Jpn. J. Infect. Dis., 57, 229-235, 2004

Invited Review Terrestrial Distribution of the Lyme Borreliosis Agent burgdorferi Sensu Lato in East Asia Toshiyuki Masuzawa* Department of Microbiology and COE Program in the 21st Century, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka 422-8526, Japan (Received September 3, 2004) CONTENTS: 1. Introduction 4. B. burgdorferi sl complex found in Japan 2. Lyme borreliosis agents and their classification 5. Geographical distribution of B. burgdorferi sl in 3. Relationship to the Borrelia burgdorferi sl complex East Asia 3-1. Transmission 6. Geographical distribution of B. burgdorferi sl in 3-2. Vectors the boundary between Asia and Europe 3-3. Reservoir 7. Conclusion 3-4. Clinical manifestation SUMMARY: Lyme borreliosis is the most prevalent -borne 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, 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 . 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 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.

the patients reported in the USA has steadily increased, and 1. Introduction more than 18,000 cases have been reported (8). Similarly, in Lyme borreliosis is the most prevalent tick-borne zoonosis Europe a high incidence rate has been reported from Central- and an important emerging infection in Europe, North Eastern Europe. The incidences per 100,000 people in Slovenia America, and Far Eastern countries (1-3). It was known and Austria are 120 and 130, respectively (9). initially as Lyme arthritis in the USA in the 1970s, and the town of Old Lyme in Connecticut, USA, was the initial focus 2. Lyme borreliosis agents and their classification of juvenile rheumatoid arthritis (4). Then, it became apparent that the arthritis was a late manifestation of tick-transmitted, Borreliae are roughly classified into two groups: relapsing multisystemic disease, which had been recognized in Europe fever-associated Borrelia spp. and Lyme borreliosis-related 100 years ago. The pathogen, a kind of spirochete, was discov- Borrelia spp. (B. burgdorferi sl spp. complex) (10). Relapsing ered by Burgdorfer and colleague (5) on the American vector fever-associated Borrelia spp., including Borrelia coriaceae, tick (former Ixodes dammini), and was are transmitted by soft ticks of the . The named Borrelia burgdorferi (B. burgdorferi sensu stricto [ss]) avian borreliosis agent, Borrelia anserina, is also carried by by Johnson et al. (6). Ixodes ricinus complex ticks are the soft tick Argas persicus. Other relapsing-fever-associated infected with the spirochete B. burgdorferi sensu lato (sl) spp. Borrelia spp. are Borrelia recurrentis, transmitted by the hu- complex, which causes the disease (7). To date, the number of man body louse Pediculus humanus, and the bovine borreliosis agent Borrelia theileri, transmitted by Rhipicephalus evertsi *Corresponding author: Mailing address: Department of Micro- and Boophilus spp. (10). Specificity occurs between Borrelia biology and COE Program in the 21st Century, University of spp. and tick spp. Lyme-borreliosis-related Borrelia are trans- Shizuoka School of Pharmaceutical Sciences, Yada 52-1, Shizuoka mitted by hard ticks of the Ixodes spp. complex. Initially, the 422-8526, Japan. Tel: +81-54-264-5710, Fax: +81-54-264-5715, agent occurs as one species. To date, the agents have been E-mail: [email protected] grouped in the B. burgdorferi sl spp. complex (7) and have been further classified into at least 11genomospecies (Fig. 1) This article is an Invited Review based on a lecture presented at (11), including the human pathogens B. burgdorferi ss, the 5th Infectious Diseases Forum at the National Institute of Borrelia garinii (7) and Borrelia afzelii (12). Infectious Diseases, Tokyo, 19-20 January 2004. B. burgdorferi sl is Gram-negative, microareophilic,

