Quantification of the human granulocytic ehrlichiosis agent based on analysis of rRNA isolated from control and infected HL-60 cells

Human granulocytic ehrlichiosis (HGE) is an emerging vector-borne disease c aused by an Ehrlichia species similar or identical to E. equi and E. phagoc ytophila. Previous studies have shown that the pathogen can be cultivated i n vitro in permissive cells such as human promyelocytic HL-60 leukemia cell s. The mechanism(s) of its infection and propagation in target cells, howev er, is not well understood, due in part to lack of a method capable of quan titatively determining the amount of the infectious agent. Although several assays currently exist for the HGE agent, they are mostly qualitative and have a number of limitations. In this report, size differences between prok aryotic and eukaryotic rRNAs are utilized to quantitatively assay the HGE a gent in HL-60 cells. By comparing the integrated intensity of agarose gel r esolved HGE-specific rRNA in host cells, with identically prepared and anal yzed rRNA isolated from known quantities of E. coli (JM 109), it is possibl e to calculate the E. coli-equivalence of the HGE agent present in HL-60 ce lls according to the equation: Y (E. coli, in viable cells x 10(8)) = -2.57 3 + 0.11X (% infection by the HGE agent in HL-60 cells), The method describ ed is reproducible, sensitive, and is not limited by availability of antise ra. Furthermore, since the assay has no designer primer and repeated amplif ication requirements, it can be easily disseminated to and standardized in other laboratories. (C) 1999 Academic Press.