Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet

Abstract

To identify ehrlichial agents in Boophilus microplus ticks, DNA samples of B. microplus collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of B. microplus from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5'-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of Anaplasma marginale, an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of Ehrlichia chaffeensis, an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the E. canis group of the genus EHRLICHIA: Sequence comparison of the 16S rRNA gene with the members of the genus Ehrlichia reveals that the novel ehrlichial agent detected in B. microplus ticks is a new species of the genus Ehrlichia and is most closely related to E. chaffeensis.

FIG. 1.

The different fragments of 16S rRNA genes amplified with different pairs of primers from the tick DNA specimens. The italicized words represent the names of primers, and the bold lines represent the sizes of the PCR products.

FIG. 2.

A highly variable region of sequence located at the 5′ end of the 16S rRNA gene revealed by multiple alignment of 16S rRNA gene sequences of Ehrlichia spp. in the E. canis genogroup. C. ruminantium, Cowdria ruminantium.

FIG. 3.

Phylogenetic relationships between Ehrlichia sp. strain Tibet and other members of the genus Ehrlichia. The tree was constructed using the neighbor-joining program in the software package CLUSTALX.