Genetic Diversity of Borrelia burgdorferi Sensu Lato Isolated in Far Eastern Russia

One-hundred and fifty-seven Borrelia isolated from adult ticks, Ixodes persulcatus, and wild rodents, Clethrionomys rufocanus and Apodemus peninsulae, in the far eastern part of Russia were characterized and identified by restriction fragment length polymorphism (RFLP) of the 5S-23S rRNA intergenic spacer. Some isolates showed unique RFLP patterns and were determined as Borrelia garinii on the basis of a sequence analysis of the intergenic spacer amplicon and reactivity with species-specific monoclonal antibodies (MAbs). 86.5 and 12.7% of the tick isolates, and 74.2 and 12.9% of the rodent isolates were determined as Borrelia garinii and Borrelia afzelii, respectively, but no Borrelia burgdorferi sensu stricto was detected. This finding is similar to the results obtained from Borrelia surveys of I. persulcatus and wild rodents in Hokkaido, Japan.     

Lipid Composition of Novel Shewanella Species Isolated from Far Eastern Seas

A comparative study of the lipid composition of 26 strains (including type strains) of marine Gammaproteobacteria belonging to the genera Shewanella, Alteromonas, Pseudoalteromonas, Marinobacterium, Microbulbifer, and Marinobacter was carried out. The bacteria exhibited genus-specific profiles of ubiquinones, phospholipids, and fatty acids, which can serve as reliable chemotaxonomic markers for tentative identification of new isolates. The studied species of the genus Shewanella were distinguished by the presence of two types of isoprenoid quinones, namely, ubiquinones Q-7 and Q-8 and menaquinones MK-7 and MMK-7; five phospholipids typical of this genus, namely, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), lyso-PE, and acyl-PG; and the fatty acids [15:0, 16:0, 16:1 (n-7), 17:1 (n-8), i-13:0, and i-15:0]. The high level of branched fatty acids (38–45%) and the presence of eicosapentaenoic acid (4%) may serve as criteria for the identification of this genus. Unlike Shewanella spp., bacteria of the other genera contained a single type of isoprenoid quinone: Q-8 (Alteromonas, Pseudoalteromonas, Marinobacterium, and Microbulbifer) or Q-9 (Marinobacter). The phospholipid compositions of these bacteria were restricted to three components: two major phospholipids (PE and PG) and a minor phospholipid, bisphosphatidic acid (Alteromonas and Pseudoalteromonas) or DPG (Marinobacterium, Microbulbifer, and Marinobacter). The bacteria exhibited genus-specific profiles of fatty acids.     

Lyme Disease Borrelia spp. in Ticks and Rodents from Northwestern China

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.     

Polymerase Chain Reaction Analysis of Borrelia Species Isolated in Japan

Primer reactivities of 25 Borrelia burgdorferi sensu lato isolates from the ticks, Ixodes persulcatus and I. ovatus, in Japan and 10 isolates in Europe and North America were investigated. The methods used in this study were the polymerase chain reaction (PCR) on the flagellin structural gene (fla), the outer surface protein A gene (osp A) and the outer surface protein B gene (osp B), and the restriction fragment length polymorphism (RFLP) analysis of PCR products from osp A and osp B, The flagellin PCR primer set reacted with all the Borrelia strains tested. Four genospecies, B. burgdorferi sensu stricto, B. garinii, B. afzelii and B. japonica, were differentiated by PCR using osp A and osp B primers combined with RFLP analysis. Some Japanese isolates from I. persulcatus were identified as B. garinii or B. afzelii. The other isolates from I. persulcatus did not fit in any of the 4 genospecies. These results suggested that Japanese isolates from I. persulcatus are highly heterogeneous in their osp A and osp B structures. Furthermore, PCR primers targeting fla are applicable to the gene diagnosis for Lyme disease in Japan and osp A and osp B primers can be used to classify B. burgdorferi sensu lato isolates into genospecies by PCR and RFLP analyses.     

Distribution of Borrelia Species Associated with Lyme Disease in the Subalpine Forests of Nagano Prefecture,Japan

We surveyed the natural distribution of Borrelia species associated with Lyme disease in the subalpine forests of Nagano prefecture, Japan, during 1993-94. Tick-derived isolates (n = 112) from Ixodes persulcatus and rodent-derived isolates (n = 55) from Apodemus argenteus, Apodemus speciosus, Eothenomys andersoni, Eothenomys smithii, and Microtus montebelli were classified by rRNA gene restriction fragment length polymorphism analysis (RFLP ribotyping). Ribotype group IV (an intraspecific variant of Borrelia garinii) was predominant among the tick isolates. It was also isolated repeatedly from the rodents. Ribotype group III (Borrelia afzelii) was detected in low frequencies among the tick and rodent isolates. The data suggest that humans are likely to be exposed to the group IV when they are bitten by I. persulcatus ticks.     

