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Journal of Clinical Microbiology, September 2001, p. 3031-3039, Vol. 39, No. 9
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.9.3031-3039.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Hisashi Inokuma,1,2 Philippe Brouqui,2 Michel Drancourt,2 and Didier Raoult2,*

Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Yamaguchi University, 753-8515 Yamaguchi, Japan,1 and Unité des Rickettsies, Faculté de Médecine, Université de la Méditerranée, Marseille Cédex 5, France2

Received 25 January 2001/Returned for modification 8 April 2001/Accepted 30 May 2001

The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia 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, and Neorickettsia helminthoeca) have been determined by degenerate 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) 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 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 genes 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 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-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (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 of E. muris and the 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 digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.

* Corresponding author. Mailing address: Unité des Rickettsies, Faculté de Médecine, 27 bd. Jean Moulin, 13385 Marseille Cédex 5, France. Phone: (33)-4-91-32-43-75. Fax: (33)-4-91-83-03-90. E-mail: Didier.Raoult{at}medecine.univ-mrs.fr.

Journal of Clinical Microbiology, September 2001, p. 3031-3039, Vol. 39, No. 9
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.9.3031-3039.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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