2.1. Disease outbreaks, sampling, and bacterial isolation
After the first report of E. ictaluri infection in red tilapia (Oreochromis sp.) in a northern province of Vietnam (Dong et al., 2019), similar disease outbreaks with massive mortalities continually occurred in various tilapia farms in nine provinces in northern Vietnam between February 2019 and March 2021. Epidemiological investigations and fish sampling were conducted at 26 affected farms (Table 1), including 5 earth-pond farms and 21 cage-culture farms located in 9 provinces in northern Vietnam (Figure 1).
During the study, information on the source of fish stocking (imported or domestic) and the estimation of fish mortality caused by E. ictaluri was obtained by interviewing the farmers. In each affected farm, the water temperature was measured at the time of fish sampling using a water quality meter (YSI Professional Plus, YSI Incorporated, Ohio, USA). Ten to fifteen diseased fish were collected, placed in sterile sealed plastic bags, and transferred to the laboratory of the Department of Aquatic Environment and Fish Pathology, Faculty of Fisheries, Vietnam National University of Agriculture (VNUA) in a cold box (below 4 °C) for microbial analysis. The clinical signs and gross features of all the diseased fish were observed and recorded.
Bacteria were isolated from the head kidney, spleen, and liver of the affected fish using brain heart infusion agar (BHIA). The plates were then incubated at 28 °C for 48 h. Twenty-six representative isolates corresponding to 26 disease outbreaks were selected and preserved in brain heart infusion broth (BHIB) containing 20% glycerol and kept at –80 °C for further examination.