Toshiyuki Masuzawa

Lyme borreliosis is the most prevalent tick-borne zoonosis and an important emerging infection in Europe, North America, and Far Eastern countries. The geographical distribution of Borrelia spp. and the relationship between Borrelia spp. and tick spp. in East Asian countries have been studied. In Northern Asian countries, Ixodes persulcatus carries Eurasian-type Borrelia garinii (20047 type), Borrelia afzelii, and Asian-type B. garinii (variant NT29), whereas Borrelia burgdorferi sensu stricto has not been isolated. In contrast, Asian-type B. garinii has not been found in the European vector tick Ixodes ricinus. These Borrelia spp. cause Lyme borreliosis in their respective countries. The specificity between Borrelia spp. and tick spp. has been confirmed from studies in the Moscow region, which is a sympatric region for the tick spp. I. persulcatus and I. ricinus. In Southeast Asian countries including the southernmost island of Okinawa, the Borrelia valaisiana-related genomic group is carried by Ixodes granulatus. In Japan, a similar borrelia strain Am501, is transmitted by Ixodes columnae. Ixodes ovatus transmits Borrelia japonica but not other species. On other hand, in central China and Nepal, the ticks harbor Borrelia sinica. It is believed that these molecular epidemiological surveys will provide useful information for the diagnoses and prevention of Lyme borreliosis in these countries.

An expedition across the Asian part of the Black Sea coast and national parks of Northern Turkey was organized in the summer of 2001 to investigate the presence of Borrelia burgdorferi sensu lato (s.l.), Lyme borreliosis agent, and Anaplasma phagocytophilum, human granulocytic ehrlichiosis agent, in wild mice. A total of 65 Apodemus flavicollis, Apodemus sylvaticus, Microtus epiroticus, Crocidura suaveolens and Mus macedonicus, were captured. Two out of 22 Apodemus sylvaticus specimens were seropositive for B. afzelii by enzyme-linked immunosorbent assay as confirmed by Western blotting, however cultures of skin and bladder samples from all small mammals in Barbour–Stoenner–Kelly's medium-II remained negative for B. burgdorferi s.l. All sera tested were negative for Anaplasma phagocytophilum by indirect immunofluorescent assay. The prevalence of B. burgdorferi s.l. and Anaplasma phagocytophilum is low in wild mice of the Asian part of Northern Turkey.

Previously, a novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium, which infests tortoises (Testudo graeca), by using Barbour–Stoenner–Kelly (BSK) II medium; the tick samples were taken from the Istanbul area in northwestern Turkey [ Güner et al. (2003) . Microbiology 149, 2539–2544]. Here is presented a detailed characterization of the spirochaete. Electron microscopy revealed that strain IST7T is morphologically similar to other spirochaetes of the genus Borrelia and possesses 15 to 16 flagellae that emerge from both polar regions. PFGE analysis revealed the genome to comprise a linear chromosome of approximately 1 Mb; two large linear plasmids of approximately 145 and 140 kb, and several small plasmids ranging from 50 to 20 kb in size were also found. The 16S rRNA gene sequence of this Borrelia isolate exhibited 99·4 to 99·8 % identity with other strains isolated from H. aegyptium and less than 99 % similarity with those of other Borrelia species. A phylogenetic tree, generated from 16S rRNA gene sequences, demonstrated that the spirochaete isolates from H. aegyptium clustered together and branched off from both Lyme-disease-related and relapsing-fever-associated Borrelia species. A single copy of the rrs gene was detected in the genome of strain IST7T by Southern hybridization. DNA–DNA hybridization results showed that strain IST7T was distinct from Lyme-disease-related Borrelia, Borrelia burgdorferi and the relapsing-fever-associated species Borrelia hermsii. The G+C content of strain IST7T is 30·0 mol%. From these genetic features, a novel Borrelia species, Borrelia turcica sp. nov., is proposed; the type strain is IST7T (=JCM 11958T=DSM 16138T).

