Inokuma H, Brouqui P, Drancourt M, Raoult D
Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Yamaguchi University, 753-8515 Yamaguchi, Japan.
J Clin Microbiol. 2001 Sep;39(9):3031-9. doi: 10.1128/JCM.39.9.3031-3039.2001.
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.
已通过简并PCR和基因组步移法确定了13种埃立克体属物种（查菲埃立克体、犬埃立克体、鼠埃立克体、最近从卵形硬蜱中检测到的一种埃立克体物种、反刍动物考德里氏体、嗜吞噬细胞埃立克体、马埃立克体、人类粒细胞埃立克体病[HGE]病原体、边缘无形体、中央无形体、腺热埃立克体、里氏埃立克体和蠕虫新立克次体）柠檬酸合酶基因（gltA）的序列。埃立克体属的gltA基因长度为1197 bp（腺热埃立克体和里氏埃立克体）至1254 bp（边缘无形体和中央无形体），该基因的GC含量从30.5%（从卵形硬蜱中检测到的埃立克体物种）到51.0%（中央无形体）不等。埃立克体属物种间gltA核苷酸序列的同源性百分比为49.7%（里氏埃立克体与中央无形体）至99.8%（HGE病原体与马埃立克体）。推导的氨基酸序列的同源性百分比为44.4%（腺热埃立克体与鼠埃立克体）至99.5%（HGE病原体与马埃立克体），而16S rRNA基因的同源性范围为83.5%（里氏埃立克体与从卵形硬蜱中检测到的埃立克体物种）至99.9%（HGE病原体、马埃立克体和嗜吞噬细胞埃立克体）。由gltA核苷酸序列或氨基酸序列构建的系统发育树的结构与从16S rRNA基因序列推导的结构相似，但显示出更高的自展值。基于对埃立克体属gltA序列的比对分析，设计了两组引物，分别用于扩增蜱传埃立克体属和新立克次体属埃立克体基因组（蠕虫新立克次体、腺热埃立克体和里氏埃立克体）。除鼠埃立克体和与从卵形硬蜱中分离出的密切相关的埃立克体（需要对PCR产物进行序列分析）外，蜱传埃立克体属物种可通过AcsI和XhoI的限制性片段长度多态性（RFLP）模式进行特异性鉴定。同样，新立克次体属埃立克体基因组物种可通过RcaI消化的RFLP模式进行特异性鉴定。如果得到证实，该技术将有助于快速鉴定埃立克体属物种。
