Vertical transmission of Edwardsiella ictaluri in laboratory zebrafish revealed by highly-sensitive single molecule RNA in situ hybridization.

Edwardsiella ictaluri (E. ictaluri) is a significant bacterial pathogen responsible for severe losses in freshwater aquaculture and poses emerging concerns for laboratory zebrafish (Danio rerio) colonies. Although previous studies have suggested the potential for intergenerational or vertical transmission of E. ictaluri, direct experimental evidence supporting this hypothesis has remained lacking. In this study, we developed a hybridization chain reaction-enhanced single-molecule fluorescence in situ hybridization (HCR-smFISH) assay that enables highly sensitive, single-cell- and single-molecule-level detection of E. ictaluri within host tissues. Using an established zebrafish infection model, we characterized the tissue tropism and spatial distribution of the pathogen in key immune and metabolic organs, including the spleen, gills, and liver. Notably, whole-embryo imaging provided direct evidence of maternally mediated transmission, demonstrating that bacterial signal adhere to the chorion surface of eggs. Subsequent disinfection trials demonstrated that NaClO surface sterilization effectively eliminated egg-associated bacteria and alleviated infection-induced inflammatory responses in the progeny. Collectively, these findings fill a critical knowledge gap regarding the vertical transmission of E. ictaluri and offer practical guidance for disease prevention, broodstock management, and the establishment of specific pathogen-free zebrafish lines in research and aquaculture settings.