3.5. Virulence test and histopathology
The experimental challenge using four multi-drug resistant E. ictaluri isolates, Ed.HB-02, Ed.HD-09, Ed.YB-08, and Ed.TB-07 (Table 1), resulted in LD50 values of 42, 54, 46, and 61 CFU/fish, respectively (Figure 4). Overall, the mortality rates were dose-dependent. The fish that received high doses (106–107 CFU/fish) showed 77%–97% mortality within 3 days and reached almost 100% at 5–6 days post infection (dpi). The fish that died on day 3 or earlier showed visceral decay, fluid accumulation in the fish abdomen, and unclear visceral white spots. After 3 dpi, all dead fish exhibited clear white spots in the viscera, similar to those of the naturally infected fish collected from ponds/cages (Figure 5). In the groups injected with lower doses (101–102 CFU/fish), white spots clearly appeared in the spleen, head kidney, and posterior kidney after 4–5 dpi and in the liver after 10 dpi. Noticeably, apart from some affected fish with darker color, most infected fish in the challenge test showed no obvious external clinical signs. Bacterial isolation from the internal organs of the infected fish resulted in dominant pinpoint colonies, which were identical to the colony morphology of E. ictaluri and tested positive by species-specific assay (data not shown). No bacteria were recovered from the clinically healthy fish in the control groups.
The histopathological manifestation of E. ictaluri -infected fish reached consensus at a similar challenge dose among the four bacterial isolates used. The lesions accurately reflected the gross features of the affected organs. Severe multifocal necrosis and pyogranulomas were observed in the spleen and kidneys (Figure 6 A, C). At high magnification, splenic focal necrosis surrounded by collagenous fibers, infiltration of inflammatory cells, the presence of basophilic rod-shaped bacterial clumps, and pyknosis and karyorrhexis was clearly observed (Figure 6B). Similarly, the kidneys of infected fish exhibited pyogranulomas, multifocal necrosis, and hyaline droplet accumulation in the kidney tubular epithelium (Figure 6D). The affected livers showed severe congestion, hepatic lipidosis, and tissue degeneration with an occasional presence of multifocal necrotic areas (Figure 6E-F). The brains of diseased fish also exhibited severe congestion and inflammation in the primitive meninges and periventricular gray zone of the optic tectum (Figure 6 G-H).