Serum-free media for the in vitro cultivation of Cowdria ruminantium

The in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, was first achieved in 1985; since then, most groups working with this culture system have used media which were supplemented with serum and, in addition, most of them contained tryptose phosphate broth. These undefined products vary from batch to batch and often fail to support the growth of C. ruminantium. We are therefore working towards the development of a completely chemically defined medium for Cowdria culture. We attempted the propagation of the Welgevonden stock of C. ruminantium in bovine endothelial cell cultures in a variety of serum-free culture media. Four synthetic media gave unsatisfactory results, these were: SFRE-199, Iscove's modified Dulbecco's medium, Dulbecco's modified Eagle's medium, and Leibovitz L-15. These media were all supplemented with a proprietary solution A (components solution A of the HL-1 medium kit, containing transferrin, testosterone, sodium selenite, ethanolamine, saturated and unsaturated fatty acids, and stabilizing proteins). Three other serum-free media did support the growth of C. ruminantium: a modified HL-1 medium, Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12), and RPMI 1640. The chemical composition of DME/F-12 and RPMI 1640 are published, but not that of the HL-1 medium. Each of these media was supplemented with proprietary solution A. Various supplements were investigated as alternative to the incompletely specified solution A; bovine lipoproteins and bovine transferrin were identified as essential supplements which effectively replaced compound solution A. C. ruminantium was propagated in the three growth-supportive media for at least 10 passages.