(L.) chagasi by indirect ELISA, according to Lima et al. (2003), and simultaneously positivity in rapid test rK39 and in PCR amplification of Leishmania spp. DNA in spleen tissue. A group of 6 healthy dogs, both males and females, from a nonendemic area (Londrina, State of Paraná, Brazil) were included in the study as negative controls. These dogs were serum negative for L. (L.) chagasi by indirect ELISA ( Lima et al., 2003), negative in the rapid test rK39 and in PCR amplification of Leishmania spp. DNA in spleen tissue. Samples of spleen from both groups were removed by surgical excision. The dogs were

premedicated with the combination of morphine (0.4 mg kg−1 IM) and acepromazine (0.05 mg kg−1 IM). Fifteen minutes later, propofol (4.0 mg kg−1 IV) and midazolam (0.1 mg kg−1 IV) were used for induction. Immediately, the dogs were positioned in dorsal recumbency and anesthesia was maintained Small Molecule Compound Library MLN2238 purchase with isoflurane (1.5 V%). The heart and respiratory rates, the systolic arterial blood pressure and end-tidal CO2 measurements were monitored during all anesthetic procedure. The samples were maintained

in RPMI 1640 supplemented with 10% (v/v) fetal calf serum (Sigma) at 4 °C and processed immediately to evaluate apoptosis. From each dog, 4 ml of blood was collected from the cephalic veins, clotted at room temperature for 4 h and subsequently centrifuged to extract the serum. The serum samples were stored at–20 °C prior to analysis and additional blood samples were collected with sodium EDTA and processed immediately to evaluate

apoptosis. To perform this test, blood samples were collected by venipuncture and centrifuged and the serum was separated. The procedure of the test was performed in accordance with the manufacturer’s recommendations. DNA obtained from spleen samples was extracted by freezing and thawing the cells 3 times and washing them in 1× SSC buffer solution (NaCl 3 M, sodium citrate 0.3 M, pH 7.0). For cell lysis and protein digestion, 300 μl of lysing solution was added (10% SDS in 0.2 M sodium acetate) together with 20 μg/ml proteinase K. Samples were incubated at 56 °C for 2 h and the DNA was extracted using the phenol/chloroform/isoamyl alcohol method (25:24:1), according to Sambrook whatever et al. (1989). After extraction, DNA was resuspended in 50 μl TE (10 mM Tris–HCl pH 8.0, 1 mM EDTA pH 8.0) and incubated for 3 min at 60 °C. The material was stored at −20 °C until used. The 13A (3′-GTG GGG GAG GGG CGT TCT-5′) and 13B (3′-ATT TTA CAC CAA CCC CCA GTT-5′) primers were used (Rodgers et al., 1990) to amplify a 120 bp fragment located in the constant region of the kinetoplast minicircles in all Leishmania species. The PCR was performed in a 60 μl volume containing 30 pmol of each primer (Invitrogen®), 0.2 mM DNTPs (Invitrogen®), 1.5 mM MgCl2 (Invitrogen®), 5 U Taq DNA Polymerase (Invitrogen®), 50 nM buffer solution, milliQ water and DNA.