Identification of a p28 gene in Ehrlichia ewingii: Evaluation of gene for use as a target for a species-specific PCR diagnostic assay

PCR was used to amplify a 537-by region of an Ehrlichia ewingii gene encodi ng a homologue of the 28-kDa major antigenic protein (P28) of Ehrlichia cha ffeensis. The E. ewingii p28 gene homologue was amplified from DNA extracte d from whole blood obtained from four humans and one canine with confirmed cases of infection. Sequencing of the PCR products (505 bp) revealed a part ial gene with homology to outer membrane protein genes from Ehrlichia and C owdria spp.: p30 of Ehrlichia cams (less than or equal to 71.3%), p28 of E. chaffeensis (less than or equal to 68.3%), and map] of Cowdria ruminantium (67.3%). The peptide sequence of the E. ewingii partial gene product was d educed (168 amino acids) and the antigenicity profile was analyzed, reveali ng a hydrophilic protein with less than or equal to 69.1% identity to P28 o f E. chaffeensis, less than or equal to 67.3% identity to P30 of E. canis, and less than or equal to 63.1% identity to MAPI of C. ruminantium. Primers were selected from the E. ewingii p28 sequence and used to develop a speci es-specific PCR diagnostic assay. The p28 PCR assay amplified the expected 215-bp product from DNA that was extracted from EDTA-treated blood from eac h 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 a ssay was evaluated and determined to detect as few as 38 copies of the p28 gene.