Since cattle are its principal intermediate host ( Dubey et al., 1996), the parasite causes important economic losses in the cattle industry ( Anderson et al., 2000). Serological methods are commonly used in epidemiological

studies of N. caninum in animals. Methods include the indirect fluorescent antibody test (IFAT) ( Dubey et al., 1988), direct agglutination, Western blotting, and enzyme-linked immunosorbent assays (ELISAs) ( Dubey and Schares, 2006). In IFAT, which is considered the reference technique for N. caninum serology ( Dubey and Schares, 2006), intact tachyzoites are used as antigens. The test detects antibodies directed against antigens present on the cell surface of the parasite ( Bjorkman and Uggla, 1999), although reactivity with other coccidian parasites might be possible i.e. Toxoplasma CH5424802 in vivo gondii ( Dubey et al., 2003). Also, the assay involves subjectiveness in the scoring of results ( Pare et al., 1995), is labor-intensive, and does not lend itself

to large-scale investigations. ELISAs, in contrast, are feasible at larger scales and do not involve subjective interpretation. Single antigens, particularly the parasite’s surface proteins, as an alternative for tachyzoite extracts, Bcl-2 inhibitor are suitable candidates for the development of more specifics tests. A number of dense-granule antigens of N. caninum have been identified to date, including NcGRA1, NcGRA2, NcGRA6, NcGRA7 (NCDG1/Nc-p33), NcPI-S, and NTPase ( Howe and Sibley, 1999 and Morris et al., 2004), some of which have been tested for neosporosis diagnosis. NcGRA2, NcGRA6, and NcGRA7 have been prepared as recombinant antigens to be used in ELISA for diagnosing neosporosis in cattle and canine population

( Lally et al., 1996 and Liu et al., 2007). The surface protein NcSRS2 is common to both tachyzoite and bradyzoite stages and is of potential use for the serological during diagnosis of N. caninum infection ( Ahn et al., 2003, Gaturaga et al., 2005, Ghalmi et al., 2009, Hemphill and Gottstein, 1996 and Nishikawa et al., 2001). The available ELISAs lack in validation data, and some of these tests are only partially published ( Dubey and Schares, 2006). The purpose of the present study was to standardize an indirect ELISA which uses the C-terminal domain of protein NcSRS2 to diagnose N. caninum in cattle, and compare its performance with the IFAT. N. caninum, strain NC-1 ( Dubey et al., 1988) was used to prepare an antigen formulation for IFAT. The parasites were propagated in Vero cells maintained in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), at 37 °C with 5% CO2. When 80% of the Vero cells that had been infected with N. caninum tachyzoites shows CPE (citopatic effect), the cell monolayers were removed by scraping, twice washed with phosphate-buffered saline (PBS) solution, and then centrifuged at 1000 × g for 10 min.