OprI is an outer membrane lipoprotein from Pseudomonas aeruginosa, and when fused to a recombinant antigen, will exert adjuvant properties by engaging Toll-like receptor 2, leading to dendritic cell activation. Previous studies have shown that the Neospora caninum (Nc) antigens NcPDI, NcROP2 and NcROP40 are implicated in host cell interactions and are promising vaccine candidates. In two independent experiments, the efficacy of a polyvalent vaccine formulation composed of OprI-NcPDI, OprI-NcROP2 and OprI-NcROP40 (collectively named O-Ags) was assessed in non-pregnant and pregnant Balb/c mouse models challenged with tachyzoites of the high-virulence isolate Nc-Spain7. Parameters that were investigated were clinical signs, fertility, parasite burden in adult mice, humoral and cellular immune responses at different time-points prior to and after challenge infection, vertical transmission and post-natal survival of offspring mice, all to explore potential correlations with efficacy. Vaccination of mice with O-Ags induced a mixed Th1/Th2 immune response in adult mice and led to significantly increased protection against cerebral infection. Vaccination with O-Ags also resulted in reduced vertical transmission, and postnatal disease in offspring was significantly inhibited at a rate not observed in mice infected with a high-virulence isolate to date. However, O-Ags mixed with TLR ligands targeting TLR3 and TLR7, which are known to induce clear Th1-biased responses, or vaccination with OprI fused to the non-N. caninum antigen ovalbumin (OprI-OVA) did not confer protection.