Consensus Sequence on the Genes Encoding the Major Outer Surface Proteins (OspA and OspB) of Borrelia garinii Isolate

Japanese Lyme borrelias classified as ribotype IV is predominant among isolates derived from clinical specimens, reservoir rodents and Ixodes persulcatus ticks, and has been characterized as Borrelia garinii. These B. garinii isolates have antigenic and genetic features apparently different from North American, European and other Asian isolates, especially in major outer surface proteins A (OspA) and B (OspB). In this study, we cloned and sequenced the genes encoding OspA and OspB from B. garinii strain FujiP2 (ribotype IV strain) isolated from I. persulcatus in Shizuoka, Japan. A sequence analysis revealed significant differences to the previously published sequences of ospA and ospB of B. burgdorferi sensu lato. The open reading frames of ospA and ospB consist of 822 and 888 nucleotides corresponding to the proteins of 273 and 295 amino acids, with molecular weights of 29,643 and 31,786 daltons, respectively. The most interesting finding is that the two osp genes share a consensus 282 bp sequence in their carboxy-terminal portions and that the ospB gene is flanked by a 282 bp-long direct repeat sequence. The deduced amino-acid (aa) sequences of OspA and OspB of strain FujiP2 showed 60.1% homology, and have overall similarities of 70.5%, 70.3% and 75.6% to OspAB proteins of B. burgdorferi sensu stricto strain B31, Borrelia afzelii strain ACA1 and Borrelia garinii strain Ip90, respectively.     

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.     

Identification of Borrelia burgdorferi Isolated in Korea Using Outer Surface Protein A (OspA) Serotyping System

Two characteristic strains (935T, 934U) of B. burgdorferi isolated from Ixodes persulcatus and a wild rodent (Apodemus agrarius) in Korea were selected and analyzed by an immunoblot method using the monoclonal antibodies directed to different epitopes of outer surface protein A (OspA). The reactive pattern of strain 934U with these monoclonal antibodies was identical to that of strains belonging to B. afzelii and that of strain 935T was different from other isolates. Monoclonal antibody (5TEE3) which is specific to strain 935T did not react with any other Western and Japanese isolates. So, it was suggested that there exist at least two groups of B. burgdorferi in Korea. One could be classified as B. afzelii and the other is a divergent group from three known species of B. burgdorferi sensu stricto, B. garinii and B. afzelii.     

DNA‐based identification and OspC serotyping in cultures of Borrelia burgdorferi s.l. isolated from ticks collected in the Moravia (Czech Republic)

Two different genetic loci, flaB and ospC, were employed to assign genospecies and OspC phylogenetic type to 18 strains isolated from ticks collected in Pisárky, a suburban park in the city of Brno, Czech Republic. The RFLP analysis revealed three different genospecies (B. afzelii, B. garinii, and B. valaisiana). Three samples from the collection contained more than one genospecies. In the other 15 strains, nucleotide sequences of flaB and ospC were determined. The following phylogenetic analysis assigned 12 isolates to genospecies B. garinii and three to B. afzelii. These isolates were further subdivided into seven distinct ospC groups. The most related OspC types were G2, G4, and G5 (B. garinii) and A3 and A8 (B. afzelii).     

Borrelia japonica in Nature: Genotypic Identification of Spirochetes Isolated from Japanese Small Mammals

Spirochetes were isolated from earlobe tissues of shrews (Sorex unguiculatus, Sorex caecutiens, and Crocidura dsinezumi), voles (Clethrionomys rufocanus), and mice (Apodemus argenteus and Apodemus speciosus) captured in various localities in Japan. The isolates were identified as Borrelia japonica by rRNA gene restriction fragment length polymorphism analysis. The data suggest that these small mammals are candidates of reservoir hosts for B. japonica.     

Granulocytic Ehrlichia infection in Ixodid ticks and mammals in woodlands and uplands of the U.K.