Pulsed Field Gel Electrophoresis Analysis of Borrelia burgdorferi Sensu Lato Isolated in Japan and Taxonomic Implications with Lyme Disease Spirochetes

Genomic DNAs of Borrelia burgdorferi sensu lato isolates obtained in Japan sharing different rRNA gene ribotypes were digested with rare-cutter restriction endonucleases and the fragments obtained were separated by pulsed field gel electrophoresis (PFGE). The sizes of large restriction cleavage bands with MluI endonuclease were quite similar among isolates in each ribotype group. On the other hand, the PFGE profiles obtained with the other enzymes (NruI, Sal I or SplI) were rather divergent, and Japanese isolates were distinguishable from the United States and European isolates. The Japanese isolates classified as ribotypes group II (Borrelia garinii) and III (B. afzelii) showed different PFGE patterns from that of European isolates. The isolates grouped into ribotype IV revealed distinctively different PFGE profiles. These results indicate that the Japanese isolates may be genetically divergent and distinct from the United States and European isolates.     

Comparative Studies on Borrelia afzelii Isolated from a Patient of Lyme Disease,Ixodes persulcatus Ticks,and Apodemus speciosus Rodents in Japan

The genospecies Borrelia afzelii was isolated from a patient of Lyme disease in Hokkaido, Japan, for the first time, by culturing the minced erythema lesion in BSK II medium. Two analytical methods, rRNA gene restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) using the specific primer set to amplify the 16S rRNA gene, revealed that this clinical isolate belongs to the group of B. afzelii. In our culture collection of spirochetes, part of the isolates from Ixodes persulcatus ticks, and from Apodemus speciosus rodents, were also classified as B. afzelii. These results strongly suggest that the agent pathogenic to humans is maintained in “rodent-tick” transmission cycle.     

In Vitro Antibiotic Susceptibilities of Borrelia Isolates from Erythema Migrans Lesion of Lyme Disease Patients in Japan

Antibiotic susceptibilities of twelve borrelial isolates from skin of patients with erythema migrans (EM) and ticks (Ixodes persulcatus and I. ovatus) in Japan were examined by in vitro microdilution MIC method and macrodilution MBC method. Nine EM isolates and 3 tick isolates were susceptible to amoxicillin, erythromycin, and minocycline. MICs for Japanese isolates were 0.038–0.30 μg/ml, < 0.012 μg/ml, and < 0.012–0.05 μg/ml, respectively. MBCs were as follows: 0.038–0.88 μg/ml, < 0.012–0.10 μg/ml, and <0.025–0.78 μg/ml, respectively. These antibiotics could be recommended for treatment of patients in early stage of Lyme disease in Japan.     

Negative Finding in Cross-Protective Activity of Japanese Borrelia Isolates against Infection with Three Species of Lyme Disease Borrelia in Outbred Mice

Outer surface protein A (OspA) is the most promising candidate for a component vaccine against Lyme disease caused by Borrelia burgdorferi sensu lato. Active cross-protection using a whole-cell vaccine prepared from strains belonging various OspA serotypes observed in Japan and worldwide was examined. No cross-protection was obtained by heterologous OspA-serotype vaccines. Since OspA is a highly variable protein expressed by Borrelia, this suggests that immunologically different OspA serotypes need to be combined for the development of an effective vaccine in Japan.     

Characterization of Borrelia Species Isolated from Ixodid Ticks,Ixodes ovatus

Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.     

Lyme disease Borrelia spp. in ticks and rodents from northwestern China.

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.     

Trichosporon Species Isolated from Guano Samples Obtained from Bat-Inhabited Caves in Japan

Yeasts from caves have rarely been examined. We examined yeasts collected from bat guano samples from 20 bat-inhabited limestone and volcanic caves located in 11 prefectures in Japan. Of ~700 yeast-like colonies, nine Trichosporon species were recovered from 15 caves. Two of these were known species, and the remaining seven are potentially novel species, based on molecular phylogenetic analyses. In addition to Trichosporon species, identifiable strains of eight ascomycetous yeasts and one basidiomycetous yeast were recovered at frequencies of 5 to 35%. Our findings suggest that Trichosporon spp. are the major yeast species in bat guano in Japan and that bat guano is a potentially rich source of previously undescribed yeast species.     

分离自东北地区主要蔬菜根茎上的真菌

为明确蔬菜土传病害病原菌的种类及筛选有益真菌,对采自我国东北地区主要蔬菜的根茎样本进行了组织分离和形态学鉴定,共鉴定出19个属30个种或变种,其中镰孢菌属为优势属,共分离得到10个种。研究结果表明：葫芦科蔬菜上分离的真菌种类最多,为11个属17个种。从采集地来看,在黑龙江和辽宁两省分离到的真菌数量较多,分别为12个属1...     

