Pattern of recognition of Besnoitia besnoiti tachyzoite and bradyzoite antigens by naturally infected cattle.

Bovine besnoitiosis is caused by the protozoan parasite Besnoitia besnoiti. Many recent cases have been described in different European countries, which may be indicative of expansion of the disease in the next few years. Many infected animals remain asymptomatic; therefore, serological tests are essential tools for diagnosis. The objective of the present work was to identify B. besnoiti tachyzoite and bradyzoite immunodominant antigens (IDAs). IDAs were recognised by SDS-PAGE under reducing conditions and Western blot analysis. Positive sera from symptomatic (n=18) and asymptomatic (n=18) cattle came from herds endemically infected by B. besnoiti and were confirmed positive by IFAT, whereas negative sera (n=4) came from besnoitiosis-free herds and were also confirmed negative by IFAT. Up to 28 tachyzoite antigens in the range of 8.5-190.8 kDa were recognised. Based on the frequency of recognition, six IDAs (14.2, 33, 37.1, 39.6, 46.3 and 190.8 kDa) were identified. The 37.1 kDa antigen was recognised by 100% of sera, usually as an intense band. On the other hand, 30 bradyzoite antigens in the range of 8.5-187.9kDa were detected. Seven bradyzoite IDAs (8.5, 15.1, 16.8, 19.0, 34.7, 38.6 and 124.4 kDa) were identified and two of them (15.1 and 16.8 kDa) were considered the most immunogenic ones. Additionally, sera from animals with clinical symptoms recognised a significantly higher number of bradyzoite antigens. Finally, significant cross-reactions with other closely related apicomplexan parasites were not detected. This is the first description of B. besnoiti bradyzoite antigens. In addition, the identification of tachyzoite and bradyzoite IDAs may be useful for the development of vaccines and diagnostic tools for differentiating between acute and chronic infections. Further proteomic studies are needed in order to identify stage-specific proteins.