Characterization of spirochetes isolated from ticks (Ixodes tanuki, Ixodes turdus, and Ixodes columnae) and comparison of the sequences with those of Borrelia burgdorferi sensu lato strains.

Ixodes persulcatus serves as a tick vector for Borrelia garinii and Borrelia afzelii in Japan; however, unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 from Ixodes tanuki, 6 from Ixodes turdus, and 1 from Ixodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction fragment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that these Borrelia strains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates of I. tanuki and C. rufocanus as group Hk501 and all isolates of I. turdus as group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme disease Borrelia species known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 from I. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.     

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.     

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.     

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.     

Diversity of Borrelia burgdorferi sensu lato in Russian Far East

Thirty strains of Borrelia burgdorferi sensu lato have been isolated from Ixodes persulcatus ticks and from skin lesions of Lyme disease patients in the Russian Far East from 1997 to 2003. We amplified full-length outer surface protein A (ospA) gene of all strains. BLAST search and following phylogenetic analysis showed that strains form four well-defined groups. Four strains belong to Borrelia afzelii species. Other strains distributed into tree major groups, identified as Borrelia garinii. Indeed, based on the ospA gene comparison, phylogenetic relationship of these groups among each other does not differ from relationship among other previously defined groups inside B. burgdorferi sensu lato genogroup, such as B. afzelii or Borrelia bissettii. Further investigations of genetic and serologic properties of the strains belonging to those groups are required in order to clarify their taxonomic status.     

Genetic characterization of pathogenic Borrelia, group A14S, isolated in Ukraine

Borrelia Ir-5215, isolated from ticks Ixodes ricinus in Ukraine (the Crimean autonomous region), was identified by the method of the polymorphism of the fragment length of the restriction amplicon of rRNA spacer region 5S-23S. Its Msel-restriction profile was relatively similar to that of B. afzelii. The sequencing of spacer region rrf (5S)-rrl (23S) and 16S rRNA gene, as well as the analysis of the similarity of nucleotide sequences, obtained in the course of these study, revealed the differences between Borrelia sp, lr-5215 and six European species of Borrelia burgdorferi sensu lato and a high level of similarity (more than 95.1% for 5S-23S rRNA and 99.4% for 16S rRNA gene) to three known representatives of genome group A14S (Borrelia spp. A14S, I-77 and PC-Rq17). This suggests that isolated Borrelia lr-5215 is a new representative of pathogenic B. burgdorferi sensu lato genome group A14S, which is spread, together with Central Europe, also in southern Ukraine.     

Genetic features of Borrelia miyamotoi transmitted by Ixodes persulcatus

The definition and molecular typing of Borrelia miyamotoi transmitted by the Ixodes persuccatus ticks was based on the partial sequencing of the 16S rRNA, p66, and glpQ genes. All the B. miyamotoi analyzed sequences of the 16S rRNA, glpQ, and p66 gene fragments from I. persulcatus were identical and had 99.9-100% similarity to corresponding genes of the B. miyamotoi strain FR64b isolated in Japan. The analyzed amino acid sequences revealed that the 66 protein B. miyamotoi in the site corresponding to the surface-exposed domain contained considerable difference from the Borrelia hermsii, the typical member tick-born relapsing fever, as from Borrelia lonestari transmitted by the Ixodes ticks.     

Genetic Analysis of Borrelia burgdorferi Sensu Lato in Korea Using Genomic Hybridization and 16S rRNA Gene Sequence Determination

Nine Borrelia burgdorferi sensu lato isolated in Korea were subjected to genomic hybridization using 16S rRNA gene probe and specific restriction patterns (HindIII and EcoRV) led these nine Borrelia into five subtypes. The evolutionary relationships of the five isolates corresponding to five RFLP groups were measured through the sequence determination of 16S rRNA gene and phylogenetic analysis. The isolates 935T (group I), 934U and 17Y (Group IIa, IIb) were well clustered with B. garinii and B. afzelii. 5MT and 9MT strains (Group IIIa and Group IIIb) formed a common branch shared with B. afzelii cluster although the evolutionary distance was rather long. So, most of B. burgdorferi sensu lato in Korea was B. afzelii or B. afzelii-related group and some minor group such as B. garinii also existed.     

Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis.

We studied 48 Borrelia isolates that were associated with Lyme borreliosis or were isolated from ticks and identified three DNA relatedness groups by using the S1 nuclease method. The three DNA groups (genospecies) were associated with specific rRNA gene restriction patterns, protein electrophoresis patterns, and patterns of reactivity with murine monoclonal antibodies. Genospecies I corresponded to Borrelia burgdorferi sensu stricto since it contained the type strain of this species (strain ATCC 35210); this genospecies included 28 isolates from Europe and the United States. Genospecies II was named Borrelia garinii sp. nov. and included 13 isolates from Europe and Japan. Genospecies III (group VS461) included seven isolates from Europe and Japan.     

Metascardovia criceti Gen. Nov., Sp. Nov., from hamster dental plaque

A novel microorganism, Metascardovia criceti gen. nov., sp. nov., was isolated from dental plaque of golden hamsters fed with a high-carbohydrate diet. The three isolated strains, OMB104, OMB105, and OMB107, were Gram-positive, facultative anaerobic rods that lacked catalase activity. Analyses of the partial 16S rRNA and heat-shock protein 60 (HSP60) gene sequences of these isolates indicated that they belonged to the family Bifidobacteriaceae. However, in contrast to Bifidobacterium, one of the genera under this family, these isolates grew under aerobic conditions, and the DNA G + C contents were lower (53 mol%) than those of Bifidobacterium. On the basis of phylogenetic analyses using phenotypic characterization, and partial 16S rRNA and HSP60 gene sequences data, we propose a novel taxa, Metascardovia criceti for OMB105(T) (type strain=JCM 13493(T)=DSM 17774(T)) for this newly described isolate.     

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.     

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.     

A Novel Obligate Intracellular Gamma-Proteobacterium Associated with Ixodid Ticks,Diplorickettsia massiliensis,Gen. Nov., Sp. Nov

Background

Obligate intracellular bacteria of arthropods often exhibit a significant role in either human health or arthropod ecology.

Methodology/Principal Findings

An obligate intracellular gamma-proteobacterium was isolated from the actively questing hard tick Ixodes ricinus using mammalian and amphibian cell lines. Transmission electron microscopy revealed a unique morphology of the bacterium, including intravacuolar localization of bacteria grouped predominantly in pairs and internal structures composed of electron-dense crystal-like structures and regular multilayer sheath-like structures. The isolate 20B was characterized to determine its taxonomic position using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that this strain belongs to the family Coxiellaceae, order Legionellales of Gamma-proteobacteria, and the closest relatives are different Rickettsiella spp. The level of 16S rRNA gene sequence similarity between strain 20B and other recognized species of the family was below 94.5%. Partial sequences of the rpoB, parC and ftsY genes confirmed the phylogenetic position of the new isolate. The G+C content estimated on the basis of whole genome analysis of strain 20B was 37.88%. On the basis of its phenotypic and genotypic properties, together with phylogenetic distinctiveness, we propose that strain 20B to be classified in the new genus Diplorickettsia as the type strain of a novel species named Diplorickettsia massiliensis sp. nov.

Conclusions/Significance

Considering the source of its isolation (hard tick, often biting humans) the role of this bacterium in the pathology of humans, animals and ticks should be further investigated.     

An Ixodes minor and Borrelia carolinensis enzootic cycle involving a critically endangered Mojave Desert rodent

