Characterization of Ehrlichia Species from Ixodes ovatus Ticks at the Foot of Mt. Fuji, Japan

Abstract

A total of 390 adult ticks (288 Ixodes ovatus and 102 I. persulcatus) collected at the foot of Mt. Fuji and two near cities in Shizuoka prefecture, Japan, were examined for Ehrlichia infection by isolation with laboratory mice from whole tick tissues. Ehrlichial DNAs were detected from the spleens of mice inoculated with tissues from I. ovatus, but not I. persulcatus. The prevalence of ehrlichiae in the ticks was estimated to be ca. 3%. The 16S rDNA analysis revealed that the sequences of 8 ehrlichial isolates (termed “Shizuoka” isolates) obtained were identical, and they were very similar, but not identical, to those of two Ehrlichia species strain variants recently isolated in Japan, followed by Ehrlichia chaffeensis in the US. Analysis of parts of the omp‐1 multigene family specific for monocytic ehrlichiosis agents showed that the Shizuoka isolates were distinct from other ehrlichial organisms. The Shizuoka isolates caused death in immunocompetent laboratory mice, suggesting that they are highly pathogenic in mice. The data show that the Shizuoka isolates are likely to be a new strain variant of Ehrlichia species in Japan. Further characterization and surveillance will be required in Japan due to the presence of these human ehrlichiosis agent‐like organisms.

Molecular Identification of Ehrlichia Species and ‘Candidatus Neoehrlichia mikurensis’ from Ticks and Wild Rodents in Shizuoka and Nagano Prefectures, Japan

“…All ticks collected were dissected, and the whole-tissue specimens of 4 to 15 ticks were pooled and homogenized in sucrose-phosphate-glutamate buffer (SPG) as described previously (5). Each pooled-tick homogenate of 0.5 ml was inoculated intraperitoneally into 5-weekold ddY male mice (Japan SLC, Inc., Hamamatsu, Japan).…”

“…The nested PCRs were performed by the procedure described previously (5). The primers used for the amplification of the omp-1 multigene family (5,(12)(13)(14) were conP28-F1 and conP28-R1 for the first step, and conP28-F2 and conP28-R2 for the second step, as described previously (5).…”

“…HF565, Anan and Shizuoka, but the omp-1 paralog sequences were quite distinctive (5). Two variants of HF565 and Shizuoka showed high virulence to laboratory mice, but the Anan variant showed avirulence (5,20). Cheng et al found that some paralogous genes in the omp-1 multigene family of E. chaffeensis human isolates are missing on the genomes of individual isolates (2), although these isolates have identical sequences of 16S rDNA.…”

“…Each pooled-tick homogenate of 0.5 ml was inoculated intraperitoneally into 5-weekold ddY male mice (Japan SLC, Inc., Hamamatsu, Japan). All inoculated mice were treated with 0.16 mg of cyclophosphamide/g of body weight at 0, 4 and 8 days after the inoculation of tick homogenates (5). On day 9 to 21 after the inoculation of tick homogenates, the mice were sacrificed and the spleens were harvested for the preparation of total DNA.…”

Occurrence of Ehrlichia canis in free-living primates of the genus Callithrix

“…After DNA extraction, the DNA samples were stored at -20 °C for further analysis. Real time PCR analysis was performed in a step one real-time PCR system (Applied Biosystems, USA), using the primers ER-R1 (5'-GGAGGTAATGCACCAGCC-3') and ER5-3 (5'-GTTAGAGTTCCTTGATGG-3') which identify the specific 16S ribosomal gene for Ehrlichia genus organisms (INAYOSHI et al, 2004). E. canis DNA and deionized sterile water were used as positive and negative controls, respectively.…”