Characterization of Neospora caninum microneme protein 10 (NcMIC10) and its potential use as a diagnostic marker for neosporosis.

Improvements in the serological diagnosis of neosporosis are needed to differentiate acute versus chronic Neospora caninum infections. In the present study, N. caninum microneme protein 10 (NcMIC10), similar to other microneme proteins, was shown to be released in a calcium-dependent manner. NcMIC10 may be discharged during active invasion of host cells by the parasite, and thus represent an excellent marker for the diagnosis of neosporosis. In order to test this hypothesis, recombinant NcMIC10 (rNcMIC10) was expressed in Escherichia coli, and polyclonal antibodies were generated against non-overlapping fragments of the protein. A capture ELISA was developed using these antibodies, and was found to be highly accurate and reproducible with a detection range of 10-10,000 pg/ml. The anti-rNcMIC10 antibodies used in this study did not cross-react with the Toxoplasma gondii antigens. NcMIC10 was detected by the ELISA in sera of 9 out of 10 goats (90%) experimentally infected with N. caninum tachyzoites. In general, goats infected with a lower dose (10(4)) of the parasite displayed a peak in NcMIC10 levels between weeks 4 and 5 post infection. Goats infected with a higher parasite dose (10(6)) displayed a more rapid increase in NcMIC10 levels. In most animals, NcMIC10 decreased to undetectable levels by week 6 post infection. This is the first circulating Neospora antigen-based assay which may complement the existing antibody-based assays for a rapid and cost-effective definitive diagnosis of neosporosis in livestock.