229 Species North Europe/ America West Russia East RussiaChina Nepal Japan Research (TIGR) and co-workers (13,14), and the total B. andersonii I. dentatus genome size was found to be 1,521 kbp. The ribosomal RNA I. spinipalpis B. bissettii △ (rRNA) gene of B. burgdorferi sl is unique with two copies I. pacificus B. burgdorferi ○ I. scapularis each of the 23S rRNA gene (rrl) and 5S rRNA gene (rrf)

B. lusitaniae being tandemly repeated but only one copy of the 16S rDNA gene (rrs) being located on the chromosome (15, 16). An B. garinii (Eurasian type) ○ I. ricinus intergenic spacer (about 250bp) between rrfA and rrsB is B. afzelii ○ I. persulcatus I. persulcatus highly variable and polymorphic (Fig. 3). PCR-restriction B. garinii (Asian type) ○ fragment length polymorphism (RFLP) analysis by Postic et

B. valaisiana △ I. ricinus I. columnae al. (17) showed a system of discrimination for B. burgdorferi sl that has become a widely used typing system (18). The B. valaisiana-related I. granulatus I. granulatus advantage of this method is that no amplification occurs with B. sinica I. ovatus the -associated Borrelia spp. because there is B. japonica I. ovatus no tandem repeat. Using this method, isolates of B. garinii B. tanukii I. tanuki are further divided into two subgroups: the B. garinii RFLP B. turdi I. turdus pattern B isolated from Europe and Asia (Eurasian-type B. Fig. 1. Genomospecies of Borrelia burgdorferi sensu lato and the garinii) and the RFLP pattern C of B. garini, which is only relationship between Borrelia spp. and vector tick spp. ○ and △ indicate found in Asia (Asian-type B. garinii). the species pathogenic to human and suspected of causing disease, respectively. 3. Relationship to the B. burgdorferi sl complex 3-1. Transmission The B. burgdorferi sl complex is transmitted by ixodid ticks spp. during feeding for over more than 48 h. Usually, ixodid ticks have a 2-year life cycle including three stages, larva, nymph, and adult, and at each stage, the tick takes blood from a variety of vertebrates. During feeding, Borrelia is transmit- ted from reservoir hosts to ticks. Borrelia can survive in

1.0μ m 0.5μ m the tick midgut during the digestion process and after tick- molting, the borrelia is transmitted to infested host from ticks during its blood-feeding. Since transovarial trans- Fig. 2. The morphology of B. burgdorferi sensu lato. Periplasmic flagella mission of Borrelia has rarely been observed, the agent is and its insertion points are shown. maintained between the ticks and reservoir animals (enzootic transmission cycle). 3-2. Vectors rrfB rrfA rrlA t-RNA rrlB rrs The major vectors are I. scapularis and in the USA, I. ricinus in Europe, and (Fig. RIS2 BB421 mag BB423 BB424 4) in Western Europe and Northern Asia (19). The American RIS1 PCR vector ticks, I. scapularis and I. pacificus harbor B. burgdorferi Primer RIS1; CTGCCGAGTTCGCCCCAGT ss, whereas I. ricinus is the prominent vector for transmitting RIS2; TCCTAGGCATTCACCATA five species, B. burgdorferi ss, B. garinii, B. afzelii, Borrelia (bp) 300 Amplicon (ca.250bp) Dra I RFLP lusitaniae (20), and Borrelia valaisiana (21), in European countries. Among these species, B. burgdorferi, B. afzelii, and

Mse I or Dra I 100 B. garinii are pathogenic to humans. On the other hand, the pathogenicity of B. lusitaniae and B. valaisiana still remains to be elucidated (18). The American tick Ixodes dentatus is RFLP analysis a restricted vector, and only Borrelia andersonii has been 20 ABCD

