Following the description in Japan of Japanese spotted fever, caused by Rickettsia japonica, a search for the vector of this disease led to the isolation of several rickettsiae from various tick species. Sixty-three rickettsial isolates were obtained from six different tick species, and six type strains were described by PCR and monoclonal antibody testing. We identified these six strains by amplification and sequencing of the genes encoding 16S rRNA and citrate synthase. We confirmed that the isolates from Dermacentor taiwanensis and Haemaphysalis flava ticks were R. japonica isolates. In Ixodes ovatus, Ixodes persulcatus, and Ixodes monospinosus, we identified a Rickettsia identical or closely related to Rickettsia helvetica, a species that is pathogenic for humans and that to date has only been found in Europe. Finally, we identified a new genotype of unknown pathogenicity, genotype AT, that was isolated from Amblyomma testudinarium ticks and that is closely related to a Slovakian genotype obtained from Ixodes ricinus ticks.Rickettsioses are emerging infectious diseases caused by rickettsiae, which are obligate intracellular gram-negative bacteria associated with arthropod parasites. These arthropod vectors (ticks, fleas, mites, and insects) can transmit rickettsiae to mammals and humans. The rickettsioses share characteristic clinical features, including fever, headache, rash, and sometimes eschar (tache noire) formation at the site of the tick bite. The number of representatives of the genus Rickettsia and the number of newly described rickettsioses have increased in recent decades as a result of the development of an improved cell culture isolation technique and the extensive use of bacterial detection and identification based on molecular biology techniques (14). Comparison of the sequences of PCR-amplified fragments of the genes encoding 16S rRNA (ribosomal DNA [rDNA]) (16), citrate synthase (gltA) (17), or the rOmpA outer membrane protein (ompA) has become a reliable method for the identification of Rickettsia spp. (15).The discovery of Japanese spotted fever by Mahara (9) in 1984 began the study of spotted fever group (SFG) rickettsioses in Japan. The causative agent was identified and named Rickettsia japonica (22). The bacterium was apparently detected in three genera and six species of ticks (9). This stimulated the search for rickettsiae in ticks (19); and since 1993, 63 strains have been isolated from six tick species: Amblyomma testudinarium (25 isolates) (5, 25), Dermacentor taiwanensis (1 isolate) (20, 24), Haemaphysalis flava (1 isolate) (6), Ixodes monospinosus (1 isolate) (4), Ixodes ovatus (33 isolates), and Ixodes persulcatus (2 isolates) (4). These isolates were tested by using two monoclonal antibodies (MAbs) and the immunoperoxidase reaction. One of the two MAbs, which had a broad spectrum of reactivity, reacted with all SFG rickettsiae, and the other MAb was specific for R. japonica (12). These isolates were also tested by PCR with primer sets derived from gltA, ompA, and ompB. The glt...

A survey was performed for Lyme disease borrelia in the southern part of China, in Zhejiang, Sichuan and Anhui provinces, along the Yangtze River valley, in May of 1997 and 1998. Twenty isolates from Ixodes granulatus, Ixodes ovatus, Apodemus agrarius and Niviventer confucianus were obtained. These isolates were characterized by RFLP of the 5S-23S rDNA intergenic spacer, sequence analysis of the intergenic spacer, 16S rDNA and flagellin gene, DNA-DNA hybridization analysis, SDS-PAGE and Western blotting with mAbs. Six isolates from A. agrarius, five from I. granulatus collected in Zhejiang province and one from N. confucianus in Sichuan province were highly similar to strains 10MT and 5MT isolated in Korea and classified as Borrelia valaisiana. Four isolates from A. agrarius and I. granulatus collected in Zhejiang province generated unique RFLP patterns and phylogenetic analysis of the 16S rDNA and flagellin gene sequences suggested that the isolates should be classified as B. valaisiana. Furthermore, three isolates (CMN1a, CNM2, CMN3T) from N. confucianus captured in Sichuan province and one (CWO1) from I. ovatus in Anhui province showed lower 165 rDNA sequence similarity (less than 99.0%) to sequences of previously described Lyme disease-related Borrelia species. DNA-DNA hybridization results revealed that strains CMN3T and CMN1a were clearly distinct from all other known Lyme disease Borrelia species. Electron microscope observation showed the spirochaetes to be morphologically similar to those of Borrelia, but the cells contained only four periplasmic flagella inserted at each end of the spirochaetes. Based on these results, a new Borrelia species, Borrelia sinica sp. nov., is proposed. Strain CMN3T is the type strain of this new species.

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Here, we describe for the first time the prevalence and genetic properties of Bartonella organisms in wild rodents in Japan. We captured 685 wild rodents throughout Japan (in 12 prefectures) and successfully isolated Bartonella organisms from 176 of the 685 rodents (isolation rate, 25.7%). Those Bartonella isolates were all obtained from the rodents captured in suburban areas (rate, 51.8%), but no organism was isolated from the animals captured in city areas. Sequence analysis of rpoB and gltA revealed that the Bartonella isolates obtained were classified into eight genetic groups, comprising isolates closely related to B. grahamii (A-I group), B. tribocorum and B. elizabethae (B-J group), B. tribocorum and B. rattimassiliensis (C-K group), B. rattimassiliensis (D-L group), B. phoceensis (F-N group), B. taylorii (G-O group), and probably two additional novel Bartonella species groups (E-M and H-P). B. grahamii, which is one of the potential causative agents of human neuroretinitis, was found to be predominant in Japanese rodents. In terms of the relationships between these Bartonella genetic groups and their rodent species, (i) the A-I, E-M, and H-P groups appear to be associated with Apodemus speciosus and Apodemus argenteus; (ii) the C-K, D-L, and F-N groups are likely implicated in Rattus rattus; (iii) the B-J group seems to be involved in Apodemus mice and R. rattus; and (iv) the G-O group is probably associated with A. speciosus and Clethrionomys voles. Furthermore, dual infections with two different genetic groups of bartonellae were found in A. speciosus and R. rattus. These findings suggest that the rodent in Japan might serve as a reservoir of zoonotic Bartonella infection.

