The identification and determination of toxin genes of Vibrio strains causing hemorrhagic disease on red drum (Sciaenops ocellatus) using PCR

Data were collected from 30 strains of Vibrio and sampled on different organs (brain, hemorrhagic site and digestive tract) of Sciaenops ocellatus infection. The results showed that the nucleotide sequences 16S rRNA region are highly similar to those of V. alginolyticus, V. azureus, V. fluvialis, V. natriengens and V. orientalis, which were published on Genbank and other, ranging from 98.05 to 100%. The digestive tract has the most common Vibrio strains (V. alginolyticus [16] V. azureus [7] and V. fluvialis). Thereout, 25 of 30 strains of Vibrio contained 1 to 3 toxin genes, except V. parahaemolyticus. Six parameters were used to measure the DNA polymorphism of 33 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 17.980 × 10–3 ± 0.003 (Pi), respectively (P < 0,05). The G + C content above 1434 sites positions of nucleotide sequences accounted 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.


Introduction
More than 100 Vibrio spp. have been reported and are predominantly associated with a variety of marine, estuarine, or other aquatic habitats (Janda 2015). Red drum (Sciaenops ocellatus) was originally discovered 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 and Sun 2011). In recent years, red drum (S. ocellatus) mortalities have been associated with Streptococcus iniae infection (Eldar et al. 1999), (Mmanda et al. 2014). Seven Vibrio strains (including 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) causing hemorrhagic disease in red drum (S. ocellatus) had only the tlh gene, and none of the Vibrio strains had tdh and trh genes (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 SoVip gene is 2570 bp in length and contains six exons and five introns. The open reading frame of 1065 bp is flanked by a 50 untranslated Open Access *Correspondence: nguyenquanglinh@hueuni.edu.vn; phamthihaiyen@hueuni.edu.vn region (UTR) of 34 bp and a 30 UTR of 350 bp and the fish pathogen Edwardsiella tarda but is downregulated by the fish pathogens Listonella anguillarum and Streptococcus iniae (Dang et al. 2010). Toxin gene neutrality was tested by three methods (Tajima's D test-Methods for sequence analysis of candidate genes obtained; Fu and Li's D* and F* test, Fu's Fs-statistic P values being significant or not), (Tajima 1989), (Fu and Li 1993) and (Fu 1995), they indicated an excess of low frequency polymorphisms relating to expectation, evidence for a deficiency of alleles, as expected from a recent population bottleneck and the evolution of the studied 30 strains bacteria Vibrio, was balancing selection, sudden contraction, rare alleles appeared in populations with low frequency. The studied population had a few individuals showing large differences in comparison with other individuals. The study aims to identify and determine toxin genes in Vibrio infected red drum; hence, an understanding of Vibrio spp. infected on fish to cause Vibriosis in aquatic animals in brackish and marine water.

Collection of fish disease
In this study, data were collected from the field and stored at −20 °C, and we used thirty strains of bacteria with different morphologies isolated from three different organs in fish (S. ocellatus) that have hemorrhagic disease ( Fig. 1) in Thua Thien Hue Province, Vietnam, based on medium TCBS (thiosulphate citrate bile salt sucrose).

Total DNA extraction method
The DNA extraction method presented in this paper is an improved method of phenol/chloroform extraction according to the method of (Neumann et al. 1992). We eliminated the step to use SDS/lysozyme or proteinase K and extraction of cells directly by phenol. To extract the DNA from bacteria isolated from hemorrhagic disease in fish, a 1 mL cell suspension was centrifuged at 8.000 rpm for 2 min 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 and 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 vortex-mixing step of 60 s. The samples were subsequently centrifuged at 13.000 rpm for 5 min at 4 °C to separate the aqueous phase from the organic phase. A total of 160 µl of the upper aqueous phase was transferred to a clean 1.5 ml tube. Forty microliters of TE buffer was added to 200 µl, mixed with 100 µl of chloroform and centrifuged for 5 min at 13,000 rpm at 4 °C. The 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. A total of 160 µl of the upper aqueous phase was transferred to a clean 1.5 ml tube. Then, 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 13.000 rpm at 4 °C. Then, 150 µl of the 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 A 260 /A 280 ratios and the A 260 values to determine protein impurities and DNA concentrations according to (Neumann et al. 1992).

