Tick identification is important in control of tick-born diseases because tick-borne pathogens are often transmitted by specific tick species. In this study, we determined partial DNA sequences of the mitochondrial 16S rDNA gene (mt-rrs) for ticks including 7 genera and 39 species, and these ticks were allocated to 113 sequence types. Of the 39 species of ticks, 36 species (92.3%) were distinguishable by phylogenetic analysis of mt-rrs. This result suggests that species identification of ticks based on mt-rrs is a viable alternative to morphological identification. In order to establish a DNA database for identification of ixodid and argasid ticks in Japan, we deposited all sequence data in GenBank (from AB819156 to AB819268).
Abstract We retrospectively confirmed 2 cases of human Anaplasma phagocytophilum infection. Patient blood samples contained unique p44/msp2 for the pathogen, and antibodies bound to A. phagocytophilum antigens propagated in THP-1 rather than HL60 cells. Unless both cell lines are used for serodiagnosis of rickettsiosis-like infections, cases of human granulocytic anaplasmosis could go undetected.
Abstract Borrelia miyamotoi , a member of the tick-borne relapsing fever spirochetes, shows a serum-resistant phenotype in vitro. This ability of B. miyamotoi may contribute to bacterial evasion of the host innate immune system. To investigate the molecular mechanism of serum-resistance, we constructed a membrane protein-encoding gene library of B. miyamotoi using Borrelia garinii strain HT59G, which shows a transformable and serum-susceptible phenotype. By screening the library, we found that bom1093 and bom1515 of B. miyamotoi provided a serum-resistant phenotype to the recipient B. garinii . These B. miyamotoi genes are predicted to encode P35-like antigen genes and are conserved among relapsing fever borreliae. Functional analysis revealed that BOM1093 bound to serum vitronectin and that the C-terminal region of BOM1093 was involved in the vitronectin-binding property. Importantly, the B. garinii transformant was not serum-resistant when the C terminus-truncated BOM1093 was expressed. We also observed that the depletion of vitronectin from human serum enhances the bactericidal activity of BOM1093 expressing B. garinii , and the survival rate of BOM1093 expressing B. garinii in vitronectin-depleted serum is enhanced by the addition of purified vitronectin. Our data suggests that B. miyamotoi utilize BOM1093-mediated binding to vitronectin as a mechanism of serum resistance.
In Eurasia, the geographically most widespread ixodid tick species of the bat families Rhinolophidae Gray, Vespertilionidae Gray, and Miniopteridae Dobson were considered to belong to four species, Ixodesvespertilionis Koch, I.collaris Hornok, I.ariadnae Hornok, and I.simplex Neumann. Previous data attest that bat-associated tick species from Eastern Asia show remarkable genetic difference from the above four tick species, but in the absence of detailed morphological comparison these were regarded as conspecific. In this study we compensate for this lack of data on three bat-associated tick species, reporting their morphological comparison, as well as molecular and phylogenetic relationships. According to the results we describe the females of three tick species new to science, i.e., I.nipponrhinolophi Hornok & Takano, sp. nov., I.fuliginosus Hornok & Takano, sp. nov., and I.fujitai Hornok & Takano, sp. nov. In case of all three new tick species the cytochrome c oxidase subunit (coxI) gene showed remarkably high sequence differences from the species that they previously were thought to belong to, well exceeding the average limit delineating ixodid tick species. This, as well as observed morphological differences fully justify their taxonomical status as new species.
To the Editor: Tick-borne relapsing fever (TBRF) is caused by infection with spirochetes belonging to the genus Borrelia. We previously reported a human case of febrile illness suspected to be TBRF on the basis of serologic examination results; the vector most likely was a genus Carios tick that had fed on a seabird colony (1). However, surveillance of ticks in the area did not identify Borrelia spp. in any of the Carios ticks sampled (2). In 2007 and 2008, a borreliosis investigation was conducted on Kutsujima Island (35.71′N, 135.44′E) because a bird-associated tick, genus Carios, inhabits this island. During the investigation, 77 Carios ticks (55 nymphs, 11 adult males, and 11 adult females) were collected from colonies of seabirds: Swinhoe's storm petrel (Oceanodroma monorhis) and streaked shearwater (Calonectris leucomelas). Identification of tick species as C. sawaii was based on tick morphology and rrs gene sequence analysis of the tick mitochondrion DNA (2). Total DNA was extracted from the ticks by using a DNeasy Tissue Kit (QIAGEN, Germantown, MD, USA). For the detection of Borrelia DNA, PCR designed was based on the flagellin gene (flaB) according to Sato et al. (3). To check for contamination and amplicon carryover, we used blank tubes as a negative control for each experiment. Of 77 C. sawaii ticks that were positive by PCR of tick genes (2), 25 (14 nymphs, 6 adult males, 5 adult females) were positive for Borrelia DNA by PCR of flaB.
