Detection of novel piroplasmid species and Babesia microti and Theileria orientalis genotypes in hard ticks from Tengchong County, Southwest China.

To reveal the genetic diversity of Babesia microti and Theileria orientalis in Southwest China, we conducted a molecular survey of piroplasms in hard ticks in a China-Myanmar border county. Host infesting and questing ticks were collected from Tengchong County in 2013 and 2014. Piroplasm infection in ticks was detected by PCR, and then, phylogenetic analysis was conducted to study the genetic diversity of the pathogens identified in ticks. All in all, six piroplasm species comprising of B. microti; B. orientalis; a novel Babesia species designated Babesia sp. Tengchong, China; T. orientalis; T. luwenshuni; and an as yet undescribed piroplasmid species referred to as Piroplasmid sp. Tengchong, China, have been identified after screening goat- and cattle-attached ticks. In addition, B. bigemina has been identified by screening questing ticks. Phylogenetic analysis based on the 18S rRNA and partial ß-tubulin gene revealed two novel potentially zoonotic genotypes designated B. microti Tengchong-Type A and B. The T. orientalis genotypes identified in the present study represent the seven known genotypes 1-5, 7, and N3 as revealed by phylogenetic analysis of 18S rRNA and MPSP genes. Importantly, an additional genotype designated N4 has also been identified in this study, which brings the number of recognized T. orientalis genotypes to a total of twelve. Thus, besides the two novel species, Babesia sp. Tengchong, China, closely related to Babesia species isolated from yak and Piroplasmid sp. Tengchong, China, our study demonstrates that additional novel B. microti and T. orientalis genotypes exist in Southwest China.

Development of a SNP barcode to genotype Babesia microti infections.

Babesia microti is tick-borne disease that is an emerging threat to public health due to increasing prevalence and expanding geographic range. Detection and constant surveillance of babesiosis is imperative for predicting pathogen expansion. Leveraging our whole genome sequence (WGS) analyses of B. microti, we developed a single nucleotide polymorphism (SNP)-based high resolution melt (HRM) surveillance tool. We developed our HRM assay using available sequence data and identified 775 SNPs. From these candidate SNPs, we developed a 32-SNP barcode that is robust and differentiates geographically distinct populations; it contains SNPs that are putatively neutral, located in nuclear, mitochondrial, and apicoplastal regions. The assays are reproducible and robust, requiring a small quantity of DNA (limit of detection as low as 10 pg.). We analyzed the performance of our HRM assay using 26 B. microti clinical samples used in our WGS study from babesiosis endemic regions in the United States. We identified a minimal barcode consisting of 25 SNPs that differentiate geographically distinct populations across all clinical samples evaluated (average minor allele frequency > 0.22). Supporting our previous WGS findings, our 25-SNP barcode identified distinct barcode signatures that segregate B. microti into two lineages: Northeast and Midwest, with the Northeast having three subpopulations: Connecticut/Rhode Island, Nantucket, and the R1 reference group. Our 25-SNP HRM barcode provides a robust means genetic marker set that will aid in tracking the increasing incidence and expanding geographic range of B. microti infections.

Wide Distribution and Genetic Diversity of Babesia microti in Small Mammals from Yunnan Province, Southwestern China.

BACKGROUND: Babesia, usually found in wild and domestic mammals worldwide, have recently been responsible for emerging malaria-like zoonosis in infected patients. Human B. microti infection has been identified in China, primarily in the Southwest along the Myanmar border but little direct surveillance of B. microti infection in rodents has been carried out here (Yunnan province). In this region, a diverse topographic range combined with tropical moisture sustains a high biodiversity of small mammals, which might play important role on Babesia transmission. METHODS: Small mammals were captured in 141 sample locations from 18 counties located Yunnan Province, and screened for B. microti-like parasites infection by a nested PCR to target 18S rRNA gene of Babesia, plus directly sequencing for positive samples. Univariate and multivariate forward stepwise logistic regression analysis was used to access the association between infections and some related risk factors. RESULTS: Infection with Babesia microti was confirmed in 2.4% (53/ 2204) of small mammals. Significant differences in prevalence rates of B. microti were observed based on variations in forest, agricultural, and residential landscapes. Furthermore, adult small mammals had higher prevalence rates than younger, pubertal mammals. The near full-length 18S rRNA gene revealed that there were two types of B. microti, Kobe and Otsu, which demonstrate the genetic diversity and regional distribution. CONCLUSIONS: There exists a wide distribution and genetic diversity of endemic B. microti in Southwestern China, warranting further investigations and monitoring of clinical disease in individuals presenting with Babesia like symptoms in these areas.

