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.