Fig. 3. The unique rDNA organization in B. burgdorferi sensu lato. 23S rRNA gene (rrlA and rrlB) and 5S rRNA gene (rrfA and rrfB) Ixodes persulcatus are tandemly repeated. Since the intergenic spacer between rrfA and rrlB is highly variable, the Borrelia spp. can be differentiated by RFLP analysis of the spacer. Representative RFLP patterns digested by the restriction enzyme DraI are illustrated. Pattern A, B. burgdorferi ss; Pattern B, Eurasian-type B. garinii (20047-type); Pattern C, Asian-type B. garinii (variant NT29 type); and Pattern D, B. afzelii. helical-shaped bacteria with 7 to 11 periplasmic flagella (10). Its length varies from 10 to 20 μm, and its width varies from Female Male 0.2 to 0.5 μm (Fig. 2). The organism has a unique genomic organization of one linear chromosome 910 kb in size and a Fig. 4. The vector tick Ixodes persulcatus found in Northern Asia. variety of 12 linear and 9 circular plasmids. The genome Left, adult female; right, adult male. The scale on the left indicates sequences were determined by the Institute of Genetic 1 mm.

230 isolated from this species. From these findings, the vector Japan. The other rare tick vectors, I. tanuki and I. turdus for B. burgdorferi sl is not strictly species-specific, whereas harbor Borrelia tanukii, and Borrelia turdi, respectively a molecular epidemiological study of the Japanese vector ticks (39,40), while the Japanese strain Am501, which has only spp. and Borrelia spp. contradicts this assumption (see Sec- been isolated from I. columnae, is now considered to be B. tion 4). valaisiana (21). The pathogenicity of these Borrelia remains 3-3. Reservoir to be determined. In the USA, B. andersonii and Borrelia bissettii specifi- cally reside in the cottontail rabbit (22) and wood mice (23), 5. Geographical distribution of B. burgdorferi sl respectively. In Europe, B. burgdorferi ss, B. afzelii, and B. in East Asia garinii OspA-serotype 4 are maintained in small mammals B. burgdorferi sl spp. have been well characterized in such as mice and voles, whereas the reservoir for other OspA Japan and Western Europe, whereas the status of Lyme serotypes of B. garinii and B. valaisiana are birds (24, 25). borreliosis is not yet clear in areas between Europe and Similarly, in Japan, isolates from birds or tick-infested birds Japan. To clarify this, surveys in Eastern Asia and Eastern have been identified as B. garinii, whereas Borrelia from small European countries have been conducted from 1995 to the mammals have been identified as B. afzelii and B. garinii. present (Fig. 5), and included Far-Eastern Russia (Khabarovsk, The characterization of these B. garinii indicated that B. Vladivostok, and Yuzhno-Sakhalinsk), western Russia (Mos- garinii variant ribotype IV (26, most are Asian-type B. garinii) cow), Turkey (Trakya, European side of Turkey), northern is maintained in bird and rodents, in contrast to isolates China (Yakeshi, Inner Mongolia), southern China (Zhejiang, from birds, which have been classified as belonging to the B. Sichuan, and Anhui provinces), Uighur in western China, garinii ribotype II (Eurasian-type B. garinii). These specific Korea, Taiwan, and Kinmen island, which is located offshore relationships are due to the sensitivity of these Borrelia spp. from Hisamen, Fujian province in China. Ticks were collected to host complements (27,28). Eurasian-type B. garinii is by flagging vegetation in woodland areas, and rodents were resistant to the serum of birds, whereas it is sensitive to the captured with Sherman live traps. Borrelia was cultured from serum of rodents. Similar to this, B. afzelii is resistant to the tick-midguts and ear-punched in BSKII medium. RFLP analy- serum of rodents and is sensitive to the serum of birds. sis of the 5S-23S rRNA intergenic spacer sequence was used 3-4. Clinical manifestation to determine the Borrelia spp. Furthermore, isolates were also In Japan, among the 20 identified species of ixodid ticks, characterized by sequence analysis of the intergenic spacer, Borrelia spp. have been isolated from I. persulcatus, Ixodes 16S rDNA (rrs) and the flagellin gene (flaB). ovatus, Ixodes tanuki, Ixodes turdus, and Ixodes columnae B. burgdorferi sl isolated from I. persulcatus collected in (2). Lyme borreliosis causes multisystemic disorders includ- the Far-Eastern regions of Russia, Inner Mongolia in north- ing skin symptoms, arthritis, neurological abnormalities, eastern China, Uighur in western China, and the northern part circulatory disturbances, conjunctivitis, muscular pain, and of South Korea were identified as Eurasian-type B. garinii gastrointestinal complaints (1,3). The initial symptom of Lyme (20047 type), as well as the Asian-type (variant NT29 type) borreliosis, erythema migrans (EM), is observed in 70 to 80% and B. afzelii (41-46). In contrast, the borrelia isolates of patients in the USA (3). A relationship exists between obtained from wild rodent species such as Apodemus clinical manifestations and causative Borrelia spp. In the USA, peninsulae captured in Khabarovsk and Inner Mongolia, as is characterized by arthritis, whereas in Euro- well as A. peninsulae, Apodemus agrarius, and Crocidura pean countries, neuroborreliosis and late skin manifestations lasiura from the northern part of South Korea, were identified such as achrodermatitis chronica atrophicans (ACA) are com- mon. The relationship between B. garinii and neuroborreliosis, and B. afzelii and ACA, respectively, has been suggested (29). Specific relationships between Borrelia spp., tick spp., reservoir animals and their clinical manifestations have been ① determined to exist based on several findings obtained from ② molecular epidemiological analyses of Borrelia isolates. Since ④ the distribution of Borrelia spp. and clinical symptoms of the B. afzelii ③ patient can be predicted from the distribution of tick spp. and B. garinii B. japonica ⑪ reservoir animals spp., molecular epidemiological surveys ⑤ provide useful information for diagnosis and prevention of Lyme borreliosis. ⑨ ⑧ ⑥ B. sinica ⑩ B. valaisiana-related ⑬ 4. B. burgdorferi sl complex in Japan ⑦