Prevalence, genetic identity and vertical transmission of Babesia microti in three naturally infected species of vole, Microtus spp. (Cricetidae).

BACKGROUND: Vertical transmission is one of the transmission routes for Babesia microti, the causative agent of the zoonotic disease, babesiosis. Congenital Babesia invasions have been recorded in laboratory mice, dogs and humans. The aim of our study was to determine if vertical transmission of B. microti occurs in naturally-infected reservoir hosts of the genus Microtus. METHODS: We sampled 124 common voles, Microtus arvalis; 76 root voles, M. oeconomus and 17 field voles, M. agrestis. In total, 113 embryos were isolated from 20 pregnant females. Another 11 pregnant females were kept in the animal house at the field station in Urwitalt until they had given birth and weaned their pups (n = 62). Blood smears and/or PCR targeting the 550 bp 18S rRNA gene fragment were used for the detection of B. microti. Selected PCR products, including isolates from females/dams and their embryos/pups, were sequenced. RESULTS: Positive PCR reactions were obtained for 41% (89/217) of the wild-caught voles. The highest prevalence of B. microti was recorded in M. arvalis (56/124; 45.2%), then in M. oeconomus (30/76; 39.5%) and the lowest in M. agrestis (3/17; 17.7%). Babesia microti DNA was detected in 61.4% (27/44) of pregnant females. Vertical transmission was confirmed in 81% (61/75) of the embryos recovered from Babesia-positive wild-caught pregnant females. The DNA of B. microti was detected in the hearts, lungs and livers of embryos from 98% of M. arvalis, 46% of M. oeconomus and 0% of M. agrestis embryos from Babesia-positive females. Of the pups born in captivity, 90% were born to Babesia-positive dams. Babesia microti DNA was detected in 70% (35/50) of M. arvalis and 83% (5/6) of M. oeconomus pups. Congenitally acquired infections had no impact on the survival of pups over a 3-week period post partum. Among 97 B. microti sequences, two genotypes were found. The IRU1 genotype (Jena-like) was dominant in wild-caught voles (49/53; 92%), pregnant females (9/11; 82%) and dams (3/5; 60%). The IRU2 genotype (Munich-like) was dominant among B. microti positive embryos (20/27; 74%) and pups (12/17; 71%). CONCLUSION: A high rate of vertical transmission of the two main rodent genotypes of B. microti was confirmed in two species of naturally infected voles, M. arvalis and M. oeconomus.

Babesia microti from humans and ticks hold a genomic signature of strong population structure in the United States.

BACKGROUND: Babesia microti is an emerging tick-borne apicomplexan parasite with increasing geographic range and incidence in the United States. The rapid expansion of B. microti into its current distribution in the northeastern USA has been due to the range expansion of the tick vector, Ixodes scapularis, upon which the causative agent is dependent for transmission to humans. RESULTS: To reconstruct the history of B. microti in the continental USA and clarify the evolutionary origin of human strains, we used multiplexed hybrid capture of 25 B. microti isolates obtained from I. scapularis and human blood. Despite low genomic variation compared with other Apicomplexa, B. microti was strongly structured into three highly differentiated genetic clusters in the northeastern USA. Bayesian analyses of the apicoplast genomes suggest that the origin of the current diversity of B. microti in northeastern USA dates back 46 thousand years with a signature of recent population expansion in the last 1000 years. Human-derived samples belonged to two rarely intermixing clusters, raising the possibility of highly divergent infectious phenotypes in humans. CONCLUSIONS: Our results validate the multiplexed hybrid capture strategy for characterizing genome-wide diversity and relatedness of B. microti from ticks and humans. We find strong population structure in B. microti samples from the Northeast indicating potential barriers to gene flow.

Ixodes persulcatus Ticks as Vectors for the Babesia microti U.S. Lineage in Japan.

