[Show abstract][Hide abstract] ABSTRACT: Abstract DNA sequences encoding the GroES and GroEL proteins of Orientia tsutsugamushi were amplified by the PCR and sequenced. Pairwise alignment of full-length groES and groEL gene sequences indicated high sequence similarity (90.4-100% and 90.3-100%) in O. tsutsugamushi, suggesting that these genes are good candidates for the molecular diagnosis and phylogenetic analysis of scrub typhus. Comparisons of the 56-kD type-specific antigen (TSA) protein gene and the groES and groEL genes showed that genotypes based on the 56-kD TSA gene were not related to a cluster containing the groES and groEL genes in a dendrogram, suggesting that a gene rearrangement may be associated with homologous recombination in mites.
[Show abstract][Hide abstract] ABSTRACT: Abstract Babesiosis is a tick-borne protozoan disease affecting many mammalian species worldwide and is caused by the intra-erythrocytic multiplication of the apicomplexa from the Babesia genus. The present study aimed to detect the presence of Babesia sp. in 13 American mink from Hokkaido, Japan. One of 13 animals was positive in nested PCR targeting the 18S ribosomal RNA (SSU rDNA) and subunit 7 (eta) of the chaperonin-containing t-complex polypeptide 1 (CCT7) genes from the genera Babesia and Theileria, belonging to the order piroplasmida. Sequencing of the PCR product of SSU rDNA revealed 99% homology to the isolates of Babesia sp. SAP#131 found in raccoons in Hokkaido, while that of the CCT7 gene showed 80% homology to the isolates of Babesia gibsoni in dog as determined by BLAST analysis. We refer to the cognate sequence as Babesia sp. NV-1. Phylogenetic analyses of SSU rDNA and CCT7 genes from Babesia sp. NV-1 revealed them to be most closely related to the Babesia sp. SAP#131 from raccoon from Hokkaido and to dog B. gibsoni, respectively.Here we demonstrate the first molecular evidence of the Babesia sp. NV-1 parasite in feral American mink (Neovison vison) in Hokkaido, Japan.
Journal of Parasitology 09/2012; 99(2). DOI:10.1645/GE-3185.1 · 1.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed serological tools for the detection of hantavirus-specific antibodies and hantavirus antigens in shrews. The work was focussed to generate Thottapalayam virus (TPMV)-specific monoclonal antibodies (mAbs) and anti-shrew immunoglobulin G (IgG) antibodies. The mAbs against TPMV nucleocapsid (N) protein were produced after immunization of BALB/c mice with recombinant TPMV N proteins expressed in Escherichia coli, baculovirus and Saccharomyces cerevisiae-mediated expression systems. In total, six TPMV N-protein-specific mAbs were generated that showed a characteristic fluorescent pattern in indirect immunofluorescence assay (IFA) using TPMV-infected Vero cells. Out of the six mAbs tested, five showed no cross-reaction to rodent-associated hantaviruses (Hantaan, Seoul, Puumala, Tula, Dobrava-Belgrade and Sin Nombre viruses) in IFA and enzyme-linked immunosorbent assay (ELISA), although one mAb reacted to Sin Nombre virus in IFA. None of the mAbs cross-reacted with an amino-terminal segment of the shrew-borne Asama virus N protein. Anti-shrew-IgG sera were prepared after immunization of rabbits and BALB/c-mice with protein-G-purified shrew IgG. TPMV-N-protein-specific sera were raised by immunisation of Asian house shrews (Suncus murinus) with purified yeast-expressed TPMV N protein. Using these tools, an indirect ELISA was developed to detect TPMV-N-protein-specific antibodies in the sera of shrews. Using an established serological assay, high TPMV N protein specific antibody titres were measured in the sera of TPMV-N-protein-immunized and experimentally TPMV-infected shrews, whereas no cross-reactivity to other hantavirus N proteins was found. Therefore, the generated mAbs and the established ELISA system represent useful serological tools to detect TPMV, TPMV-related virus antigens or hantavirus-specific antibodies in hantavirus-infected shrews.
