Indentification and determination of toxin genes of Vibrio strains caused hemorrhagic disease on red drum (Sciaenops ocellatus) by PCR method

: In this study, we isolated thirty strains of Vibrio from three different organs (brain, hemorrhagic site and digestive system) of Sciaenops ocellatus disease. The results showed that nucleotide sequences 16S rRNA region are highly, similar to those of V. alginolyticus , V. azureus , V. fluvialis and V. orientalis is published on Genebank, ranging from 98.05 to 100 %. The digestive system has the most common Vibrio strains ( V. alginolyticus , V. azureu s and V. fluvialis ). Thereout, We found 25/30 strains of Vibrio containing from 1 to 3 toxin genes. None of V. parahaemolyticus present. Six parameters were used to measure the DNA polymorphism of thirty-three homologous DNA sequences in this Vibrio bacteria population. The results indicated that, number of separate polymorphic sites (S), total number of mutant sites (Eta), number of haplotype (h), haplotype diversity (Hd), average number of nucleotide differences (k), nucleotide diversity (Pi) were 98 (S) 103 (Eta), 9 (h), 0.887±0.032 (Hd), 25.789 (k) and 17.980x10-3±0.003 (Pi), respectively (P < 0,05). The G+C content above 1434 sites positions of nucleotide sequences accounts for 0.542. The phylogenetic tree showed that these strains are divided into six groups. As observed, the appearance of isolated Vibrio on 3 organs of fish ( S. ocellatus ) hemorrhagic are V. azureus (27,67 %), V. alginolyticus (50 %), V. orientalis (6,67 %) and V. fluvialis (16,67 %). Through this result, we found that the diversity of Vibrio species that appeared on the red drum was used in the 16S rRNA region and the presence of toxin genes in these Vibrio species. fish In this study, we described the identification and determination of toxin gene neutrality which was tested basing on three methods (Tajima, 1989), (Fu & Li, 1993) and (Fu, 1995) showing that there has been an excess of low frequency polymorphisms relating to expectation, evidence for a deficiency of alleles, as would be expected from a recent population bottleneck and the evolution of the studied 30 strains bacteria Vibrio population was balancing selection, Abstract In this study, we isolated thirty strains of Vibrio from three different organs (brain, hemorrhagic site and digestive system) of Sciaenops ocellatus disease. The results showed that nucleotide sequences 16S rRNA region are highly, similar to those of V. alginolyticus , V. azureus , V. fluvialis and V. orientalis is published on Genebank, ranging from 98.05 to 100 %. The digestive system has the most common Vibrio strains ( V. alginolyticus , V. azureu s and V. fluvialis ). Thereout, We found 25/30 strains of Vibrio containing from 1 to 3 toxin genes. None of V. parahaemolyticus present. Six parameters were used to measure the DNA polymorphism of thirty-three homologous DNA sequences in this Vibrio bacteria population. The results indicated that, number of separate polymorphic sites (S), total number of mutant sites (Eta), number of haplotype (h), haplotype diversity (Hd), average number of nucleotide differences (k), nucleotide diversity (Pi) were 98 (S) 103 (Eta), 9 (h), 0.887±0.032 (Hd), 25.789 (k) and 17.980x10-3±0.003 (Pi), respectively (P < 0,05). The G+C content above 1434 sites positions of nucleotide sequences accounts for 0.542. The phylogenetic tree showed that these strains are divided into six groups. As observed, the appearance of isolated Vibrio on 3 organs of fish ( S. ocellatus ) hemorrhagic are V. azureus (27,67 %), V. alginolyticus (50 %), V. orientalis (6,67 %) and V. fluvialis (16,67 %). Through this result, we found that the diversity of Vibrio species that appeared on the red drum was used in the 16S rRNA region and the presence of toxin genes in these Vibrio species. 10 14

