Heparinized blood diluted 1:5 in complete culture medium consisting of DMEM (Dulbecco's Modified Eagle Medium, Eurobio, France) supplemented with 5% fetal calf serum (Hyclone, Perbio, France), 2 mM L-glutamine, 100 U/mL penicillin and 50 mg/mL streptomycin (Eurobio, France) was stimulated with 10 mg/mL CONA (Sigma, France), or with 50 ng/mL of PMA (Sigma, France) and 1 mg/mL of ionomycin (Sigma, France) or 1 mg/mL LPS from Escherichia coli (Sigma, France). Cytokine content was measured in supernatants using ELISA tests as already described [41] . Briefly, purified fractions of anti-swine IL-2, IL-4, IFNc (clones A150D 3F1, A155B 16F2 and A151D 5B8 respectively, Biosource, France) and IL10 (clone 148801, R and D System, France) were used as capture antibodies, in conjunction with the biotinylated anti-swine IL-2, IL-4, IL10 and IFNc monoclonal antibodies (clones A150D 8H10, A155B 15C6 and A151D 13C5, respectively, Biosource, Clinisciences, France) or anti-swine polyclonal antibody (goat anti-porcine IL-10, R and D System, France). Streptavidin-horseradish peroxidase (Biosource) and TMB (Fermentas, MD, USA) were used for detection. Absorbance was read at 450 nm using an ELISA plate reader (Spectra thermo, Tecan, NC, USA) and the Biolise 2.0 data management software. Recombinant pig IL-2, IL-4, IL10 and IFNc were used as standards. The detection limits were 700 pg/ mL, 60 pg/mL, 90 pg/mL and 100 pg/mL for IL-2, IL-4, IL10 and IFNc, respectively. Results were expressed as pg of cytokine/ mL.