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1.  Human Babesiosis in Japan: Epizootiologic Survey of Rodent Reservoir and Isolation of New Type of Babesia microti-Like Parasite 
Journal of Clinical Microbiology  2001;39(12):4316-4322.
We have carried out epizootiologic surveys at various sites in Japan to investigate wild animals that serve as reservoirs for the agents of human babesiosis in the country. Small mammals comprising six species, Apodemus speciosus, Apodemus argenteus, Clethrionomys rufocanus, Eothenomys smithii, Crocidura dsinezumi, and Sorex unguiculatus, were trapped at various places, including Hokkaido, Chiba, Shiga, Hyogo, Shimane, and Tokushima Prefectures. Animals harboring Babesia microti-like parasites were detected in all six prefectures. Inoculation of their blood samples into hamsters gave rise to a total of 20 parasite isolates; 19 were from A. speciosus, and the other 1 was from C. rufocanus. Sequencing of the parasite small-subunit rRNA gene (rDNA) sequence revealed that 2 of the 20 isolates were classified as Kobe type because their rDNAs were identical to that of the Kobe strain (the strain from the Japanese index case). The other 18 isolates were classified as a new type, designated the Hobetsu type, because they all shared an identical rDNA sequence which differed significantly from both that of Kobe-type isolates and that of northeastern United States B. microti (U.S. type). The parasites with Kobe-, Hobetsu- and U.S.-type rDNAs were phylogenetically closely related to each other but clearly different from each other antigenically. The isolates from rodents were demonstrated to be infective for human erythrocytes by inoculation into SCID mice whose erythrocytes had been replaced with human erythrocytes. The results suggest that a new type of B. microti-like parasite, namely, the Hobetsu type, is the major one which is prevalent among Japanese wild rodents, that A. speciosus serves as a major reservoir for both Kobe- and Hobetsu-type B. microti-like parasites, and that C. rufocanus may also be an additional reservoir on Hokkaido Island.
doi:10.1128/JCM.39.12.4316-4322.2001
PMCID: PMC88542  PMID: 11724838
2.  Tick Surveillance for Relapsing Fever Spirochete Borrelia miyamotoi in Hokkaido, Japan 
PLoS ONE  2014;9(8):e104532.
During 2012–2013, a total of 4325 host-seeking adult ticks belonging to the genus Ixodes were collected from various localities of Hokkaido, the northernmost island of Japan. Tick lysates were subjected to real-time PCR assay to detect borrelial infection. The assay was designed for specific detection of the Relapsing fever spirochete Borrelia miyamotoi and for unspecific detection of Lyme disease-related spirochetes. Overall prevalence of B. miyamotoi was 2% (71/3532) in Ixodes persulcatus, 4.3% (5/117) in Ixodes pavlovskyi and 0.1% (1/676) in Ixodes ovatus. The prevalence in I. persulcatus and I. pavlovskyi ticks were significantly higher than in I. ovatus. Co-infections with Lyme disease-related spirochetes were found in all of the tick species. During this investigation, we obtained 6 isolates of B. miyamotoi from I. persulcatus and I. pavlovskyi by culture in BSK-M medium. Phylogenetic trees of B. miyamotoi inferred from each of 3 housekeeping genes (glpQ, 16S rDNA, and flaB) demonstrated that the Hokkaido isolates were clustered with Russian B. miyamotoi, but were distinguishable from North American and European B. miyamotoi. A multilocus sequence analysis using 8 genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) suggested that all Japanese B. miyamotoi isolates, including past isolates, were genetically clonal, although these were isolated from different tick and vertebrate sources. From these results, B. miyamotoi-infected ticks are widely distributed throughout Hokkaido. Female I. persulcatus are responsible for most human tick-bites, thereby I. persulcatus is likely the most important vector of indigenous relapsing fever from tick bites in Hokkaido.
doi:10.1371/journal.pone.0104532
PMCID: PMC4128717  PMID: 25111141
3.  Microbial Population Analysis of the Salivary Glands of Ticks; A Possible Strategy for the Surveillance of Bacterial Pathogens 
PLoS ONE  2014;9(8):e103961.
Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.
doi:10.1371/journal.pone.0103961
PMCID: PMC4121176  PMID: 25089898
4.  Borrelia burgdorferi sensu lato prevalence in tick populations in Estonia 
Parasites & Vectors  2013;6:202.
