Citrate synthase gene sequence: a new tool for phylogenetic analysis and identification of Ehrlichia

The sequence of the citrate synthase gene (gltA) of 13 ehrilichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia spec ies recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia ph agocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] age nt, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, and Neorickettsia helminthoeca) have been determined by degenerat e PCR and the Genome Walker method. The ehrlichial gltA genes are 1,197 bp (E. sennetsu and E. risticii) to 1,254 bp (A. marginale and A. centrale) lo ng, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected fro m I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nuc leotide sequences among ehrlichial species were 49.7% (E. risticii versus A . centrale) to 99.8% (HGE agent versus E. equi). The percent identities of deduced amino acid sequences were 44.4% (E. sennetsu versus E. muris) to 99 .5% (HGE agent versus E. equi), whereas the homology range of 16S rRNA gene s was 83.5% (E. risticii versus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, and E. phagocytophila). The architecture of t he phylogenetic trees constructed by gltA nucleotide sequences or amino aci d sequences was similar to that derived from the 16S rRNA gene sequences bu t showed more-significant bootstrap values. Based upon the alignment analys is of the ehrlichial gltA sequences, two sets of primers were designed to a mplify tick-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (N. helmi nthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymor phism (RFLP) patterns of AcsI and XhoI with the exception of E. muris and t he very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroup Ehrlichia species were specifically identified by RFLP patterns of RcaI dig estion. If confirmed this technique will be useful in rapidly identifying E hrlichia spp.