Onderstepoort Journal of Veterinary Research, 69:147-153 (2002) The Kumm isolate of Ehrlichia ruminantium: In vitro isolation, propagation and characterization

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the KOmm isolate that resisted all attempts at in vitro culture until the successful exper-iment described here. In one experiment white blood cells were harvested from heparinized blood derived f(Om a sheep infected with the KOmm isolate. The cells were added to DH 82 cells and incu-bated at 37 °C. The high metabolic activity of the DH 82 cells necessitated that cell growth be re-tarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment another sheep was infected, using a higher dose of the same batch of KOmm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E2). Ten days after culture initiation only the E2 cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (KOmm-1) was less virulent for mice than that of the sec-ond cultured isolate (KOmm-2) which killed all mice. Upon molecular characterization with E. rumi