Background
Estonia is located in a unique area of co-distribution of Ixodes ricinus and I. persulcatus, which are the main tick vectors of Borrelia burgdorferi sensu lato. In the last decade, the incidence rate of Lyme borreliosis in Estonia has increased dramatically up to 115.4 per 100,000 in 2012. Here we present the first survey of the presence, the prevalence and genetic characteristics of B. burgdorferi s.l. complex spirochetes in the tick population in Estonia.
Methods
During the years 2006–2009, 2833 unfed Ixodes ricinus and I. persulcatus were collected from 43 sites in 7 counties in mainland Estonia as well as in 10 sites on the Saaremaa Island. DNA samples from ticks were analyzed individually using nested PCR of the ribosomal 5S-23S spacer region followed by bidirectional sequencing.
Results
The overall estimated prevalence of B. burgdorferi s.l was 9.7% and varied from 4.9% to 24.2% on the mainland and to 10.7% in Saaremaa Island. Ixodes persulcatus ticks showed significantly higher prevalence rates compared to that in I. ricinus-16.3% and 8.2%, respectively. The most prevalent genospecies was B. afzelii which was detected in 53.5% of Borrelia-positive ticks, followed by B. garinii and B. valaisiana with 26.2% and 5.5%, respectively. Also, B. bavariensis and B. burgdorferi s.s. DNA in single I. ricinus ticks were detected. Borrelia afzelii, B. garinii and B. valaisiana were detected in both tick species. Two genetic subgroups of B. garinii (NT29 and 20047) and two genetic subgroups of B. afzelii (NT28 and VS461) were found to be circulating in all studied regions as well as in both tick species, except B. garinii subgroup NT29, which was found only in I. persulcatus ticks.
Conclusions
In the current study we detected the circulation of five B. burgdorferi s.l. genospecies and estimated the prevalence in ticks in different regions of Estonia. Detection and genetic characterization of Borrelia genospecies, especially those of public health importance, in the natural foci may help assessing high risk areas of human exposure to B. burgdorferi s.l.
doi:10.1186/1756-3305-6-202
PMCID: PMC3716901  PMID: 23837798
Ticks; Borrelia burgdorferi sensu lato; Ixodes; Lyme disease
5.  A novel approach, based on BLSOMs (Batch Learning Self-Organizing Maps), to the microbiome analysis of ticks 
The ISME Journal  2013;7(5):1003-1015.
Ticks transmit a variety of viral, bacterial and protozoal pathogens, which are often zoonotic. The aim of this study was to identify diverse tick microbiomes, which may contain as-yet unidentified pathogens, using a metagenomic approach. DNA prepared from bacteria/archaea-enriched fractions obtained from seven tick species, namely Amblyomma testudinarium, Amblyomma variegatum, Haemaphysalis formosensis, Haemaphysalis longicornis, Ixodes ovatus, Ixodes persulcatus and Ixodes ricinus, was subjected to pyrosequencing after whole-genome amplification. The resulting sequence reads were phylotyped using a Batch Learning Self-Organizing Map (BLSOM) program, which allowed phylogenetic estimation based on similarity of oligonucleotide frequencies, and functional annotation by BLASTX similarity searches. In addition to bacteria previously associated with human/animal diseases, such as Anaplasma, Bartonella, Borrelia, Ehrlichia, Francisella and Rickettsia, BLSOM analysis detected microorganisms belonging to the phylum Chlamydiae in some tick species. This was confirmed by pan-Chlamydia PCR and sequencing analysis. Gene sequences associated with bacterial pathogenesis were also identified, some of which were suspected to originate from horizontal gene transfer. These efforts to construct a database of tick microbes may lead to the ability to predict emerging tick-borne diseases. Furthermore, a comprehensive understanding of tick microbiomes will be useful for understanding tick biology, including vector competency and interactions with pathogens and symbionts.
doi:10.1038/ismej.2012.171
PMCID: PMC3635243  PMID: 23303373
BLSOMs; emerging diseases; metagenomics; microbiomes; symbionts; ticks
6.  Genetic Identification of Rickettsiae Isolated from Ticks in Japan 
Journal of Clinical Microbiology  2002;40(6):2176-2181.
