SEROLOGICAL DIAGNOSIS OF BOVINE NEOSPOROSIS BY NEOSPORA-CANINUM MONOCLONAL ANTIBODY-BASED COMPETITIVE-INHIBITION ENZYME-LINKED-IMMUNOSORBENT-ASSAY

Neospora caninum, a protozoan parasite closely related to Toxoplasma g ondii, causes abortion and congenital infection in cattle. To investig ate specific methods of antemortem diagnosis, the antibody responses o f infected cows were evaluated by immunoblot assay and competitive inh ibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monocl onal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A 4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unre active to antigens of two closely related apicomplexan protozoa, Toxop lasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induce d abortion revealed at minimum 14 major antigens ranging from 11 to 17 5 kDa. Although the recognized antigens varied from cow to cow, antige ns of 116, 65, and 25 kDa were detected in all cows with abortion conf irmed to he caused by N. caninum. The binding of MAb 4A4-2 to N. canin um tachyzoite antigen was consistently inhibited by sera from Neospora -infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experi mentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sa rcocystis hirsuta, which had cross-reactive antibodies recognizing mul tiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate ep itope on a single N. caninum tachyzoite surface antigen that is recogn ized consistently and specifically by Neospora-infected cattle.