Substrate utilization by Ehrlichia sennetsu and Ehrlichia risticii separated from host constituents by renografin gradient centrifugation

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Abstract
The in vitro metabolic activities of two monocytic species of Ehrlichia were investigated. The Miyayama strain of Ehrlichia sennetsu and two strains of Ehrlichia risticii, isolated in Illinois and Maryland, were cultivated in a P388D1 mouse macrophage cell line. The ehrlichia particles from heavily infected cultures were separated from host constituents by a Renografin gradient centrifugation procedure modified from those employed for rickettsiae and chlamydiae. The metabolic activities of the isolated ehrlichiae were measured by their formation of CO2 after incubation for 1 h or longer at 34 degrees C with 14C-labeled substrates. Of the substrates tested, glutamine was utilized most vigorously. The greatest activity was obtained at pH 7.2 to 8.0, while the activity rapidly declined at pH below 7. The most favorable buffer was one that contained 0.05 M potassium phosphate as well as 0.2 M sucrose, thus affording some osmotic protection. Glutamate was utilized to a much lesser extent than glutamine, and glucose was not utilized at all. No consistent differences in metabolic activities among the three strains were observed.