2.4. 16S rRNA and gyrB amplification, sequencing, and phylogenetic analysis
Nine representative isolates (Ed.HB-02, Ed.TQ-06, Ed.HD-09, Ed.TB-07, Ed.YB-08, Ed.BN-04, Ed.HY-06, Ed.SL-07, and Ed.HNa-02) originating from nine different provinces were selected for further genetic analysis. The16S rRNA and gyrB genes were amplified using the universal bacterial primer (27F/1525R, ~1500 bp) (Weisburg et al., 1991) and gyrB primer (1245F/1949R, 1860 bp) (Griffin et al., 2014); and the purified PCR products were sequenced (Macrogen, Seoul, Korea). Bacterial species identification was performed using a Basic Local Alignment Search Tool (BLAST) nucleotide search on the GenBank database. The nucleotide sequences of 16S rRNA and gyrBgenes of representative E. ictaluri isolates in this study and closely related sequences retrieved from GenBank were aligned using ClustalW (Thompson et al., 1994). Phylogenetic trees were then constructed using the neighbor-joining method with a bootstrap of 1000 replicates (Saitou & Nei, 1987) performed by MEGA 10 software (Kumar et al., 2018).