Following the description in Japan of Japanese spotted fever, caused by Rickettsia japonica, a search for the vector of this disease led to the isolation of several rickettsiae from various tick species. Sixty-three rickettsial isolates were obtained from six different tick species, and six type strains were described by PCR and monoclonal antibody testing. We identified these six strains by amplification and sequencing of the genes encoding 16S rRNA and citrate synthase. We confirmed that the isolates from Dermacentor taiwanensis and Haemaphysalis flava ticks were R. japonica isolates. In Ixodes ovatus, Ixodes persulcatus, and Ixodes monospinosus, we identified a Rickettsia identical or closely related to Rickettsia helvetica, a species that is pathogenic for humans and that to date has only been found in Europe. Finally, we identified a new genotype of unknown pathogenicity, genotype AT, that was isolated from Amblyomma testudinarium ticks and that is closely related to a Slovakian genotype obtained from Ixodes ricinus ticks.
doi:10.1128/JCM.40.6.2176-2181.2002
PMCID: PMC130657  PMID: 12037083
7.  Natural history of Zoonotic Babesia: Role of wildlife reservoirs 
Graphical abstract
Highlights
► All zoonotic Babesia utilize mammals, primarily wildlife, as reservoirs. ► Reservoirs and/or ticks are unknown for many zoonotic Babesia species. ► Molecular characterization of Babesia from human patients is needed. ► Continued surveillance of potential reservoirs and ticks should be conducted.
Babesiosis is an emerging zoonotic disease on all inhabited continents and various wildlife species are the principal reservoir hosts for zoonotic Babesia species. The primary vectors of Babesia are Ixodid ticks, with the majority of zoonotic species being transmitted by species in the genus Ixodes. Species of Babesia vary in their infectivity, virulence and pathogenicity for people. Various factors (e.g., increased interactions between people and the environment, increased immunosuppression, changes in landscape and climate, and shifts in host and vector species abundance and community structures) have led to an increase in tick-borne diseases in people, including babesiosis. Furthermore, because babesiosis is now a reportable disease in several states in the United States, and it is the most common blood transfusion-associated parasite, recognized infections are expected to increase. Because of the zoonotic nature of these parasites, it is essential that we understand the natural history (especially reservoirs and vectors) so that appropriate control and prevention measures can be implemented. Considerable work has been conducted on the ecology of Babesia microti and Babesia divergens, the two most common causes of babesiosis in the United States and Europe, respectively. However, unfortunately, for many of the zoonotic Babesia species, the reservoir(s) and/or tick vector(s) are unknown. We review the current knowledge regarding the ecology of Babesia among their reservoir and tick hosts with an emphasis of the role on wildlife as reservoirs. We hope to encourage the molecular characterization of Babesia from potential reservoirs and vectors as well from people. These data are necessary so that informed decisions can be made regarding potential vectors and the potential role of wildlife in the ecology of a novel Babesia when it is detected in a human patient.
doi:10.1016/j.ijppaw.2012.11.003
PMCID: PMC3862492  PMID: 24533312
Piroplasms; Tick-borne; Wildlife; Zoonoses
8.  Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes Ticks in the Baltic Regions of Russia 
Journal of Clinical Microbiology  2001;39(6):2237-2242.
The presence and distribution of Ehrlichia spp. and Borrelia burgdorferi sensu lato was demonstrated among ixodid ticks collected in the Baltic regions of Russia, where Lyme borreliosis is endemic. A total of 3,426 Ixodes ricinus and 1,267 Ixodes persulcatus specimens were collected, and dark-field microscopy showed that 265 (11.5%) I. ricinus and 333 (26.3%) I. persulcatus ticks were positive. From these samples, 472 dark-field-positive and 159 dark-field-negative ticks were subjected to PCR and subsequent reverse line blot hybridization. Fifty-four ticks (8.6%) carried Ehrlichia species, and 4 (0.6%) carried ehrlichiae belonging to the Ehrlichia phagocytophila complex, which includes the human granulocytic ehrlichiosis agent. The E. phagocytophila complex and an Ehrlichia-like species were detected only in I. ricinus whereas Ehrlichia muris was found exclusively in I. persulcatus, indicating a possible vector-specific infection. Borrelia garinii was found predominantly in I. persulcatus, but Borrelia afzelii was evenly distributed among the two tick species. Only two I. ricinus ticks carried B. burgdorferi sensu stricto, while Borrelia valaisiana and a newly identified B. afzelii-like species were found in 1.7 and 2.5% of all ticks, respectively. Of the dark-field-positive ticks, only 64.8% yielded a Borrelia PCR product, indicating that dark-field microscopy may detect organisms other than B. burgdorferi sensu lato. These observations show that the agent of human granulocytic ehrlichiosis may be present in ticks in the Baltic regions of Russia and that clinicians should be aware of this agent as a cause of febrile disease.
doi:10.1128/JCM.39.6.2237-2242.2001
PMCID: PMC88117  PMID: 11376063
9.  Identification of Parasitic Communities within European Ticks Using Next-Generation Sequencing 
Background
Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents.
