Comparative Toll-like receptor profiling reveals breed-specific innate immune architecture following Newcastle disease vaccination.

The present study investigated the expression profiles of six TLR genes (TLR2A, TLR3, TLR4, TLR5, TLR7 and TLR21) in peripheral blood mononuclear cells (PBMCs) following genotype II ND vaccination across fifteen genetically diverse chicken populations representing exotic, indigenous, fancy, synthetic and broiler parent lines. Relative gene expression was quantified by qPCR, and receptor expression data were integrated into five novel composite immune indices, namely the Viral Recognition Index (VRI), Bacterial Recognition Index (BRI), Immune Diversity Score (IDS), Shannon TLR Diversity Index (STDI), and Immune Breadth Index (IBI). Principal component analysis (PCA), hierarchical clustering and correlation analyses were further employed to characterize breed-specific innate immune architecture. Significant breed-dependent variation (P < 0.001) was observed for all six TLRs, demonstrating substantial genetic diversity in early pathogen recognition. Indigenous breeds, particularly Aseel Red and Kadaknath, exhibited broader activation of bacterial and viral recognition receptors, whereas White Frizzle showed the highest TLR2A expression and Frizzle displayed comparatively greater TLR5 and TLR7 expression among the fancy breeds. Synthetic populations exhibited intermediate but line-specific immune activation, while the broiler parent line (BCSFL) consistently demonstrated comparatively lower expression across most TLRs. PCA demonstrated pronounced breed-specific clustering of innate immune responses, with PC1 alone explaining 55.9% of the total variance, suggesting that coordinated TLR expression constituted the major source of immunological variation among chicken breeds. While correlation and hierarchical clustering analyses identified coordinated activation of bacterial (TLR2A, TLR4 and TLR5) and viral (TLR3, TLR7 and TLR21) sensing pathways in indigenous populations, whereas the Shannon TLR Diversity Index remained relatively conserved across most breeds. ND vaccine-induced TLR mediated immunity is characterized by functional specialization rather than hierarchical superiority, with distinct chicken genetic groups exhibiting differential investment in pathogen recognition pathways. The composite immune indices proposed herein provide a quantitative systems-level framework for evaluating innate immune architecture and represent promising hypothesis-generating biomarkers for comparative immunogenetics, precision breeding, and vaccine evaluation in poultry, warranting further validation through independent populations and virulent NDV challenge studies.