No 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.

Dendritic cells (DCs) represent the first line defence of the innate immune system following infection with pathogens. We exploratively addressed invasion and survival ability of Neospora caninum, a parasite causing abortion in cattle, in mouse bone marrow DCs (BMDCs), and respective cytokine expression patterns. Immature BMDCs were exposed to viable (untreated) and nonviable parasites that had been inactivated by different means. Invasion and/or internalization, as well as intracellular survival and proliferation of tachyzoites were determined by NcGRA2-RT-PCR and transmission electron microscopy (TEM). Cytokine expression was evaluated by reverse transcription (RT)-PCR and cytokine ELISA. Transmission electron microscopy of DCs stimulated with untreated viable parasites revealed that N. caninum was able to invade and proliferate within BMDCs. This was confirmed by NcGRA2-RT-PCR. On the other hand, no viable parasite organisms were revealed by TEM when exposing BMDCs to inactivated parasites (nonviability demonstrated by NcGRA2-RT-PCR). Cytokine expression analysis (as assessed by both RT-PCR and ELISA) demonstrated that both viable and nonviable parasites stimulated mBMDCs to express IL-12p40, IL-10 and TNF-alpha, whereas IL-4 RNA expression was not detected. Thus, exposure of mBMDCs to both viable and nonviable parasites results in the expression of cytokines that are relevant for a mixed Th1/Th2 immune response.

C57BL/6 mice were infected with Neospora caninum tachyzoites during pregnancy, yielding a transplacental infection of developing fetuses. Subsequently, congenitally infected newborn mice were treated either once or three times with toltrazuril (or placebo) at a concentration of 31.25 mg compound per kg body weight. Both toltrazuril and placebo treatment had no negative effect on newborns, as noninfected treated pups developed normally without differences in mortality and morbidity to matching nontreated control animals. Already one application of toltrazuril was significantly (p<0.01) able to delay the outbreak of neosporosis in newborn mice, when compared to placebotreated infected controls. We found significantly higher proportion of surviving newborns in one-time-toltrazuriltreated and three-time-toltrazuril-treated groups (34% and 54%, respectively) when compared to one-time-placebotreated and three-time-placebo-treated groups (14% and 30%, respectively). There was no significant difference (p= 0.2) in the proportion of surviving pups between one-timetoltrazuril and three-time-toltrazuril treatment. However, the number of diseased and Neospora-positive pups (46% and 47%, respectively) was markedly reduced after three-timetoltrazuril treatment compared to all other groups. Threetime-treatment also resulted in the highest antibody (IgG, IgG2a) response. Pharmacokinetic analyses using individual serum samples revealed that, although toltrazuril was absorbed and metabolized to toltrazuril sulfone by newborn mice, medicated animals exhibited an unexpected rapid turnover (half-life time) of the compound. Toltrazuril and the metabolite were also found in brain tissues, indicating that passage of the blood-brain barrier occurred. In conclusion, we could show that three times treatment with toltrazuril had a high impact on the course of infection in congenitally N. caninum-infected newborn mice.

Abstract:In the present work, we optimized a recently established NcGRA2-RT-PCR based on RNA to detect live Neospora caninum parasites in tissue, and compared the results with the conventional inoculation of diagnostic specimen onto cell culture. C57BL/6 mice were experimentally infected with Nc-1 tachyzoites and subsequently euthanized 6 or 12 days post infection (dpi). Selected organs were used to search for parasites by (i) PCR using genomic DNA (gDNA), (ii) PCR using cDNA and (iii) in vitro inoculation of cell culture. At 6 dpi, Neospora-gDNA was detected in 34 out of 36 organs. Viable parasites were detected in 11 (NcGRA2-RT-PCR) and 15 (in vitro cultivation) out of 36 organs. Comparison of NcGRA2-RT-PCR and in vitro detection gave a fair agreement (kappa 0.29), whereas comparison of PCR using gDNA and RT-PCR or in vitro detection resulted in a slight agreement (kappa 0.05 and 0.08, respectively) only. At 12 dpi, parasite gDNA was found in 10 out of 36 organs. In 7 of these organs viability of parasites was confirmed with NcGRA2-RT-PCR and growth of parasites in cell culture. Comparison of NcGRA2-RT-PCR and in vitro detection gave a substantial agreement (kappa 0.8), whereas comparison of PCR using gDNA and RT-PCR or in vitro detection resulted in a moderate agreement (kappa 0.59 and 0.77, respectively). As NcGRA2-RT-PCR is almost as sensitive as in vitro cultivation in detecting live parasites, it represents a fast, easy and safe method of viable parasite detection, and thus an attractive alternative to the in vitro cultivation approach.