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Plant Sciences


Granulocytic Ehrlichia was isolated from canine blood obtained from animals 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 revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocyticEhrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.

Members of the genusEhrlichia include species which have a tropism for mononuclear phagocytes (E. canis, E. chaffeensis,E. muris, E. sennetsu, and E. risticii) (33) and those which infect granulocytes (E. ewingii [33], E. phagocytophila [10, 33], E. equi[14, 26], and the recently discovered agent of human granulocytic ehrlichiosis [HGE] [3, 7]). Disease caused by granulocytic Ehrlichia (GE) is manifested by fever, lethargy, thrombocytopenia, and death, and many species from diverse geographical locations have shown evidence of natural infection, including horses (25, 27, 29, 38), dogs (24,34, 35), small mammals (39, 40), and humans (4,15).

The similar host range and near identity of the 16S rRNA genes ofE. phagocytophila, E. equi, and the HGE agent (7) have raised the possibility that these organisms represent a single species (2, 28). In addition, Dumler et al. (9) have shown that they share significant antigenicity by immunofluorescence and immunoblot assays. Objective methods for species classification, e.g., molecular genetic analysis, have not been readily available, primarily because of an inability to culture these ehrlichiae in vitro. However, we (reference 43 and unpublished data) and others (13) have recently demonstrated successful cultivation of GE isolates from dogs and humans, respectively.

In this paper, we describe the use of purified GE obtained from in vitro culture of infected HL60 cells, a promyelocytic human cell line, to generate a genomic DNA library for expression screening with sera obtained from dogs experimentally infected with GE. The screening resulted in the isolation of recombinant clones containing complete genes encoding three putative proteins of GE, GE 160, GE 130, and GE 100 (named for apparent molecular mass in kilodaltons). One of these proteins, the 100-kDa protein, is similar in both glutamic acid content and repeated amino acid structure to an immunodominant 120-kDa E. chaffeensis protein (45). Both the 100- and 130-kDa granulocytic Ehrlichia proteins share some amino acid sequence homology to the 120-kDa E. chaffeensis protein.