A recent epidemiological survey revealed a high preva- ⑫ lence of Borrelia in I. persulcatus and I. ovatus, the most common tick spp. to infect humans in Japan (30-32). I. Fig. 5. Geographical distribution of B. burgdorferi sl and sites surveyed persulcatus carries B. afzelii and B. garinii (33-36), whereas in East Asia from 1995 to the present. 1, Sakhalin; 2, Khabarovsk; 3, Vladivostok; 4, Yakeshi; 5, Chunchon, I. ovatus only transmits Borrelia japonica (37,38). B. Odaesan, Chungiu, and Haenam in Korea; 6, Okinawa; 7, Kinmen, burgdorferi sl spp. isolated from patients in Japan include B. Taichung, and Alishan in Taiwan; 8, Hangzhou; 9, Mt. Huangshan; garinii and B. afzelii, but not other Borrelia spp. (33,36). 10, Nanchuan; 11, Urumci; 12, Thailand, 13, Nepal. Furthermore, Moreover, Japanese B. garinii can be further classified into surveys in the Moscow region (Russia), Trakya (the western part of Turkey) and Serbia-Montenegro have been completed or are under two subtypes: a Eurasian-type (20047 type) found in Europe way. Other B. burgdorferi sl spp., B. tanukii, B. turdi, and B. valaisiana and Asia, and an Asian-type (variant NT29 type) found in (strain Am501 isolated from I. columnae) were found in Japan. B. Asia (17,39). B. burgdorferi ss has not yet been found in tanukii was also found to be present in Nepal (unpublished result).

