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

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 realtime 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 mg/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.

NcGRA2-RT-PCR to Detect Live Versus Dead Parasites in Neospora caninum-Infected Mice

“…Nevertheless, distinction between live and dead organisms is an important tool, for example, in testing chemotherapeutical efficacy or in assessing infectiosity of carrier animals or organs. Recently, a RT-PCR based on the NcGRA2 gene was established in view to distinguish between parasiticidal and parasitostatic efficacy of given compounds in cell-culture-based assays [8].…”

“…The use of immunocompromised animals to detect viable parasites is the most sensitive technique so far. However, as detection of viable parasites in tissue from infected animals by NcGRA2-RT-PCR was tested for the first time, we used in vitro cultivation -in reference to [8] -to obtain information about sensitivity and handling procedure of the new technique. In a further future step, NcGRA2-RT-PCR will be compared with inoculation of diagnostic materials (obtained from naturally infected bovines) into immunocompromised mice.…”

“…Detection of parasite-specific cDNA by NcGRA2-RT-PCR was done as recently described [8] with N. caninumspecific primers NcGRA2-F1 and NcGRA2-R2. PCR was performed using 25 pmol of each primer and 1 l cDNA in a final volume of 25 l.…”

“…Recently, we described a novel PCR that was established as a useful tool to distinguish between live and dead parasites in cell culture following a chemotherapeutic impairment of parasite viability [8]. NcGRA2-RT-PCR was specific for N. caninum and sensitive enough to detect 0.1 parasites equivalents per reaction [8].…”

“…NcGRA2-RT-PCR was specific for N. caninum and sensitive enough to detect 0.1 parasites equivalents per reaction [8]. In the present work, we compared the NcGRA2-RT-PCR for detection of live parasites ex vivo in organs from experimentally infected animals with the conventional approach to inoculate diagnostic material into appropriate cell culture subsequently maintained for 4 weeks.…”