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Infect Immun, April 1998, p. 1356-1363, Vol. 66, No. 4
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Molecular Cloning and Sequencing of Three Granulocytic Ehrlichia Genes Encoding High-Molecular-Weight Immunoreactive Proteins

James R. Storey,1 Linda A. Doros-Richert,1 Cindy Gingrich-Baker,1 Kenneth Munroe,1 Thomas N. Mather,2 Richard T. Coughlin,1 Gerald A. Beltz,1 and Cheryl I. Murphy1,*

Aquila Biopharmaceuticals, Inc., Worcester, Massachusetts 01605,1 and Center for Vector-Borne Disease, University of Rhode Island, Kingston, Rhode Island 028812

Received 31 October 1997/Returned for modification 8 January 1998/Accepted 27 January 1998

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 granulocytic Ehrlichia 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.

* Corresponding author. Mailing address: Aquila Biopharmaceuticals, 365 Plantation St., Worcester, MA 01605. Phone: (508) 797-5777, ext. 188. Fax: (508) 797-4014. E-mail: molbio{at}splusnet.com.




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