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 .