Methodology/Principal Findings
We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species.
Conclusions/Significance
Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.
Author Summary
Diseases transmitted by ticks have diverse etiology (viral, bacterial, parasitic) and are responsible for high morbidity and mortality rates around the world, both in humans and animals. The emergence or re-emergence of tick-borne diseases is increasingly becoming a problem as the geographical distribution of several tick species is expanding, as well as the numbers of potential or known tick-borne pathogens are constantly evolving. It is thus necessary to know which microorganisms circulate within communities of this major vector to ensure adequate epidemiological surveillance. In this study, we evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce, without a priori, an inventory of both predicted and non-expected parasites carried by Ixodes ricinus, the most prevalent human biting tick in France. Our findings suggest that NGS strategies could be used to produce an inventory of live parasites residing in ticks from a selected area, thereby expanding our knowledge base of tick-associated parasites.
doi:10.1371/journal.pntd.0002753
PMCID: PMC3967966  PMID: 24675738
10.  Epidemiological Survey of Babesia Species in Japan Performed with Specimens from Ticks Collected from Dogs and Detection of New Babesia DNA Closely Related to Babesia odocoilei and Babesia divergens DNA 
Journal of Clinical Microbiology  2003;41(8):3494-3498.
Detection and analysis of Babesia species from ticks recovered from dogs in Japan were attempted by PCR and nucleotide sequence analysis based on the 18S rRNA gene, respectively. A total of 1,136 ticks were examined for Babesia DNA by 18S rRNA-based PCR and nucleotide sequencing. Partial sequences of Babesia canis vogeli DNA were detected from six ticks in Aomori, Nara, Hiroshima, Oita, and Okinawa Prefectures; and Babesia gibsoni Asia-1 DNA was also detected in four ticks in Osaka, Hiroshima, Miyazaki, and Okinawa Prefectures. Unique sequences of 1,678 bp were also obtained from Ixodes ovatus ticks in Akita and Fukui Prefectures. The sequences were similar to those of Babesia odocoilei (97.7%) and Babesia divergens (97.6%). This is the first report of the detection of DNA belonging to this group in Japan.
doi:10.1128/JCM.41.8.3494-3498.2003
PMCID: PMC179768  PMID: 12904344
11.  Tickborne Pathogen Detection, Western Siberia, Russia 
Emerging Infectious Diseases  2005;11(11):1708-1715.
Ixodes and Dermacentor ticks harbor Borrelia, Anaplasma/Ehrlichia, Bartonella, and Babesia species.
Ixodes persulcatus (n = 125) and Dermacentor reticulatus (n = 84) ticks from Western Siberia, Russia, were tested for infection with Borrelia, Anaplasma/Ehrlichia, Bartonella, and Babesia spp. by using nested polymerase chain reaction assays with subsequent sequencing. I. persulcatus ticks were infected with Borrelia burgdorferi sensu lato (37.6% ± 4.3% [standard deviation]), Anaplasma phagocytophilum (2.4% ± 1.4%), Ehrlichia muris (8.8% ± 2.5%), and Bartonella spp. (37.6% ± 4.3%). D. reticulatus ticks contained DNA of B. burgdorferi sensu lato (3.6% ± 2.0%), Bartonella spp. (21.4% ± 4.5%), and Babesia canis canis (3.6% ± 2.0%). Borrelia garinii, Borrelia afzelii, and their mixed infections were observed among I. persulcatus, whereas B. garinii NT29 DNA was seen in samples from D. reticulatus. Among the I. persulcatus ticks studied, no Babesia spp. were observed, whereas B. canis canis was the single subspecies found in D. reticulatus.
doi:10.3201/eid1111.041195
PMCID: PMC3367347  PMID: 16318722
Ixodes persulcatus; Dermacentor reticulatus; Borrelia burgdorferi sensu lato; Anaplasma phagocytophilum; Ehrlichia muris; Bartonella; Babesia canis canis; nested PCR; sequencing; research
12.  Anaplasma phagocytophilum–infected Ticks, Japan 
Emerging Infectious Diseases  2005;11(11):1780-1783.