Determination of toxin gene
The presence of toxin genes in Vibrio spp. strains was determined through the presence of genes encoding toxic proteins (tlh, tdh, trh and toxR), which are based on specific primers for these genes (Table 1). PCR procedure: 50 ng of total DNA, 10 pmol of each primer, 25 µl PCR master mix 2 × (2.4 mM dNTPs each, 0.3 units Taq DNA polymerase, Promega, USA), and sterile distilled water (total volume of 50 µL). PCR amplification was performed in an MJ Mini ™ Thermal Cycler (Bio-Rad, USA) as follows: 94 °C for 3 min; followed by 30 cycles at 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min; and a final cycle of 72 °C for 7 min. PCR products were used for electrophoresis on a 1% agarose gel using standard electrophoresis procedures in TAE 1X buffer with ethidium bromide dye, and electrophoresis images were read by a direct UV reading system (UV-transilluminator, Model: DyNa Light).

16S rRNA gene amplification and sequencing
PCR was performed to amplify the 16S rRNA region originating from the genome with a pair of 16S primers: 27F: AGA GTT TGATCMTGG CTC AG and 1492R: TAC GGY TAC CTT GTT ACG ACTT (Jeremy A Frank et al. 2008). PCR was performed on Applied Biosystems-Life Technologies-Thermo Fisher Scientific-USA with a reaction component of 25 µl PCR master mix 2 × (2.4 mM dNTPs 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 was amplified with the following thermal cycle: 95 °C/5 min; 30 cycles × (95 °C/60 s; 57 °C/50 s; 72 °C/60 s); 72 °C/10 min. Aliquots (10 µl) of PCR products were electrophoresed and visualized in 1% agarose gels using standard electrophoresis procedures in TAE 1X buffer with ethidium bromide dye, and electrophoresis images were read by a direct UV reading system (UV transilluminator, Model: DyNa Light). Partial 16S rRNA genes of selected isolates in each site were sequenced by MACROGEN, Republic of Korea (dna.macrogen.com). Finally, the 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 were purified with Isolate II PCR and Gel (Bioline) kits. Then, they were 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 nucleotide sequences were arranged based on the Clustals program (Thompson et al. 1997) and edited by using BioEdit 7.0.5 software (Hall 1999). Finally, the 16S rRNA sequence of the isolation was compared with that of other microorganisms using BLAST (http:// www. ncbi. nlm. nih. gov/ BLAST/ Blast. cgi). The DNA polymorphism analysis was based on eight parameters, including the 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), and nucleotide diversity (Pi), considered polymorphic measurements in the population (Rozas et al. 2005). Neutrality was tested based on three methods, Tajima's D test (Tajima 1989), Fu and Li's D * and F* test (Fu and Li 1993) and Fu's (Fu 1995), using DNASP 6.0 software. A phylogenetic tree showing genetic relationships was built by MEGA X software (Molecular Evolution Genetics Analysis) based on the UPGMA method (Sneath and 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) is shown next to the branches (J 1985). The tree was 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 (Tamura et al. 2004) and are in units of the number of base substitutions per site. This analysis involved 48 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1434 positions in

PCR result
The PCR products of the 16S rRNA region were purified with Isolate II PCR and Gel (Bioline) kits. Then, they were 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 16S rRNA region was approximately 1450 bp for the remaining 30 isolated bacterial strains based on TCBS medium. The BLAST result on NCBI was used to verify and compare with the sequences of the Vibrio spp. with accession number GenBank (Table 2) showed that the nucleotide sequences obtained were highly similar to those of V. alginolyticus, V. azureus, V. fluvialis and V. orientalis, ranging from 98,05 to 100% ( Table 2).
The results indicated that all PCR products of the 16S rRNA region in the 30 isolated bacterial strains based on medium TCBS showed a single band with a 100% amplification rate. All samples gave high DNA concentrations and are clearly seen. The obtained size was approximately 1.500 bp, which is in line with the initial expected size (Fig. 2).