Microtus californicus scirpensis is an endangered, isolated subspecies of California vole. It requires water pools and riparian bulrush (Schoenoplectus americanus) and occupies some of the rarest habitat of any North American mammal. The minimally vegetated, extremely arid desert surrounding the pools is essentially uninhabitable for Ixodes species ticks. We describe an enzootic cycle of Borrelia carolinensis in Ixodes minor ticks at a site 3500 km distant from the region in which I. minor is known to occur in Tecopa Host Springs, Inyo County, eastern Mojave Desert, California. Voles were live‐trapped, and ticks and blood samples queried by PCR and DNA sequencing for identification and determination of the presence of Borrelia spp. Between 2011–2013, we found 21 Ixodes minor ticks (prevalence 4–8%) on Amargosa voles and Reithrodontomys megalotis. DNA sequencing of 16S rRNA from ticks yielded 99% identity to I. minor. There was 92% identity with I. minor in the calreticulin gene fragment. Three ticks (23.1%), 15 (24%) voles, three (27%) house mice, and one (7%) harvest mice were PCR positive for Borrelia spp. Sequencing of the 5S‐23S intergenic spacer region and flagellin gene assigned Amargosa vole Borrelia strains to B. carolinensis. Ixodes minor, first described in 1902 from a single Guatemalan record, reportedly occurs only in the southeast American on small mammals and birds. The source of this tick in the Mojave Desert and time scale for introduction is not known but likely via migratory birds. Borrelia strains in the Amargosa ecosystem most closely resemble B. carolinensis. B. carolinensis occurs in a rodent‐I. minor enzootic cycle in the southeast U.S. although its epidemiological significance for people or rodents is unknown. The presence of a tick and Borrelia spp. only known from southeast U.S. in this extremely isolated habitat on the other side of the continent is of serious concern because it suggests that the animals in the ecosystem could be vulnerable to further incursions of pathogens and parasites.     

Isolation and characterization of a novel Borrelia group of tick-borne borreliae from imported reptiles and their associated ticks

The members of the genus Borrelia are transmitted by arthropods and known to be infectious to vertebrates. Here we found isolates and DNAs belonging to the Borrelia turcica and unknown Borrelia species from imported reptiles and their ectoparasites. The Borrelia strains were isolated from blood and multiple organs of exotic tortoises, and were experimentally infectious to captive-bred tortoises. These findings suggest that these tortoises may be a candidate as the reservoir host of the Borrelia species. In this study, the Borrelia strains were also isolated from and/or detected in hard-bodied ticks, Amblyomma ticks and Hyalomma ticks. In some of these ticks, immunofluorescence imaging analysis revealed that the Borrelia had also invaded into the tick salivary glands. Accordingly, these ticks were expected to be a potential vector of the Borrelia species. Sequencing analyses of both housekeeping genes ( flaB gene, gyrB gene and 16S rDNA gene) and 23S rRNA gene-16S rRNA gene intergenic spacer region revealed that these Borrelia strains formed a monophyletic group that was independent from two other Borrelia groups, Lyme disease Borrelia and relapsing fever Borrelia . From these results, the novel group of Borrelia comprises the third major group of arthropod-transmitted borreliae identified to date.     

Genetic diversity and the absence of regional differences of Borrelia garinii as demonstrated by ospA and ospB gene sequence analysis

Unfed adult Ixodes persulcatus ticks were collected from four locations of Nagano and Hokkaido in Japan. Infected Borrelia garinii were investigated by PCR-RFLP of the ospA and ospB gene sequences. The primer set amplified an approximately 1.6-kb DNA fragment (0.7-kb in some strains), and BsrI, BstYI, or NlaIII digestion of the product resulted in six distinctively different PCR-RFLP groups and two independent borrelial strains. The representatives in each PCR-RFLP group and individuals from the borrelial strains were sequenced, and their deduced amino acid sequences were aligned. A neighbor-joining phylogenetic analysis showed that the B. garinii OspA or OspB sequences were each divided into three major clusters including isolates from both the Nagano and Hokkaido locations. There was no local difference in OspA/B sequences between Nagano and Hokkaido. The osp gene of Borrelia burgdorferi sensu lato is highly heterogeneous, and this was also confirmed by our sequence analysis. Some strains of the different PCR-RFLP groups had closely related OspA sequences, while the OspB sequences of these strains were quite different. These findings suggested intraspecies gene exchange and recombination events between the two genes in B. garinii.     

