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Journal of Clinical Microbiology, November 2001, p. 3871-3876, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.3871-3876.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification of a p28 Gene in Ehrlichia ewingii: Evaluation of Gene for Use as a Target for a Species-Specific PCR Diagnostic Assay

Asiya A. Gusa,1 Richard S. Buller,2 Gregory A. Storch,2 Mark M. Huycke,3,4 Linda J. Machado,3,4 Leonard N. Slater,3,4 Steven L. Stockham,5 and Robert F. Massung1,*

Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia1; The Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis,2 and Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri---Columbia, Columbia,5 Missouri; and Division of Infectious Diseases, Department of Medicine, University of Oklahoma Health Sciences Center,3 and Department of Veteran's Affairs Medical Center,4 Oklahoma City, Oklahoma

Received 8 May 2001/Returned for modification 3 July 2001/Accepted 12 August 2001

PCR was used to amplify a 537-bp region of an Ehrlichia ewingii gene encoding a homologue of the 28-kDa major antigenic protein (P28) of Ehrlichia chaffeensis. The E. ewingii p28 gene homologue was amplified from DNA extracted from whole blood obtained from four humans and one canine with confirmed cases of infection. Sequencing of the PCR products (505 bp) revealed a partial gene with homology to outer membrane protein genes from Ehrlichia and Cowdria spp.: p30 of Ehrlichia canis (<= 71.3%), p28 of E. chaffeensis (<= 68.3%), and map1 of Cowdria ruminantium (67.3%). The peptide sequence of the E. ewingii partial gene product was deduced (168 amino acids) and the antigenicity profile was analyzed, revealing a hydrophilic protein with <= 69.1% identity to P28 of E. chaffeensis, <= 67.3% identity to P30 of E. canis, and <= 63.1% identity to MAP1 of C. ruminantium. Primers were selected from the E. ewingii p28 sequence and used to develop a species-specific PCR diagnostic assay. The p28 PCR assay amplified the expected 215-bp product from DNA that was extracted from EDTA-treated blood from each of the confirmed E. ewingii infections that were available. The assay did not produce PCR products with DNA extracted from E. chaffeensis-, E. canis-, or E. phagocytophila-infected samples, confirming the specificity of the p28 assay for E. ewingii. The sensitivity of the E. ewingii-specific PCR assay was evaluated and determined to detect as few as 38 copies of the p28 gene.

* Corresponding author. Mailing address: Centers for Disease Control and Prevention, 1600 Clifton Rd., MS G-13, Atlanta, GA 30333. Phone: (404) 639-1082. Fax: (404) 639-4436. E-mail: rfm2{at}cdc.gov.

Journal of Clinical Microbiology, November 2001, p. 3871-3876, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.3871-3876.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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