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

Abstract

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 (< or =71.3%), p28 of E. chaffeensis (< or =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 < or =69.1% identity to P28 of E. chaffeensis, < or =67.3% identity to P30 of E. canis, and < or =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.

FIG. 1

Comparison of E. chaffeensis (E. chaf.) p28-19 gene (Arkansas strain; accession no. U72291) to the E. ewingii p28 genes (C-1, OK-1, OK-2, MO-1, and MO-3). Dots represent sequence agreement, letters represent nucleotide differences, and the tilde symbol indicates that the sequence was not available. All primers used in this study are indicated by arrows for orientation.

FIG. 2

Deduced amino acid sequences of E. ewingii P28 (C-1, OK-1, OK-2, MO-1, and MO-3) compared with the amino acid sequences of the most-homologous copies of E. chaffeensis (E. chaf.) P28 (Arkansas P28-19; accession no. U72291), E. canis P30 (Oklahoma; accession no. AF078553), and C. ruminantium MAP1 (Nyatsanga; accession no. U50834).

FIG. 3

Species-specific p28 PCR assay results for E. ewingii with primers EEM2F and EEM1R. The first five lanes show PCR products obtained with the E. ewingii-infected template DNAs (lane 1, C-1; lane 2, OK-1; lane 3, OK-2; lane 4, MO-1; and lane 5, MO-3). The remaining lanes show PCR products amplified from DNAs from samples containing E. chaffeensis (lanes 6 to 10), E. canis (lane 11), and E. phagocytophila (lane 12) as well as positive (+) and negative (−) controls. The expected size (215 bp) is indicated. Lanes M show size standards (HaeIII digest of phage φX174 DNA).

FIG. 4

Sensitivity of the species-specific p28 PCR assay for E. ewingii with primers EEM2F and EEM1R. A 10-fold dilution series of pMAP template amplified by the p28 PCR assay is shown (lane 1, 3.84 × 10⁷ copies of pMAP, through lane 8, 3.84 copies of pMAP). The expected size (215 bp) is noted. Lanes M show size standards (HaeIII digest of phage φX174 DNA).