231 as either Asian-type B. garinii or B. afzelii. Asian-type B. sensitivities of Borrelia to the sera of their reservoir host garinii and B. afzelii are naturally maintained between I. including rodent serum lysed B. valaisiana, but this is not the persulcatus and wild mammals. These observations have also case from bird serum (27,28). The findings that B. valaisiana- been made for the northern part of Japan. related groups in East Asia are present on wild rodents and In the middle of South Korea (Choongju), where I. insectivores further accentuates the phenotypic differences persulcatus is not found, Ixodes nipponensis is the main tick between B. valaisiana isolates found in Europe and Asia. The vector of these Borrelia spp. Although I. nipponensis is com- group Borrelia was isolated from I. granulatus (11,48), a tick monly found in Japan, no isolate has been found here. The sp. that inhabits the southern islands of Japan, Korea, and genetic differences between isolates of I. persulcatus and I. Southeast Asian countries where the B. valaisiana-related nipponensis remain to be investigated. group was found. Classification of this group of Borrelia Other Korean isolates of I. nipponensis collected at the remains to be determined. southern tip of the Korean Peninsula have been tentatively From a survey in the southern part of China (Sichuan and classified as belonging to the B. valaisiana-related group Anhui provinces along the Yangtze river valley), a unique (45). We have obtained similar borrelia isolates from rodents borrelia was isolated from I. ovatus and the wild rodent (Rattus losea, Mus formosanus) captured in Taiwan (Taichung Niviventer confucianus. These isolates showed low similarity and Kinmen Island) (47), from the tick, Ixodes granulatus of their rrs sequence to the sequences of previously described and wild rodents (A. agrarius and Niviventer sp.) captured in Borrelia spp., and can be distinguished by RFLP analysis of Zhejiang and Sichuan provinces located in the Yangtze river their 5S-23S rRNA intergenic spacer sequences. The borrelia valley in China (11), and from wild mammals (Suncus was named Borrelia sinica, a new member of the of Lyme murinus, Mus caroli, Rattus norvegicus, and Crocidura borreliosis-related Borrelia spp. (11). This species is distrib- watasei) in the Okinawa archipelago, the southern-most uted in central China and Nepal (unpublished results) but its islands of Japan (48). Strain Am501, isolated from the tick pathogenicity is unknown. The geographical distribution of I. columnae in Japan, is closely related to B. valaisiana Borrelia spp. in East Asia is summarized in Figure 5. B. garinii isolated from I. ricinus in Europe (21,39,45). However, these and B. afzelii are distributed into far eastern Russia, northern Asian isolates are divergent from B. valaisiana from Europe China, western China, Korea, and Japan. On the other hand, as shown by a phylogenetic tree constructed using flaB (Fig. B. valaisiana-related species are distributed in southern parts 6) and rrs data (data not shown) (48). Blackbirds and pheas- of Asia, such as south China, Taiwan, the southern tip of the ants have been identified as reservoir hosts for a B. valaisiana Korean Peninsula, Thailand, and the southernmost island in Europe (49,50), but these strains have not been isolated of Japan, Okinawa. B. sinica, transmitted by I. ovatus, is from wild mammals. A correlation exists between the distributed in central China and Nepal, whereas I. ovatus transmits B. japonica in Japan.

