stressful. The industry faces serious threat owing to a variety of diseases problem, especially caused by vibrosis.
Vibriosis is one of the most serious bacterial diseases affecting in the fish culture (Liao et al., 1996) and a major disease problem in mariculture causing high mortality and severe economic loss in many production countries (Goarant et al., 1999). Vibriosis has also been recorded in cultured of groupers (Lee, 1995). This disease can cause more than 70% mortality in maricultured grouper in the disease season.
Recently, various molecular methods have been developed for the detection of this pathogen (Urakawa et al., 1999a, b) and some studies have also been carried out to determine the causative agent of vibriosis in the grouper from geographically different regions (Lee, 1995; Yii et al., 1999), which confirmed the persistent occurrence of vibriosis among groupers.
Molecular based-approaches have been successfully applied, such as RFLP (Restriction Fragment Length Polymorphism) for rapid grouping of vibrios (Urakawa et al., 1999a, b; Radjasa et al., 2001).
Recently, repetitive sequence-based PCR (rep-PCR) has also been used to group a number of bacterial isolates that produced complex fingerprint profiles from gram negative bacteria. However, this technique has not been employed to estimate the diversity of the causative agent of vibriosis among groupers. Recently, Radjasa et al. (2007b) successfully applied repetitive-PCR for grouping marine psychrotrophic bacteria collected from deep-sea waters of Makasar strait, Indonesia as well as the bacterial symbionts of sponge Haliclona sp. (Radjasa et al., 2007c).
To best our knowledge, there has no report been documented so far on describing diversity of vibrios, the causative agent of vibriosis associated with groupers from Indonesian marine culture using molecular tools. The research regarding the molecular diversity of vibrios as the causative agents is important for creating health management of groupers culture. In this study, we reported the phylogenetic diversity of vibrios bacteria as a causative agent vibriosis isolated from groupers from Karimunjawa Islands, Indonesia assessed by 16S rDNA approach.
MATERIALS AND METHODS
Sampling of Grouper Fish
The moribund groupers were collected from cage cultures at Karimunjawa Islands,
Java Sea, Indonesia (Fig. 1) and identified as tiger grouper
(Epinephelus fuscoguttatus); humpbeck grouper (Cromileptus
altivelis) and coral cod (Plectropomus maculates). After collection,
groupers were put into the containers and immediately brought to the Marine
Centre Laboratory of Fisheries and Marine, Science Faculty, Diponegoro University
in Jepara, North Java for bacterial isolation.
Bacterial Isolation
Bacteria were isolated directly from the external lesion and kidney of groupers
by streak plate on TCBS agar (composition (g L-1): Peptone from casein
5.0; peptone from meat 5.0; yeast extract 5.0; sodium citrate 10.0; sodium thiosulfate
10.0; ox bile 5.0; sodium cholate 3.0; sucrose 20.0; sodium chloride 10.0; iron
(III) citrate 1.0; thymol blue 0.04; bromothymol blue 0.04 and agar-agar 14.0).
| Fig. 1: | Sampling site for the collection of grouper fish from Karimunjawa Islands, Java Sea, Indonesia |
Bacterial isolation was also conducted from the inner part of kidney and part of tissue lesions, which were scraped off with a sterile knife. The resultant tissues were serially diluted, spread on TCBS agar medium and were incubated at room temperature for 24-48 h. On the basis of morphological features, colonies were randomly picked and purified by making streak plates (Madigan et al., 2000).
Repetitive-PCR
The procedure was carried out according to a method previously described
by Radjasa et al. (2007b). In the rep-PCR, BOX
A1R (5’-CTACggCAAggCgACgCTgACg-3’) (Versalovic
et al., 1994) was used. The REP 1R-I and REP 2-I primers contain
the nucleotide inosine (I) at ambiguous positions in the REP consensus. PCR
reaction contained of 1 μL DNA template (diluted 100x), 1 μL primer,
7.5 μL Megamix Royal dan sterile water up to total volume of 15 μL.
