Citrate Synthase Gene Sequence: a New Tool for Phylogenetic Analysis and Identification of Ehrlichia

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Abstract
The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis,Ehrlichia canis, Ehrlichia muris, anEhrlichia species recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia phagocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] agent, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, andNeorickettsia helminthoeca) have been determined by degenerate PCR and the Genome Walker method. The ehrlichialgltA genes are 1,197 bp (E. sennetsu andE. risticii) to 1,254 bp (A. marginaleand A. centrale) long, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected from I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nucleotide sequences among ehrlichial species were 49.7% (E. risticii versusA. 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 genes was 83.5% (E. risticiiversus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, andE. phagocytophila). The architecture of the phylogenetic trees constructed by gltA nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial gltA sequences, two sets of primers were designed to amplify tick-borneEhrlichia and Neorickettsia genogroupEhrlichia (N. helminthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of AcsI and XhoI with the exception ofE. muris and the very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroupEhrlichia species were specifically identified by RFLP patterns of RcaI digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.