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NcGRA2 as a molecular target to assess the parasiticidal activity of toltrazuril against Neospora caninum
M Strohbusch1, N Müller, A Hemphill
1Institute of Parasitology, University of Berne, Laenggass-Strasse 122, CH-3012 Berne, Switzerland.
Abstract:
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 microg/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.
Insights
A new molecular assay distinguishes live from dead Neospora caninum parasites by measuring specific mRNA levels. This quantitative real-time RT-PCR method aids in evaluating drug efficacy for treating bovine neosporosis.
Area of Science:
- Veterinary Parasitology
- Molecular Biology
- Drug Discovery
Background:
- Bovine neosporosis, caused by Neospora caninum, lacks effective treatments, with current drug efficacy assessments relying on conventional methods.
- Existing in vitro assays for Neospora caninum infection struggle to differentiate between live and dead parasites, hindering drug development.
Purpose of the Study:
- To develop and validate a novel molecular assay for distinguishing live from dead Neospora caninum parasites.
- To establish a quantitative real-time RT-PCR (qRT-PCR) assay for monitoring parasite viability.
- To differentiate between parasitostatic and parasiticidal drug effects.
Main Methods:
- Quantitative real-time RT-PCR (qRT-PCR) targeting the NcGra2 gene of Neospora caninum.
- Monitoring NcGra2 mRNA levels in infected cells treated with toltrazuril.
- Validation using infected cell cultures treated for 2-20 days with toltrazuril.
Main Results:
- The NcGRA2-RT-real time PCR assay successfully detected NcGra2 transcripts in live Neospora caninum parasites.
- A 10-day toltrazuril treatment showed parasitostatic activity (presence of NcGra2 transcripts).
- A 14-day toltrazuril treatment resulted in the absence of NcGra2 transcripts, indicating parasiticidal activity, confirmed by lack of proliferation after drug withdrawal.
Conclusions:
- A novel molecular assay using NcGra2 gene expression via qRT-PCR can accurately assess Neospora caninum viability.
- This assay effectively distinguishes between parasitostatic and parasiticidal drug effects, crucial for developing new treatments for bovine neosporosis.
- The developed assay holds significant potential for accelerating drug discovery and development for Neospora caninum infections.
