Genetic diversity and toxic genes analysis of vibrio spp. isolated from white leg shrimp and marine fishes cultured in Tam G iang lagoon in Thua Thien Hue province, Vietnam

Objective: This study was done to report the results of genetic diversity and toxic genes analysis of Vibrio pathogen isolated from white leg shrimp and marine fishes cultured in Thua Thien Hue province, Vietnam. Methods/statistical analysis: Pathogen Vibrio spp. were isolated from shrimps and fishes, and were identified by 16S rRNA sequencing. The presence of toxin genes in Vibrio spp. strains were determined through the presence of genes encoding toxic proteins (pirAvp, pirBvp, tlh, tdh and trh) based on specific primers for these genes. Genetic diversity of Vibrio strains was analysed by RAPD technique. Findings: A total of 120 Vibrio colonies from shrimps (with Acute Hepatopancreatic Necrosis Disease) and fishes (with hemorrhagic disease) cultured in Tam Giang lagoon in Thua Thien Hue, Vietnam were isolated. Of which, 14/54 strains from shrimps had pirAvp and pirBvp genes and 18/66 strains from fishes had tlh gene, and none of Vibrio strains had tdh and trh genes. Randomly amplified polymorphic DNA (RAPD) analysis of 36 Vibrio samples showed the 148 polymorphic DNA fragments from ten random primers. The genetic diversity is high within studied species. In there, V. parahaemolyticus has the highest diversity level (h=0.1645 and I=0.2695) while V. shilonii is lowest (h=0.0136) and I=0.0207). The degree of genetic differentiation among species is also high (Gst=0.4827). Genetic identity between V. parahaemolyticus and V. vulnificus (0.9545) is highest while between V. shilonii and V. harveyi (0.7416) is lowest. The dendrogram also showed that V. parahaemolyticus is closely related to V. vulnificus whereas V. shilonii and V. harveyi have large distance. Application/improvements: This study is aimed to provide scientific data as the basis for the study and production of rapid diagnostic kits in the future.


Introduction
Vibrio spp. are found in all over the world in marine and estuarine environments. In fish, this genus is present as a part of the normal intestinal microflora. Some Vibrio spp. (such as V. anguilarum, V. harveyi, and V. parahaemolyticus) can cause dangerous diseases in shrimp (1) .
In shrimp, Acute Hepatopancreatic Necrosis Disease (AHPND) has caused remarkable mortality (up to 100%) in populations of shrimp cultured in South East Asian and Latin American countries. This disease is referred to as early mortality syndrome (EMS). AHPND is caused by several Vibrio species, which secretes proteins similar to Photorhabdus insect-related (Pir) toxins (PirAvp and PirBvp) (1,2) . Vibrio spp. are also mainly pathogenic to brackish water and marine fish. The distribution of vibriosis is worldwide and causes economic loss to the aquaculture industry (3) .
The Tam Giang lagoon in Thua Thien Hue province of Vietnam is the biggest lagoon in the Southeast Asia. Aquaculture has developed in the Tam Giang Lagoon since the late 1970s, and has become the most important livelihood activity since the early 1990s. However, aquaculture production decreased continuously since 2009 until now. The main reason is because of the disease outbreak, natural disasters, climate change and the environment issues, especially water pollution has serious impacts on the health of the aquatic animals. In addition, the increased intensity of the flood seriously affects aquaculture production through changing of water quality and salinity. Many farmers lost their production due to serious Vibrio bacterial and viral shrimp diseases related to increasing water temperature (4,5) .
Randomly amplified polymorphic DNA (RAPD) is a PCR-based genotyping technique, using random primers to detect changes in the DNA sequence. According to Behura et al (2015), RAPD was a reliable and fast technique for discriminating between the species of Vibrio, therefore, this is a powerful tool for these prawn pathogens study (6) .
In Thua Thien Hue, to our knowledge there is limited published data available on the genetic diversity and toxic genes analysis of Vibrio isolated from white leg shrimp and marine fishes cultured. This study is therefore aimed to provide scientific data as the basis for the study and production of rapid diagnostic kits.

