Fig. 3

IFN-γ production in stimulated splenocyte supernatants and IFN-γ and IL-2 expression in stimulated splenocytes. Chickens were vaccinated in ovo on ED18 and received a second vaccination 14 days ph. Vaccines consisted of 15 µg of BPL inactivated H9N2 virus administered alone (BPL), with AddaVax™ (BPL + Add), or with 2 µg CpG ODN 2007 (BPL + CpG). One group received just PBS as a negative control (PBS). Five spleens from 5 birds per group were collected 10 days post secondary vaccination. Splenocytes were isolated and seeded in 48 well plates and stimulated with 1 μg/ml of BPL inactivated H9N2 virus. Supernatants were collected a 48 and 72 h after stimulation and supernatant IFN-γ concentrations were quantified. IFN-γ expression in splenocytes was quantified at b 12 h and c 24 h post-stimulation and IL-2 expression was quantified at d 12 h and e 24 h post-stimulation. Gene expression was quantified relative to β-actin expression. Group means that share the same letter did not differ significantly. Standard error of the mean is indicated with error bars. Data was analyzed using Duncan’s Multiple Range Test and differences in means were considered significant if p < 0.05