BACKGROUND: Neospora caninum is an intracellular parasitic protozoon that can infect pregnant animals and cause symptoms such as miscarriage, stillbirth and mummified fetuses. It is one of the main causes of miscarriage in bovines. Apical membrane antigen (AMA) and dense granule protein (GRA) are two major antigenic proteins of N. caninum. METHODS: In this study, NcGRA9 recombinant subunit vaccine and Ad5-NcAMA1 recombinant adenovirus vaccine were prepared and used to immunize C57BL/6 mice and Yanbian yellow cattle. RESULTS: IgG, IgG1, IgG2a, IgG2b, IgA and IgE antibodies and interferon (IFN-γ), interleukin (IL-4) and tumor necrosis factor (TNF-α) cytokines were significantly higher in immunized mice than in the control group (P <
0.0001). The biochemical indexes showed that vaccination had no effect on hepatic and renal functions. The survival rate was 70% in mice immunized with NcGRA9 vaccine, 75% in mice immunized with Ad5-NcAMA1 vaccine, 85% in the combined immunization group and 10% in the control group. The parasite load in the brain and liver tissues of the immunized groups was significantly lower than in the control group, as detected by fluorescence quantitative PCR (P <
0.0001). In cattle, IgG, IgG2a, IgG2b, IgA, IgM and IgE antibodies and IFN-γ, IL-4 and TNF-α cytokines were significantly higher in the immunized groups than in the control group (P <
0.0001). Combined immunization with NcGRA9 + Ad5-NcAMA1 was significantly better than immunization with either vaccine alone. CONCLUSIONS: The biochemical indexes showed that the vaccine had no effect on the liver and kidney functions of cattle. Our results indicate that combined immunization with NcGRA9 + Ad5-NcAMA1 may be a candidate for bovine neosporosis vaccination.