Bacterial ghost cells as tools for the stimulation of PRR-mediated immune-transcription in zebrafish larvae

Bacterial derivatives and mimetics are receiving attention as an emerging immunostimulant in animal health, aquaculture, poultry, and immunotherapy due to their ability to enhance innate immune responses in a non-specific manner. Bacterial ghosts (BG) possess an inherent repertoire of surface-associated antigens and pathogen-associated molecular patterns (PAMPs) that confer strong immunostimulatory potential. To determine the immunomodulatory and developmental effect of bacterial ghost cells (GCs) derived from a Gram-positive bacterium, Staphylococcus aureus, prepared using Lugol’s iodine-based protocol, Danio rerio (zebrafish) embryos were exposed to different concentrations of GCs for 6h, 12 h and 24 h. Time- and dose-dependent changes in zebrafish embryo morphology were assessed microscopically, while the immunostimulatory potential of GCs was evaluated through gene expression analysis using qRT-PCR. Overall, the results demonstrated that even at the highest concentration and longest exposure duration, GCs did not induce harmful effects or developmental abnormalities in the larvae. Gene expression analysis confirmed the immunostimulatory activity of GCs with upregulation of major innate immune signalling genes such as tlr2, nod2, nfkb and cytokines such as tnfa and il1b . These findings highlight the importance of small animal models such as zebrafish for understanding the in vivo behaviour of GCs, where complex interactions among multiple cell types contribute to the systemic response following treatment.