2.1. IgY production, extraction and purification

Trypomastigotes forms of T. cruzi (strain Y) was used as antigen to immunize the chickens, according the method described by ^{Sampaio et al. (2014b)}, with some adaptations for T. cruzi, as follows below: cryopreserved trypomastigotes were inoculated into mice for reactivation and multiplication of strain, and blood samples were collected at the peak of parasitemia. In order to separate the parasites, blood was diluted in DMEM medium (1:1 v/v), followed by centrifugation (400 g for 10 min). The supernatant, rich of trypomastigotes, was collected and the amount of antigen was determined, aliquoted, and cryopreserved until use.

The chicken immunization protocol was as described by ^{Sampaio et al. (2014b)}. Briefly, a solution containing trypomastigotes of T. cruzi was added to incomplete Freund's adjuvant (Difco®) at a 1:1 ratio, with a final volume of 1.0 mL. It was injected intramuscularly at five different sites of the pectoral muscle of two 25-week-old New Hampshire chickens. The interval between immunizations was set as 10 days (days 0, 10, 20, 30, 40, 50, 70 and 80), totaling eight inoculations, being 2.4 × 10⁴ trypomastigotes for the first 4 inoculations and 7.2 × 10³ trypomastigotes of T. cruzi for the other 4 inoculations. Chicken eggs were collected from the third week post-immunization and stored at 4 °C until they were processed. One chicken was not immunized and its eggs were used as controls.

Extraction was performed by the method of precipitation of polyethylene glycol 6000 (PEG-6000) (^{Polson et al., 1980}). Firstly, the yolk was gently separated and its fat was removed using PBS (1:2 ratio) and PEG 6000 at a final concentration of 3.5%. This mixture was held in a roller mix for 30 min and then centrifuged for 80 min at 4.000 rpm and 4 °C. The supernatant was removed, filtered in filter paper (28 μm), and the final volume measured. PEG 6000 was once again added (8.5%), and the solution kept in a roller mix for 10 min, and centrifuged for 30 min at 4000 rpm and 4 °C. In this stage the supernatant was discarded and the pellet was dissolved in 10 mL of PBS. A final step using PEG 6000 (12%) was performed as described previously. The final pellet was dissolved in 2.4 mL of PBS, and this solution was dialyzed overnight in saline solution (0.1%) at 4 °C. A final dialyzation was performed for three more hours, the extract was collected, placed in tubes, stored at − 20 °C, and lyophilized until further use.

The specific concentration of IgY anti-T. cruzi was measured according to the Lambert-Beer's law using an extinction coefficient of 1.33 for IgY (^{Polson et al., 1980}). The protein content of the samples was measured by the method of ^{Bradford (1976)}, with Coomassie blue using bovine serum albumin as standard, and monitored by measuring the maximum absorbance of the solution at 595 nm.