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Introduction
More than 100 Vibrio spp. have been reported and are predominantly associated with a variety of marine, estuarine, or other aquatic habitats (Janda, Newton, & Bopp, 2015). Red drum (Sciaenops ocellatus) was discovered originally in the Atlantic Ocean and the Gulf of Mexico; it was introduced into China in 1991 and since then it has been cultured extensively in several provinces in China (Zhang & Sun, 2011). In recent years, red drum (S. ocellatus) mortalities associated with Streptococcus iniae infection (Eldar, A., et al., 1999), (Mmanda et al., 2014). There were seven Vibrio strains (includings V. vulnificus HM-TA-D2-L2-V2; V. vulnificus HM-TA-G2-V1-D2; V. brasiliensis HM-X-13/6; V. cholerae V-13/6; V. parahaemolyticus HM-17/6; V. cholerae HM-V-13/6 and V. vulnificus HM-X-13/6) deterted to cause hemorrhagic disease in red drum (S. ocellatus) had only tlh gene and none of Vibrio strains had tdh and trh genes (Hoang Tan Quang et al., 2020). The research identified this fish (S. ocellatus) viperin gene (SoVip) and analyzed its expression in relation to bacterial challenge. The complete gene of SoVip is 2570 bp in length and contains six exons and five introns. The open reading frame of SoVip is 1065 bp, which is flanked by a 50 untranslated region (UTR) of 34 bp and a 30 UTR of 350 bp and the fish pathogen Edwardsiella tarda but down regulated by the fish pathogens Listonella anguillarum and Streptococcus iniae (Dang, Zhang, Hu, & Sun, 2010). In this study, we described the identification and determination of toxin gene neutrality which was tested basing on three methods (Tajima, 1989), (Fu & Li, 1993) and (Fu, 1995) showing that there has been an excess of low frequency polymorphisms relating to expectation, evidence for a deficiency of alleles, as would be expected from a recent population bottleneck and the evolution of the studied 30 strains bacteria Vibrio population was balancing selection,

Abstract
In this study, we isolated thirty strains of Vibrio from three different organs (brain, hemorrhagic site and digestive system) of Sciaenops ocellatus disease. The results showed that nucleotide sequences 16S rRNA region are highly, similar to those of V. alginolyticus, V. azureus, V. fluvialis and V. orientalis is published on Genebank, ranging from 98.05 to 100 %. The digestive system has the most common Vibrio strains (V. alginolyticus, V. azureus and V. fluvialis). Thereout, We found 25/30 strains of Vibrio containing from 1 to 3 toxin genes. None of V. parahaemolyticus present. Six parameters were used to measure the DNA polymorphism of thirty-three homologous DNA sequences in this Vibrio bacteria population. The results indicated that, number of separate polymorphic sites (S), total number of mutant sites (Eta), number of haplotype (h), haplotype diversity (Hd), average number of nucleotide differences (k), nucleotide diversity (Pi) were 98 (S) 103 (Eta), 9 (h), 0.887±0.032 (Hd), 25.789 (k) and 17.980x10-3±0.003 (Pi), respectively (P < 0,05). The G+C content above 1434 sites positions of nucleotide sequences accounts for 0.542.
The phylogenetic tree showed that these strains are divided into six groups. As observed, the appearance of isolated Vibrio on 3 organs of fish (S. ocellatus) hemorrhagic are V. azureus (27,67 %), V. alginolyticus (50 %), V. orientalis (6,67 %) and V. fluvialis (16,67 %). Through this result, we found that the diversity of Vibrio species that appeared on the red drum was used in the 16S rRNA region and the presence of toxin genes in these Vibrio species. Keywords: Sciaenops ocellatus, TDH, TRH, TLH, ToxR, Toxin gene, Vibrio.

Collection of fish disease
In this study, we used thirty strains of bacteria with different morphologies isolated from three different organs in the fish (S. ocellatus) that have hemorrhagic disease (Fig 1) in Thua Thien Hue province, Vietnam, basing on the medium TCBS (Thiosulphate Citrate Bile Salt Sucrose).

Total DNA extraction method
The DNA extraction method presented in this paper is an improved method of the standard phenol/chloroform method (Neumann, Pospiech, & Schairer, 1992). We eliminated the lysis step that uses SDS/lysozyme or proteinase K, and lysed cells directly by phenol. To extract the DNA from bacteria isolated from hemorrhagic disease in fish, 1 mL cell suspension was centrifuged at 8000 rpm for 2 minutes, for the collection of pellet cells. After removing the supernatant, the cells were washed with 400 µl STE Buffer (100 mM NaCl, 10 mM Tris/ HCl, 1 mM EDTA, pH 8.0) twice, then centrifuged at 8000 rpm for 2 min. The pellets were resuspended in 200 µl TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0). After this,100 µl Tris-saturated phenol (pH 8.0) was added to these tubes, followed by a vortexmixing step of 60 s. The samples were subsequently centrifuged at 13000 rpm for 5 min at 4°C to separate the aqueous phase from the organic phase. 160 µl upper aqueous phase was transferred to a clean 1.5 ml tube. 40 µl TE buffer was added to make 200 µl and mixed with 100 µl chloroform and centrifuged for 5 min at 13000 rpm at 4°C. Lysate was purified by chloroform extraction until a white interface was no longer present; this procedure might have to be repeated two to three times. 160 µl upper aqueous phase was transferred to a clean 1.5 ml tube. 40 µl TE and 5 µl RNase (at 10 mg/ml) were added and incubated at 37°C for 10 min to digest RNA. Then 100 µl chloroform was added to the tube, mixed well and centrifuged for 5 min at 13000 rpm at 4 °C. 150 µl upper aqueous phase was transferred to a clean 1.5 ml tube. The aqueous phase contained purified DNA and was directly used for the subsequent experiments or stored at 20°C. The purity and yield of the DNA were assessed spectrophotometrically by calculating the A260/A280 ratios and the A260 values to determine protein impurities and DNA concentrations (Neumann et al., 1992).

