5% (148/155) of animals were females and 71.6% (111/155)

were over one year old. Serum samples were stored at −20 °C until being tested for the presence of antibodies against T. gondii and N. caninum. All procedures were performed according to the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation and this study received approval from the Ethical Committee of the Institution. T. gondii tachyzoites (RH strain) were maintained by intraperitoneal serial passages in outbred Swiss mice at regular 48 h intervals ( Mineo et al., 1980). Mouse peritoneal exudates were harvested when the majority of tachyzoites were extracellular, and then washed twice (720 × g, 10 min, 4 °C) in phosphate-buffered saline (PBS, pH 7.2). The resulting pellet was resuspended see more in PBS for antigen preparation. N. caninum tachyzoites (Nc-1 strain) were maintained in Vero cells cultured in RPMI 1640 medium supplemented with 2% heat-inactivated calf fetal Neratinib supplier serum in a 5% CO2 atmosphere at 37 °C and harvested by scraping off the cell monolayer after 2–3 days of infection ( Silva et al., 2007). Tachyzoites were purified

by forcible extrusion through a 26-gauge needle to lyse any remaining intact host cells, which were also removed by centrifugation at low speed (45 × g) for 1 min at 4 °C. The supernatant containing parasite suspension was collected and then washed twice (720 × g, 10 min, 4 °C) in PBS and the resulting pellet was resuspended in PBS for antigen preparation. N. caninum and T. gondii whole antigens were prepared according to Camargo (1964). Parasite suspensions were treated with 1% formaldehyde for 30 min at room temperature. After washing in PBS, parasites were dry-fixed in microscopic slides and stored at −20 °C until being used in IFAT. N. caninum and T. gondii soluble antigens were prepared as described elsewhere ( Silva et al., 2007). Parasite suspensions

were treated with protease inhibitors (aprotinin, 10 μg/mL; leupeptin, 50 μg/mL; phenyl-methylsulfonyl fluoride [PMSF], 1.6 mM) and then lysed by five freeze–thaw (liquid nitrogen else and water bath at 37 °C) cycles and further by ultrasound (six 60 Hz cycles for 1 min each) on ice. After centrifugation (10,000 × g, 30 min, 4 °C), supernatants were collected and the protein concentration was determined ( Lowry et al., 1951). Different batches were done for antigen preparation and pooled together to obtain the required protein concentration. Soluble antigen aliquots were stored at −20 °C until being used in ELISA. IFATs were carried out to detect IgG antibodies to T. gondii (IFAT-Tg) and N. caninum (IFAT-Nc) as described elsewhere ( Figliuolo et al., 2004). Slides containing formolyzed tachyzoites were incubated with sheep sera at serial twofold dilutions starting from 1:64 to 1:8192 (for IFAT-Tg) or 1:50 to 1:3200 (for IFAT-Nc) in PBS.