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The recent isolation of Wolbachia pipientis in the continuous cell line Aa23, established from eggs of a strain of the Asian tiger mosquito Aedes albopictus, allowed us to perform extensive characterization of the isolate. Bacterial growth could be obtained in C6/36, another A. albopictus cell line, at 28°C and in a human embryonic lung fibroblast monolayer at 28 and 37°C, confirming that its host cell range is broader than was initially thought. The bacteria were best visualized by Diff-Quik and May-Grunwald-Giemsa staining. Proteins from 213 to 18 kDa with two major protein bands of 65 and 25 kDa were observed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. By Western blotting with specific polyclonal mouse and rabbit antisera, dominant immunoreactive antigens were found at approximately 100, 80, and 30 kDa. The genome size was calculated to be 1,790 17 kb by pulsed-field gel electrophoresis. The sequence of the citrate synthase gene (gltA )o fW. pipientis was determined by gene walking. Its position in the phylogenetic tree constructed with gltA confirmed that found in a phylogenetic tree constructed with 16S rRNA genes and that it belongs in the subgroup of the class Proteobacteria and that it is closely related to but independent from the genera Ehrlichia, Anaplasma, and Neorickettsia. Bacteria of the genus Wolbachia are maternally inherited and are known to infect a wide range of arthropods and filarial nematodes (31, 37). The first description of Wolbachia was made in 1924, when it was detected in the ovaries of the mosquito Culex pipiens and classified as an unnamed Rickettsia (11). The bacterium was subsequently named Wolbachia pipi- entis (10), to honor Wolbach, who first made these observa- tions. On the basis of its association with arthropods and its intracellular location, it was classified in the family Rickettsi- aceae, tribe Wolbachieae (36). The advent of molecular tools for phylogenetic and taxonomic studies, especially 16S rRNA gene sequencing, has dramatically modified classification of the bacteria belonging to the family Rickettsiaceae (35, 36). W. pipientis was found to be in the subgroup of the class Pro- teobacteria and closely related to the genus Rickettsia (7). On the basis of analysis of 16S rRNA genes, groESL, and surface protein genes, the members of the order Rickettsiales have recently been reorganized in the families Rickettsiaceae and Anaplasmataceae (7). W. pipientis was then included in the family Anaplasmataceae with the genera Ehrlichia, Neorickett- sia, and Anaplasma. The increasing use of gene sequencing for the characterization of insect endosymbionts has demonstrated the extreme diversity of the Wolbachia genus (21). Phylogeny based on genes such as ftsZ and wsp has revealed the existence of four major clades within the genus: clades A and B in insects, mites, and crustaceans and clades C and D in filarial

Detection and analysis of Babesia species from ticks recovered from dogs in Japan were attempted by PCR and nucleotide sequence analysis based on the 18S rRNA gene, respectively. A total of 1,136 ticks were examined for Babesia DNA by 18S rRNA-based PCR and nucleotide sequencing. Partial sequences of Babesia canis vogeli DNA were detected from six ticks in Aomori, Nara, Hiroshima, Oita, and Okinawa Prefectures; and Babesia gibsoni Asia-1 DNA was also detected in four ticks in Osaka, Hiroshima, Miyazaki, and Okinawa Prefectures. Unique sequences of 1,678 bp were also obtained from Ixodes ovatus ticks in Akita and Fukui Prefectures. The sequences were similar to those of Babesia odocoilei (97.7%) and Babesia divergens (97.6%). This is the first report of the detection of DNA belonging to this group in Japan.

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 Acs I and Xho I 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 Rca I digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.

We describe here the clinical significance of coinfection with Theileria orientalis and Babesia ovata in cattle. Anemia status in a herd of dairy cattle in Japan was investigated in relation to infection with these parasites. Our findings indicate that while B. ovata infection might not be the primary cause of anemia in the cattle, it may contribute to the clinical development of anemia in animals coinfected with both B. ovata and T. orientalis .

Detection and analysis of Babesia species from ticks recovered from dogs in Japan were attempted by PCR and nucleotide sequence analysis based on the 18S rRNA gene, respectively. A total of 1,136 ticks were examined for Babesia DNA by 18S rRNA-based PCR and nucleotide sequencing. Partial sequences of Babesia canis vogeli DNA were detected from six ticks in Aomori, Nara, Hiroshima, Oita, and Okinawa Prefectures; and Babesia gibsoni Asia-1 DNA was also detected in four ticks in Osaka, Hiroshima, Miyazaki, and Okinawa Prefectures. Unique sequences of 1,678 bp were also obtained from Ixodes ovatus ticks in Akita and Fukui Prefectures. The sequences were similar to those of Babesia odocoilei (97.7%) and Babesia divergens (97.6%). This is the first report of the detection of DNA belonging to this group in Japan.