IgY was purified by the water-dilution method as previously described [19] . In order to obtain a representative sampling of antibody production along the weeks after immunization, eggs laid weekly from each hen were pooled prior to IgY extraction. Then, egg white was removed and egg yolk was diluted 10-fold in deionized ultrapure water adjusted to pH 5.0-5.2 with 1N HCl and homogenized thoroughly. After centrifugation at 10.0006g for 25 min at 4uC, the supernatant was collected, consisting of lipid-free fraction (S1). S1 was then precipitated with the addition of 19% sodium sulphate (w/v). After centrifugation (10.0006g, 25 min, 4uC), the pellet was retrieved and represented the IgYenriched fraction (P2). P2 samples were resuspended and dialyzed against PBS to eliminate residual salt. Additionally, P2 samples were submitted to exclusion-size gel chromatography using Sephacryl S-300 column (GE Healthcare, Uppsala, Sweden), at a flow rate of 3 mL/min, and the IgY-enriched protein fraction was determined by 280 nm readings. Actual protein concentration was measured by BCA kit (Sigma) and samples were stored at 220uC until use. This protocol was also applied to extract IgY antibodies from the eggs of hens immunized in house with N. caninum soluble antigen, as well as from commercially available egg yolk powder containing anti-Eimeria spp.(E. acervulia, E. maxima e E. tenella) IgY antibodies (Supracox, Investigación Aplicada, Sociedad Anónima de Capital Variable, Puebla, Mexico). Possible cross- Figure 3 . Immunohistochemistry for T. gondii detection using polyclonal IgY anti-STAg antibodies. Paraffin-embedded brain sections of mice chronically infected with ME-49 strain were incubated with IgY anti-STAg and rabbit IgG anti-IgY conjugated to fluorescein isothiocyanate (FITC). (A and B) , segmented parasitophorus vacuoles were detected into host cell cytoplasm around the DAPI-stained nucleus (blue). (C and D), IgY anti-STAg antibodies strongly recognized antigens on outer walls of tissue cysts and free tachyzoites (C, arrow). doi:10.1371/journal.pone.0040391.g003 reactivity of the purified IgY antibodies to mouse proteins were ruled out by immunoblots (Fig. S2) .