Transfection efficiency of three target siRNAs for ChTLR15. A To ensure the transfection efficiency of three target siRNAs for ChTLR15, the concentrations of terminal fluorescently labeled siRNA (FAM Negative Control) and Lipofectamine 2000 were optimized according to the manufacturer’s instructions. The final concentration of 50 nM siRNA and 5 μL of Lipofectamine 2000 were chosen. The transfection effects were observed by fluorescence microscopy (left, 10 × 40) and light microscopy (right, 10 × 40). B Three ChTLR15 siRNAs (siChTLR15#1, siChTLR15#2 and siChTLR15#3) were transfected into DF1 monolayer cells (1.5 × 106 cells per well) in 6-well plates. At 24 h post-transfection, DF1 cells in each well were stimulated with purified E. tenella sporozoites (4 × 105 sporozoites per well) for 2 h. DF1 cells transfected with negative control (NC) siRNA and stimulated with (NC + stim) or without (NC + Unstim) purified sporozoites were designed as controls. The interference efficiency of three ChTLR15 siRNAs was compared by quantifying the mRNA level of ChTLR15. siChTLR15#1 showed the highest interference efficiency and was chosen for subsequent experiments. Highly significant differences (p < 0.01) between numbers with different capital letters. Significant differences (p < 0.05) between numbers with different lowercase letters. No significant difference (p > 0.05) between numbers with the same letter.