F. noatunensis subsp. orientalis uptake by zebrafish leukocytes in vitro. (A) Representative dot plot before (Pre-sort) and after (Post-sort) cell sorting of leukocytes harvested from adult zebrafish kidney incubated for 1 h with Fno-FITC. Bacterial uptake was quantified by flow cytometry as the percentage of cells with increased FITC fluorescence. (B to C′) Ultrastructural TEM analysis of zebrafish kidney leukocytes with internalized F. noatunensis subsp. orientalis (Fno) after 1 h of incubation. In macrophages (B and C), bacteria reside mostly in tight phagosomes (B′) and only rarely in larger phagolysosomes (C′) with additional luminal cargo. Black arrowheads indicate phagosomal/phagolysosomal membrane. Bars, 2 μm (B to C) and 500 nm (B′ to C′).

Cumulative survival and immune response of adult zebrafish immunized with MVs and infected with F. noatunensis subsp. orientalis. (A) Kaplan-Meier representation of cumulative survival of adult zebrafish vaccinated with 20 μg MVs and infected with 10⁶ CFU F. noatunensis subsp. orientalis (P value, 0.001 [log rank test]). (B) Histological examination by hematoxylin-eosin (HE) staining of spleen from unvaccinated challenged fish 7 dpc (panel a) and MV-vaccinated fish challenged with F. noatunensis subsp. orientalis at 7 dpc (panel b). Arrowheads indicate granulomatous structures. Bars, 150 μm (magnification, ×10). (C and D) Transcriptional response of selected cellular markers of kidney from MV-vaccinated fish before challenge (C) and after challenge (D) as measured by RT-qPCR. Bars represent means ± standard deviations (SD) of relative expression levels compared to those for the control (PBS injected). Relative expression levels were normalized to the expression of eef1a1l1. Asterisks indicate significant upregulation (****, P < 0.0001; ***, P < 0.001).