, 2012) and toxoplasmosis in sheep and humans (Hide et al., 2009). Despite the efforts of previous studies to confirm this transmission route in horses (Duarte et al., 2004 and Locatelli-Dittrich et al., 2006), many points are still unclear, including the relationship between the level of antibodies in mares and the frequency of vertical transmission of

these agents in the Sarcocystidae family. Therefore, the aim of study was to correlation levels of antibodies in mares with pre colostral foals seropositive and assess the level and distribution of antibodies against Neospora spp., S. neurona and T. gondii, in mares and pre colostral foals an the parturition We obtained 181 samples from mares, without clinical history of neurological and reproductive Neratinib manufacturer diseases, and their newborns, in Rio

Grande do Sul, Brazil. The blood was drawn by jugular puncture from mares during parturition and from their newborns before colostrum intake. The whole blood was centrifuged at 250 × g for 10 min to separate serum, which was stored at −20 °C until tested. This research was licensed by the Ethics and Animal Experimentation Federal University of Santa Maria, with number 81/2009. Neospora caninum (NC-1 strain) and Erastin molecular weight S. neurona (SN-37R) tachyzoites were maintained under the same conditions by the continuous passage of HeLa cells and CV-1 cells, respectively, at 37 °C and 5% CO2 in RPMI medium supplemented with 25 mM HEPES, 2 mM of l-glutamine, 3 mM sodium bicarbonate and antibiotic/antimycotic solution (penicillin 100 IU/mL, streptomycin 100 μg/mL and amphotericin B 0.25 g/mL; Gibco). T. gondii (RH) tachyzoites were maintained in BALB/c mice by serial passage for 48–72 h ( Mineo et al., 1980). This maintained licensed by the Ethics and Animal Experimentation Federal University of Uberlandia, with number 029/2012. A parasite suspension was washed two times (720 × g, 10 min, 4 °C) in phosphate-buffered saline 0.01 M (PBS, pH 7.2), treated with protease inhibitors (Complete, Roche) and then subjected to ten freeze–thaw cycles and sonication Isotretinoin (60 Hz,

90% amplitude, in ice bath). After centrifugation (10,000 × g, 30 min, 4 °C), the supernatant was collected and filtered through 0.22 μm membrane (Millex, Millipore, USA). The supernatant, containing soluble antigens of N. caninum (NLA), S. neurona (SnLA) or T. gondii (STAg), was collected and the protein concentration was estimated using the Bradford assay. Aliquots were stored at −20 °C until use. Indirect ELISAs were carried out to detect IgG antibodies as described elsewhere Silva et al. (2007), with modifications. In summary, high-binding microtiter plates were coated with NLA, SnLA or STAg (10 μg/ml) in 0.06 M carbonate buffer (pH 9.6) overnight at 4 °C. The plates were then washed three times with PBS containing 0.