The U.S. lineage, one of the major clades in the Babesia microti group, is known as a causal agent of human babesiosis mostly in the northeastern and upper midwestern United States. This lineage, however, also is distributed throughout the temperate zone of Eurasia with several reported human cases, although convincing evidence of the identity of the specific vector(s) in this area is lacking. Here, the goal was to demonstrate the presence of infectious parasites directly in salivary glands of Ixodes persulcatus, from which U.S. lineage genetic sequences have been detected in Asia, and to molecularly characterize the isolates. Five PCR-positive specimens were individually inoculated into hamsters, resulting in infections in four; consequently, four strains were newly established. Molecular characterization, including 18S rRNA, ß-tubulin, and CCT7 gene sequences, as well as Western blot analysis and indirect fluorescent antibody assay, revealed that all four strains were identical to each other and to the U.S. lineage strains isolated from rodents captured in Japan. The 18S rRNA gene sequence from the isolates was identical to those from I. persulcatus in Russia and China, but the genetic and antigenic profiles of the Japanese parasites differ from those in the United States and Europe. Together with previous epidemiological and transmission studies, we conclude that I. persulcatus is likely the principal vector for the B. microti U.S. lineage in Japan and presumably in northeastern Eurasia. IMPORTANCE: The major cause of human babesiosis, the tick-borne blood parasite Babesia microti, U.S. lineage, is widely distributed in the temperate Northern Hemisphere. However, the specific tick vector(s) remains unidentified in Eurasia, where there are people with antibodies to the B. microti U.S. lineage and cases of human babesiosis. In this study, the first isolation of B. microti U.S. lineage from Ixodes persulcatus ticks, a principal vector for many tick-borne diseases, is described in Japan. Limited antigenic cross-reaction was found between the Japan and United States isolates. Thus, current serological tests based on U.S. isolates may underestimate B. microti occurrence outside the United States. This study and previous studies indicate that I. persulcatus is part of the B. microti U.S. lineage life cycle in Japan and, presumably, northeastern Eurasia. This report will be important for public health, especially since infection may occur through transfusion, and also to researchers in the field of parasitology.

Identification and functional study of a novel 2-cys peroxiredoxin (BmTPx-1) of Babesia microti.

Babesia microti is an emerging human pathogen and the primary causative agent of human babesiosis in many regions of the world. Although the peroxiredoxins (Prxs) or thioredoxin peroxidases (TPx) enzymes of this parasite have been sequenced and annotated, their biological properties remain largely unknown. Prxs are a family of antioxidant enzymes that protect biological molecules against metabolically produced reactive oxygen species (ROS) and reduce hydrogen peroxide (H2O2) to water in both eukaryotes and prokaryotes. In this study, TPx-1 cDNA was cloned from B. microti (designated BmTPx-1). Recombinant BmTPx-1 (rBmTPx-1) was expressed in Escherichia coli as a histidine fusion protein and purified using Ni-NTA His bind resin. To test the defense capacity of enzymatic antioxidants against the effect of ROS, a mixed-function oxidation system was utilized with the recombinant BmTPx-1 protein. A decreased ability of rBmTPx-1 to donate electrons to the thioredoxin (Trx)/TrxR reductase system was clarified by reaction with H2O2. These results suggest that BmTPx-1 has a great impact on protecting parasites from oxidative stress in the erythrocytic stage.

The PCR detection and phylogenetic characterization of Babesia microti in questing ticks in Mongolia.

Babesia microti is a tick-transmitted zoonotic hemoprotozoan parasite. In the present study, we investigated B. microti infection in questing ticks in Mongolia. A total of 219 questing ticks were collected from three different Mongolian provinces (Bayan-Olgii, Khovsgol, and Selenge). Of these, 63 from Selenge were identified as Ixodes persulcatus, while the remaining 156 (from all three provinces) were identified as Dermacentor nuttalli. When the tick DNA samples were screened using a B. microti-specific nested PCR, 19 (30.2%) of the 63 I. persulcatus ticks were found to be B. microti-positive. The parasite was not detected in D. nuttalli. Subsequently, the 18S rRNA, cox1, and tufA sequences of B. microti were amplified, sequenced, and subjected to phylogenetic analyses. Sequencing analyses showed that the Mongolian 18S rRNA, cox1, and tufA sequences were 99.6-100%, 96.7-97.2%, and 94.7-95.3% homologous, respectively, with B. microti R1 strain US-type sequences from humans. In the phylogenetic analyses, the Mongolian cox1 and tufA sequences were found to be separate lineages, which formed sister-clades to the R1 strain sequences, while all of the Mongolian B. microti 18S rRNA sequences were clustered within US-type clade containing several other sequences of human origin. In conclusion, in addition to reporting the presence of B. microti for the first time in questing ticks in Mongolia, the present study found that Mongolian I. persulcatus ticks were infected with US-type B. microti. These findings warrant large-scale studies to detect and characterize B. microti in ticks, small mammals, and humans. Such studies should provide us with a better understanding of zoonotic Babesia epidemiology in Mongolia.

Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani.

Babesia spp. are obligate protozoan parasites of red blood cells. Transmission to humans occurs through bites from infected ticks or blood transfusion. Infections with B. microti account for the majority of the reported cases of human babesiosis in the USA. A lower incidence is caused by the more recently described species B. duncani. The current gold standard for detection of Babesia is microscopic examination of blood smears. Recent PCR-based assays, including real-time PCR, have been developed for B. microti. On the other hand, molecular assays that detect and distinguish between B. microti and B. duncani infections are lacking. Closely related species of Babesia can be differentiated due to sequence variation within the internal transcribed spacer (ITS) regions of nuclear ribosomal RNAs. In the present study, we targeted the ITS regions of B. microti and B. duncani to develop sensitive and species-specific droplet digital PCR (ddPCR) assays. The assays were shown to discriminate B. microti from B. duncani and resulted in limits of detection of ~10 gene copies. Moreover, ddPCR for these species were useful in DNA extracted from blood of experimentally infected hamsters, detecting infections of low parasitemia that were negative by microscopic examination. In summary, we have developed sensitive and specific quantitative ddPCR assays for the detection of B. microti and B. duncani in blood. Our methods could be used as sensitive approaches to monitor the progression of parasitemia in rodent models of infection as well as serve as suitable molecular tests in blood screening.

Prevalence of tick-borne pathogens in an urban park in Rome, Italy.

INTRODUCTION: Limited information is available about the presence of tick-borne pathogens in urban parks in Italy. To fill this gap, ticks were collected in a public park in Rome over a 1-year period and screened by molecular methods for tick-borne pathogens. RESULTS AND CONCLUSION: The most abundant tick species were Rhipicephalus turanicus and Ixodes ricinus. The predominant pathogens detected were Borrelia. burgdorferi sensu lato (36%), Rickettsia spp. (36%), and Coxiella burnetii (22%). Among less frequently detected pathogens, Babesia microti was detected for the first time in Italy, with a prevalence of 4%. Neither Bartonella spp. nor Francisella tularensis were detected. With regard to co-infections, the most frequent double and triple infections involved Rickettsia spp., B. burgdorferi sl., and C. burnetii.. A positive correlation was detected between pathogens and I. ricinus. Further studies are needed in order to assess risk associated with tick-borne pathogens in urban areas.

Not "out of Nantucket": Babesia microti in southern New England comprises at least two major populations.

BACKGROUND: Deer tick-transmitted human babesiosis due to Babesia microti appears to be expanding its distribution and prevalence in the northeastern United States. One hypothesis for this emergence is the introduction of parasites into new sites from areas of long-standing transmission, such as Nantucket Island, Massachusetts. METHODS: We developed a typing system based on variable number tandem repeat loci that distinguished individual B. microti genotypes. We thereby analyzed the population structure of parasites from 11 sites, representing long-standing and newly emerging transmission in southern New England (northeastern United States), and compared their haplotypes and allele frequencies to determine the most probable number of B. microti populations represented by our enzootic collections. We expected to find evidence for a point source introduction across southern New England, with all parasites clearly derived from Nantucket, the site with the most intense longstanding transmission. RESULTS: B. microti in southern New England comprises at least two major populations, arguing against a single source. The Nantucket group comprises Martha's Vineyard, Nantucket and nearby Cape Cod. The Connecticut/Rhode Island (CT/RI) group consists of all the samples from those states along with samples from emerging sites in Massachusetts. CONCLUSIONS: The expansion of B. microti in the southern New England mainland is not due to parasites from the nearby terminal moraine islands (Nantucket group), but rather from the CT/RI group. The development of new B. microti foci is likely due to a mix of local intensification of transmission within relict foci across southern New England as well as long distance introduction events.

