NcGRA2 as a molecular target to assess the parasiticidal activity of toltrazuril against Neospora caninum

• Published in: Parasitology Vol. 135; no. 9; pp. 1065 - 1073
• Main Authors: STROHBUSCH, M., MÜLLER, N., HEMPHILL, A., GREIF, G., GOTTSTEIN, B.
• Format: Journal Article
• Language: English
• Published: Cambridge, UK Cambridge University Press 01.08.2008
• Subjects: Animals; Biological and medical sciences; Cattle; Cattle Diseases - drug therapy; Cattle Diseases - parasitology; cell-culture assay; Coccidiosis - drug therapy; Coccidiostats - pharmacology; Coccidiostats - therapeutic use; Fundamental and applied biological sciences. Psychology; General aspects; General aspects and techniques. Study of several systematic groups. Models; Invertebrates; Neospora - drug effects; Neospora caninum; Parasites; Polymerase Chain Reaction; Protozoan Proteins - genetics; Protozoan Proteins - isolation & purification; RT-PCR; toltrazuril; Triazines - pharmacology; Triazines - therapeutic use; cell-culture assay; toltrazuril; RT-PCR; Neospora caninum; Polymerase chain reaction; Protozoa; Cell culture; Apicomplexa; Parasite; Activity; Toltrazuril
• ISSN: 0031-1820; 1469-8161
• DOI: 10.1017/S0031182008004599

The treatment of Neospora caninum infection in the bovine host is still at an experimental stage. In contrast to the in vivo situation, 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. The NcGra2 gene of N. 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 system in vitro was achieved using Neospora-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 of NcGRA2-transcripts, whereas after a 14-day treatment period no NcGRA2-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 against N. caninum, with a view to distinguishing between parasiticidal and parasitostatic efficacy of given compounds.