To characterize the Borrelia spp., we sequenced amplified fragments of the flaB gene and the 16S ribosomal RNA (16SrRNA) gene of Borrelia spp. in a tick and compared the results with those of representative Borrelia spp. The primers BflaPBU and BflaPCR (3) for flaB and the 4 PCR primers (Technical Appendix) for 16SrRNA were used for direct sequencing and/or amplification. DNA sequence (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AB491928,term_id:225703023,term_text:AB491928}}AB491928) of a 294-bp amplified fragment of flaB showed the following nucleotide similarities with those of Borrelia spp.: B. turicatae (98.98%), B. parkeri (98.30%), Borrelia sp. Carios spiro-1 (98.64%), and Borrelia sp. Carios spiro-2 (98.30%). DNA sequence (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AB491930,term_id:225703027,term_text:AB491930}}AB491930) of a 1,490-bp amplified fragment of 16SrRNA showed the following nucleotide similarities with those of Borrelia spp.: B. turicatae (99.60%), B. parkeri (99.53%), and Borrelia sp. Carios spiro-2 (99.45%). Borrelia sp. Carios spiro-1 and Carios spiro-2, which were recently identified in C. kelleyi in the United States, have been classified into TBRF Borrelia (4,5). The Borrelia sp. found in this study, designated as Borrelia sp. K64, was closely related to B. turicatae but was distinct from other TBRF Borrelia spp. (Technical Appendix).
To observe Borrelia spp. in tick tissues, we performed an indirect fluorescence assay (IFA) according to methods described by Fisher et al. (6), with minor modifications. A tick that was negative by PCRs of flab and 16SrRNA was used as a negative control. The IFA of the tick salivary gland and midgut was conducted by using acetone for fixation, goat anti-Borrelia sp. polyclonal immunoglobulin (Ig) G (1:100; KPL, Inc., Gaithersburg, MD, USA) as the primary antibody, and Alexa fluor 488-labeled rabbit antigoat IgG (1:200, Invitrogen, Carlsbad, CA, USA) as the secondary antibody. Our analysis showed a spirochete, which was stained by anti-Borrelia spp. antibody, in salivary gland and midgut tissue (Technical Appendix). However, no spirochetes were detected by IFA in the negative control (data not shown).
We also attempted to isolate Borrelia spp. from tick specimens by using Barbour-Stoenner-Kelly medium (7). The motility of Borrelia-like organisms in the medium was initially observed by using dark-field microscopy. The Borrelia-like organisms were identified as Borrelia sp. K64 by sequencing of PCR-amplified fragments of flaB and 16SrRNA genes from the cultured medium. However, these Borrelia organisms were found for only 2 weeks after inoculation (data not shown).
The vertebrate reservoir hosts of TBRF Borrelia are usually rodents but can be a variety of other animals (8). Although competence as a reservoir has not been determined for birds, infection of an owl with a TBRF Borrelia sp. has been reported (9). The vertebrate host of the spirochete has not yet been determined. Given our results, it is possible that seabirds are potential vertebrate hosts for Borrelia spp.
In Japan, relapsing fever is a neglected infectious disease because it was not reported during 1956–1998 (10). In this study, we detected a Borrelia sp. in C. sawaii, and the spirochete we characterized is closely related to B. turicatae. Although the human health implications of infections caused by Borrelia spp. are not yet known, the findings from this study should contribute to the epidemiologic investigation of TBRF in Japan.
Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fever." One of the candidate SFG Rickettsia species is "Candidatus Rickettsia kotlanii," which was first detected in Haemaphysalis concinna in Hungary in 2006. However, its precise phylogenetic position in the SFG is not clear because only single-gene sequence-based phylogenetic analyses were performed using very limited genes. Here, we present the complete genome sequences of two Japanese "Ca. R. kotlanii" isolates, which differed only by a 135 bp insertion/deletion (InDel). Using these genomes and publicly available whole genome sequences of other Rickettsia species, the precise phylogenetic position of "Ca. R. kotlanii" in Rickettsia was determined to be in a clade of the SFG. The phylogenetic relationships and average nucleotide identity of "Ca. R. kotlanii" relative to the other species indicated that "Ca. R. kotlanii" is an independent taxon in the SFG. Notably, although the genomes of the two isolates were almost identical, the isolates were obtained from different tick species in different regions and years, suggesting extremely low genomic diversity in "Ca. R. kotlanii." While the genome of "Ca. R. kotlanii" is the smallest in the transitional group and SFG Rickettsia sequenced to date, we identified genes uniquely present or absent in "Ca. R. kotlanii," but most were apparently degraded. Therefore, analyses of differences at the sequence (single nucleotide polymorphisms and small InDels) or gene expression level will be required to understand the functional or physiological features unique to "Ca. R. kotlanii."
The ecologies of Lyme disease Borrelia spp. are very specific to location, as they are dependent upon the spirochete species and genotypes, the vectors and the host vertebrates present. In Hokkaido, Japan, where two human pathogenic, Lyme disease Borrelia spp. are present, and human cases are reported annually, the ecologies have been poorly studied. Our goal was to determine whether variation in borrelial infection rates among rodent species sharing an environment, is due to immunological or ecological differences. To this end, we examined the relationships between tick burden and borrelial infection, by including examination of agreement between nested PCR, as a test for infection, and serology, as a test for exposure. We collected 868 rodents, comprised of four species commonly found in Hokkaido, and tested for infection rates with Borrelia spp. using PCR for the borrelial flaB gene, seroprevalence of Borrelia afzelii and Borrelia garinii using ELISA, and attachment of ticks by direct counts. We noted a correlation between differential nymph and larval burdens and the borrelial infection rates found among the four rodent species. Furthermore, there was significant correlation between infection and seroprevalence of B. afzelii and B. garinii (P<0.01), between infection and Ixodes persulcatus nymph burden (P<0.01), and between seroprevalence and I. persulcatus nymph burden (P<0.01). The close agreement among rodent species seroprevalences with infection rates and tick burdens suggest the differences in infection rates of Borrelia spp. may largely be a direct consequence of differential exposure to vectors.
