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 caused by an Ehrlichia species similar or identical to E. equi and E. phagocytophila. Previous studies have shown that the pathogen can be cultivated in vitro in permissive cells such as human promyelocytic HL-60 leukemia cells. The mechanism(s) of its infection and propagation in target cells, however, is not well understood, due in part to lack of a method capable of quantitatively 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 prokaryotic and eukaryotic rRNAs are utilized to quantitatively assay the HGE agent in HL-60 cells. By comparing the integrated intensity of agarose gel resolved HGE-specific rRNA in host cells, with identically prepared and analyzed rRNA isolated from known quantities of E. coli (JM 109), it is possible to calculate the E. coli-equivalence of the HGE agent present in HL-60 cells according to the equation: Y (E. coli, in viable cells x 10(8)) = -2.573 + 0.11X (% infection by the HGE agent in HL-60 cells). The method described is reproducible, sensitive, and is not limited by availability of antisera. Furthermore, since the assay has no designer primer and repeated amplification requirements, it can be easily disseminated to and standardized in other laboratories.
Wu
J M
JM
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
Whyzmuzis
C A
CA
Bertone
M G
MG
Zhou
B S
BS
Hsieh
T C
TC
D002452:Cell Count; D004536:Ehrlichia / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D016873:Ehrlichiosis / Q000097:blood / Q000175:diagnosis / Q000382:microbiology*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D004996:Ethidium; D006098:Granulocytes / Q000382:microbiology* / Q000473:pathology; D018922:HL-60 Cells; D006801:Humans; D008970:Molecular Weight; D009518:New York; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics; D012335:RNA, Ribosomal / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D004996:Ethidium