Determination of toxin gene
The agarose gel electrophoresis of PCR products determined the presence of the trh, tdh, tlh and toxR genes at bands 269 bp, 500 bp, 450 bp and 367 bp, respectively (Fig. 2). We found that 25/30 strains of Vibrio contained at least 1 toxic gene, whereas 5 isolates carried 3 toxin genes. However, none of these isolates consisted of all virulence toxin genes ( Table 2). The results clearly indicated the presence of virulence toxins (trh, tdh and tlh) and a regulator toxin (toxR). Among them, 18 isolates presented tlh, while only 2 isolates were found to carry tdh gen.

Sequencing and analyzing genetic relationships
Six parameters, including the number of polymorphic sites (S), total number of mutant sites (Eta), number of haplotypes (h), haplotype diversity (Hd), average number of nucleotide differences (k), and nucleotide diversity (Pi), were used to evaluate the diversity of the 30 studied Vibrio strains. As shown in Table 4, ninety-eight separate polymorphic positions (S) created 103 mutant positions (Eta) shown in 30 studied bacterial strains. Vibrio were classified into nine types of haplotypes (h) with a haplotype diversity coefficient accounting for 0.887 ± 0.032 (Hd), an average number of nucleotide differences of 25.789 (k), and a nucleotide diversity coefficient of 17.980 × 10 -3 ± 0.003 (Pi). All indicators were processed with statistical significance p < 0,05. The G + C content above 1434 site positions of nucleotide sequences accounted for 0.542 (Table 3). Three methods, namely, Tajima's D test, Fu and Li's D* and F* test, and Fu's Fs, were used to test neutrality. The results in Table 4 indicated that a negative Tajima's D signifies an excess of low-frequency polymorphisms compared with the initial expectation (statistical significance: not significant, p > 0.10). Meanwhile, a positive value of F S (13.659) is evidence for a deficiency of alleles, as would be expected from a recent population bottleneck (Strobeck's S statistic: 0.000). In addition, Fu and Li's F * (statistical significance 0.10 > p > 0.05) and the value of Fu and Li's D* (statistical significance: **, P < 0.02) both yielded positive results, which showed that the evolution of the 30 studied bacterial strains in the Vibrio population was balancing selection and sudden contraction; in other words, rare alleles appeared in populations with low frequency, and 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 isolated from three different parts of the fish (S. ocellatus)   using the UPGMA method. Figure 3, these strains are divided into six groups. Among these, group I includes the strains of isolated Vibrio that are closely related to V. azureus. These strains are mainly concentrated in the digestive system and are 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 that are closely related to Vibrio orientalis. Group VI consists of 4 strains, concentrated in the digestive system and having a close genetic relationship with V. fluvialis (Fig. 3). As observed, the isolated Vibrio on 3 organs of red drum fish showing signs of hemorrhagic bleeding were V. azureus (27,67%), V. alginolyticus (50%), V. orientalis (6,67%) and V. fluvialis (16,67%).