Infectivity and Early Antibody Response to Borrelia burgdorferi Sensu Lato Isolated in Japan in Outbred Mice

Borrelia burgdorferi sensu lato isolated from Ixodes ovatus (B. japonica), I. persulcatus and patients with erythema migrans (EM) in Japan were determined on infectivity and arthritis induction-activity in outbred mice. Infectivity of B. japonica was weak and did not induce the development of footpad swelling by subcutaneous (s.c.) inoculation into the footpad. Challenged strain, NO129-M of B. japonica, to ddY mice were reinoculated to the mice at various cell numbers (1 × 10-1 × 10⁶ cells/mouse). The strain isolated from the mouse did not reinfect ddY mice and did not induce the production of specific antibody to the homologous strain. On the other hand, strains from I. persulcatus and patients with EM in Japan infected the mice and induced a serious inflammatory response in Borrelia-inoculated footpad as well as strains belonging to the three genospecies, B. burgdorferi sensu stricto, B. garinii, and B. afzelii, related to Lyme disease, from North America and Europe. The mice were infected with 10 cells of strain HP1 isolated from I. persulcatus in Hokkaido and of strain 297 isolated from a patient in the U.S.A. by subcutaneous inoculation into the hind footpad, or by intradermal inoculation into the back. Antigens of ca. 20, 23–24 (Osp C), 29, 39, 41 (flagellin) and 45 kDa reacted with the pooled sera from mice inoculated with strains HP1 and 297, but Osp A and Osp B did not.     

Species of Borrelia distinguished by restriction site polymorphisms in 16S rRNA genes

Abstract Three phyletic groups of Borrelia associated with Lyme disease, B. burgdorferi, B. garinii and group VS461 can be distinguished from each other and other species of Borrelia by Bfa I restriction site polymorphisms in PCR amplified 16S rRNA genes. One strain isolated from an Ixodes pacificus tick in California that was previously unclassifiable was distinguishable from B. burgdorferi by an Mnl I restriction site polymorphism.     

Detection of Borrelia burgdorferi sensu lato by PCR in questing Ixodes ricinus larvae from the Dutch North Sea island of Ameland

In May 1995, 61 Ixodes ricinus larvae were collected from vegetation on the Dutch North Sea island of Ameland. Fifty-seven lysates (94%) were analysed for the presence of Borrelia DNA by the polymerase chain reaction (PCR), which amplified the intergenic spacer region between the 5S and 23S rRNA genes. Three samples (5%) were positive and hybridization of the PCR product to genomic group-specific probes revealed that two larvae were infected with Borrelia afzelii and group VS116, respectively and one larval tick carried a mixed infection of B. afzelii and Borrelia garinii. Previously, we showed that these three genomic groups were present in adult and nymphal ticks collected on Ameland. Transovarial transmission may be an important factor in the establishment of these genomic groups in the local tick population.     

Prevalence and distribution of Borrelia and Babesia species in ticks feeding on dogs in the U.K.

Ticks were collected during March–July 2015 from dogs by veterinarians throughout the U.K. and used to estimate current prevalences and distributions of pathogens. DNA was extracted from 4750 ticks and subjected to polymerase chain reaction and sequence analysis to identify Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) and Babesia (Piroplasmida: Babesiidae) species. Of 4737 ticks [predominantly Ixodes ricinus Linneaus (Ixodida: Ixodidae)], B. burgdorferi s.l. was detected in 94 (2.0%). Four Borrelia genospecies were identified: Borrelia garinii (41.5%); Borrelia afzelli (31.9%); Borrelia burgdorferi sensu stricto (25.5%), and Borrelia spielmanii (1.1%). One Rhipicephalus sanguineus Latreille (Ixodida: Ixodidae), collected from a dog with a history of travel outside the U.K., was positive for B. garinii. Seventy ticks (1.5%) were positive for Babesia spp. Of these, 84.3% were positive for Babesia venatorum, 10.0% for Babesia vulpes sp. nov., 2.9% for Babesia divergens/Babesia capreoli and 1.4% for Babesia microti. One isolate of Babesia canis was detected in a Dermacentor reticulatus (Ixodida: Ixodidae) tick collected from a dog that had recently travelled to France. Prevalences of B. burgdorferi s.l. and Babesia spp. did not differ significantly between different regions of the U.K. The results map the widespread distribution of B. burgdorferi s.l. and Babesia spp. in ticks in the U.K. and highlight the potential for the introduction and establishment of exotic ticks and tick‐borne pathogens.