Detection Of 16S RDNA Of Ehrlichia Chaffeensis-like Organism And Anplasma Marginale In Boophilus Microplus Collected In Tibet

Ehrlichioses caused by Ehrlichia spp. are zoonoses occurring mainly in tropical and subtropical areas. Ehrlichia are small, coccoid, often pleomorphic, intracytoplasmic organism. The vectors for most ehrlichial agents are ticks. The aim of the present study was to identify ehrlichial pathogens in Boophilus microplus as well as other ticks by PCR and DNA sequencing analyses. The study would provide an understanding of the natural distribution of ehrlichial organisms in order to make a risk assessment for acquiring ehrlichial infection in particular geographic regions and, therefore, to facilitate proactive preventive measures of ehrlichiosis in our country.A nested PCR specific for tick-transmitted Ehrlichia spp. was established with 2 pairs of primers designed based on the ehrlichial 16S rDNA sequences. It was proved that this nested PCR was more sensitive and specific than a single-step PCR. The DNA samples of Boophilus microplus and other ticks collected from Tibet, Sichuan, and Jilin (each sample was prepared from two ticks) were detected by the nested PCR. Seventeen of 43 pools (37%) of Boophilus microplus from Tibet were positive for the presence of Ehrlichia, but 44 pools of Boophilus microplus from Sichuan, 46 pools of Ixodes persulcatus, 38 pools of Dermacentor silvarm, and 32 pools of Haemaphysalis japonica from Jilin were all negative. The 5'-end fragments (~450bp) of 16S rDNA of the positive samples in nested PCR were cloned and the cloned fragments were sequenced. Two different 16S rDNA were identified in the positive samples, one identical to that of Anplasma marginale andothers most similar to that of E. chaffeensis. A species-specific primer was designed for amplifying the complete 16S rRNA gene of the E. chaffeensis-like organism and the amplified fragments was sequenced as well. By using the DNAsis version 2.5, a phylogenetic tree was constructed according to the levels of similarity between E. chaffeensis-like organism and related bacteria. The data of homology analysis based on 16S rRNA gene sequence comparisons indicated that it belonged to E. canis genogroup and was closely related to E. chaffeensis (level of similarity: 97.6%).Our study reveals that E. chaffeensis-like organism and A. marginale are carried by Boophilus microplus from the area in Tibet; the E. chaffeensis-like organism may be a new species of Ehrlichia (Ehrlichia sp. Tibet); it also suggests human and animal ehrlichiosis may occur in the regions where ticks carrying E. chaffeensis-like organism are existing.