Figure 2

Cytokine levels in lung lavage fluid and Chlamydia psittaci-specific proliferative responses of peripheral blood mononuclear cells (PBMCs). Groups of chickens were immunized and boosted IN or IM as described in the materials and methods section. (A) Two weeks after the booster immunization, the amount of IFN-γ, IL-2, IL-12 and IL-10 cytokines contained in lung lavage fluids from chickens immunized IN or IM (6/group) was measured using single cytokine assay kits. The concentration of the cytokines in each sample was obtained by extrapolation from a standard calibration curve generated simultaneously. Each cytokine ELISA assay was performed twice with similar results. The data shown is one of those two assays and the bars represent the mean cytokine concentrations (± S.D.) in samples from six chickens in each group. (B) PBMCs obtained from six chickens/group by Lymphoprep density gradient centrifugation 2 weeks after the booster immunization were restimulated in vitro with 10⁶ IFU of UV-irradiated C. psittaci EBs (C. psittaci antigen) for 24 h at 37℃ in 5% CO₂. Antigen-specific proliferative responses were determined using the 5-Bromo-2’-deoxy-uridine (BrdU) cell proliferation assay kit (Abcam, Beijing, China) as described in the Materials and Methods section. BrdU incorporation was detected by addition of anti-BrdU antibody and the absorbance was read at 450 nm. Results are expressed as the stimulation index (SI), the ratio between absorbance values of stimulated and non-stimulated cells. The bars represent the mean SI values (± S.D.) of samples from six chickens in each group and the data shown is one of two independent lymphoproliferative assays with similar results. Significant differences between groups were evaluated by One-way ANOVA with Tukey’s post multiple comparison test at p* < 0.05, p* < 0.01 and p*** < 0.001).