Amplifications were performed with a thermal cycler model Gene Amp PCR System 9700 with the following temperature conditions: initial denaturation at 95°C for 5 min; 30 cycles of denaturation (92°C for 1 min), annealing (50°C for 1, 5 min), extension (68°C for 8 min) and final extension at 68°C for 10 min. Five microliter aliquot PCR products were run using electrophoresis on 1% ethibium bromade gel by using 1X TBE buffer.
Grouping of Isolates
Grouping was carried out according to a method of Radjasa
et al. (2007c) by making matrixes from the positions of bands on
the gel which were then analyzed by using Free Tree program by using UPGMA method
for constructing the tree. Resampling was performed by bootstrapping with 1000
replications.
PCR Amplification and Sequencing of 16s rRNA Gene Fragments
PCR amplification was carried out according to method of Radjasa
et al. (2007a). Two primers, GM3F (5’-AGAGTTTGATCMTGGC-3’)
and GM4R (5’-TACCTTGTTACGACTT-3’) were used to amplify nearly complete
16S rRNA gene (Muyzer et al., 1995). Genomic
DNA of causative agent of vibriosis-strains for PCR analysis were obtained from
cell materials taken from an agar plate, suspended in sterile water (Sigma,
Germany) and subjected to five cycles of freeze (-80°C) and thaw (95°C).
PCR amplification of partial 16S rRNA gene of bacteria, purification of PCR
products and subsequent sequencing analysis were performed according to the
method of Radjasa et al. (2007b). The determined
DNA sequences of strains were then compared for homology to the BLAST database.
Koch Postulate Test
Between two-three isolates representing each branch in the dendogram constructed
from the result of rep-PCR analysis were used in the test against healthy groupers
(45 days old with an average of 7 cm in length).
Test was performed in 10 L aquariums, in which each aquarium was filled with 9 L filtered seawater and 5 groupers, respectively and was acclimated for 7 days before the injection. Aliquots of 0.2 mL culture of each causative agent in the logarithmic growth phase (ca. 109 cells mL-1), was injected in intraperitonial of tiger grouper (Epinephelus fuscoguttatus). Control was carried out on tiger grouper with an injection of 0.2 mL saline phosphate buffer. Mortality of groupers was observed in time series of 6, 9, 12, 15, 18, 21, 24, 30, 39 and 54 h incubation on each aquarium.
RESULTS
Characteristics of the Bacterial Isolates
The clinical features of moribund groupers affected by vibriosis from Karimunjawa
Islands, Indonesia were dark skin, pale gill, haemorhagic area near mouth and
the base of the fins, haemorhage in the kidney, liver and intestinal wall, ascites
in the body cavity, yellow wish-bloody fluid; empty stomach, skin lesion and
ulcer in the surface skin.
| Table 1: | Characteristics of isolates associated with groupers from Karimunjawa Islands, Indonesia |
Bacterial isolation resulted in a total of 32 vibrio isolates (JT01-JT32) obtained from both of skin lesion and ulcer of surface skin (external lesion) as well as from kidney of moribund groupers (Table 1).
Repetitive-PCR Analysis
Based on the repetitive-PCR result and constructed dendogram of the causative
agents of the groupers, eight groups were formed. Eight different isolates representing
different groups (Fig. 2) as well as the result of confirmation
as the causative agent using Koch Postulate, were further selected for DNA sequencing.
Sequencing of Representative Causative Agents
Based on molecular characterization it is shown that all isolates are the
members of the genus Vibrio as presented in the Table 2.
Phylogenetic Analysis
A comparison of the 16S rRNA gene sequence of representative strains of
JT 02; JT 07: JT 10; JT 13; JT 20; JT 24; JT 27 and JT 31 with sequences from
GenBank demonstrated that these isolates are affiliated with vibrionales within
the family Vibrionacea (Fig. 3) with a homology in the range
of 97-100%, respectively.
| Fig. 2: | Dendrogram of bacterial isolates associated with groupers (JT.02, JT 07; JT 10, JT 13, JT 20, JT 27 and JT 31) were selected for further DNA sequencings |
| Table 2: | Molecular characterization of 8 representatives of the causative agents |
| Table 3: | Pathogenicity test of the causative agents of vibriosis in groupers |
| No mortality was observed in the control | |
Koch Postulate Test
Based on the result of the test, it is indicated that eight isolates were
confirmed as the main causative agents of vibrisosis of groupers from Karimunjawa
Islands, Indonesia (Table 3).