Determination of toxin gene
The presence of toxin genes in Vibrio spp. strains were determined through the presence of genes encoding toxic proteins (tlh, tdh, trh and toxR) which is based on specific primers for these genes ( 3 on 1% agarose gel, using standard electrophoresis procedures in TAE 1X buffer with Ethydium bromide dye and read electrophoresis images by direct UV reading system (UV-transillumnator, Model: DyNa Light).  (Marlina et al., 2007).

16S rRNA Gene Amplification and Sequencing
Performing PCR reaction to amplify the 16S rRNA region, originating from genome with a pair of 16S primers: 27F: AGAGTTTGATCMTGGCTCAG and 1492R: TACGGYTACCTTGTTACGACTT (Jeremy A Frank et al., 2008). The PCR reaction is performed on the Applied Biosystems -Life Technologies -Thermo Fisher Scientific -USA with a reaction component of 25 µl PCR master mix 2 × (2.4 mM dNTP each, 0.3 units Taq DNA polymerase), 10 pmol of 27F primer, 10 pmol of 1492 primer, 1 µl of total DNA (50 ng/µl) and sterile distilled water to a final volume of 50 µl. The 16S rRNA gene region is amplified with the following thermal cycle: 95°C/5 minutes; 30 cycles x (95°C/60 seconds; 57°C/50 seconds; 72°C/60 seconds); 72°C/10 minutes. Aliquots (10 µl) of PCR products were electrophoresed and visualized in 1% agarose gels using standard electrophoresis procedures in TAE 1X buffer with Ethydium bromide dye and read electrophoresis images by direct UV reading system (UV-transillumnator, Model: DyNa Light). Partial 16S rRNA genes of selected isolates in each site were sequenced by MACROGEN, Republic of Korea (dna.macrogen.com). Finally, 16S rRNA sequence of the isolation was compared with that of other microorganisms using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi).

Sequencing and analyzing genetic relationships
The PCR products of the 16S rRNA region are purified with Isolate II PCR and Gel (Bioline) kits. Then, they are sequenced directly by the dideoxy termination method on the ABI PRISM ® 3100 Avant Genetic Analyzer (Applied Biosystems) at Maccrogen Company, Korea (dna.macrogen.com).
The DNA polymorphism analysis is based on eight parameters including number of separate polymorphic sites (S), total number of mutant sites (Eta), number of haplotypes (h), haplotype diversity (Hd), average number of nucleotide differences (k), nucleotide diversity (Pi) are considered as a polymorphic measurement in the population (J. Rozas & R. Rozas, 2005). Neutrality is tested based on three methods, Tajima's D test (Tajima, 1989), (Fu Y.X., & Li, Ư.H.,1993) and (Fu, Y. X., 1995) using DNASP 6.0 software.
Phylogenetic tree showing genetic relationship will be built by MEGA X software (The Molecular Evolution Genetics Analysis), based on methods of UPGMA method (Sneath & Sokal, 1973). The optimal tree with the sum of branch length equal to 0.08795656 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, J., 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Koichiro Tamura, Masatoshi Nei, & Kumar, 2004) and are in the units of the number of base substitutions per site. This analysis involved 48 nucleotide sequences. All ambiguous positions were removed for each sequence pair   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  61  62  63  64  65 4 (pairwise deletion option). There were a total of 1434 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar S., Stecher G., Li M., Knyaz C., & K., 2018).

PCR result
The results indicated that all PCR products of the 16S rRNA region in the studied of 30 isolated strain bacteria based on medium TCBS showed a single band with 100% amplification rate. All samples gave high DNA concentration and are clearly seen. The obtained size was approximately 1500 bp, which goes in line with the initial expected size (Fig.  2).
The PCR products of the 16S rRNA region are purified with Isolate II PCR and Gel (Bioline) kits. Then, they are sequenced directly by the dideoxy terminator method on the ABI PRISM ® 3100 Avant Genetic Analyzer (Applied Biosystems) at Maccrogen Company, Korea (dna.macrogen.com). The results of the 16S rRNA region were about 1450 bp for the remaining 30 isolated strain bacteria based on medium TCBS. The BLAST result on NCBI was used to verify and compare with the sequences of the Vibrio spp. with accession number Genebank (Table 2) showed that the nucleotide sequences obtained were highly similar to those of the V. alginolyticus, V. azureus, V. fluvialis and V. orientalis, ranging from 98,05 to 100 % ( Table 2).