Sample collection
Seventy samples of shrimp (body weight of 0,64 g ± 0,22 and length of 4,37 cm ±0.29 Hepatopancreatic Necrosis Disease syndromes were collected from white-leg shrimp (Litopenaeus vannamei) at farms in Dien Hai (n=8), Dien Mon (n=12) and Dien Huong (n=15) communes (Phong Dien district), Phu Thuan town (n=15) (Phu Vang district) and Loc Binh commune (n=20) (Phu Loc district) ( Figures 1 and 2 ). Sampling was done with live shrimps which showed signs of pathology: stopped eating; swim slowly; pale, atrophy, and toughness in the liver and empty intestine. The outer surface of the shrimp body was disinfected with 70 • alcohol before bacterial isolation. Vibrio sp. was isolated from their liver and pancreas, then samples were cultured on TCBS medium (Thiosulfate Citrate Bile Sucrose Agar) at 30 • C for 24 hours. Thirty-two samples of fishes including seabass (Lates calcarifer, n=12), red drum (Sciaenops ocellatus, n=15) and grouper (Epinephelus fuscoguttatus, n=5) with haemorrhage were collected from cages (in Hai Duong communes and Thuan An town, Phu Vang district) had typical signs: stopped eating, and swimming slowly (Figure 1 and 2). The external signs of three fish species were hemorrhage on body, abdomen, fin erosion, tailless condition and protruding eyes. The fish was disinfected with 70 • alcohol and cleaned, using the sterile implants for sampling bacteria from the hemorrhage areas on the body and bacterial samples in the heart, liver, kidneys, intestines, spleen and brain for culturing on TCBS medium, incubated at 30 • C for 24 hours.
After 24 hours, the development of colonies was checked. The types of colonies on plates were distinguished based on color, shape and size of colonies. The dominant colonies were transferred to TCBS medium to select pure colonies for further studies.
A total of 120 Vibrio spp. colonies were isolated on TBSC medium from shrimps (54 colonies) and fishes (66 colonies) in Thua Thien Hue province, Vietnam for toxic genes determination.

DNA isolation
Vibrio colonies were cultured in alkaline saline peptone water (ASPW) with 2% peptone and 2% NaCl, pH 8.6, shaking speed of 180 rpm for 18 hours at 30 o C. Cells were collected by centrifugation at 13,000 rpm for 1 minute at 4 • C. Total genomic DNA was extracted using AquaPure Genomic DNA Isolaton Kit (Cat. 732-6340, Bio-rad) according to the manufacturer's instructions and then stored at 4 • C. Total DNA concentration was determined using a photo spectrometer at 260/280 nm. Genomic DNA was diluted to a final concentration of 50 ng/µL for PCR amplification. https://www.indjst.org/

Determination of toxin gene
The presence of toxin genes in Vibrio spp. strains were determined through the presence of genes encoding toxic proteins (pirAvp, pirBvp, tlh, tdh and trh) based on specific primers for these genes ( Table 1).
PCR procedure: 50 ng of total DNA, 10 pmol of each primer, 6 µL 2× Go Taq ® Green Master Mix (M7502, Promega, USA), and sterile distilled water (total volume of 12 µL). PCR amplification was performed in MJ Mini TM Thermal Cycler (Bio-Rad, USA) as follow: 95 • C for 10 minutes; followed by 30 cycles at 95 • C for 30 seconds, 53 • C for 30 seconds, and 72 • C for 1 minute; last cycle was 72 • C for 10 minutes. PCR products were used for electrophoresis on 0.8% agarose gel, stained by SafeView TM Classic Nucleic Acid Stain (Applied Biological Materials Inc., Canada) and determined by Ultra Slim LED Illuminator system. https://www.indjst.org/  (1) pirBvp

Vibrio identification
Vibrio strains with pirAvp and pirBvp genes (from shrimps) or tlh, tdh and trh genes (from fishes) were identification based on the sequencing of 16S rDNA genes ( Table 2 and 3). The total DNA was used as a template for the 16S rDNA amplification using the primer pair of 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). PCR products are purified and sequenced at Firstbase company (Malaysia). The nucleotide sequences of 16S rDNA were used for species identification with BLAST tool on Genbank. Phylogenetic tree was built with MEGA X software.

