Hens (n = 4) were immunized by muscular route with emulsion composed of STAg and Freund's adjuvant, according to previously described protocol [46] . Primary immunization was performed with 100 mg of STAg in 250 mL of PBS and equal volume of Freund's complete adjuvant (Sigma). Two boosters were performed at 15 day intervals, with 100 mg of STAg plus Freund's incomplete adjuvant. In parallel, Neospora caninum (n = 4) and PBS (n = 2) -immunized hens were maintained for parasite-specific and Figure 1 . Purification of egg yolk antibodies. (A) Separation of the water soluble fraction (S1) from a lipid-rich precipitate (P1), after the incubation of crude egg yolk with acid water (pH 5.0-5.2); S1 precipitation by salting-out (19% Na 2 SO 4 ) produced an enriched IgY pellet (P2) and a supernatant with contaminants (S2). (B): Purity degree of IgY samples determined by SDS-PAGE (8%), demonstrating that salting-out protocol produced high purity IgY antibodies. (C) Size-exclusion chromatography of the P2 fraction, with peak of IgY between 13th and 19th fractions, where (D) the 14th fraction presented the highest degree of purity. (E): IgY enrichment conferred by Slot-blot assay, IgY was detected until the concentration of 0.01 mg of protein (box). Bovine sera albumin (BSA) was used as negative control. doi:10.1371/journal.pone.0040391.g001 irrelevant IgY purification, respectively. Hens were monitored daily for adverse effects and individual laid eggs were daily collected and stored at 4uC until further processing.