Isolation and Propagation of ERGardel Virulent Strain

Infected blood from ERG-infected goats was loaded on finite cultures of bovine aortic endothelial cells (BAE) to isolate the ERGardel virulent strain (ERGvir). ERGvir was then routinely propagated in BAE as described elsewhere [36 (link),37 (link)]; ERGvir samples from passages up to passage 44 were used throughout this study, as they have been proven to be highly infectious in previous studies [38 (link)]. In order to obtain an attenuated ERG strain, ERGvir was cultivated over 230 passages in BAE cells, as suggested by Martinez (1987)[14 ]. When 80% cell lysis was observed (at 120hpi for ERGvir and 96hpi for ERGatt, S1 Fig), supernatant and cellular debris containing infectious ER elementary bodies were harvested and then used to (i) infect a freshly confluent monolayer or (ii) to be purified using a multistep centrifugation methodology [39 (link)]. Purified ERs were stored in SPG [39 (link)] at −80°C with a “Complete EDTA-free” anti-protease cocktail (Roche, Germany) prior to proteomic analysis. Prior to in vivo assays, purified ER were stored in SPG in liquid nitrogen [38 (link)].
Growth of ERGvir and ERGatt was monitored by phase-contrast light microscopy and quantified as previously described [17 (link)].