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1 Ohara Research Laboratory, Ohara General Hospital, Fukushima 960-0195, Japan
2 Unité des rickettsies, IFR 48, CNRS UMR 6020, Faculté de médecine, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France
3 Department of Pathological Sciences, Faculty of Medical Sciences, University of Fukui, Matsuoka, Fukui 910-1193, Japan
4 Aizawa Hospital, 2-5-1 Honjo, Matsumoto 390-8510, Japan
Correspondence
Pierre-Edouard Fournier
pierre-edouard.fournier{at}medecine.univ-mrs.fr
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These authors contributed equally to this work. 
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, gltA, ompB and sca4 gene sequences of strain IO-1T are AF394906, AF394901, DQ110870 and DQ110869, respectively.
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) as the agent of Japanese spotted fever (Mahara et al., 1985) has motivated the study of spotted fever group (SFG) rickettsiae in ticks. To date, 63 SFG rickettsial strains have been isolated from ticks in this country (Fujita et al., 1999). Among these, only three Rickettsia species with validly published names have been identified, namely, R. japonica, Rickettsia helvetica and Rickettsia tamurae. R. japonica has been identified in several tick species including Haemaphysalis longicornis, Haemaphysalis flava, Haemaphysalis formosensis, Haemaphysalis hystricis, Dermacentor taïwanensis and Ixodes ovatus (Mahara, 1997; Fournier et al., 2002). R. helvetica has been found in Ixodes monospinosus and Ixodes persulcatus (Fournier et al., 2002) and R. tamurae has been isolated from Amblyomma testudinarium (Fournier et al., 2006). In June 1993, a rickettsial isolate named IO-1T was obtained from an I. ovatus nymph collected in Oozaso, in the Fukushima prefecture (Fujita et al., 1999). Subsequently, 32 additional isolates that exhibited gltA gene sequences identical to that of the IO-1T isolate have been obtained from I. ovatus ticks in Japan. To date, this rickettsia has not been found in any other tick species. A preliminary mouse serotyping assay and sequencing of the entire 16S rRNA (rrs) and gltA genes demonstrated that Rickettsia sp. strain IO-1T exhibits a unique serotype and unique genotypic characteristics (Takada et al., 2001; Fournier et al., 2002). An electron microscopic study of Rickettsia sp. strain IO-1T demonstrated that it multiplied in the cytoplasm of L929 cells, although a few bacteria were seen in intranuclear location (Yano et al., 2004). However, the ability of this rickettsia to stimulate actin-based motility, as described for other SFG rickettsiae (Heinzen et al., 1993), has not been investigated to date. We herein demonstrate that this rickettsia fulfils the requirements for its proposal as a novel species (Fournier et al., 2003; Raoult et al., 2005).
Five IO-1-like rickettsial isolates obtained from Japanese I. ovatus ticks were included in our study. These included strain IO-1T described above, strain IO-2 from the Kagoshima prefecture (isolated in 1994), strain IO-12 from the Fukushima prefecture (1997), strain IO-25 from the Akita prefecture (1997) and strain IO-38 from Awaji island (2001). For strain IO-1T, we first retrieved rrs and gltA nucleotide sequences from GenBank (Table 1). We then extracted DNA from strain IO-1T and the other four isolates using the QIAmp tissue kit (QIAGEN), according to the manufacturer's instructions. PCR amplification and sequencing of the 5' and 3'-ends of the ompA gene and the complete ompB and sca4 genes were attempted from all five isolates using previously described primers and conditions (Fournier et al., 1998; Roux & Raoult, 2000; Sekeyova et al., 2001). In addition, rrs and gltA genes were amplified from strains IO-2, IO-12, IO-25 and IO-38. No ompA PCR product could be obtained from any of the studied isolates. However, whether this negative result is the result of the absence of the ompA gene in IO-1-like isolates or the incapacity of the primers used to amplify the gene is not yet known. In contrast, PCR products of the expected sizes were obtained for the other genes. Sequences were edited by removal of regions of ambiguity at the 5' and 3' ends so that their lengths were 1417, 1047, 4845 and 2946 bp for rrs, gltA, ompB and sca4, respectively. The five isolates exhibited identical sequences for each of the four genes. For further tests, we selected the IO-1T strain as representative of these five isolates. The phylogenetic relationships of Rickettsia sp. strain IO-1T with all Rickettsia species with validly published names for which rrs, gltA, ompB and sca4 gene sequences are available (Table 1) were evaluated for each gene using the neighbour-joining and maximum-parsimony methods within the MEGA 3.1 software (Kumar et al., 2004) and the maximum-likelihood method within the PHYLIP software package (Felsenstein, 1989). For