We report Anaplasma phagocytophilum infection of Ixodes persulcatus and I. ovatus ticks in Japan. Unique p44/msp2 paralogs (and/or 16S rRNA genes) were detected in tick tissues, salivary glands, and spleens of experimentally infected mice. These findings indicate the public health threat of anaplasmosis in Japan.
doi:10.3201/eid1111.050407
PMCID: PMC3367361  PMID: 16318739
Anaplasma phagocytophilum; anaplasmosis; Ixodes; Japan; p44 protein; 16S ribosomal RNA; dispatch
13.  Wild Cervids Are Host for Tick Vectors of Babesia Species with Zoonotic Capability in Belgium 
Abstract
Babesiosis is a tick-borne disease caused by different species of intraerythrocytic protozoan parasites within the genus Babesia. Different species of Babesia are described as potentially zoonotic and cause a malaria-like disease mainly in immunocompromised humans. Interest in the zoonotic potential of Babesia is growing and babesiosis has been described by some authors as an emergent zoonotic disease. The role of cervids to maintain tick populations and act as a reservoir host for some Babesia spp. with zoonotic capability is suspected. To investigate the range and infection rate of Babesia species, ticks were collected from wild cervids in southern Belgium during 2008. DNA extraction was performed for individual ticks, and each sample was evaluated for the absence of PCR inhibition using a PCR test. A Babesia spp. genus-specific PCR based on the 18S rRNA gene was applied to validated tick DNA extracts. A total of 1044 Ixodes ricinus ticks were collected and 1023 validated samples were subsequently screened for the presence of Babesia spp. DNA. Twenty-eight tick samples were found to be positive and identified after sequencing as containing DNA representing: Babesia divergens (3), B. divergens-like (5), Babesia sp. EU1 (11), Babesia sp. EU1-like (3), B. capreoli (2), or unknown Babesia sp. (4). This study confirms the presence of potentially zoonotic species and Babesia capreoli in Belgium, with a tick infection rate of 2.7% (95% CI 1.8,3.9%). Knowledge of the most common reservoir source for transmission of zoonotic Babesia spp. will be useful for models assessing the risk potential of this infection to humans.
doi:10.1089/vbz.2011.0722
PMCID: PMC3319931  PMID: 22214270
Babesia species; Belgium; Cervids; Tick; Zoonotic
14.  Survey of Rodents and Ticks in Human Babesiosis Emergence Area in Japan: First Detection of Babesia microti-Like Parasites in Ixodes ovatus 
Journal of Clinical Microbiology  2004;42(5):2268-2270.
Babesia microti-like parasites were detected for the first time in Ixodes ovatus in Hyogo Prefecture, Japan, where two reported types of B. microti-like parasites were recognized in many rodents. Of 80 adult I. ovatus ticks collected, 5 possessed the reported type and 1 possessed a new type of B. microti-like parasite.
doi:10.1128/JCM.42.5.2268-2270.2004
PMCID: PMC404679  PMID: 15131209
15.  Antigenic properties of Borrelia burgdorferi isolated from Ixodes ovatus and Ixodes persulcatus in Hokkaido, Japan. 
Journal of Clinical Microbiology  1991;29(8):1568-1573.
Spirochete strains HP3 and HO14, isolated from Ixodes persulcatus and I. ovatus in Hokkaido in 1989, were the first isolates of Borrelia burgdorferi, the etiological agent of Lyme disease, to be recognized in Japan. Antigenic properties of the Japanese strains were compared with those of the strains isolated in the United States (B31 and 297) and Europe (IRS, P/Gau, P/Bi, 2/B45, and 3/B56) by Western blotting (immunoblotting), by using monoclonal antibodies (MAbs) against strains B31 and P/Bi. The Japanese strains reacted with MAb U40 against the 41-kDa antigen. MAb E34a31 against Osp A reacted with all the strains tested except for strain HP3. Furthermore, MAb U31b against Osp A reacted with all the American and European strains but did not react with the Japanese strains. When MAbs against Osp B were used, MAb E34b reacted only with European strains and MAb U34b reacted only with the American strains. However, neither showed reactivity to two Japanese strains. MAb E60 against 60-kDa antigen reacted with all the U.S. and European strains and strain HP3 but did not react with Japanese strain HO14. These results indicate that the antigenicity of the Japanese strains isolated from two species of ixodid ticks is different from that of the strains isolated in the United States and Europe. It is suggested that the Japanese strains are much more suitable than the U.S. or European strains as the antigen source for the serodiagnosis of Lyme disease in Japan.
Images
PMCID: PMC270164  PMID: 1761676
16.  Prevalence and diversity of Babesia spp. in questing Ixodes ricinus ticks from Norway 
Parasites & Vectors  2012;5:156.
