Anti-N. caninum IgG response in serum. IgG response in intravenously challenged animals (A) and in IV and SC challenged animals with 104 Nc-Spain7 tachyzoites (B). Each point represents the mean + SD at the different sampling times for each group. Data beyond day 28 pi are not included because several animals did not maintain pregnancy and were therefore sacrificed. Serum levels of total IgG antibodies against N. caninum are expressed as a relative index percent (RIPC), according to the formula: RIPC = (OD405 sample − OD405 negative control)/(OD405 positive control − OD405 negative control) × 100. The humoral immune response shown in figure was analysed using two-way ANOVA of repeated measures. For significant differences between infected groups, (*) indicates P < 0.05 and (****) indicates P < 0.0001.