شناسایی باکتری‏ های جنس Vibrio جداسازی شده از ماهی آکواریومی گوپی (Poecillia reticule) در استخرهای پرورش ماهی کاشان، اصفهان، ایران

Introduction

More than twenty thousand species of freshwater ornamental fish exist in the world. Guppy fish (Poecillia reticule) is one of the most familiar ornamental fish in many countries, including Iran. The length of the fish is up to 7 cm. The fish swims in mid water. The guppy fish can coexist with all other fish. Examples of guppy fish are snakeskin, red tail, yellow tail, singaporean, blue tail, rabbit tail and glass- belly (1).

All fish are in the exposure of infections caused by different kinds of bacteria, fungi and viruses (2), however bacteria particularly Gram- negative species, are the most common agents of infectious diseases in ornamental fish (3). Environmental factors such as stress, poor quality of water, sewage pollution, unhygienic handling of fish and polluted aquaculture feeding can result in increasing of susceptibility to infections among fish (4 & 5). However, immune system deficiency of fish enhances the morbidity of infection (6).

Streptococcus, Mycobacterium, Aeromonas, Pseudomonas, Edwardsiella, Yersinia and Vibrio are the most infectious bacterial genera in fish (2). Vibrio spp. can cause septicemia in fish. The disease will be adversed by temperature change or environmental stress. It is estimated that vibrios consist approximately 60% of total heterotrophic bacteria in aquarium and are from opportunistic fish pathogens (7). As Vibrio species especially Vibrio cholera is from powerful human pathogens and possibly transmitted among aquarists in countries which are outside cholera endemic areas (8), it is necessary to be considered in aquaculture healthcare systems in our country.

During the last decade, biologists have employed different molecular techniques in phylogenetics, evolution, and population diversity. Analysis of 16S (small subunit) rDNA and, more recently, 16S internal transcribed spacer (ITS) has been used greatly in these studies on prokaryotic microorganisms (9).

The aim of the present study was to detect Vibrio species isolated from ornamental guppy fish using biochemical and molecular species.

Materials and methods

Samples and isolation of the bacteria: In a period of time from January to September 2012, skin, gill, kidney and brain tissue samples were obtained from symptomatic guppy in five aquaculture ponds in Kashan, Iran. The samples obtained from a total of 40 symptomatic guppy fish. The included symptoms were: ulcer, pop eye, cloudy eye, dropsy, columnaris, mouth rot, tail rot and bloated stomach. The samples inoculated on enriched culture media including brain heart infusion broth, tripticase soy broth and blood agar. The culture media were incubated at 32°C for 24 hours. The isolated bacteria were identified using conventional biochemical analysis (10 & 11).

Due to the effect of water quality on the general health of fish; the amount of dissolved oxygen (DO), acidity (pH) and five- day biochemical oxygen demand (BOD) of the pond water was measured on the sites of sampling.

DNA extraction and sequence amplification: DNA content of media was extracted using Roche Applied Science DNA extraction kit. Pairs of primers were used according to the specific genome sequences of the biochemical detected bacteria. The characteristics of primers and information about amplification by polymerase chain reaction (PCR) are shown in table 1. A mixture consists of 1X PCR buffer, 0.2 mM dNTP mix, 1.5 mM MgCl₂, 0.5 mM of each primer, 1 unit.ml^-1 Taq DNA polymerase and 1 mg of the extracted DNA was used for each amplification reaction. The products analyzed by gel electrophoresis using 1% agarose.

Sequence analysis of the amplified fragments: The final products have been sequenced by ABI3730XL system (Bioneer Corporation, Korea) and aligned in BLAST data base. Also the content of amplified fragment was analyzed by Gene runner program version 3.05. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 (13).

Results

Water quality parameters: The amount of dissolved oxygen (DO), pH and five- day biochemical oxygen demand (BOD) of the pond water are shown in table 2.

The isolated bacteria: All isolates showed similarity to Vibrio spp., according to morphological and biochemical analysis. Detection of bacteria using biochemical tests is shown in table 3. The amount of 5% of samples (2 fish) was infected to Vibrio spp., according to biochemical tests. Both infected fish were obtained from the same pond.

Molecular detection: As the results of molecular amplification, the 750bp fragment amplicon of Vibrio spp. 16SrDNA were obtained (Fig. 1). Both samples were detected as positive by PCR.

Fig. 1- The amplified fragment for Vibrio spp. 16SrDNA. The amplicon was 750 bp in length.

Table 3- The morphological and biochemical characteristics of the isolated bacteria, which were similar to Vibrio spp.

Table 1: Primer sequences and characteristics of amplification procedures.

Table 2- The average of water quality measures in sampling ponds shows low quality of water for fish culturing.

Table 4- The nucleotide sequence of fragments amplified from Vibrio spp.

Fig. 2. Phylogenic tree confirmed the isolate Vibrio cholera FJ147 after alignment in MEGA version 4.