Time course, dose–response curves for Neospora caninum cytotoxicity. N. caninum caused cytotoxicity to endothelial cells in a dose-dependent manner. Cells were cultured in 96-well plate and were infected with N. caninum at different MOIs as described in methods. The supernatants were collected at the indicated time points, and were assayed for the cytosolic enzyme lactate dehydrogenase released by cells, using the CytoTox96 nonradioactive cytotoxicity assay. Data are expressed as mean ± SEM of three independent experiments performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 versus the control group, respectively.