Temporal changes in viability and infectivity of aerobically stored Ascaridia galli eggs: Experimental and epidemiological implications

The infectivity of Ascaridia galli eggs is central to experimental parasitology and understanding parasite transmission in poultry. While embryonation and larval motility are commonly used indicators of egg viability, their relationship with actual infectivity is poorly defined, particularly as eggs age during laboratory storage. This study evaluated temporal changes in the viability and infectivity of A. galli eggs aerobically stored at 25 °C for 3, 6, 12, 18, 24, 30 and 36 weeks from time of collection. Viability was assessed using morphological integrity, in ovo larval motility, and hatch-staining assays, while infectivity was determined through experimental infections in day-old chickens (n = 24 per egg storage period (SP)). For all SP groups, infective doses were based on the number of morphologically viable eggs or motile larvae administered (300 eggs/bird). Across all in vitro measures, eggs maintained high viability for up to 12 weeks (78-91%), then progressively declined to 19% by 36 weeks, with an average loss of about 2% per week. Infection outcomes closely mirrored in vitro viability: total worm counts per bird decreased from 9 to 10 worms for 3-12-week-old eggs to 2 worms for 36-week-old eggs. Correspondingly, infectivity fell from 3.04 to 3.35% in younger eggs (3-12 week) to 0.68% in the oldest eggs. By 8 weeks post-infection, adult worms accounted for 90-95% of populations from young-egg infections, compared with only 48-52% in infections from older eggs, which contained up to 51% immature worms. Adult worm size and reproductive potential also declined with egg age, and sex ratios became slightly male-biased in older cultures. Empirically derived conversion factors (CF_v) provided a quantitative link between in vitro viability and infectivity within the viable egg subpopulation, while adjusted CFₜ extended this estimate to the total egg population, with linear interpolation enabling estimation for SP-specific infectivity. These findings underscore that not all viable-looking eggs are infective and highlight a defined period of optimal infectivity for experimental use. Overall, the study informs best practices for aerobic storage of A. galli eggs in the laboratory and provides a framework for applying in vitro viability data to experimental infections and modelling transmission risk in poultry systems.