Purification and storage of Ehrlichia ruminantium

Ehrlichia ruminantium Gardel virulent strain (passages 17, 19, and 24) and ERGatt (passages 240, 242, and 244) variants were grown in bovine aortic endothelial cells (BAECs), as described elsewhere (Marcelino et al., 2015 (link)). When 80% cell lysis was observed, infectious and extracellular EBs were harvested and then used to (i) infect a new confluent monolayer of BAEC or (ii) to be purified using a multistep centrifugation methodology (Peixoto et al., 2007 (link)). Purified EBs were stored at -80°C with a “Complete EDTA-free” anti-protease cocktail (Roche, Germany) and anti-phosphatases (Roche, Germany) prior to proteomic analysis.