Discussion
In this study, we isolated 30 strains of Vibrio from three different organs (brain, hemorrhagic site and digestive tract) of S. ocellatus. The results showed that the nucleotide sequence 16S rRNA regions are highly similar to those of V. alginolyticus, V. azureus, V. fluvialis and V. orientalis published in GenBank, ranging from 98.05 to 100%.
The digestive system has the most common Vibrio species (V. alginolyticus, V. azureus). None of the V. parahaemolyticus species were present in the samples, and the same report identified that Vibrio species were isolated from cultured olive flounder (Paralichthys olivaceus) on Jeju Island, South Korea, and none of the V. parahaemolyticus species were also present (Sohn et al. 2019), as reported by (Quang et al. 2020). The V. parahaemolyticus strain is a pathogen causing shrimp disease, (Dao et al. 2014), (Khanh et al. 2019). The presence of toxic genes related to the hemolysin of fish was found in various Vibrio sp. Meanwhile, approximately 50% of isolates consisted of toxin operon genes. All V. parahaemolyticus isolates contained the toxR genes, but the trh gene did not exist in clams (Corbicula moltkiana) (Marlina et al. 2007). Our data confirmed that three isolates carried both toxR and trh genes, including isolates that exhibited high similarity to V. fluvialis, V. alginolyticus, and V. orientalis. TDH is an enzyme that lyses human red blood cells on Wagatsuma blood agar plates, which is referred to as the Kanagawa phenomenon positive. Another toxin produced by Kanagawa phenomenon-negative Vibrio strains is the tdh-related hemolysin (trh) toxin encoded by the trh gene (Al-Othrubi et al. 2011). Thermolabile hemolysin (tlh) is another Vibrio enterotoxin that causes blood cell lysis in infected fish, and tlh is encoded by the tlh gene (Hasrimi et al. 2018). Among the 4 toxin genes (toxR, tdh, trh and tlh) investigated from Vibrio spp. causing hemorrhagic disease in S. ocellatus, the results showed that the frequency of the toxR gene was detected in the 15 isolates using PCR assay, lowest of tdh gene was 2 isolates, trh gene was 9 isolates and the highest of tlh was 18 isolates using PCR assay. In addition, the frequency of toxin gene occurrence also showed that there were 5/30 Vibrio strains none carrying the toxin gene (code number: YHTH12; YHTH47; YVL11; YVL26 and YVL84), 10/30 strains carrying only 1 toxin gene, 11/30 strains carrying 2 toxin genes and 4 strains carrying 3 toxin genes (V. alginolyticus strain 3-31, code number YHTH44 (toxR, trh and . a PCR product of gene tdh. b PCR product of gene tlh. c PCR product of the toxR gene and d PCR product of the trh gene tlh); V. alginolyticus strain 3-31, code number YVL22 (toxR, trh and tlh); V. alginolyticus strain 3-5, code number YVL24 (toxR, tdh and tlh) and V. orientalis strain 5-13, code number YVL42 (toxR, trh and tlh)). None of Vibrio carried all 4 toxin genes. All Vibrio strains isolated from three marine fish species (S. ocellatus, Lates calcarifer and Epinephelus fuscoguttatus) carried only one tlh gene (Quang et al. 2020). According to Long et al. 2019, we isolated and identified the V. parahaemolyticus 01 strain in Thua Thien Hue Province, Vietnam, causing ulcer disease in S. ocellatus. The full-length thermolabile hemolysin (tlh) gene (1257 bp), encoding the antigen thermolabile hemolysin toxin (tlh) of Vibrio sp. was cloned and sequenced successfully. Sequence analysis of cloned genes showed complete similarity to the V. parahaemolyticus strain (GenBank: AY289609.1) (Long et al. 2019). We further examined the presence of virulence genes homologous to those in V. cholerae (toxR, toxS, VPI and ace); toxR was found in 16 V. alginolyticus strains, and toxS was found in 17 strains out of 34. Indicated in two species (Dicentrarchus labrax) and (Sparus aurata). Positive amplification of the virulence pathogenicity island (VPI) was produced by 12 V. alginolyticus strains. Finally, the expected amplification fragment was found in 7 V. alginolyticus isolates. Thus, the pathogenicity of V. alginolyticus may be the result of a combination of all these factors (Kahla-Nakbi et al. 2009).
Six parameters were used to evaluate the diversity of the 30 studied Vibrio bacterial strains. The results showed that ninety-eight separate polymorphic positions (S) created 103 mutant positions (Eta) shown in 30 studied Vibrio strains classified into nine types of haplotypes (h) with a haplotype diversity coefficient accounting for 0.887 ± 0.032 (Hd), an average number of nucleotide differences of 25.789 (k), and a nucleotide diversity coefficient of 17.980 × 10 -3 ± 0.003 (Pi). All indicators were processed with statistical significance p < 0.05. The G + C content above 1434 site positions of nucleotide sequences accounted for 0.542. Neutrality was tested based on three methods (Tajima's D test, Fu and Li's D* and F* test, Fu's Fs) showing that there was an excess of low frequency polymorphisms relative to expectation, evidence for a deficiency of alleles, as would be expected from a recent population bottleneck and the evolution of the studied 30 Vibrio bacteria 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. The phylogenetic tree showed the genetic relationship of 30 Vibrio strains using the UPGMA method (bootstrap = 1000) and showed that these strains were divided into six groups. As observed, the isolated Vibrio on 3 hemorrhagic organs of fish (S. ocellatus) were V. azureus (27,67%), V. alginolyticus (50%), V. orientalis (6,67%) and V. fluvialis (16,67%).