Serodiagnosis of canine and bovine neosporosis using a recombinant truncated NCDG1 protein in an enzyme-linked immunosorbent assay

In the search for antigens with serodiagnostic potentials, the Neospora caninum immunodominant dense granule antigen, NCDG1 homologue was obtained after immunoscreening a N. caninum tachyzoite cDNA library. The hydrophobic signal peptide of this gene was truncated and expressed in E. coli as a soluble fusion protein tagged with glutathione sepharose transferase (GST). With mouse anti-NCDG1t antibodies, NCDG1 was localized as granular, punctuate structures in the anterior and posterior portions of the N. caninum tachyzoites with an expected 33kda molecular weight. The recombinant antigen (GST-NCDG1t) retained its immunoreactivity in immune sera from N. caninum experimentally-infected dogs, and showed no cross reactivity with Toxoplasma gondii and other dog-related parasites such as Babesia gibsoni, Babesia canis canis, Babesia canis rossi, Babesia canis vogeli and Leishemania infantum. For large-scale screening of canine and bovine populations, the NCDG1t was evaluated for its diagnostic potential using enzyme linked immunosorbent assay (ELISA). The NCDG1t-ELISA clearly distinguished between N. caninum-positive and negative control dog and cow serum samples. Using NCDG1t-ELISA, 33 (23%) canine and 40 (40%) bovine and 9 (9.6%) canine and 9 (8.24%) bovine serum samples from selected areas in Japan and China tested positive, respectively. Test specificity for canine assay was demonstrated when NCDG1t-ELISA showed no reactivity with antibodies from dog sera experimentally infected with other parasites. In addition, 39 ELISA positive dog serum samples from Japan and China were confirmed positive by Western blot analysis while three samples were shown to be negative, showing 93% agreement between the two methods. For the bovine assay, a gold standard panel of bovine sera for ELISA was established to assess its diagnostic performance in terms of sensitivity and specificity. Although the specificity of NCDG1t-ELISA with respect to Western blot defined negative reference cow sera was high (97.6%), false-negatives were observed. But with a calculated sensitivity of 84.4%, NCDG1t-ELISA proved to be relatively reliable for the diagnosis of bovine neosporosis. However, the combined application of two recombinant antigens (e.g. NCDG1 and Nc SAG1) in the same immunoassay, is worth considering to further improve the sensitivity of NCDG1t-ELISA for diagnosis of bovine neosporosis. To date, this is the first N. caninum dense granule antigen-based ELISA for the diagnosis of canine neosporosis. Furthermore, this is the first reported survey of canine neosporosis in Nanjing and Yanbian provinces in China; and constitutes the second report in Japan using IFAT