Genetic diversity
Genomic DNA were used as template for RAPD analysis, including 14 strains with pirABvp genes, 18 strains with tlh gene and 4 non-toxic genes strains (Table 2 and 3). PCR reactions were carried out according to Quang et al (2016). Each reaction contained 10 µl 2× PCR master mix (GoTaq Green Master Mix 2X, Promega, USA), 20 pmol each primers and 50 ng genomic DNA in a 20 µL final volume. Amplification was performed in a thermocycler (SimpliAmp, ThermoFisher Scientific, USA) under the following conditions: 3 min at 95 • C; followed by 42 cycles for 1 min of denaturing at 92 • C; 1 min of annealing at 36 • C and a 2 min extension at 72 • C; a final extension for 10 min at 72 • C. PCR products were separated on a 1.4% agarose gel for 5 hr at 40 V (8) . Samples with lack of bands in agarose gel were replicated twice to get confirmation. Ten random decamer oligonucleotide primers (Operon Technologies, USA) were selected for evaluation (Table 4).
PCR-RAPD amplicons were scored as presence (1) or absence (0) of a band in each sample. The sizes of the RAPD markers were estimated by using the DNA size standard (GeneRuler 1kb DNA Ladder, Thermo Scientific). The genetic identity and genetic distance between populations were expressed using Nei's (1972) genetic distance (9) . Genetic parameters were calculated as the total genotype diversity in populations (Ht), total genotype diversity within populations (Hs), mean coefficient of gene differentiation (Gst), and estimate of gene flow (Nm); number of polymorphic bands, observed number of alleles (na), effective number of alleles (ne), Nei's (1973) gene diversity (h), Shannon's information Index (I) for RAPD data using the POPGENE software ver. 1.31 (10) .

Isolation of pathogenic Vibrio
From 120 Vibrio samples, we found that 14 strains (from shrimps) and 18 strains (from fishes) had pirAvp and pirBvp genes or tlh gene, respectively ( Tables 2 and 3). V. parahaemolyticus is the most popular species that cause diseases in shrimp and fish, and has both pirABvp and tlh genes. V. shilonii displayed only in shrimp while V. vulnificus, V. harveyi and V. cholerae appeared only in fish.
In shrimp, the pVPA3-1 plasmid causes AHPND was identified in V. parahaemolyticus strain 13-028/A3, it consists of genes that encode mobilization proteins, replication enzymes, virulence-associated proteins, and proteins similar to Pir toxins. These Pir toxin-like proteins are encoded by 2 genes (pirA-and pirB-like), also known as pirAvp and pirBvp genes (1) . In Thua Thien Hue province, 14/54 samples from shrimp had pirABvp genes (25.93%), including V. parahaemolyticus, V. shilonii, V. communis and V. furnissii species. The bacterium V. shilonii was found in the first time in Vietnam. V. shilonii had been found to be a cause of bleaching in the coral Oculina patagonica (11) , in recent paper, V. shilonii caused AHPND was found in Ecuador (1) In fish, the majority of Vibrio strains isolated were not pathogenic. The pathogenic strains are those that produce thermostable https://www.indjst.org/ direct haemolysin (TDH) toxin. 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 (12) . Thermolabile hemolysin (TLH) is an another Vibrio enterotoxin that cause blood cell lysis in infected fish, TLH is encoded by tlh gene (13) . In this study, 18 2015), none of the eight AP-positive AHPND V. parahaemolyticus strains possesses the genes for the conventional virulence factors affecting humans, such as tdh, trh and type III secretion system 2 (14) . In Bangladesh, Ahmmed et al. (2019) found tlh, pirA and pirB genes from two AHPND positive V. parahaemolyticus strains isolated from shrimp (Penaeus monodon) (15) . The previous studies in Malaysia showed that the most virulent of the non-cholera vibrios (V. vulnificus) have various virulence factors that facilitate the development of clinical disease (16) . V. vulnificus also have been implicated in fish diseases such as septicemia (3) . In our study, V. vulnificus (6/18) was the most popular patogen species in fish.
Vibrio spp. that caused disease in shrimp and marine fish are widely distributed in brackish and saline waters worldwide. They exist in the aquatic environment and can be harmful to shrimp and fish in favorable conditions.
In shrimp, the presence of Vibrio spp. such as V. parahaemolyticus, V. alginolyticus, V. vulnificus, etc. are likely to cause AHPND (17) . The disease has caused huge losses, high mortality, and a very high risk for shrimp farming from 2010 to the https://www.indjst.org/  (18) . Diseases caused by Vibrio bacteria appear all year round but often out break when high water temperature and in high salinity waters, somehow the disease can still occur in the estuaries or for a number of freshwater fish species. Although bacteria can be infected in different parts, they often attack the heart and muscles of the fish. Because they have a strong ability to attack the muscular system, hence it is very serious and difficult to treat, bacteria can cause chronic diseases in adults. If the disease occurs acute, the consequences of damage are very heavy.
In fish larvae, fry or fingerlings, when seriously infected they can be killed up to 50%. In adult fish, the rate of damage is lower but the fish will develop anorexia or stop eating or ceases in growth and at harvest it is possible to observe necrotic wounds on the skin and muscles of the fish. When the fish were infected with Vibrio, fish eats less or stops eating, they swim on the surface layer and around the cage edge. On the fish body, often appear various small reddish ulcers with the skin bulging surrounding it and a lot of viscous. The internal organs, liver, kidney, spleen with hemorrhage would have occurred and empty gastrointestinal tract (5) . Restrepo et al. (2018) reported that the gene transfer capacity of Vibrio species goes beyond the clade classification, creating new Vibrio pathogenesis and has major implications for the spread of emerging diseases (19) . In our study, ten Vibrio species contain toxic genes (pirA, pirB, and tlh), they seem to be the results of gene transfer of among Vibrio species in Vietnam sea.

