The Diversity of Vibrios Associated with Vibriosis in Pacific White Shrimp (Litopenaeus vannamei) from Extensive Shrimp Pond in Kendal District, Indonesia

Vibriosis out breaks frequently occur in extensive shrimps farming. The study were commenced to find out the clinical signs of white shrimp that was infected by the Vibrio and to identify the bacterial associated with vibriosis in the pacific white shrimp, Litopenaeus vannamei. Bacterial isolates were gained from hepatopancreas and telson of moribund shrimps that were collected from extensive shrimp ponds of Kendal District, Indonesia and cultured on Thiosulfate Citrate Bile Salts Sucrose Agar (TCBSA). Isolates were clustered and identified using repetitive sequence-based polymerase chain reaction (rep-PCR). Three representative isolates (SJV 03, SJV 05 and SJV 19) were amplified with PCR using primers for 16S rRNA, and sequence for further identification. The clinical signs of shrimps affected by vibrio were pale hepatopancreas, weak of telson, dark and reddish coloration of smouth, patches of red colour in part of the body on the carapace, periopods, pleuopods, and telson. A total of 19 isolates were obtained and belong to three groups of genus Vibrios. Result of the 16S DNA sequence analysis, the vibrio found in this study related to vibriosis in white shrimps from extensive shrimp ponds of Kendal were closely related to Vibrio harveyi (SJV 03); V. parahaemolyticus (SJV 05) and V. alginolyticus (SJV 19).


Introduction
Pacific white shrimp (Litopenaeus vannamei) is an important aquaculture species that has high economic value for export commodities from Kendal. This shrimp farms in this area usually use intensive and extensive technology to increase its production. However, inadequate intensive shrimp management or low quality management of extensively pond will cause disease problems, such as bacteria [1] and mass mortality [2]. Outbreaks of bacterial diseases, such as vibriosis, are still a problem in shrimp farming [3][4]. Vibrio  characterized by pale or reddish carapace, pale hepatopancreas, root and redish of telson; red and root antenna [1]. Although this is a normal part of the bacterial flora in the environment of estuary and marine waters [4][5], some species of vibrio are considered opportunistic pathogens against finfish, shellfish and shrimp [5]. Therefore, the genus Vibrio can cause production failure due to vibriosis in shrimp culture [3][4][6], resulting on death at the stage of zoe, larvae and adult [7]. Various studies have been conducted to identify vibrio associated with vibriosis in the aquaculture industry [2]. Some vibriosis agents in shrimp have been identified, such as: V. harveyi [8][9]; V. alginolyticus [2]; V. parahaemolyticus [2] [7]; V. fluvialis, V. damsela and V. vulnificus [7]. Previous studies using biochemical methods to identify vibrio diversity in this area. Hence, the classification of bacteria with molecular approaches should be applied to develop early detection of disease caused by viruses, bacteria, including vibriosis in shrimp [9].

Sample of Shrimp
A total 31 moribound pacific white shrimp (L. vannamei), suspected having vibriosis infection, were collected from extensive brackish water pond in Kendal district located on Wakak River, Kendati, Indonesia. The identification of pacific white shrimp with vibriosis is based on the clinical symptoms of references suggested by Lighter [6]. This research applies exploratory method with purposive random sampling. The isolation of bacteria in the sample was done at the Aquaculture Laboratory of Fisheries and Marine Science Faculty of Diponegoro University.

Isolation of Bacteria.
The vibrio bacteria were isolated from hepatopancreas and telson from pacific white shrimp (L. vannamei) with streak plate methods on Thiosulfate Citrate Bile Salts Sucrose Agar (TCBSA) and it incubated at room temperature for 24-48 hours [10]. The colonies were randomly drawn and purified by making streak plates based on their morphological features [11].

Repetitive-PCR
Rep-PCR was done based on the method previously described by [10] which has been modified by [13]. BOX AIR (5'-CTACggCAAggCgACgCTgACg-3') was applied in the rep-PCR. The REP 1R-I and REP 2-I primers contain the nuclutide inosine (I) at ambigious potitions in the REP consensus. PCR reaction contained of 1 μL DNA template (diluted 100x), 1 μL primer, 7.5 μL Megamix Royal and sterile water up to a total volume of 05 μL.
Amplification was performed by thermal rotor model of Gene Amp PCR 9700 with the following temperature conditions as follows: initial denaturation at 95ºC for 5 minutes; 30 cycles of denaturation (92° C for 1 minute), annealing (50ºC for 1.5 minutes), extension (68ºC for 8 minutes) and final extension at 68ºC for 10 minutes. Five microliter aliquot PCR products were carried out using electrophoresis on 1% bromade gel ethers using 1X TBE buffer . The result was visualized in UV trans-illuminator and the image was captured by the gel documentation system (Biorad) [9].

