Amino acid content of cell cultures infected with Cowdria ruminantium propagated in a protein-free medium.

The in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, was first achieved in 1985. Culture media were usually supplemented with serum and tryptose phosphate broth, both undefined components, contributing to great variability. Recently, we reported about the propagation of stocks of C. ruminantium in a protein-free culture medium referred to as SFMC-23, which is chemically fully defined. To clarify whether the amino acid composition in SFMC-23 is adequate for the in vitro propagation of Cowdria, the Welgevonden stock was propagated in SFMC-23 medium. After a 3-day culture period, samples were taken from uninfected and infected bovine endothelial cell cultures. They were analyzed for free amino acids by the Pico Taq reversed-phase HPLC precolumn derivatization method. Eighteen different amino acids were examined. A considerable decrease in concentration was observed with proline (29%) and glutamine (62%). Further dramatic changes were observed with amino acids which accumulated in the culture medium: aspartic acid, serine, asparagine, tryptophane, glycine, and alanine. The concentration of alanine increased by approximately 660%. The concentrations of all other amino acids analyzed remained within a 25% range, either increasing or decreasing. These results suggest that only glutamine may run short during in vitro cultivation. It seems more likely that accumulation of various amino acids may impact negatively on long-term Cowdria propagation.