MOLECULAR-CLONING AND SEQUENCING OF 3 GRANULOCYTIC EHRLICHIA GENES ENCODING HIGH-MOLECULAR-WEIGHT IMMUNOREACTIVE PROTEINS

Granulocytic Ehrlichia was isolated from canine blood obtained from an imals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revea led that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia a nd screened with pooled sera from tick-challenged, granulocytic Ehrlic hia-infected dogs. Several clones were isolated and sequenced. Three c omplete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with co nvalescent-phase sera from dogs and human patients recovering from HGE . This approach will be useful for identifying candidate diagnostic an d vaccine antigens for granulocytic ehrlichiosis and aid in the classi fication of geno-group members.