Background
Ixodes ricinus ticks transmit Babesia species to vertebrate hosts. Using molecular tools we were able to detect the presence of this piroplasmid in its vector. The aims of this study were to investigate the prevalence and identity of Babesia species in questing ticks collected in various areas of Norway.
Methods
DNA from questing l. ricinus ticks were examined with a realtime PCR for the presence of Babesia. Positive samples of tick DNA were identified to species using PCR, and sequence analysis.
Results
From a total of 1908 questing l. ricinus ticks, 17 (0.9%) indicated the presence of Babesia spp. after realtime-PCR screening. Ixodes ricinus harbouring Babesia spp. was detected in 9 out of 22 localities. Further molecular analyses of DNA from these positive ticks indicate the presence of Babesia venatorum, B. divergens, B. capreoli and a currently undescribed Babesia in Norwegian ticks. The most prevalent was B. venatorum found in 71% of the positive ticks.
Conclusions
A total of 17 out of 1908 (0.9%) ticks were positive for Babesia. Our data confirm that there are several Babesia species in ticks in Norway. Babesia venatorum was the most prevalent. This species has a zoonotic potential and may cause human babesiosis following a tick bite.
doi:10.1186/1756-3305-5-156
PMCID: PMC3439691  PMID: 22862883
Babesia spp; Questing Ixodes ricinus; Zoonosis; Piroplasmosis; Realtime PCR; Prevalence; Sequencing
17.  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.
PMCID: PMC91932  PMID: 10698761
18.  Detection and Genetic Characterization of Relapsing Fever Spirochete Borrelia miyamotoi in Estonian Ticks 
PLoS ONE  2012;7(12):e51914.
During the years 2008–2010 I. ricinus and I. persulcatus ticks were collected from 64 sites in mainland Estonia and on the island Saaremaa. Presence of B. miyamotoi was found in 0.9% (23/2622) of ticks. The prevalence in I. persulcatus and I. ricinus ticks differed significantly, 2.7% (15/561) and 0.4% (8/2061), respectively. The highest prevalence rates were in found South-Eastern Estonia in an area of I. persulcatus and I. ricinus sympatry and varied from 1.4% (1/73) to 2.8% (5/178). Co-infections with B. burgdorferi s.l. group spirochetes and tick-borne encephalitis virus were also revealed. Genetic characterization of partial 16S rRNA, p66 and glpQ genes demonstrated that Estonian sequences belong to two types of B. miyamotoi and cluster with sequences from Europe and the European part of Russia, as well as with sequences from Siberia, Asia and Japan, here designated as European and Asian types, respectively. Estonian sequences of the European type were obtained from I. ricinus ticks only, whereas the Asian type of B. miyamotoi was shown for both tick species in the sympatric regions.
doi:10.1371/journal.pone.0051914
PMCID: PMC3522604  PMID: 23251652
19.  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%.
PMCID: PMC168014  PMID: 8779571
20.  Identification of Tick-Borne Pathogens in Ticks Feeding on Humans in Turkey 
Background
The importance of tick-borne diseases is increasing all over the world, including Turkey. The tick-borne disease outbreaks reported in recent years and the abundance of tick species and the existence of suitable habitats increase the importance of studies related to the epidemiology of ticks and tick-borne pathogens in Turkey. The aim of this study was to investigate the presence of and to determine the infection rates of some tick-borne pathogens, including Babesia spp., Borrelia burgdorferi sensu lato and spotted fever group rickettsiae in the ticks removed from humans in different parts of Ankara.
Methodology/Principal Findings
A total of 169 ticks belonging to the genus Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus were collected by removing from humans in different parts of Ankara. Ticks were molecularly screened for Babesia spp., Borrelia burgdorferi sensu lato and spotted fever group rickettsiae by PCR and sequencing analysis. We detected 4 Babesia spp.; B. crassa, B. major, B. occultans and B. rossi, one Borrelia spp.; B. burgdorferi sensu stricto and 3 spotted fever group rickettsiae; R. aeschlimannii, R. slovaca and R. hoogstraalii in the tick specimens analyzed. This is the report showing the presence of B. rossi in a region that is out of Africa and in the host species Ha. parva. In addition, B. crassa, for which limited information is available on its distribution and vector species, and B. occultans, for which no conclusive information is available on its presence in Turkey, were identified in Ha. parva and H. marginatum, respectively. Two human pathogenic rickettsia species (R. aeschlimannii and R. slovaca) were detected with a high prevalence in ticks. Additionally, B. burgdorferi sensu stricto was detected in unusual tick species (H. marginatum, H. excavatum, Hyalomma spp. (nymph) and Ha. parva).