TA1 CKA2a (C) OM50 6. Geographical distribution of B. burgdorferi sl OM43 in the boundary between Asia and Europe OS66/01 CKA3b (C) I. persulcatus is the prevalent vector in the southern forest OS115/01 zone on the Asian side of Eurasia, from the western border 997 OC1/01 OG1/01 of Russia to its far eastern frontier bordering China, Korea, OG45/01 and Japan. However, on the Western side of Eurasia, most 614 OM92/01 OR5/01 European countries and North Africa harbors I. ricinus. KR3 (K) Turkey constitutes a bridge between European countries and 1000 CKA4a (C) 5MT (K) Asia. To elucidate the distribution and dissemination of OS49 vector ticks spp. and also Borrelia spp. between Europe and 618 OS31 B. valaisiana-related Asia, a survey held in Trakya on the Black Sea coast of the OS13 OS3/01 group European side of Turkey was conducted (51). Twelve of 299 1000 TM1 (T) I. ricinus were infected with Borrelia spp. (prevalence 844 10MT (K) 992 OM58/01 rate, 4.0%). Ten pure cultures were subjected to further char- CKA3a (C) 636 acterization by sequencing analysis of their 5S-23S rDNA 830 CKA1 (C) OS28/01 intergenic spacer, rrs and the flaB gene. One B. burgdorferi 999 504 OS42 ss, two B. garinii Eurasian-type, two B. afzelii, four B. VS116 B. valaisiana lusitaniae and one B. valaisiana isolates were identified. 805 Am501 B. valaisiana Hk501 B. tanukii However, no Asian-type B. garinii was found, and all Borrelia 0.01 VS461 B. afzelii spp. known to be carried by I. ricinus were discovered among HO14 B. japonica CMN3 B. sinica the isolates. Our results provide the first evidence of the 20047 B. garinii existence of a Lyme borreliosis agent in Turkey, and the Ya501 B. turdi situation in Turkey is similar to that in other Western European PotiB2 B. lusitaniae DN127 B. bissettii countries. 505 21123 B. andersonii In Asian countries such as Japan, the far eastern part of 888 B31 B. burgdorferi B. hermsii Russia, and the northern and western part of China, B. garinii and B. afzelii are transmitted by I. persulcatus. In contrast, B. Fig. 6. A phylogenetic tree based on the flagellin gene sequence. burgdorferi ss has not been found in these regions. European The Okinawa isolates are indicated in bold type, and isolates from China, Taiwan, and Korea indicated in parentheses are abbreviated as B. garinii only generates an RFLP B pattern (Eurasian- C, T, and K, respectively. The bar represents a sequence divergence type B. garinii, type20047). B. garinii from Japan (39), far of 1.0%. eastern (42,52) and western Russia (52), and China (43,46)

232 I. ricinus (National Institute of Infectious Diseases), M. Nakamura (Okinawa Prefectural Institute), E. S. Güner (Yeditepe Uni- B. lusitaniae B. valaisiana versity, Turkey), I. G. Kharitonenkov (Moscow University, B. burgdorferi sensu stricto Russia), K. H. Park (Konkuku University, Korea), M. K. Cho and W. H. Chang (Hallym University), X. Ma (Zhejiang B. garinii (Eurasian-type) Institute of Microbiology, China), M.-J. Pan (National Taiwan University, Taiwan), L.-K. Lin (Tunghai University, Taiwan), B. afzelii E. I. Korenberg (Gamaleya Institute, Russia), and H. Wang and J. Wang (Chinese Medical University, China). B. garinii (Asian-type) This study was supported in part by Grants-in-Aid for International Cooperative Research (No. 08044310, 08041181, I. persulcatus and l0041204), Grants-in-Aid for Scientific Research (No. 08670312, 09670294, 11670267) from the Ministry of Fig. 7. B. burgdorferi sl spp. transmitted by I. persulcatus and I. ricinus. Education, Culture, Sports, Science and Technology, Japan, A relationship between tick spp. and Borrelia spp. was found in and Grants-in-Aid for Scientific Research (B) No. 13576014 Moscow, a sympatric region where both tick spp. occur. from the Japan Society for the Promotion of Science (JSPS). exhibit either pattern B, or pattern C (referred as Asian-type REFERENCES B. garinii, variant NT29) originating from I. persulcatus collected in Japan. Asian-type B. garinii has not been found 1. Steere, A. C. (2001): Lyme disease. N. Engl. J Med., among European isolates from I. ricinus (51,52). Similarly, 345, 115-125. B. burgdorferi ss has not been isolated from I. persulcatus. 2. Yanagihara, Y. and Masuzawa, T. (1997): Lyme disease These observations suggest that I. ricinus and I. persulcatus (Lyme borreliosis). FEMS Immunol. Med. Microbiol., are not suitable vectors for Asian-type B. garinii and B. 18, 249-261. burgdorferi ss, respectively. The habitat of both species 3. Steere, A. 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