Amino acid and protein depletion in medium of cell cultures infected with Cowdria ruminantium.

Cowdria ruminantium (Rickettsiales) causes heartwater in ruminants of Africa, and some islands off Africa and in the Caribbean Sea. The in vitro culture method for the organism devised in 1985, which provided for the first time a means for production of adequate quantities of live organisms and their products, is erratic and requires improvement. We studied depletion of amino acids (AAs) and major proteins in culture medium taken daily from infected and uninfected ovine and bovine vascular endothelial cell cultures. AAs of these samples were analyzed by Pico Tag reversed phase HPLC precolumn derivatization, and major proteins determined by capillary electrophoresis using a 57 cm x 75 microns fused silica tube at high pH. In both ovine and bovine cell cultures, significant depletion of arginine and glutamine occurred over a 5-day observation period regardless of whether they were infected or uninfected. This indicates that supplementation of nutrient media with these AAs might improve conditions for growth of the organism. Both AAs are essential for survival of cultured cells, and probably for the rickettsia (although the metabolism of C. ruminantium is poorly understood). Concentrations of several AAs increased in infected cultures, implying de novo synthesis and/or proteolysis on the part of the organism. In fact, several protein fractions did decrease in culture medium throughout the course of infection, while increasing or remaining unchanged in uninfected control cultures. Proteolytic activity by C. ruminantium may be essential for nitrogen metabolism by the organism. It is suggested that studies such as these will facilitate the development of a specific medium for optimal in vitro growth of the heartwater organism, and may also lead to an understanding of the metabolic stratagem of C. ruminantium. This knowledge, in turn, could reveal the mechanism for pathogenesis of heartwater, with implications for control.