Ticks and saliva collection

Rhipicephalus microplus ticks (Porto Alegre strain, free from Babesia spp. and Anaplasma spp.) were obtained from a laboratory colony maintained as previously described⁵⁷. Hereford calves were acquired from a tick-free area and infested with 10-day-old R. microplus larvae. Fully engorged female ticks were obtained after the spontaneous detachment from the calves. Ticks were rinsed with sterile distilled water, and salivation was induced by dorsal injection of 5 µL of 2% pilocarpine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in ethanol (Merck KGaA, Darmstadt, Germany)^58,59. Rm-saliva was collected using a pipette tip from ticks maintained at 28 °C in a humid chamber for approximately 3 h. Collected Rm-saliva was stored at − 80 °C upon use in the experiments. Animals used in these experiments were housed in Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Brazil. Experiments were conducted considering ethical and methodological guidelines in agreement with the Norms by the Animal Experimentation Ethics Committee of the UFRGS. The protocols were approved by the Comissão de Ética no Uso de Animais (CEUA)-UFRGS. These experiments were carried out in compliance with the ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357).