Grouping of Isolates
The grouping of isolates was performed based on the method of making matrixes from the position of bands on the gel analyzed using the Free Tree program. This program uses UPGMA method for tree creation. Resampling was done by bootstrapping, 1000 replications [10] 2.5. PCR Amplification and Sequencing of 16s rRNA Gene Fragments PCR amplification was initiated based on the method of Radjasa et al. [10][12] which has been modified by Sarjito et al. [13]. Two primers, GM3F (5'AGAGTTTGATCMTGGC-3') and GM4R (5'-TACCTTGTTACGACTT-3') were used to amplify the nearly complete 16S rRNA gene [12]. The genomic DNA of the causative agent of strain vibriosis for PCR analysis was obtained from the cell material taken from the agar plate, which was suspended in sterile water (Sigma, Germany) and subjected to five freezing cycles at -80ºC and melting at 95ºC. PCR amplification of 16S partial bacterial rRNA gene [14], PCR product purification and subsequent sequencing analysis were performed in accordance with [10] [12][13]. The sequence of DNA strains determined then compared to the homology to the BLAST database. To determine the relationship of the genus vibrios, the phylogenetic stage begins in accordance with the software Mega 1.

Result 3.1.1. Characteristic of the Bacterial Isolates
The clinical signs of shrimp affected by vibrios were pale hepatopancreas, weak of telson, dark and reddish colorozation of mouth, patches of red colour in part of the body, periopods, pleuopods and telson ( Figure 1).  Bacterial isolation resulted in a total of 19 isolates were obtained from hepatopancreas, gill, haemolimph and carapaceous wounds from affected pacific white shrimp (L. vannamei) ( Table 1) Figure.

Discussion
The genus vibrios are commonly found in marine and estuarine environments [4]. However, some species have demonstrated clinical significance for aquatic animals and are recognized as potential pathogens. Vibrio has been detected related to vibriosis in shrimp [9] [15]. Therefore, the genus vibrio has the potential to cause vibriosis in shrimp [16]. These vibrio strains are found in aquatic ecosystems, such as water or sediment [17] or waters [18]. Most Vibrio spp. commonly reported pathogens for aquatic animals, but [2] reported that Vibrio sp. 33 is an avirulent strain. The pacific white shrimp (L. vannamei) infected by the vibrio were identified by clinical signs as follows: pale hepatopancreas, weakened telson, darkness and redness colorization the mouth, and red spots on the carapace on the body, periopods, pleuopods, and telson. Similar clinical signs have been reported by Sarjito et al. [1].
The study also revealed that there were three Vibrio strains that infected pacific white shrimp in extensive brackish water pond, Kendal, i.e. Vibrio harveyi (SJV 03); V. parahaemolyticus (SJV 05) and V. alginolyticus (SJV 19). These results indicate that the vibrios diversity associated with vibriosis in pacific white shrimp, L. vannamei, was higher than the diversity reported on tiger shrimp, Penaeus monodon, [1]. However, this diversity was lower than the diversity found in grouper in Karimunjawa [13], pacific white shrimp, L. vannamei, in India [5].
V. harveyi is naturally found in marine and estuarine environments [16]. These bacteria are also known as pathogenic bacteria associated with black tiger shrimp, P. monodon, and the Pacific white shrimp L. vannamei, [20], Atlantic mediterranian invasive algae Caulerpa cylindracea [20], black tiger shrimp, P. monodon, from brackish water pond and coastal the region of India [16], marine invertebrate and vertebrate [3]. V. harveyi is also the main causative agent in luminous vibriosis [15] and vibriosis in shrimp ponds [22]. Furthermore, Thongkao [8] and Ramesh et al. [21] stated that V. harveyi is known as a bacterial pathogen in shrimp around the world that causes mortality up to 100% in the culture system.
V. parahaemolyticus has been reported as an important causative agent of disease in shrimps and resulted on mortality, and economical losses in this industry [22]. V. parahaemolyticus was also  [23].
The vibrio was also known as the causative agent of vibriosis in pacific white shrimp L. vannamei [24]. The bacteria was also identified the dominant vibrio species associated with the mass mortality of juvenile Chinese shrimp, Fenneropenaeus chinensis [25]. Meanwhile, V. alginolyticus was reported as normal bacteria of estuarine and marine environment [27]. V. alginolyticus was reported as a pathogenic bacterium in a giant tiger shrimp growing pond, P. monodon, in India. Furthermore, Bunpa et al. [26] also stated that this bacterium was important bacterial and pathogenic bacteria associated with infections caused by seafood, fish diseases, shrimp diseases, sediment and seawater.

Conclusion
Clinical signs of shrimp infected by the genus vibrio associated with vibriosis from Kendal district, Indonesia were pale of hepatopacreas weaken of telson, dark and reddish colorization mouth, patches of red colour in part of the body on the carapace, periopods, pleuopods and telson. The study found that vibrios associated with vibriosis in shrimp from Kendal's extensive brackish water were closely to Vibrio harveyi (SJV 03); V. parahaemolyticus (SJV 05) and V. alginolyticus (SJV 19)