all studied genes and using the three analysis methods, Rickettsia sp. strain IO-1T was grouped with R. helvetica with elevated bootstrap values (Fig. 1). When calculating similarity values between nucleotide sequences of strain IO-1T and Rickettsia species, transitions or transversions, not insertions or deletions, were included. For all four loci examined, strain IO-1T shared highest sequence similarity with R. helvetica (99.0, 99.0, 98.2 and 98.3 %, respectively, for the rrs, gltA, ompB and sca4 genes). However, these values were lower than the cut-offs proposed for Rickettsia species definition (Fournier et al., 2003). Therefore, on the basis of genotypic criteria, Rickettsia sp. strain IO-1T, although phylogenetically closely related to the R. helvetica species (Fig. 1), belonged to a distinct species. In addition, we compared Rickettsia sp. strain IO-1T to another Japanese rickettsia, R. tamurae, also phylogenetically related to R. helvetica (Fournier et al., 2006). We found degrees of nucleotide sequence similarity between the two rickettsiae of 98.5, 96.7, 95.7 and 91.2 %, respectively, for the rrs, gltA, ompB and sca4 genes. Therefore, the two rickettsiae were sufficiently genetically different to be classified within different species. Mouse serotyping was conducted by microimmunofluorescence (MIF) as described by Philip et al. (1978). We used as antigens Rickettsia sp. strain IO-1T, R. helvetica type strain C9P9T and R. tamurae type strain AT-1T, cultivated on Vero cells (ATCC CRL-1587) as described previously (Marrero & Raoult, 1989). Strain IO-1T caused cytopathic effects after 5 days of incubation. If the specificity difference (SPD) was 
3, the isolates were assumed to belong to different serotypes. Using serum from mice immunized with strain IO-1T, we found MIF antibody titres of 1 : 400, 1 : 50 and 1 : 100 to the homologous antigen and to R. tamurae and R. helvetica, respectively. Using serum from mice immunized with R. tamurae, we found MIF antibody titres of 1 : 400, 1 : 50 and 1 : 100 to the homologous antigen and to strain IO-1T and R. helvetica, respectively. Finally, using serum from mice immunized with R. helvetica, we found MIF antibody titres of 1 : 800, 1 : 200 and 1 : 100 to the homologous antigen and to strain IO-1T and R. tamurae antigens, respectively. On the basis of these results, the SPD between these rickettsiae was 4 between strain IO-1T and R. helvetica and 6 between strain IO-1T and R. tamurae. Therefore, the genotypic and serotypic specificity of Rickettsia sp. strain IO-1T justify its classification in a distinct species.
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Description of Rickettsia asiatica sp. nov.
Rickettsia asiatica (a.si.a'ti.ca. L. fem. adj. asiatica Asian).
Gram-negative, obligately intracellular bacterium. Grows in Vero and L929 cells at 32 °C in minimal essential medium supplemented with 2 % fetal calf serum and 2 mg L-glutamine ml–1. Non motile. 16S rRNA, gltA, ompB and sca4 gene sequencing indicate that this rickettsia is clearly different from all other recognized rickettsial species, the most closely related organisms being R. helvetica and R. tamurae. No information is available about the possible pathogenicity of this organism for vertebrate hosts. The known geographical distribution of this bacterium is restricted to Japan.
The type strain is strain IO-1T (=CSUR R2T), which was isolated from Ixodes ovatus ticks in 1993 in Fukushima prefecture, Japan (Fujita et al., 1999). The type strain has been deposited in the Collection de souches de l'Unite des rickettsies (CSUR), WHO Collaborative Center for Rickettsioses, Borrelioses and Tick-borne Infections, Marseilles, France, and is being deposited in the American Type Culture Collection (ATCC). Strains IO-2, IO-12, IO-25 and IO-38, have been deposited in the CSUR under references CSUR R4, CSUR R5, CSUR R6 and CSUR R7, respectively, and are also being deposited in the ATCC.
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ACKNOWLEDGEMENTS |
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Fournier, P.-E., Roux, V. & Raoult, D. (1998). Phylogenetic analysis of spotted fever group rickettsiae by study of the outer surface protein rOmpA. Int J Syst Bacteriol 48, 839–849.
Fournier, P.-E., Fujita, H., Takada, N. & Raoult, D. (2002). Genetic identification of rickettsiae isolated from ticks in Japan. J Clin Microbiol 40, 2176–2181.
Fournier, P.-E., Dumler, J. S., Greub, G., Zhang, J., Wu, Y. & Raoult, D. (2003). Gene sequence-based criteria for identification of new Rickettsia isolates and description of Rickettsia heilongjiangensis sp. nov. J Clin Microbiol 41, 5456–5465.
Fournier, P.-E., Takada, N., Fujita, H. & Raoult, D. (2006). Rickettsia tamurae sp. nov., isolated from Amblyomma testudinarium ticks. Int J Syst Evol Microbiol 56, 1673–1675.
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