Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4 ؉ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4؉ -and CD8 ؉ -T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-␥) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Neospora caninum is an intracellular apicomplexan protozoan parasite that causes abortion and congenital infection in cattle (21). Transplacental parasite transmission is the major mode of infection (18, 45) which persists in the herd over successive generations, causing significant economic losses due to abortion, culling, and decreased milk production (13,20,25,52,53). Recrudescence of chronic, congenitally acquired infection due to downregulation of Th1 immune responses during pregnancy may be largely responsible for transplacental N. caninum transmission (10,22,27,42). A vaccine that could control tachyzoite parasitemia and decrease congenital infection or abortion rates is needed.Studies of Toxoplasma gondii, a protozoan parasite closely related to N. caninum in biology, phylogeny, and immunogenicity, unequivocally show the importance of T lymphocytes in resistance to infection by adoptive transfer, gene knockout, and depletion studies with mouse models (19). The presence of parasite-specific CD4 ϩ and CD8 ϩ cytotoxic T lymphocytes (CTL) is also associated with immunity to natural T. gondii infection in humans (14,41). In murine models of neosporosis, a T-lymphocyte-mediated immune response, characterized by increased levels of gamma interferon (IFN-␥) and interleukin 12 (IL-12), correlates with disease resistance (5,30,33), and IFN-␥ alone inhibits tachyzoite growth and cellular invasion (24,38,51,55). In pregnant mice, protection against vertical transmission correlates with induction of parasite-specific Tlymphocyte-mediated immunity (39). Cattle infected with N. caninum produce parasite-specific, major histocompatibility complex (MHC)-restricted, CD4 ϩ CTL (50) that could reduce N. caninum infection indirectly by IFN-␥ activation of mononuclear phagocytes and directly by killing N. caninum-infected cells.To identify vaccine candidate parasite antigens for induction of CTL and IFN-␥ secretion in cattle against neosporosis abortion, this study focused on major tachyzoite glycosylphosphatidylinositol-anchored surface proteins, N. caninum surface antigen 1 (NcSAG1) and N. caninum SAG1-related sequence 2 (NcSRS2) (26, 28). The T. gondii surface pr...