Conclusions/Significance
This study investigates both the distribution of several tick-borne pathogens affecting humans and animals, and the presence of new tick-borne pathogens in Turkey. More epidemiological studies are warranted for B. rossi, which is very pathogenic for dogs, because the presented results suggest that B. rossi might have a wide distribution in Turkey. Furthermore, we recommend that tick-borne pathogens, especially R. aeschlimannii, R. slovaca, and B. burgdorferi sensu stricto, should be taken into consideration in patients who had a tick bite in Turkey.
Author Summary
Ticks are widespread in over all Turkey. Primary tick-borne diseases (TBDs), such as theileriosis, babesiosis, and anaplasmosis, affecting animals have been known for a long time in Turkey. However, TBDs have become a major concern in humans in recent years due to the recent Crimean-Congo hemorrhagic fever outbreak in Turkey. We know that some TBDs like CCHF, Lyme borreliosis, spotted fever group rickettsiosis, babesiosis and anaplasmosis exist in this geography. However, the other diseases except for CCHF are neglected in patients. In this study, we aimed to investigate Babesia spp., Borrelia burgdorferi sensu lato and spotted fever group rickettsiae in ticks removed from humans in different parts of Ankara by using PCR and sequencing. The result of this study showed that 4 Babesia species, one B. burgdorferi sensu lato, and 3 spotted fever group rickettsia are detected in ticks. The most striking result of this study is that B. rossi, which is very pathogenic for dogs, was reported for the first time from a region that is out of Africa and in Ha. parva. Therefore, B. rossi should be considered in dogs in Turkey. Furthermore, we propose that R. aeschlimannii, R. slovaca, and B. burgdorferi sensu stricto should be taken into consideration in patients who had a tick bite in Ankara.
doi:10.1371/journal.pntd.0003067
PMCID: PMC4125308  PMID: 25101999
21.  Ticks and associated pathogens collected from dogs and cats in Belgium 
Parasites & Vectors  2013;6:183.
Background
Although Ixodes spp. are the most common ticks in North-Western Europe, recent reports indicated an expanding geographical distribution of Dermacentor reticulatus in Western Europe. Recently, the establishment of a D. reticulatus population in Belgium was described. D. reticulatus is an important vector of canine and equine babesiosis and can transmit several Rickettsia species, Coxiella burnetii and tick-borne encephalitis virus (TBEV), whilst Ixodes spp. are vectors of pathogens causing babesiosis, borreliosis, anaplasmosis, rickettsiosis and TBEV.
Methods
A survey was conducted in 2008-2009 to investigate the presence of different tick species and associated pathogens on dogs and cats in Belgium. Ticks were collected from dogs and cats in 75 veterinary practices, selected by stratified randomization. All collected ticks were morphologically determined and analysed for the presence of Babesia spp., Borrelia spp., Anaplasma phagocytophilum and Rickettsia DNA.
Results
In total 2373 ticks were collected from 647 dogs and 506 cats. Ixodes ricinus (76.4%) and I. hexagonus (22.6%) were the predominant species. Rhipicephalus sanguineus (0.3%) and D. reticulatus (0.8%) were found in low numbers on dogs only. All dogs infested with R. sanguineus had a recent travel history, but D. reticulatus were collected from a dog without a history of travelling abroad. Of the collected Ixodes ticks, 19.5% were positive for A. phagocytophilum and 10.1% for Borrelia spp. (B. afzelii, B. garinii, B. burgdorferi s.s., B. lusitaniae, B. valaisiana and B. spielmanii). Rickettsia helvetica was found in 14.1% of Ixodes ticks. All Dermacentor ticks were negative for all the investigated pathogens, but one R. sanguineus tick was positive for Rickettsia massiliae.
Conclusion
D. reticulatus was confirmed to be present as an indigenous parasite in Belgium. B. lusitaniae and R. helvetica were detected in ticks in Belgium for the first time.
doi:10.1186/1756-3305-6-183
PMCID: PMC3688525  PMID: 23777784
Ticks; Dermacentor reticulatus; Dogs; Cats; Belgium; Borrelia; Anaplasma; Rickettsia
22.  Granulocytic Ehrlichiae in Ixodes persulcatus Ticks from an Area in China Where Lyme Disease Is Endemic 
Journal of Clinical Microbiology  2000;38(11):4208-4210.
