Novel tools for the diagnosis and differentiation of acute and chronic bovine besnoitiosis.

Diagnosis of acute bovine besnoitiosis is a major diagnostic problem. We developed diagnostic tests to serologically diagnose and differentiate acute and chronic cases of bovine besnoitiosis using affinity purified antigens of Besnoitia besnoiti tachyzoites in immunoblots and in both, a conventional ELISA and an avidity ELISA. Sera of acutely and chronically infected cattle were investigated using these tests. Acutely infected cattle initially recognised an antigen of 74 kDa relative molecular mass, followed by reactions with increasing intensity against 81 and 28 kDa antigens. In addition, faint reactions against antigens with 36, 37, 39 and 42 kDa molecular mass started soon after seroconversion and increased over time. An antigen of 45 kDa molecular mass was transiently recognised early after infection but not or only weakly in the chronic stage. At least two antigens, the 39 and the 42 kDa antigens, seem to be located on the surface of B. besnoiti tachyzoites as determined by biotinylation. Affinity purified antigen was used to establish an APure-BbELISA which showed excellent sensitivity (100%) relative to a serological reference system in naturally, most likely chronically, infected cattle. Specificity was also high (99.8%) as determined in cattle from herds with Neospora caninum-associated abortions. The antibody levels in APure-BbELISA were correlated with the parasite load in the skin or the mucous membrane of the vestibulum vaginae as determined by real-time PCR. In acute cases of bovine besnoitiosis (confirmed by the detection of low avidity IgG in the APure-BbELISA) first specific antibodies were detected by ELISA in all animals except one, at the same time or earlier than in the serological reference system. The detection of parasite DNA in skin by real-time PCR was clearly superior to serological analysis in detecting infected cattle during acute besnoitiosis.