A case of Rickettsia heilongjiangensis infection in Japan was identified in a 35-year-old man who had rash, fever, and eschars. Serum contained R. heilongjiangensis antibodies, and eschars contained R. heilongjiangensis DNA. R. heilongjiangensis was also isolated from ticks in the suspected geographic area of infection.

Rickettsia sp. strain AT-1 T was isolated from Amblyomma testudinarium ticks in Japan in 1993. Comparative analysis of sequences obtained from 16S rRNA, gltA, ompA, ompB and sca4 gene fragments demonstrated those from AT-1 T to be markedly different from those of other members of the spotted fever group. Using mouse serotyping, it was also observed that Rickettsia sp. strain AT-1 T was different from other Rickettsia species with validly published names. Such genotypic and phenotypic characteristics warrant its classification as a representative of a novel species, for which the name Rickettsia tamurae sp. nov. is proposed, with the type strain AT-1 T (=CSUR R1 T).

Spurred by the recent isolation of a novel hantavirus, named Imjin virus (MJNV), from the Ussuri white-toothed shrew (Crocidura lasiura), targeted trapping was conducted for the phylogenetically related Asian lesser white-toothed shrew (Crocidura shantungensis). Pair-wise alignment and comparison of the S, M and L segments of a newfound hantavirus, designated Jeju virus (JJUV), indicated remarkably low nucleotide and amino acid sequence similarity with MJNV. Phylogenetic analyses, using maximum likelihood and Bayesian methods, showed divergent ancestral lineages for JJUV and MJNV, despite the close phylogenetic relationship of their reservoir soricid hosts. Also, no evidence of host switching was apparent in tanglegrams, generated by TreeMap 2.0β.

Borrelia sp. prevalence in ticks on migratory birds was surveyed in central Japan. In autumn, a total of 1,733 birds representing 40 species were examined for ticks. A total of 361 ticks were obtained from 173 birds of 15 species, and these ticks were immature Haemaphysalis flava (94.4%), Haemaphysalis longicornis, Ixodes columnae, Ixodes persulcatus, Ixodes turdus, and an unidentified Ixodes species. Of these, 27 juveniles of H. flava on Turdus pallidus, Turdus cardis, or Emberiza spodocephala, 2 juveniles of I. persulcatus on T. pallidus, and 1 female H. flava molted from a T. pallidus-derived nymph were positive for the presence of Borrelia by Barbour-Stoenner-Kelly culture passages. In spring, a total of 16 ticks obtained from 102 birds of 21 species were negative for the spirochete. Isolates from 15 ticks were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis; all isolates were identified as Borrelia garinii with pattern B/B based on the previous patterning. According to the intergenic spacer sequences, 2 of 15 isolates, strains Fi14f and Fi24f, were highly similar to B. garinii strains 935T of Korea and ChY13p of Inner Mongolia, China, respectively. These findings indicate that Lyme disease-causing B. garinii may have been introduced to Japan by migratory birds from northeastern China via Korea. Additionally, a case of transstadial transmission of B. garinii from nymph to adult H. flava suggests that the infected H. flava may transmit Borrelia to large animals.Lyme disease is primarily caused by three genomic species, Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii (2, 4). B. garinii and B. afzelii are widely distributed from Europe to the Far East including Japan, while B. burgdorferi sensu stricto is prevalent in North America and has been confirmed in part of Europe. B. burgdorferi sensu lato is mainly transmitted by some tick species of the Ixodes ricinus complex, and these ticks infest both mammals and birds (1,9,10,13,19,25).Concerning the prevalence of Borrelia in ticks feeding on birds, B. garinii has been isolated from Ixodes persulcatus on Emberiza spodocephala and Turdus chrysolaus in Hokkaido, Japan (20, 24), and B. burgdorferi sensu stricto, B. garinii, B. afzelii, and Borrelia andersonii have been isolated from Ixodes dentatus, I. ricinus, Ixodes scapularis, Ixodes uriae, and other ticks, which infest a large number of bird species in Europe and North America (6,13,15,27,28,30). Furthermore, Haemaphysalis leporispalustris, detected on some bird species, was reported to be a reservoir of B. burgdorferi sensu stricto in North America (13,26).B. afzelii is transmitted between I. persulcatus and field rodents, and B. garinii is transmitted between I. persulcatus and migratory birds or rodents, in Hokkaido, Japan (24, 25). However, there has not been a survey of Borrelia in migratory birds which travel directly between the Asiatic continent and Japan. In this survey, we examined the Borrelia prevalence in juvenile ticks remo...