These specimens were used for evaluation of the sensitivity and specificity of ITS1 TD PCR. From groups A and B, blood samples for PCR and HCT were collected after treatment with trypanocides, on 1, 2, 3, 4, 6, 10, 16, 23, 30 and 44 days post-treatment.

These specimens were used for evaluation as a “test of cure” of ITS1 TD PCR. Blood was drawn from the jugular vein into K2-EDTA vacutainer tubes. The blood was stored at −80 °C for subsequent DNA extraction. For parasite detection, blood was drawn into heparinised collection tubes, transferred to 6 heparin-containing capillary tubes and centrifuged for 6 min at 13,000 × g. The buffy coat was examined under a microscope for the Selleck BIBF-1120 presence of living trypanosomes according to Woo (1970). For assessing the specificity of the PCR primers, non-infected blood collected on heparin or on Na2-EDTA from bovine, goat, dog, horse, human and mouse was used. Total genomic DNA was extracted from 200 μl of blood using the High Pure PCR Template Purification Kit (Roche Applied Sciences) according to the manufacturer’s instructions, except that bound DNA was eluted with 60 μl elution buffer instead of 200 μl. Purified DNA was stored at −80 °C. Each round of DNA extraction included a negative control (PCR-grade water) and a positive

control (parasite DNA-spiked ZD1839 blood) alongside the bovine blood specimens. For determination of the analytical sensitivity, trypanosomes were grown in mice and parasites were counted in a Uriglass cell counting chamber. Since bovine blood was not readily available at the ITM in Antwerp, 10-fold serial dilutions of parasites were prepared

in 1 ml volumes of ice-cold freshly collected naïve human or mouse blood. Two hundred μl Parvulin of the thus prepared blood series were subjected to DNA purification as described above. For assessment of analytical specificity, trypanosomes were grown in mice. Trypanosomes were separated from the blood by anion exchange chromatography (Lanham and Godfrey, 1970) and subjected to DNA purification with the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s instructions. ITS1 primers for detection of the Trypanosoma genus were described in Claes et al. (2007). Primer sequences are: ITS1-Forward 5′-TGT AGG TGA ACC TGC AGC TGG ATC, ITS1-Reverse 5′-CCA AGT CAT CCA TCG CGA CAC GTT. PCR assays were performed in a Biometra T3000 cycler (Germany). Each reaction contained a final volume of 50 μl, including 5 μl of template DNA, 200 μM of each dNTP (Eurogentec), 0.2 μM of each primer (Biolegio), 1 unit of Hot Star Taq Plus DNA polymerase (Qiagen), 1× Coral Load PCR buffer, and 0.1 mg/ml acetylated bovine serum albumin (Promega).