2.3 Sampling
Captures and biological sample collection in the Pantanal occurred between June 2011 and January 2015 and included blood collection and ear tissue biopsy. Blood was collected through venipuncture of saphenous vein, using syringes and/or Vacuntainer® tubes with and without anticoagulant.
From blood samples collected in tubes containing EDTA, approximately 0.6–1.0 ml of blood was aseptically inoculated into two tubes containing Novy-Mc Neal-Nicole medium (NNN) with a liver infusion tryptose medium (LIT) overlay (hemoculture). Serum was obtained after blood centrifugation (10 min at 3,500 rpm) from tubes without anticoagulants and stored at -20C°. Ear tissue was biopsied (1.0 cm fragment) and immediately stored in plastic tubes containing 99% ethanol. Samples were stored in a portable cooler with ice and taken to a field laboratory, where they were preprocessed and stored, to be later sent to laboratory analyses.
In the Cerrado, biological samples were obtained from road-killed animals encountered between January and March of 2014, on three main state and federal highways (BR 262, 163 and 267). Highways were monitored every 15 days by driving a car slowly with two people looking for carcasses. For every animal encountered, a necropsy was performed, and biological samples were collected and associated with the GPS position of each of the road-killed animal.
Two tissue fragments of approximately 2 cm diameter from spleen, liver, lungs and one fragment (1 cm) of the ear were collected and stored in separated tubes with ethanol 99% and transported to the laboratories for molecular analysis. Only samples derived from carcasses there were not in apparent autolysis were collected.