Archives of Virology 07/2012; 157(11). DOI:10.1007/s00705-012-1405-9 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: We demonstrate here the identification and phylogenetic characterization of Babesia microti (B. microti)-like parasite detected from a splenectomized Japanese macaque (Macaca fuscata fuscata) at a facility for laboratory animal science. On Day 133 after splenectomy, intra-erythrocytic parasites were found on light microscopic examination, and the level of parasitemia reached 0.3% on blood smear. Molecular characterization of the parasite using nested-polymerization chain reactions targeting the 18S rRNA, β-tubulin, and subunit 7 (eta) of the chaperonin-containing t-complex polypeptide 1 (CCT7) genes were identified as a B. microti-like parasite, designated the Japanese Macaque Babesia-1 (JM-1).
The American journal of tropical medicine and hygiene 10/2011; 85(4):635-8. DOI:10.4269/ajtmh.2011.11-0035 · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Babesia microti, the primary causal agent of human babesiosis in North America, was thought to distribute in Europe in association with ixodid ticks and rodents. Recent analyses of β-tubulin and the eta subunit of the chaperonin-containing t-complex protein 1 (CCT7) genes revealed discrete clusters (a species-complex comprised of at least 4 taxa for the U.S., Kobe, Munich, and Hobetsu). To further assess the micro-evolutionary history and genetic variability within the taxon, we combined a set of 6 introns from the CCT7 gene to use as a rapidly evolving DNA marker. Phylogenetic and comparative sequence analyses subdivided the U.S. taxon into 3 geographic subclades--North America, western to central Eurasia, and northeastern Eurasia (≥ 98% bootstrap supports for each node). The Kobe taxon, which occurs only in a few geographic foci of Japan, could further be subdivided into 2 subgroups (100% support). The Munich and Hobetsu taxa, common to Europe and Japan, respectively, exhibited little or no pairwise sequence divergence among geographically diverse samples, suggesting an extreme population bottleneck during recent history. Despite the small sample size, this study provides a better understanding of the micro-evolutionary relationships and the genetic variability present within each lineage of the B. microti-group.
[Show abstract][Hide abstract] ABSTRACT: Tula virus (TULV) and Puumala virus (PUUV) are hantaviruses carried by the bank vole (Myodes glareolus) and European common vole (Microtus arvalis), respectively. PUUV is a causative agent of hemorrhagic fever with renal syndrome (HFRS), while TULV is thought to be apathogenic to humans. The N-terminal regions of the N proteins from TULV and PUUV were expressed and applied as enzyme-linked immunosorbent assay (ELISA) antigens. Colonized Japanese grass voles (Microtus montebelli) and BALB/c mice were used for experimental inoculation of the vole-borne hantaviruses TULV and PUUV. Voles and mice showed significant antibody production toward both viruses, but these antisera showed little cross-reactivity between TULV and PUUV in the immunofluorescence antibody assay and ELISA. In contrast, sera from patients with HFRS caused by PUUV exhibited high cross-reactivity against the TULV antigen, and sera from a natural rodent reservoir showed moderate cross-reactivity against the heterologous antigen, indicating that the antigenic cross-reactivity between TULV and PUUV differs in sera from rodents and humans.
[Show abstract][Hide abstract] ABSTRACT: A frozen-stored blood clot of a wild brown bear cub Ursus arctos yesoensis that had been captured in Hokkaido, Japan was examined for piroplasma infection using polymerase chain reaction (PCR). Two 18S ribosomal RNA gene (SSU rDNA) sequences were generated. One 1565-bp sequence showed the highest similarity with B. gibsoni (95.9% identity) but, phylogenetically, was found to belong to a distinct lineage. The other sequence (1709-bp) could not be definitively assigned to a described taxon, sharing only limited homology to the closest named species (90.1% identity with C. felis). In order to enhance information obtained from the SSU rDNA sequence, further detection and sequence analysis of the CCTeta gene sequence were done revealing the simultaneous presence of three closely related genotypes (all in a monophyletic lineage) within a single bear host. This finding suggested the possibility that a new Babesia species (Babesia sp. UR1) might have been maintained in nature in wild brown bears. While the parasite's biology is yet unknown, to our knowledge, this is, excepting the single case documentation in 1910 of a hemoparasite in a bear at Russian zoo, the first reported case of piroplasms inhabiting a bear species.