A total of 372 adult Ixodes persulcatus ticks were collected from vegetation in a forest area of Heilongjiang Province in northeastern China, where Lyme disease is known to be endemic. The ticks were examined for the presence of granulocytic ehrlichiae by heminested PCR with primers derived from the 16S rRNA gene. Of 310 ticks obtained from the Dahe forestry farm, two pools (each containing 5 ticks) were found positive, with a minimum infection rate of 0.6%. Ehrlichial DNA was also detected in one female (1.6%) of 62 ticks collected from the Yulin forestry farm. The overall minimum infection rate of the 372 I. persulcatus adults was 0.8%. The nucleotide sequences of 919-bp PCR products from the three positive tick specimens were identical to each other and very closely related to the members of the Ehrlichia phagocytophila genogroup. This is the first identification of granulocytic ehrlichiae in ticks in Asia and the first report of infection in I. persulcatus anywhere.
PMCID: PMC87564  PMID: 11060091
23.  Prevalence of Tick-Borne Pathogens in Ixodes ricinus and Dermacentor reticulatus Ticks from Different Geographical Locations in Belarus 
PLoS ONE  2013;8(1):e54476.
Worldwide, ticks are important vectors of human and animal pathogens. Besides Lyme Borreliosis, a variety of other bacterial and protozoal tick-borne infections are of medical interest in Europe. In this study, 553 questing and feeding Ixodes ricinus (n = 327) and Dermacentor reticulatus ticks (n = 226) were analysed by PCR for Borrelia, Rickettsia, Anaplasma, Coxiella, Francisella and Babesia species. Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). Pathogens with low prevalence rates in ticks were Anaplasma phagocytophilum (2.2%), Coxiella burnetii (0.9%), Francisella tularensis subspecies (0.7%), Bartonella henselae (0.7%), Babesia microti (0.5%) and Babesia venatorum (0.4%). On a regional level, hotspots of pathogens were identified for A. phagocytophilum (12.5–17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%), suggesting established zoonotic cycles of these pathogens at least at these sites. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Identified hotspots of infected ticks should be included in future surveillance studies, especially when F. tularensis ssp. and C. burnetii are involved.
doi:10.1371/journal.pone.0054476
PMCID: PMC3551763  PMID: 23349900
24.  Quantitative PCR for Detection of Babesia microti in Ixodes scapularis Ticks and in Human Blood 
Vector Borne and Zoonotic Diseases  2013;13(11):784-790.
Abstract
Babesia microti, the primary cause of human babesiosis in the United States, is transmitted by Ixodes scapularis ticks; transmission may also occur through blood transfusion and transplacentally. Most infected people experience a viral-like illness that resolves without complication, but those who are immunocompromised may develop a serious and prolonged illness that is sometimes fatal. The geographic expansion and increasing incidence of human babesiosis in the northeastern and midwestern United States highlight the need for high-throughput sensitive and specific assays to detect parasites in both ticks and humans with the goals of improving epidemiological surveillance, diagnosis of acute infections, and screening of the blood supply. Accordingly, we developed a B. microti-specific quantitative PCR (qPCR) assay (named BabMq18) designed to detect B. microti DNA in tick and human blood samples using a primer and probe combination that targets the 18S rRNA gene of B. microti. This qPCR assay was compared with two nonquantitative B. microti PCR assays by testing tick samples and was found to exhibit higher sensitivity for detection of B. microti DNA. The BabMq18 assay has a detection threshold of 10 copies per reaction and does not amplify DNA in I. scapularis ticks infected with Babesia odocoilei, Borrelia burgdorferi, Borrelia miyamotoi, or Anaplasma phagocytophilum. This highly sensitive and specific qPCR assay can be used for detection of B. microti DNA in both tick and human samples. Finally, we report the prevalence of B. microti infection in field-collected I. scapularis nymphs from three locations in southern New England that present disparate incidences of human babesiosis.
doi:10.1089/vbz.2011.0935
PMCID: PMC3822370  PMID: 24107203
Babesia microti; Ixodes scapularis; Babesiosis; Quantitative PCR; Babesia odocoilei; Ticks
25.  Lyme Disease Borrelia spp. in Ticks and Rodents from Northwestern China 
Applied and Environmental Microbiology  2001;67(11):5161-5165.
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.
doi:10.1128/AEM.67.11.5161-5165.2001
PMCID: PMC93285  PMID: 11679340

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