RAPD analysis
A total of 20 RAPD primers were assayed for their specificity in detecting Vibrio species, ten primers were highly reproducible and found suitable for use in RAPD-PCR. These selected primers generated total of 148 amplified bands, ranging in size from approximately 200 (OPB-18) to 4000 bp (OPB-01 and OPN-03), and the number of PCR products of each primer varied from 11 to 17 ( Table 4). All of bands were polymorphic (with a mean of 14.80 bands per primer). OPG-17 is the best primer for Vibrio with 100% genotypes were amplified (15 bands) ( Figures 3 and 4). In this study, the percentage of polymorphic bands (PPB) of each population was ranged from 25.80% (V. shilonii) to 100% (V. parahaemolyticus and V. vulnificus) ( Table 5

Genetic diversity
Data for observed number of alleles (na), effective number of alleles (ne), Nei's (1973) genetic diversity (h), Shannon's information index (I), for all the Vibrio species were analyzed using ten RAPD primers and their respective values were found as 2.0000, 1.2885, 0.2046, and 0.3435, respectively (Table 6). V. shilonii displayed the lowest value of Nei's (1973) gene diversity (h=0.0136) and Shannon's information index (I=0.0207), whereas the highest population was V. parahaemolyticus (h=0.1645 https://www.indjst.org/    Table 6). The average diversity within populations (Hs) was 0.1114, which accounted for 51.72% of the total diversity found in the populations (Ht = 0.2154). The mean coefficient of gene differentiation (Gst) value of 0.4827 indicated a very high degree of genetic differentiation among species. The gene flow (Nm) was 0.5359, which indicated that it was small among the species (  Table 7).
The values of genetic identity and genetic distance between Vibrio species were given in Table 8. The analyzed data indicated https://www.indjst.org/ that the values of genetic identity between populations were low, ranging from 0.7416 to 0.9545. V. parahaemolyticus and V. vulnificus had the highest genetic identity (0.9545) while V. shilonii and V. harveyi had the lowest genetic identity (0.7416). The values of genetic distance between populations were high, varying from 0.0465 to 0.2989 and the two species of V. shilonii and V. harveyi have the largest genetic distance (0.2989).

Cluster analysis
In the genetic similarity dendrogram constructed on the basis of comparative analysis of the total loci obtained with the ten RAPD primers, the Vibrio species gets distributed based on their genetic distance ( Figure 5). The dendrogram showed that V. parahaemolyticus was closely related to V. vulnificus whereas V. shilonii and V. harveyi had large distance.

Conclusion
In our study, 120 Vibrio samples were isolated from white leg shrimp and fishes cultured in Tam Giang lagoon in Thua Thien Hue province (Vietnam). In shrimp samples, 14/54 strains had pirAvp and pirBvp genes and in fish samples, 18/66 strains had tlh gene, and none of Vibrio strains had tdh and trh genes. RAPD analysis of 36 Vibrio samples showed genetic identity between V. parahaemolyticus and V. vulnificus is highest while between V. shilonii and V. harveyi is lowest. V. shilonii had the most different RAPD pattern than other species. https://www.indjst.org/