Identification and characterization of Neospora caninum tachyzoite antigens useful for diagnosis of neosporosis.

The purpose of the present study was to identify antigens of the protozoan Neospora caninum that could be useful for the diagnosis of neosporosis in domestic animals. As revealed by immunoblotting, immune sera from a wide range of animal species exhibited a similar recognition pattern of four major and several minor N. caninum antigens. In contrast to preinoculation sera, all tested immune sera recognized nonreduced immunodominant 17-, 29-, 30-, and 27-kDa antigens. A 46-kDa protein which showed faint recognition by preimmune sera also exhibited a strong response by immune sera. Immunolocalization of the four immunodominant N. caninum antigens was investigated by immunogold electron microscopy using monospecific polyclonal antisera. The 17-kDa antigen appears to be associated with the body part of the rhoptries, while the 29- and 30-kDa antigens were associated with the dense granules, network, and limiting membrane of the parasitophorous vacuole. Studies were also conducted to compare antibody responses to N. caninum and the related protozoan Toxoplasma gondii. Although N. caninum and T. gondii (RH strain) tachyzoites shared a few cross-reacting antigens, the immunodominant antigens of both parasites were not recognized by heterologous sera. Also, immunogold staining with rabbit anti-Neospora hyperimmune serum exhibited almost no labeling of external membranes of Neospora tachyzoites compared with the very marked labeling seen when Toxoplasma tachyzoites (RH strain) were incubated with rabbit anti-Toxoplasma hyperimmune serum. These unique antigenic differences should be useful in developing a diagnostic assay for N. caninum.