Fig. 5

Neutralizing assay of the immunized sera. Serum samples were heat inactivated at 56 °C for 30 min, followed by serial dilution in 2-fold increments in Dulbecco’s modified Eagle medium (DMEM) supplemented with 2% of fetal bovine serum (FBS), and mixed 1:1 with 100 TCID₅₀ of PCV2 strain CC1 for 2 h at 37 °C. Thereafter, 100 μL of serum-virus mixture was added to 50% confluency of PK-15 cells and incubated for 1 h at 37 °C. After incubation, the serum-virus mixture was removed, and the cells were washed with PBS for three times and further incubated in DMEM supplemented with 2% of FBS at 37 °C and 5% CO₂. After incubation for 72 h, an indirect fluorescence assay (IFA) was performed to assess the neutralizing activity of the immunized sera. The results represent the means ± S.D. of three replicates of each group. P-values were calculated using two-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001