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.Ehrlichia species are known as important pathogens of medical, as well as veterinary, importance. They are intracellular microorganisms residing within the cytoplasmic vacuoles of monocytes, granulocytes, or platelets of humans and animals. Ehrlichia species elicit illnesses with fever, headache, leukopenia, and thrombocytopenia (9, 13). The genus Ehrlichia is divided into three distinct genogroups based on the similarity of the nucleotide sequence of the 16S rRNA gene: the Ehrlichia canis group (including E. canis, E. chaffeensis, E. muris, and E. ewingii), the E. phagocytophila group (E. phagocytophila, E. equi, human granulocytic ehrlichiosis [HGE] agent, and E. platys), and the E. sennetsu group (E. sennetsu, E. risticii, and the Stellantchasmus falcatus [SF] agent) (2, 15). The members of the E. sennetsu group are transmitted by aquatic vectors (16). In contrast, the members of the E. canis group and the E. phagocytophila group are both tick-borne organisms (9,15,16).

Detection of Antibodies toAnaplasma phagocytophilumandEhrlichia chaffeensisAntigens in Sera of Korean Patients by Western Immunoblotting and Indirect Immunofluorescence Assays

“…Although the I. persculcatus group that transmits A. phagocytophilum is present in South Korea, the classical tick vector for E. chaffeensis, Amblyomma americanum, is not. However, E. chaffeensis or a similar species has been detected in Amblyomma testudinarium and Haemaphysalis yeni ticks in South China (7), Boophilus microplus ticks in Tibet (27), and Ixodes ovatus ticks in Japan (23); and we have found evidence of both E. chaffeensis and A. phagocytophilum in both Haemaphysalis longicornis and I. persulcatus ticks from South Korea (17a). Although a definitive role of these causative agents and potential tick vectors in human infections cannot be assumed from these data, the presence of the causative agents and potential tick vectors with the capacity to bite humans suggests that the serological data reflect a previously unrecognized but emerging problem in South Korea and perhaps more broadly across Asia.…”

Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and Other Eubacteria in Ticks from the Thai-Myanmar Border and Vietnam

“…Within the family Anaplasmataceae, there is at present no consensus as to the degree of 16S rRNA gene dissimilarity which should be evident to distinguish two bacterial species as opposed to that which should be evident to represent natural genetic variation. A recent study suggested that 0.5% divergence in the 16S RNA gene sequence of bacteria within this family could be considered a cutoff (29); however, it seems prudent to await phylogenetic studies based on sequences of other genes before such a recommendation is accepted.…”

“…strain EBm52 was shown to be closely related to Ehrlichia sp. strain Tibet (99.9% sequence similarity) identified in B. microplus ticks collected in Tibet (29). The latter ehrlichia was recently presented as a new species within the genus Ehrlichia, based on phylogenetic analyses of the 16S rRNA gene (29).…”

Anaplasma phagocytophilum Infection in Domestic Animals in Ten Provinces/Cities of China

“…The nested PCR for A. phagocytophilum 16S rRNA genes was performed as described previously. 8 Briefly, Anaplasma genus-specific primers of outer-1 (5 -TTG AGA GTT TGA TCC TGG CTC AGA ACG-3 )/outer-2 (5 -CAC CTC TAC ACT AGG AAT TCC GCT ATC-3 ) were used in the first round of amplification, and species-specific primers of HGA1 (5 -GTC GAA CGG ATT ATT CTT TAT AGC TTG -3 )/HGA2 (5 -TAT AGG TAC CGT CAT TAT CTT CCC TAC-3 ) were used for nested PCR. A final 389-bp DNA fragment was produced.…”