Ehrlichia Culture from Blood Samples

Blood samples were processed for isolation of Ehrlichia in cell culture. To this end, the blood was aseptically collected in EDTA vacuum tubes and transported to the laboratory for mononuclear cells (MNCs) isolation, using Histopaque 1083 (Sigma-Aldrich, St. Luis, MO, USA), as previously described [19 (link)]. Cultures were initiated by seeding the MNCs in a 25 cm² culture flask containing Dulbecco’s Modified Eagle’s medium (DMEM, Sigma-Aldrich, St Louis, MO, USA) and supplemented with 20% iron-fortified Bovine Calf Serum (BCS, Sigma-Aldrich, St Louis, MO, USA). The culture flask was kept at 37 °C and 5% CO₂. Every two days, 1/5 of the primary culture medium was collected for cytologic evaluation, using Romanowsky-stained smears (NewProv, Pinhais, PR, Brazil), and fresh medium was added. After 96 hours (h) of incubation, DH82 cells were added to the primary culture of MNCs, and the supplementation of DMEM was reduced to 5% iron-fortified BCS. The culture was kept at 37 °C and 5% CO₂. Every seven days, samples were collected for cytologic evaluation and PCR. Stocks of infected DH82 cells were resuspended in cell-freezing medium (Sigma-Aldrich, St Louis, MO, USA) and frozen at −156 °C in liquid nitrogen.