Sequence and annotation of the apicoplast genome of the human pathogen Babesia microti.

The apicomplexan intraerythrocytic parasite Babesia microti is an emerging human pathogen and the primary cause of human babesiosis, a malaria-like illness endemic in the United States. The pathogen is transmitted to humans by the tick vector, Ixodes scapularis, and by transfusion of blood from asymptomatic B. microti-infected donors. Whereas the nuclear and mitochondrial genomes of this parasite have been sequenced, assembled and annotated, its apicoplast genome remained incomplete, mainly due to its low representation and high A+T content. Here we report the complete sequence and annotation of the apicoplast genome of the B. microti R1 isolate. The genome consists of a 28.7 kb circular molecule encoding primarily functions important for maintenance of the apicoplast DNA, transcription, translation and maturation of organellar proteins. Genome analysis and annotation revealed a unique gene structure and organization of the B. microti apicoplast genome and suggest that all metabolic and non-housekeeping functions in this organelle are nuclear-encoded. B. microti apicoplast functions are significantly different from those of the host, suggesting that they might be useful as targets for development of potent and safe therapies for the treatment of human babesiosis.

Molecular confirmation of the first autochthonous case of human babesiosis in Australia using a novel primer set for the beta-tubulin gene.

In 2012, the first autochthonous Australian case of human babesiosis was reported, after microscopic examinations of blood samples revealed intra-erythrocytic parasites in a hospitalized 56year-old man from NSW, who died in 2011 (Senanayake et al., 2012). Independent molecular analyses carried out in Australia and the USA, identified Babesia microti at the 18S ribosomal RNA (18S rRNA), and the beta-tubulin (ß-tubulin) gene loci. Here we present the details of a novel PCR-based assay for the ß-tubulin gene that was developed, during the original study, to corroborate the results obtained from the analysis of the 18S rDNA. The complete phylogenetic reconstruction, based on the two loci sequenced from the Australian clinical isolate, is also shown here for the first time.

Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti.

BACKGROUND: The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases. RESULTS: In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients' blood samples. CONCLUSION: Real-time quantitative PCR using specific primers and molecular beacon probes for the selected amplicon described in this study can detect three tick-borne pathogens simultaneously in an accurate manner.

A case of human babesiosis in Denmark.

We report the first human case of Babesia microti infection imported to Denmark from the United States by a 64 year old female traveller with fever of unknown origin. The case raises the possibility that Babesia-infections may be under-diagnosed, illustrates the importance of a thorough travel history and discusses important diagnostic pitfalls.

Identification and characterization of an interspersed repeat antigen of Babesia microti (BmIRA).

In this report, a novel gene encoding an interspersed repeat antigen from Babesia microti (BmIRA) was identified and described. The full-length cDNA containing an open reading frame of 1,947 bp was obtained by immunoscreening a B. microti cDNA expression library. The full-length of BmIRA gene was expressed as a GST fusion recombinant BmIRA (rBmIRA) in Escherichia coli. Sera of mice immunized with the rBmIRA detected a native parasite protein with a molecular mass of 76 kDa on Western blot analysis. The same protein was detected in the parasites by immunofluorescent antibody test (IFAT). An enzyme-linked immunosorbent assay (ELISA) using rBmIRA detected specific antibodies as early as 11 days post-infection in sera from a hamster experimentally infected with B. microti Gray stain (US type). Furthermore, a rapid immunochromatographic test (ICT) using rBmIRA detected specific antibodies in a hamster experimentally infected with B. microti from day 11 to at least day 180 post-infection. The results indicate the antibody response against the rBmIRA was maintained during the chronic stage of infection. On the other hand, an immunoprotective property of rBmIRA as a subunit vaccine was evaluated in hamsters against B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmIRA could be a useful serodiagnostic antigen for screening of B. microti infection.

Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti.

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.

Babesia microti in rodents and raccoons from northeast Florida.

