Identification of 19 Polymorphic Major Outer Membrane Protein Genes and Their Immunogenic Peptides in Ehrlichia ewingii for Use in a Serodiagnostic Assay

Ehrlichia ewingii , a tick-transmitted rickettsia previously known only as a canine pathogen, was recently recognized as a human pathogen.E. ewingii has yet to be cultivated, and there is no serologic test available to diagnoseE. ewingii infection. Previously, a fragment (505 bp) of a singleE. ewingii gene homologous to 1 of 22 genes encodingEhrlichia chaffeensis immunodominant major outer membrane proteins 1 (OMP-1s)/P28s was identified. The purposes of the present study were to (i) determine theE. ewingii omp-1 gene family, (ii) determine each OMP-1-specific peptide, and (iii) analyze all OMP-1 synthesized peptides for antigenicity. Using nested touchdown PCR and a primer walking strategy, we found 19omp-1 paralogs inE. ewingii . These genes are arranged in tandem downstream oftr1 and upstream ofsecA in a 24-kb genomic region. Predicted molecular masses of the 19 matureE. ewingii OMP-1s range from 25.1 to 31.3 kDa, with isoelectric points of 5.03 to 9.80. Based on comparative sequence analyses among OMP-1s fromE. ewingii and three otherEhrlichia spp., eachE. ewingii OMP-1 oligopeptide that was predicted to be antigenic, bacterial surface exposed, unique in comparison to the otherE. ewingii OMP-1s, and distinct from those of otherEhrlichia spp. was synthesized for use in an enzyme-linked immunosorbent assay. Plasmas from experimentallyE. ewingii -infected dogs reacted significantly with most of the OMP-1-specific peptides, indicating that multiple OMP-1s were expressed and immunogenic in infected dogs. The results support the utility of the tailored OMP-1 peptides asE. ewingii serologic test antigens.