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

Abstract

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.

Intraperitoneal and intra-nasal vaccination of mice with three distinct recombinantNeospora caninumantigens results in differential effects with regard to protection against experimental challenge withNeospora caninumtachyzoites

“…In order to further study the effects of intra-nasal vaccination on parasite viability, we employed a recently developed NcGRA2-specific RT real-time PCR (Strohbusch et al 2008 a , b ), which allows quantification of the number of viable tachyzoites in brain tissue samples of the different experimental groups. The rational behind this assay is that NcGRA2-mRNA will only be detected when viable parasites are present, since mRNA is rapidly degraded upon cell death.…”

“…To quantify the viable tachyzoites in brain samples, NcGRA2-RT-PCR was performed using the corresponding other hemisphere from each mouse (Strohbusch et al 2008 a , b ). Then 200 μl of chloroform was added to all brain samples stored in QIAzol™, and the upper phase, obtained after vigorously vortexing and centrifuging the sample for 2 min at 10 000 g , was transferred to a new tube.…”

“…Finally, cDNA was boiled at 95°C for 3 min and frozen at −80°C prior to use. The Nc-GRA2-real time PCR was performed according to Strohbusch et al (2008 a , b ). The parasite counts were calculated by interpolation from a standard curve with cDNA equivalents from 1000, 100 and 10 parasites included in each run.…”

A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts

“…Similar assays, albeit by detecting other RNA targets, have been developed earlier. For instance, one assay based on measuring of mRNA expression levels of dense granule antigen 2, a protein that is expressed in both tachyzoites and bradyzoites of N. caninum , has been used to quantify the viability of this parasite within infected host cells, and within infected tissue of experimentally infected animals ( Strohbusch et al, 2008a,b; Debache et al, 2009; Lizundia et al, 2009 ). More recently, van den Bogaart et al reported on the development and application of a duplex quantitative reverse transcriptase PCR for simultaneous assessment of drug activity against Leishmania intracellular amastigotes and their host cells, by quantifying Leishmania 18S ribosomal RNA and the human beta-2-microglobulin mRNA ( van den Bogaart et al, 2013 ).…”

Nasal administration of recombinantNeospora caninumsecreting IL-15/IL-15Rα inhibits metastatic melanoma development in lung

“…31 While supporting tumor regression, the activation of the immune system also allows Neospora elimination. Although the latter is well described in the literature, 32 we aimed to assess if the presence of lung tumor could affect Neospora distribution after intranasal administration. No difference in distribution was found between tumor bearing and healthy mice (online supplemental table 3).…”