Human babesiosis in the United States is caused most commonly by the intraerythrocytic protozoan parasite, Babesia microti . Although a few reports have described evidence of Babesia species in animals in Florida, to date Babesia microti specifically has not been reported from Florida or most other southern states. To determine if the organism is present in vertebrates in the region, small mammals were trapped and sampled at 2 sites in northeastern Florida, and DNA extracts from blood samples were screened for B. microti DNA via PCR assays targeting portions of the nuclear small subunit rRNA (18S rDNA) and beta-tubulin genes. Amplified fragments from representative samples of PCR-positive hosts were sequenced and compared phylogenetically to reference strains of Babesia species. The B. microti strains found in cotton rats ( Sigmodon hispidus ) most closely resembles B. microti sensu stricto strains that are pathogenic to humans, and strains found in raccoons ( Procyon lotor ) most closely resembles previously described raccoon-related strains of B. microti sensu lato. The results of this study suggest that B. microti is prevalent among cotton rats and raccoons at some sites in northeast Florida and may pose a risk to humans in the region.

Detection of two zoonotic Babesia microti lineages, the Hobetsu and U.S. lineages, in two sympatric tick species, ixodes ovatus and Ixodes persulcatus, respectively, in Japan.

The species Babesia microti, commonly found in rodents, demonstrates a high degree of genetic diversity. Three lineages, U.S., Kobe, and Hobetsu, are known to have zoonotic potential, but their tick vector(s) in Japan remains to be elucidated. We conducted a field investigation at Nemuro on Hokkaido Island and at Sumoto on Awaji Island, where up to two of the three lineages occur with similar frequencies in reservoirs. By flagging vegetation at these spots and surrounding areas, 4,010 ticks, comprising six species, were collected. A nested PCR that detects the 18S rRNA gene of Babesia species revealed that Ixodes ovatus and I. persulcatus alone were positive. Lineage-specific PCR for rRNA-positive samples demonstrated that I. ovatus and I. persulcatus carried, respectively, the Hobetsu and U.S. parasites. No Kobe-specific DNA was detected. Infected I. ovatus ticks were found at multiple sites, including Nemuro and Sumoto, with minimum infection rates (MIR) of ∼12.3%. However, all I. persulcatus ticks collected within the same regions, a total of 535, were negative for the Hobetsu lineage, indicating that I. ovatus, but not I. persulcatus, was the vector for the lineage. At Nemuro, U.S. lineage was detected in 2 of 139 adult I. persulcatus ticks (MIR, 1.4%), for the first time, while 48 of I. ovatus ticks were negative for that lineage. Laboratory experiments confirmed the transmission of Hobetsu and U.S. parasites to hamsters via I. ovatus and I. persulcatus, respectively. Differences in vector capacity shown by MIRs at Nemuro, where the two species were equally likely to acquire either lineage of parasite, may explain the difference in distribution of Hobetsu throughout Japan and U.S. taxa in Nemuro. These findings are of importance in the assessment of the regional risk for babesiosis in humans.

Development of real-time PCR assay for differential detection and quantification for multiple Babesia microti-genotypes.

We have developed a real-time PCR assay that can rapidly and differentially detect and quantify four genotypes of small subunit ribosomal RNA gene (SSUrDNA) of Babesia microti (Kobe-, Otsu-, Nagano- and US-types). In this assay, four genotype-specific pairs of primers targeted on internal transcribed spacer (ITS) 1 or 2 sequences were used and amplicons by each pair of primers were quantitatively detected by fluorescent SYBR Green I. The four genotype-specific pairs of primers displayed the high specificity for homologous genotype DNA. The standard curves of cycle threshold (Ct) values versus amount of target DNA per reaction (log) for all four genotypes were linear and the correlation coefficient (Rsq) values for the curves were from 0.970 to 0.997. The standard curves were almost identical even in the presence of heterologous genotype DNA. This assay could detect 10-30 fg purified DNA (equivalent to the amount of 1-5 parasite DNA) of each genotype B. microti. This assay could also detect each genotype B. microti infection in blood with 3×10(-6)%-1×10(-5)% parasitemia. This assay was applicable to field rodent and tick samples to reveal mixed infection in several samples, for which a single genotype of B. microti had been detected by direct sequencing analyses in our previous studies. This assay also seemed to be applicable to clinical human samples, showing Kobe-type positive results for the first Japanese babesiosis patient and the asymptomatic donor, both infected with Kobe-type B. microti.