Uptake of Ascaridia galli extracellular vesicles by chicken immune cells varies accordingtowormsexandinvitrocultureduration

Ascaridia galli (A. galli), a parasitic roundworm that infects chickens poses an economic burden in poultry
farming, as it causes ascaridiosis—a disease leading to reduced growth, lower egg production, and
immunosuppression. Recently, interest has grown in the parasite’s extracellular vesicles (EVs), as they
modulate host immune responses and play a key role in host-pathogen interactions. This study aimed
to optimize in vitro EV-production from A. galli and assess their uptake by chicken immune cells important for EV mediated host-pathogen communication. Adult worms were collected from infected chickens,
sex-sorted, washed, and cultured in vitro. EVs were isolated at various time points using size exclusion
chromatography and characterized. DiO-stained EVs were evaluated for uptake into chicken intestinal
epithelial cells, macrophages, peripheral blood mononuclear cells and whole blood leukocytes using flow
cytometry and confocal microscopy after 4 and 24 h incubation with the parasite derived vesicles. EVuptake increased significantly from 4 h to 24 h across all tested cell types. Female-derived EVs collected
after 24 h of worm culture gave rise to higher uptake than male-derived EVs. However, at the 40 h time
point, male EVs gave rise to greater uptake, though overall EV internalization was reduced compared to
the 24 h time point. Uptake efficiency varied depending on the EV collection time as well as the host cell
type. These findings suggest that both the sex of the worm and the duration of culture influence EV
uptake, with 24 h emerging as the optimal in vitro culture duration for production of A. galli derived
EVs with potent biological functions. The sex-specific differences highlight potential functional diversity
in EV mediated host-pathogen interactions, which need to be assessed in future studies.