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).