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

Abstract

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.

FIG. 1

Strategy for determination of the sequence of the citrate synthase gene (gltA) of the HGE agent. Primers F3 and R1b were determined after alignment of the gltA of E. coli, R. prowazekii, and B. henselae. After determination of the partial sequence, the unknown sequences of both the 3′ and 5′ ends of the gene were amplified by PCR using an adapter primer provided in the Universal Genome Walker kit and the HGE agent-specific primers based on the partial sequence. Assembly of these sequences determines the complete gltA sequence of HGE. ORF, open reading frame.

FIG. 2

Phylogenetic relationship of various Ehrlichia spp. based on the nucleotide sequences of citrate synthase gene (A) and 16S rRNA gene (B). The neighbor-joining method was used to construct the phylogenetic tree by using the ClustalW program. The scale bar represents 1% divergence. The numbers at nodes are the proportions of 1,000 bootstrap resamplings that support the topology shown.

FIG. 3

Restriction profiles obtained after AcsI (A) and XhoI (B) digestion of a portion of the citrate synthase gene amplified from 10 tick-borne ehrlichial species by PCR using consensus primers EHR-CS136F–EHR-CS778R. Lanes: M, molecular weight markers (in thousands); 1, HGE agent; 2, E. equi; 3, E. phagocytophila; 4, A. marginale; 5, A. centrale; 6, E. canis; 7, E. chaffeensis; 8, E. muris; 9, Ehrlichia sp. detected in I. ovatus; 10, C. ruminantium.

FIG. 4

Restriction profiles obtained after RcaI digestion of a portion of the citrate synthase gene amplified from three species of the Neorickettsia genogroup by PCR using consensus primer NEO-CS142F–NEO-CS730R. Lanes: M, molecular weight markers (in thousands); 1, E. sennetsu; 2, E. risticii; 3, N. helminthoeca.