Corrected optical density readings were calculated by subtracting the plate background reading. Antibodies specific for M. haemolytica were measured at week 2, 6, 10 and 12 with an in-house ELISA at the laboratory of MSD Animal

Health (Boxmeer, Netherlands). Microtitre plates were coated with M. haemolytica antigen. Serum was diluted and incubated and bound antibodies were detected after incubation with an anti-bovine serum-peroxidase conjugate as previously described ( Assié et al., 2009). At weeks 8 and 12, blood samples from a subset of calves (six randomly selected animals from each group) were collected into heparinized tubes. Peripheral blood mononuclear cells (PBMC) were isolated by centrifuging over Histopaque (Sigma) and recovered in RPMI 1640 medium (supplemented with 10% heat-inactivated foetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine). Viable cells were counted by Trypan blue exclusion buy Trametinib and resuspended at a concentration of 1 × 106 cells/ml. One millilitre of this suspension was added in duplicate to a 24-well plate. Cells were stimulated with F. hepatica excretory-secretory antigen (ES; 10 μg/ml), lipopolysaccharide (LPS; Escherichia coli 0111:B4,

Selleck FK228 Alexis; 5 μg/ml) as a bacterial antigen and toll-like receptor ligand 7/8 (TLRL; Resiquimod R848, Invivogen; 1 mg/ml) as a viral antigen. Medium and Con A (Sigma; 5 μg/ml) were used as negative and positive controls, respectively. Cell cultures were incubated for 72 h at 37 °C before supernatants were collected and stored at −20 °C for cytokine analysis. Cytokine levels were measured using commercially available

ELISA kits, or antibody pairs. Interleukin 4 (IL-4) was measured using a kit from Pierce Biotechnology in accordance with the manufacturer’s guidelines. Transforming growth factor beta (TGF-β) was measured using a human ELISA (Promega) as previously described ( Abbott et al., 2005). Nitric oxide (NO) was measured using the Griess Reagent Kit (Promega). Interleukin 10 (IL-10) and Interferon gamma (IFN-γ) were measured using commercially available isothipendyl antibody pairs as previously described ( Flynn and Mulcahy, 2008). Regression procedures were employed to comparatively assess the effect of liver fluke infection status on each of the vaccination, biochemical and cytokine variables, over a 12-week period. Prior to the analysis, the stability of the variance for each parameter was assessed, and where normality was not identified, a transformation step was applied (log transformed for PI-3 and BRSV SNT as well as GLDH and square root transformed for GGT variables). In the case of IFN-γ and BRSV, where the variance for these dependent variables could not be stabilised, a non-parametric quartile (median 50) regression model was applied. In addition, a pre-infection comparison of all variables was carried out using an independent student t-test to ensure a group effect was not present prior to commencement of the trial.