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

Data were isolated from 30 strains of Vibrio and sampled on different organs (brain, hemorrhagic site and digestive tract) of Sciaenops ocellatus infection. The results showed that nucleotide sequences 16S rRNA region are highly, similar to those of V. alginolyticus, V. azureus, V. uvialis and V. orientalis is published on Genebank, ranging from 98.05 to 100 %. The digestive tract has the most common Vibrio strains (V. alginolyticus, V. azureus and V. uvialis). Thereout, 25 of 30 strains of Vibrio contained 1 to 3 toxin genes, excepted V. parahaemolyticus. Six parameters were used to measure the DNA polymorphism of 33 homologous DNA sequences in this Vibrio bacteria population. The results indicated 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 sh (S. ocellatus) hemorrhagic are V. azureus (27,67 %), V. alginolyticus (50 %), V. orientalis (6,67 %) and V. uvialis (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, 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, Perl, Frelier, & Bercovier, 1999), (Mmanda et al., 2014). There were seven Vibrio strains (included V. vulni cus HM-TA-D2-L2-V2; V. vulni cus 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. vulni cus HM-X-13/6) 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 identi ed this sh (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 ve introns. The open reading frame of 1065 bp, which is anked by a 50 untranslated region (UTR) of 34 bp and a 30 UTR of 350 bp and the sh pathogen Edwardsiella tarda but down regulated by the sh pathogens Listonella anguillarum and Streptococcus iniae (Dang, Zhang, Hu, & Sun, 2010). Toxin genes neutrality were tested by three methods (Tajima's D test, Fu and Li's D* and F* test, Fu's Fs), (Tajima, 1989), (Fu & Li, 1993) and (Fu, 1995), they were indicated an excess of low frequency polymorphisms relating to expectation, evidence for a de ciency 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 in identi cation and determination of toxin genes of Vibrio infected on red drum, hence understanding of Vibrio spp. infected on sh to cause Vibriosis for aquatic animals in brackish and marine water.

Collection of sh disease
In this study, we used thirty strains of bacteria with different morphologies isolated from three different organs in the sh (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 phenol/chloroform according to method of (Neumann, Pospiech, & Schairer, 1992). We eliminated 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 sh, 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 Trissaturated phenol (pH 8.0) was added to these tubes, followed by a vortex-mixing 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 puri ed 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 puri ed 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 were determined through the presence of genes encoding toxic proteins (tlh, tdh, trh and toxR) which is based on speci c 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 dNTP each, 0.3 units Taq DNA polymerase, Promega, USA), and sterile distilled water (total volume of 50 µL). PCR ampli cation was performed in MJ Mini TM Thermal Cycler (Bio-Rad, USA) as follows: 94 °C for 3 minutes; followed by 30 cycles at 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute; the last cycle of 72°C for 7 minutes. PCR products were used for electrophoresis 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).

16S rRNA Gene Ampli cation and Sequencing
Performing PCR reaction to amplify the 16S rRNA region, originating from genome with a pair of 16S 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).  Table 2).
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% ampli cation 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).

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 gen Sequencing and analyzing genetic relationships    Table 4 indicated that with A negative Tajima Table 4). The phylogenetic tree shows the genetic relationship of thirty Vibrio strains which are isolated from various three different parts of the sh (S. ocellatus) using UPGMA method. Fig. 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. uvialis (Fig. 3). As observed, the appearance of isolated Vibrio on 3 organs of red drum sh showing signs of bleeding hemorrhagic are V. azureus (27,67 %), V. alginolyticus (50 %), V. orientalis (6,67 %) and V. uvialis (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 nucleotide sequences 16S rRNA region are highly similar to those of V. alginolyticus, V. azureus, V. uvialis and V. orientalis published on Genebank, ranging from 98.05 to 100%. The digestive system has the most common Vibrio species (V. alginolyticus, V. azureus and V. uvialis). None of V. parahaemolyticus presented, the same reported and identi ed that Vibrio species was isolated from cultured olive ounder (Paralichthys olivaceus) in Jeju Island, South Korea that none of V. parahaemolyticus presence too ( The present of toxic genes related to the hemolysin of sh are found in various Vibrio sp.. Meanwhile, approximately 50% of isolates consist of toxin operon gene. All V. parahaemolyticus isolates contained the toxR genes but the trh gene did not existence in clam (Corbicula moltkiana) (Marlina et al., 2007). Our data con rmed three isolates carried both toxR and trh genes including isolates exhibited highly similarity to V. uvialis, 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 trh gene (Al-Othrubi, Al zah, Son, Humin, & Rahaman, 2011). Thermolabile hemolysin (tlh) is an another Vibrio enterotoxin that cause blood cell lysis in infected sh, tlh is encoded by tlh gene (Hasrimi, Budiharjo, & Jannah, 2018). Among 4 toxin genes (toxR, tdh, trh and tlh) were 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 occurrence of toxin gene also showed that there were 5/30 Vibrio strains none carried the toxin gene (code number: YHTH12; YHTH47; YVL11; YVL26 and YVL84), 10/30 strains had only 1 toxin gene, 11/30 strains had 2 toxin genes and 4 strains carried 3 toxin genes including (V. alginolyticus strain 3-31, code number YHTH44 (toxR, trh and 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 carry all of 4 toxin genes. All Vibrio strains isolated from three marine sh species (S. ocellatus, Lates calcarifer and Epinephelus fuscoguttatus) was only carried one tlh gene present (Hoang Tan Quang  ocellatus. The full-length of thermolabile hemolysin (tlh) gene (1257 bp), encoding antigen thermolabile hemolysin toxin (tlh) of the Vibrio sp. was cloned and sequenced successfully. The sequence analysis of gene cloned shows a 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 in 17 strains out of 34. Indicated in two species (Dicentrarchus labrax) and (Sparus aurata). A positive ampli cation for the virulence pathogenicity island (VPI) was produced by 12 V. alginolyticus strains. Finally, the expected ampli cation 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, Chaieb, & Bakhrouf, 2009).
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 classi ed into nine types of haplotype (h) with haplotype diversity coe cient accounting for 0.887±0.032 (Hd), the average number of nucleotide differences is 25.789 (k), the nucleotide diversity coe cient accounts for 17.980x10 -3 ±0.003 (Pi). All indicators were processed with statistical signi cance p < 0.05. The G+C content above 1434 sites positions of nucleotide sequences account 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 de ciency 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 shows the genetic relationship of 30 Vibrio strains using UPGMA method (bootstrap = 1000) showed that these strains are divided into six groups. As observed, the appearance of isolated Vibrio on 3 organs of sh (S. ocellatus) hemorrhagic are V. azureus (27,67 %), V. alginolyticus (50 %), V. orientalis (6,67 %) and V. uvialis (16,67 % Linh analyzed the data, supervisor and wrote the paper. All authors read and approved the nal manuscript.