NcGRA2as a molecular target to assess the parasiticidal activity of toltrazuril againstNeospora caninum

Abstract

SUMMARYThe treatment ofNeospora caninuminfection in the bovine host is still at an experimental stage. In contrast to thein vivosituation, a wide range of compounds have been intensively investigated in cell-culture-based assays. Tools to demonstrate efficacy of treatment have remained conventional including morphological and cell biological criteria. In this work, we present a molecular assay that allows the distinction between live and dead parasites. Live parasites can be detected by measuring the mRNA level of specific genes, making use of the specific mRNA available in live cells. TheNcGra2gene ofN. caninum, which is known to be expressed in both tachyzoites and bradyzoites, was used to establish a quantitative real-time RT-PCR, for monitoring parasite viability. Validation of the systemin vitrowas achieved usingNeospora-infected cells that had been treated for 2–20 days with 30 μg/ml toltrazuril.NcGRA2-RT-real time PCR demonstrated that a 10-day toltrazuril-treatment exerted parasitostatic activity, as assessed by the presence ofNcGRA2-transcripts, whereas after a 14-day treatment period noNcGRA2-transcripts were detected, showing that the parasites were no longer viable. Concurrently, extended culture for a period of 4 weeks in the absence of the drug following the 14-day toltrazuril treatment did not lead to further parasite proliferation, confirming the parasiticidal effect of the treatment. This assay has the potential to be widely used in the development of novel drugs againstN. caninum, with a view to distinguishing between parasiticidal and parasitostatic efficacy of given compounds.