Neospora caninum is a tissue cyst-forming coccidium that may cause neuromuscular disorders in dogs. Infected bitches can transmit the parasite to their pups in utero. Vertical transmission may occur after primary infection during pregnancy and in subsequent pregnancies. The reason why only a few pups develop clinical neosporosis is unknown. We obtained sera from a Doberman bitch and its offspring delivered in three litters. The bitch had a titer of 1:640 in an indirect fluorescent antibody test (IFAT). At least three pups of litter A, one pup of litter B, and two pups of litter C were also seropositive for N. caninum. However, clinical neosporosis developed only in one pup of litter C, which had the highest IFAT titer (1:5,120) of all dogs examined. Western blots carried out after one-dimensional and two-dimensional separation of N. caninum tachyzoites revealed that the largest number of antigens was recognized by sera derived from the bitch. The lowest number of antigens was recognized by serum from the pup with clinical neosporosis. However, this pup uniquely recognized a major antigen with a molecular weight of about 17,000. The information collected in this study adds to our knowledge on why some pups develop clinical neosporosis and others do not.