| Fig. 3: | Phylogenetic tree based on comparative of partial 16S rRNA gene sequence analysis of Vibrio species showing the phylogenetic affiliation of strain JT 02 ; JT 07; JT 10; JT 13; JT 20; JT 24; JT 27 and JT 31. Selected sequences from the alpha subclass of Kertersia were used to root the tree. The bar indicates 2% sequence divergence |
The causative agents of vibriosis from groupers positively confirmed based on the test, in which eight isolates representing different branches were found to cause mortality on groupers in the range of 60-100% (Table 2).
DISCUSSION
We investigated marine bacteria isolated from the groupers of Karimunjawa Islands, Indonesia. The attention was focused on the occurrence of the causative agents followed by PCR-based approach, i.e., rep-PCR for estimating the richness of the causative agents of vibriosis associated with the groupers.
Understanding the causative agents of vibriosis has important implications for analysis of microbial taxonomic studies and identification, in particular for strain differentiation among the causative agents of vibriosis are essential for epidemiology and pathogenicty.
In the field of marine diseases, it is very important to identify bacterial species accurately. In addition, Somarny et al. (2002) mentioned that phenotypic identification of Vibrio sp. relies on time-consuming techniques such as studies on morphology, nutrient requirement, antibiotic resistance, enzyme comparison that have limited discriminatory powers.
Generally, the term vibriosis at grouper are described as Vibrio alginolyticus, V. parahaemolyticus, Vibrio sp. (Lee, 1995) and V. charchariae (Yii et al., 1999). Other members of the genus Vibrio are V. anguillarum, V. ordalii, V. salmonicida, V. vulnificus and V. harveyi that were also isolated from disease of fish.
Vibrio species are natural habitants of seawater and brackish water and widely distributed throughout the world. However, some species have exhibited clinical significance for aquatic animals and are recognized as potential pathogens. The large number of vibrios fish pathogens causing epizootic outbreaks in aquaculture has made it necessary to develop efficient, fast and sensitive methods for their detection. Both detection and identification of vibrios have been traditionally depended on their growth on the Thio-Sulphate Citrate Bile Salt Sucrose selective medium and subsequent characterization by biochemical tests (Diggles et al., 2000).
Vibriosis was characterized by dark skin, pale gill, exopthalmia, haemorhagic area near mouth and the base of the fins, corneal opacity and ulcers on the skin surface. Internally moribund fish also showed anemia, haemorhage in the abdominal fat, kidney and liver full of yellow-wish bloody fluid (Yii et al., 1999). The results of the present study were similar to clinical sign findings reported as shown in the Table 1. However, the corneal cavacity and exopthalmia in the moribund groupers was not detected in this study.
The results of this study revealed that the application of rep-PCR has been a reliable tool for strain rapid grouping and differentiation among the causative agents of vibriosis in groupers from Karimunjawa Islands (Fig. 2, 3). This molecular approach may be used for the analysis of other Vibrio species related to mariculture diseases.
Molecular identification of the causative agents of vibriosis (Table 2, Fig. 3) shows that the identified strains were nicely in accordance with the dendrogram constructed from the rep-PCR analysis. The identified strains include the species of V. natriegens, V. olivaceus, V. damsela, V. fortis, V. alginolitycus, V. harveyi, V. parahaemolyticus and V. carchariae (Table 2). The diverse species reported in this study has further confirmed the role of Vibrio as the dominant genus responsible for much of the observed mortality among maricultures (Jayaprakash et al., 2005).
In conclusion, the application of the rep-PCR method is useful for rapid grouping and estimation of the phylogenetic diversity of the causative agents of vibriosis from groupers with high discriminating power and offers an alternative technique for grouping of numerous of marine bacterial isolates.
ACKNOWLEDGMENTS
The authors thank Ministry of Research and Technology, Indonesia for partial financial support under Indonesian International Joint Research Grant Program (RUTI III) in the marine research area (Contract No. 10/M/Kp/I/2006/RUTI). Ocky Karna Radjasa received a research grant from International Foundation for Science (IFS), Sweden (Contract No. 3965-1).