Identification of a New Borrelia Species among Small Mammals in Areas of Northern Spain Where Lyme Disease Is Endemic

The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.     

Immunological Properties of Borrelia burgdorferi Isolated from the Ixodes ovatus in Shizuoka,Japan

Three strains of spirochetes (IKA1 to 3) were isolated from the midgut of Ixodes ovatus collected in the Ikawa region of the northern part of Shizuoka, Japan. These isolates had eight flagella, and their size and other morphological features were similar to Borrelia burgdorferi. They showed similar motility and reacted with monoclonal antibody (MAb) H9724 against borrelial flagella and with MAb H5332 against the outer surface protein A. These strains showed similar SDS-PAGE profiles to that of B. burgdorferi strain B31 and P/Bi isolated in the U.S.A. and Europe, respectively. Immunoblot with Lyme disease patient serum showed positive reactions with the flagella (41 Kilodalton, kDa), protein C (20 to 22 kDa), and outer surface protein A (29 kDa) of the isolates. Immunological properties, morphological characteristics, and epidemiological features revealed that these isolates were B. burgdorferi.     

Prevalence of Lyme Disease Borrelia spp. in Ticks from Migratory Birds on the Japanese Mainland

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.     

Genomic Analysis of Borrelia japonica Sp. Nov. Isolated from Ixodes ovatus in Japan

Genetic characteristics of 12 Borrelia isolates from the tick, Ixodes ovatus, I. persulcatus, and the rodent, Apodemus speciosus ainu, in Japan were compared to members of the three genospecies of Borrelia burgdorferi sensu lato; B. burgdorferi sensu stricto, B. garinii and group VS461. The methods used in this study were the quantitative microplate DNA hybridization assay and restriction fragment length polymorphism (RFLP) analyses of the flagellin structural genes and the 16S rRNA genes. The six isolates from I. persulcatus and A. speciosus ainu were identified as genospecies B. garinii using RFLP analysis of the flagellin and 16S rRNA genes. In contrast, RFLP analysis of the six isolates from I. ovatus indicated that they were different from the three reported genospecies. DNA homology studies confirmed the RFLP results. The six isolates from I. ovatus had DNA homologies ranging from 85 to 99%, whereas DNA relatedness of the I. ovatus isolate with strains belonging to the three genospecies was 50 to 64%. These results suggest that the strains isolated from I. ovatus in Japan differ from the three genospecies and should be classified as a new genospecies of B. burgdorferi sensu lato. We propose that strains isolated from I. ovatus should be classified as B. japonica sp. nov.     

四川东北部箭竹属一新种

甜箭竹　甜竹 (四川万源 )　新种　图 1Ｆａｒｇｅｓｉａ　ｏｓｔｒｉｎａＹｉ,ｓｐ .ｎｏｖ .ＴＹＰＥ :Ｓｉｃｈｕａｎ (四川 ) ,Ｗａｎｙｕａｎ (万源 ) ,Ｈｕａｅｓｈａｎ(花萼山 ) ,ａｌｔ.2 1 0 0ｍ ,ｔｈｉｃｋｅｔｓ ,1 998- 1 1 - 2 1 ,Ｔ .Ｐ .Ｙｉ (易同培 ) 9882 3(ｔｙｐｅ ,ＳＩＦＳ) ;ｓａｍｅｌｏｃａｌｉｔｙ ,ａｌｔ.1 980～ 2 30 0ｍ ,1 998- 1 1 - 2 1 ,Ｔ .Ｐ .Ｙｉ (易同培 ) 9882 1 (ＳＩＦＳ) .ＳｐｅｃｉｅｓＦ .ｍｕｒｉｅｒａｅ (Ｇａｍｂｌｅ)Ｙｉａｆｆｉｎｉｓ ,ｓｅｄｃｕｌｍｉｓｊｕｎｉｏｒｉｂｕｓｐｒ…     

Organization of Ribosomal RNA Genes in Borrelia burgdorferi sensu lato Isolated from Ixodes ovatus in Japan

Borrelia burgdorferi sensu lato was obtained from adult ixodid ticks, Ixodes ovatus, collected in Nagano, Japan, and was named NT112. The genomic DNA was digested with enzymes, electrophoresed, blotted and hybridized with rRNA gene probes obtained from B. burgdorferi sensu stricto B31. The results showed that the borrelial chromosome contains a single rrs (16S rRNA gene) sequence and two copies of rrl/rrf (23S/5S rRNA genes) sequences. The rrl/rrf genes were tandemly repeated at intervals of 3.2 kb and were located separately from the rrs gene on the genome. Our findings indicate that the organization of rRNA genes in Borrelia from I. ovatus ticks is identical to that of B. burgdorferi sensu stricto.