Validation of a Pre-sentient Chicken Embryo Model for Infection-driven Inflammation and Pharmacological Screenings

The chicken embryo (CE) model provides a physiologically relevant and ethically optimized system for studying infection-driven inflammation while reducing reliance on post-hatch animals. Building on a previous late-stage CE inflammation model using Salmonella Pullorum (SP), we refined the approach by inoculating embryos at 7 or 9.5 days of development (DED) via the allantoic cavity and assessing early inflammatory responses. Embryo mortality, renal histopathology, and acute-phase proteins were evaluated, revealing renal lesions and elevated SAA levels in the allantoic fluid, whereas other tissues exhibited limited alterations. Embryos inoculated at 9.5 DED were further analyzed at 11.5 and 17 DED, representing pre- and post-sentience developmental stages, respectively, using flow cytometry and cytokine gene expression analysis. Immune profiling demonstrated age-dependent modulation of lymphocyte populations, including decreased CD8⁺ T cells at 11.5 DED and increased TCRγδ⁺ cells at 17 DED, alongside developmental changes in TGF and IL-8 transcription. Pharmacological interventions with norfloxacin and indomethacin produced mechanistically distinct effects: antimicrobial treatment attenuated renal and inflammatory markers, whereas anti-inflammatory intervention alone exacerbated kidney and heart lesions. Our results define a biologically relevant developmental window in the CE in which infection with SP elicits measurable inflammatory responses while remaining prior to stages associated with sentience. This framework supports the use of the CE as a practical and ethically advantageous platform for early-stage studies of infection, inflammation, and exploratory pharmacological screening, contributing to research strategies aligned with the principles of the 3Rs.