Abstract .The prevalence of infection with Ehrlichiae of the Ehrlichia phagocytophila genogroup (the granulocytic Ehrlichiae), in questing Ixodes ricinus ticks of U.K. upland and woodland habitats, was investigated by PCR. The prevalence of infection in the three feeding stages of I. ricinus indicated that granulocytic Ehrlichiae are transmitted transstadially with no, or inefficient, transovarial transmission. The presence of infected ticks in both habitats indicates that endemic cycles of granulocytic Ehrlichia (GE) infection are maintained by both domesticated sheep and by wild reservoirs, and coexist with endemic cycles of Borrelia burgdorferi infection. Moreover, demonstration, for the first time, of GE infection in engorged Ixodes trianguliceps ticks and blood collected from wild rodents, suggests that European wild rodents are competent reservoirs. GE infection prevalence in nymphal and adult I. ricinus was significantly greater in uplands than woodlands, which is consistent with ticks of all three feeding stages feeding on reservoir-competent sheep in uplands. In one woodland studied, pheasants are important hosts for nymphal I. ricinus but are incompetent or inefficient reservoirs, so reducing GE infection prevalence in I. ricinus ticks in this habitat. 16S rRNA sequences of GE from ticks of these U.K. habitats, showed a high degree of homology with those of granulocytic Ehrlichiae isolated from humans, but also showed some evidence of genetic diversity of granulocytic ehrlichiae in the U.K. The implications of these findings, for the taxonomy of granulocytic ehrlichiae and the potential for human infections to occur in the U.K., is discussed.     

Horizontal and vertical movements of host‐seeking Ixodes pacificus (Acari: Ixodidae) nymphs in a hardwood forest

The nymph of the western black‐legged tick (Ixodes pacificus) is an important bridging vector of the Lyme disease spirochete (Borrelia burgdorferi) to humans in the far‐western United States. The previously unknown dispersal capabilities of this life stage were studied in relation to logs, tree trunks, and adjacent leaf‐litter areas in a mixed hardwood forest using mark‐release‐recapture methods. In two spatially and temporally well‐spaced trials involving logs, the estimated mean distances that nymphs dispersed ranged from ≈0.04 to 0.20 m/day on logs vs 0.11 to 0.72 m/day in litter. Prior to recapture in either trial and within the confines of the sampling grids, the greatest estimated dispersal distances by individual nymphs released on logs, and in litter 0.5 m or 1.5 m from logs, were 2.4, 3.0, and 3.0 m, respectively. Nymphs released on logs or litter tended to remain within the same biotopes in which they were freed while host‐seeking. In two simultaneous trials involving trunks spaced close‐at‐hand, nymphs released at the trunk/litter interface on all four aspects collectively dispersed a mean of 0.353 m/day on trunks vs 0.175 m/day in litter. In either trial, the greatest distances that recaptured nymphs climbed trunks, or dispersed in litter in an encircling 3‐m grid, were 1.55 m and 2.97 m, respectively. Nymphs ascending trunks did not exhibit a preference for any one aspect, and the B. burgdorferi‐infection prevalences in nymphs that climbed trunks (3.2–4.0%) did not differ significantly from those that moved horizontally into litter (10.5–17.6%). We conclude that I. pacificus nymphs use an ambush host‐seeking strategy; that they disperse slowly in all biotopes studied; that they usually continue to host‐seek in or on whatever substratum they access initially; and that B. burgdorferi‐infected nymphs are as likely to move horizontally as vertically when offered a choice.     

Effect of forest clearing on the abundance of Ixodes ricinus ticks and the prevalence of Borrelia burgdorferi s.l

Questing Ixodes ricinus L. (Acari: Ixodidae) ticks were collected on a forest trail that had been completely cleared of shrubs and ground vegetation in winter 2002 and on a nearby control uncleared forest transect in South Moravia (Czech Republic). Samples were collected each May in 2003, 2004 and 2005. Nymphal ticks were 3.4 times, 1.9 times and 1.2 times less frequent on cleared forest than on uncleared forest trails in the three respective years, whereas adult tick abundance was 27.2 times, 4.0 times and 2.2 times lower, respectively. The ticks were examined for borreliae by dark-field microscopy: prevalence of nymphal ticks infected with Borrelia burgdorferi sensu lato (12.6% to 20.0%) did not differ significantly between the cleared and uncleared trail during the 3 years. In conclusion, the habitat modification appeared to result in a decreased abundance of I. ricinus as well as a reduced frequency of infected ticks (and thus indirectly a lower potential risk of Lyme borreliosis), which lasted, however, for only 2 years. Eight cultures of borreliae isolated from the ticks were all identified as the 'ornithophilic' genomic species Borrelia garinii, possibly indicating a greater role of forest birds than that of forest rodents as the hosts of immature I. ricinus in the tick (and borrelial) colonization of the cleared part of the forest.     

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.