Abstract

This study aimed to validate the colony-count assay as a quantitative in vitro alternative to the traditional in vivo challenge method for evaluating live bacterial poultry vaccines. Live E. coli, Salmonella enteritidis, and Salmonella typhimurium vaccines were assessed for performance characteristics, estimation of measurement uncertainty, and vial-to-vial homogeneity. The assay was conducted on thirteen vials from each vaccine batch; three vials were used for determining repeatability, reproducibility, and measurement uncertainty (MU), while ten vials were used for homogeneity evaluation. The colony-count procedure demonstrated high precision with within-analyst standard deviation ranging from 0.012-0.026 log10 CFU and between-analyst standard deviation ranging from 0.0111- 0.0606 log10 CFU and relative expanded uncertainty of approximately 0.96%. One-way ANOVA and F-tests revealed no significant differences among vials for any vaccine confirming excellent batch homogeneity. These findings indicate that the colony-count method provides accurate, precise, and reproducible quantification of viable organisms in live bacterial poultry vaccines supporting its use as a scientifically robust and ethically sound in vitro alternative to animal-based challenge testing for vaccine potency assessment.

Key words: Vaccines, Colony Count, Performance Characteristics, Measurement Uncertainty, Homogeneity