2.2. Colony maintenance and feeding of the experimental groups

Ticks were kept in an incubator at 28 ± 2 °C and 85 ± 5% relative humidity. All feedings were performed in Swiss mice using feeding chambers described by Bouchard and Wikel (2005). During all procedures, mice were maintained in appropriate cages (30 × 19 × 13 cm L x W x H, maximum 5 animals/cage) and kept in a room with controlled temperature (25 ± 2 °C).

To obtain females at different physiological stages, feeding chambers were assembled into seven groups containing six mice each (females aged 6–8 week old) which were used as feeding source for the ticks (one couple with 20–30 days of fasting per mouse). The ticks were examined for attachment (6 h after being placed in contact with the host) and every 2 days when females were removed, weighed and dissected to obtain the salivary gland extract (henceforth called SGE) which was stored at −80 °C. Groups contained ticks with 2, 4, 6, 8 and 10 days of feeding, in addition to the fasting group and one with ticks that spontaneously detached from the host.

All procedures involving animals were in accordance and approved by the Ethics Committee on Animal Experimentation (CETEA/UFMG) under the protocol number 137/2011.