the Related Cyst-Forming Apicomplexans Toxoplasma gondii and

We report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulentNeospora caninum iso-lates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH andME49), all grown in human fore-skin fibroblasts. In these parasites, BKI-1294 acted with 50 % inhibitory concentrations (IC50s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninumNc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondiiME49 andN. caninumNc-Liv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Un-like in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhib-ited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experi-mentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compoun