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 phenol/chloroform according to method of (Neumann, Pospiech, & Schairer, 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, 1 mL cell suspension was centrifuged at 8.000 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 minutes. 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 minutes 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 minutes at 13.000 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 minutes to digest RNA. Then 100 µl chloroform was added to the tube, mixed well and centrifuged for 5 minutes at 13.000 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 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 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 dNTP each, 0.3 units Taq DNA polymerase, Promega, USA), and sterile distilled water (total volume of 50 µL). PCR amplification was performed in MJ MiniTM 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 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).
Table 1 Sequence of primers
Genes
|
Primer names
|
Nucleotide sequences 5’→3’
|
Size (bp)
|
References
|
toxR
|
toxR-F
|
GTCTTCTGACGCAATCGTTG
|
367
|
Luan et al., 2007; Marlina et al., 2007
|
toxR-R
|
ATACGAGTGGTTGCTGTCATG
|
tdh
|
tdh-F
|
GTAAAGGTCTCTGACTTTTGGAC
|
500
|
Luan et al., 2007; Marlina et al., 2007
|
tdh-R
|
TGGAATAGAACCTTCATCTTCACC
|
trh
|
trh-F
|
TTGGCTTCGATATTTTCAGTATCT
|
269
|
Luan et al., 2007; Marlina et al., 2007
|
trh-R
|
CATAACAAACATATGCCCATTTCC
|
tlh
|
tlh-F
|
AAAGCGGATTATGCAGAAGCACTG
|
450
|
Luan et al., 2007; Marlina et al., 2007
|
tlh-R
|
GCTACTTTCTAGCATTTTCTCTGC
|
tdh=Thermostable direct hemolysin, trh=TDH-related hemolysin, tlh=Thermolabile hemolysin, toxR=Toxin operon (Luan et al., 2007), (Marlina et al., 2007).
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, Gibson, Plewniak, Jeanmougin, & Higgins, 1997) and edited by using BioEdit 7.0.5 software (Hall, 1999). 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). The DNA polymorphism analysis was 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) considered as a polymorphic measurement in the population (J. Rozas & R. Rozas, 2005). Neutrality was tested based on three methods, Tajima’s D test (Tajima, 1989), Fu and Li’s D * and F* test (Fu & Li, 1993) and Fu's (Fu, 1995) using DNASP 6.0 software. Phylogenetic tree showing genetic relationship was 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 was shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) were 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 (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 (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).