3.2. Bacterial isolation and identification
In total, 341 infected fish collected from 26 tilapia farms (10–15 fish/farm) were subjected to bacterial isolation. A typical whitish pinpoint colony was predominantly recovered from the spleen, kidney, and liver of most diseased fish from each farm (Table 1). Twenty-six representative isolates (one per farm) were selected for further analysis. All isolates were gram-negative, rod-shaped, oxidase-negative, and catalase-positive. Other biochemical features were homogeneous among the 26 isolates and identical to the reference isolates of E. ictaluri from red tilapia (Dong et al., 2019) and striped catfish (Crumlish et al., 2002), except for Voges-Proskauer, which varied among isolates (Table 3). The specific PCR results showed that all 26 isolates were positive for both the Edwardsiella genus and E. ictaluri species, as evidenced by the presence of 848 bp and 470 bp amplicons, respectively (Figure S1).
Partial 16S rRNA and gyrB sequences (1500 bp and 1860 bp, respectively) were successfully amplified and sequenced from all nine representative isolates. Nucleotide sequences of these isolates were deposited in the GenBank database under the accession numbers MZ382896–MZ382904 for 16S rRNA and MZ576507–MZ576515 forgyrB . Nucleotide BLAST results revealed that all isolates showed 99.93%–100% and 99.41%–100% nucleotide identity with the respective 16S rRNA and gyrB sequences of the reference strains, E. ictaluri ATCC 33202 (NR024769), E. ictaluri2234 (MH540086.1), and less than 95% identity to other species in theEdwardsiella genus. Phylogenetic analysis of both 16S rRNAand gyrB gene sequences also demonstrated that nine isolates in this study were grouped in the same cluster as other E. ictaluriisolates and were separated from other species in the same genus (Figure 2).
Taken together, the results of biochemical tests, genus- and species-specific PCR, and sequencing of 16S rRNA and gyrBconfirmed that 26 bacterial isolates recovered from disease outbreaks in this study were E. ictaluri .