In 2000 and 2001, a survey was conducted of Borrelia isolated from various mammals in the southernmost islands of Japan, including Okinawa (main island), Izena, Iriomote and Ishigaki. Isolates obtained from the ear tissues of Suncus murinus (10 isolates), Mus calori (four isolates), Rattus norvegicus (one isolate) and Crocidura watasei (one isolate), were characterized by RFLP of the 5S–23S rDNA intergenic spacer and sequence analysis of the intergenic spacer, 16S rDNA and flagellin gene. While these isolates showed identical RFLPs to Borrelia valaisiana found in Korea, Taiwan and the southern and central parts of China, their RFLP patterns differed from those of B. valaisiana found in European countries, and strain Am501 isolated from Ixodes columnae in Japan. It was found that these isolates clustered with each other on a phylogenetic tree based on flagellin gene and 16S rDNA sequences, but were relatively divergent from the European B. valaisiana and strain Am501. These findings suggest that these isolates found in East Asia should be classified as a new genomospecies of Borrelia burgdorferi sensu lato.

In order to investigate the presence and prevalence of Lyme borreliosis (Lyme disease) Borrelia species, 312 unfed ticks were collected by flagging at a woodland area in Trakya, in the European side of Turkey, in May 2002. Twelve of 299 Ixodes ricinus ticks were infected with Borrelia spp., as determined by cultivation in BSK medium (prevalence rate 4.0 %). Ten pure cultures were subjected to further characterization by sequencing analysis of the 5S–23S rDNA intergenic spacer, 16S rDNA and flagellin gene. One isolate of Borrelia burgdorferi sensu stricto, two of Borrelia garinii (Eurasian type), two of Borrelia afzelii, four of Borrelia lusitaniae and one of Borrelia valaisiana were identified. However, no Asian-type B. garinii was found. Interestingly, all Borrelia species that are known to be carried by I. ricinus were discovered among the 10 isolates. These results provide the first evidence for the existence of the Lyme borreliosis agent in Turkey.

A novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium (family Ixodidae, subfamily Metastriata) using Barbour–Stoenner–Kelly (BSK) II medium. Tick samples were taken during the summer of 2000 from the Istanbul area in northwestern Turkey. Sixty-seven of 153 adults (44 %) and 72 of 185 nymphs (39 %) were infected with the novel spirochaete, whereas none of the 20 larvae examined were infected. The optimal growth temperature of the spirochaete in BSK II medium was 34–37 °C, and it could grow at 39 °C. Doubling times at 34 and 37 °C were 5·3 and 5·1 h, respectively. Six pure cultures of the spirochaete were obtained and characterized by microscopic observation, sequence analysis of the flagellin gene (flaB), SDS-PAGE and Western blotting. The spirochaete was morphologically similar to those of the genus Borrelia and contained a 41 kDa protein reactive with mAb H9724 specific to the flagellin of a Borrelia species. Polyclonal antibody raised to this spirochaete reacted with several antigen bands, whereas no bands were detected with Borrelia burgdorferi, Borrelia hermsii, Borrelia turicatae and Borrelia parkeri. The flaB sequences of the six isolates showed high similarity, with sequence similarity values ranging from 99·2 to 100 %; however, the similarity of the isolates' flaB sequences to those of the Lyme-disease-related Borrelia and relapsing-fever-associated Borrelia species was less than 90 %. These findings suggest that the unique spirochaete is a member of the genus Borrelia, and differs from previously described Borrelia species.

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.

Leptospirosis in the Philippines is an underrepresented disease. To achieve an accurate means of serodiagnosis, we demonstrated antibodies to the prevalent Leptospira serovars in sera of 71 patients from three major hospitals in Manila by the microscopic agglutination test and Western blot analysis. Sera of 53 patients contained antibody against 8 serovars poi, tarassovi, manilae, pyrogenes, australis, grippotyphosa, javanica, and autumnalis.