Determination of toxin gene
The agarose gel electrophoresis of PCR products determined the presence of trh, tdh, tlh and toxR genes at bands 269 bp, 500 bp, 450 bp and 367 bp, respectively (Fig. 2). We found 25/30 strains of Vibrio containing at least 1 toxic gene whereas 5 isolates carried out 3 toxin genes. However, none of these isolates consisted of all virulence toxins genes ( Table 2). The results clearly indicate the presence of virulence toxins (trh, tdh and tlh) and a regulator toxin (toxR). A mong them, 18 isolates presented tlh while only 2 isolates were found to be carried out tdh gene.    13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  61  62  63  64 65 Six parameters including number of polymorphic sites (S), total number of mutant sites (Eta), number of haplotypes (h), haplotype diversity (Hd), average number of nucleotide differences (k), nucleotide diversity (Pi) were used to evaluate the diversity of 30 studied Vibrio strains. As shown in table 4, ninety eight separate polymorphic positions (S) created 103 mutant positions (Eta) shown in 30 studied strain bacteria Vibrio were classified into nine types of haplotype (h) with haplotype diversity coefficient accounting for 0.887 ± 0.032 (Hd), the average number of nucleotide differences is 25.789 (k), the nucleotide diversity coefficient accounts for 17.980x10 -3 ± 0.003 (Pi). All indicators were processed with statistical significance p <0,05. The G+C content above 1434 sites positions of nucleotide sequences account for 0.542 (Table 3).  Table 4 indicated that with A negative Tajima's D signifies an excess of low frequency polymorphisms compared with initial expectation (Statistical significance: Not significant, p>0.10). Meanwhile, a positive value of FS (13.659) is the evidence for a deficiency of alleles, as would be expected from a recent population bottleneck (Strobeck's S statistic: 0.000). In addition, The Fu and Li's F * (statistical significance 0.10 > p > 0.05) and value of Fu and Li's D* (Statistical significance: **, P < 0.02) both yield positive ones, which showed that the evolution of the studied 30 strain bacteria Vibrio population was balancing selection, sudden contraction or in other words, rare alleles appeared in populations with low frequency, the studied population had very few individuals showing large differences in comparison with other individuals in the population (Table 4).
The phylogenetic tree shows the genetic relationship of thirty Vibrio strains which are isolated from various three different parts of the fish (S. ocellatus) using UPGMA method. In figure 3, these strains are divided into six groups. Among these, group I includes the strains of isolated Vibrio which are closely related to V. azureus. These strains mainly concentrate in the digestive system and hemorrhagic. Groups II, III and V consist of Vibrio strains, isolated in 3 different parts (brain, hemorrhagic and digestive system). They are closely related to V. alginolyticus. Group 4 includes two strains, isolated from the ulcer which are closely related to Vibrio orientalis. Group VI consists of 4 strains, concentrating in digestive system and having a close genetic relationship with V. fluvialis (Fig. 3). As observed, the appearance of isolated Vibrio on 3 organs of red drum fish showing signs of bleeding hemorrhagic are V. azureus (27,67 %), V. alginolyticus (50 %), V. orientalis (6,67 %) and V. fluvialis (16,67 %).

Discussion
In this study, we isolated thirty strains of Vibrio from three different organs (brain, hemorrhagic site and digestive system) of S. ocellatus. The results showed that nucleotide sequences 16S rRNA region are highly similar to those of V. alginolyticus, V. azureus, V. fluvialis and V. orientalis published on Genebank, ranging from 98.05 to 100%.
Six parameters were used to evaluate the diversity of 30 studied Vibrio bacteria strains. The result show that, ninety eight separate polymorphic positions (S) created 103 mutant positions (Eta) shown in 30 studied Vibrio strains classified into nine types of haplotype (h) with haplotype diversity coefficient accounting for 0.887±0.032 (Hd), the average number of nucleotide differences is 25.789 (k), the nucleotide diversity coefficient accounts for 17.980x10 -3 ±0.003 (Pi). All indicators were processed with statistical significance p < 0.05. The G+C content above 1434 sites positions of nucleotide sequences account for 0.542.