[Show abstract][Hide abstract] ABSTRACT: We recently reported that feral raccoons (Procyon lotor) with splenomegaly native to Japan were carriers of a Babesia microti-like parasite identical to that found in the United States, which was likely introduced to Japan from North America via raccoons imported as pets. Thus, we attempted extensive molecular survey for piroplasma infections of feral raccoon with normal spleen in Hokkaido, Japan using nested PCR that target broadly to 18S ribosomal RNA gene (SSU-rDNA) of all the parasites in the genus Babesia, Theileria, Cytauxzoon and B. microti group. Of the 348 raccoon samples analyzed, 9 gave positive signals. Cloning and phylogenetic analysis on SSU-rDNA sequences revealed that six of nine positives were found to be infected with Babesia and the remaining three with previously unreported Sarcocystis. Babesia sequences were further separated into two distantly related groups, those that reside in a novel phylogenetic group were consisted solely of four parasites found in this study, while those which included one identical sequence found in the three of our specimens were assembled together with both Babesia parasites of tick's in Japan and of raccoon's in U.S. These results may indicate that not only a B. microti-like parasite but also at least two yet undescribed Babesia species are being established in their new life cycles in the feral raccoon populations in Japan.
[Show abstract][Hide abstract] ABSTRACT: Babesia microti, the erythroparasitic cause of human babesiosis, has long been taken to be a single species because classification by parasite morphology and host spectrum blurred distinctions between the parasites. Phylogenetic analyses of the 18S ribosomal RNA gene (18S rDNA) and, more recently, the beta-tubulin gene have suggested inter-group heterogeneity. Intra-group relationships, however, remain unknown. This study was conducted to clarify the intra- and inter-group phylogenetic features of the B. microti-group parasites with the eta subunit of the chaperonin-containing t-complex polypeptide l (CCTeta) gene as a candidate genetic marker for defining the B. microti group. We prepared complete sequences of the CCTeta gene from 36 piroplasms and compared the phylogenetic trees. The B. microti-group parasites clustered in a monophyletic assemblage separate from the Babesia sensu stricto and Theileria genera and subdivided predominantly into 4 clades (U.S., Kobe, Hobetsu, Munich) with highly significant evolutionary distances between the clades. B. rodhaini branched at the base of the B. microti-group parasites. In addition, a unique intron presence/absence matrix not observable in 18S rDNA or beta-tubulin set the B. microti group entirely apart from either Babesia sensu stricto or Theileria. These results have strong implications for public health, suggesting that the B. microti-group parasites are a full-fledged genus comprising, for now, four core species, i.e., U.S., Kobe, Hobetsu, and Munich species nova. Furthermore, the CCTeta gene is an instructive and definitive genetic marker for analyzing B. microti and related parasites.
Journal of Veterinary Medical Science 02/2009; 71(1):55-68. · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite the evidence suggesting that mouse pyruvate kinase (PK) deficiency provides protection against malaria in rodents, there has been no investigation of a parallel protective effect against babesiosis caused by Babesia rodhaini. Here, we examined whether a PK-deficient co-isogenic mouse strain (CBA-Pk-1(slc)) was protected against B. rodhaini infection. We demonstrated that deficiency in pyruvate kinase correlated with a significant protective effect, with survival rates of 50%, 58% and 56% in groups inoculated with 10, 10(3) and 10(5) parasitized erythrocytes, respectively. In contrast, control CBA (CBA-Pk-1(+)) mice exhibited 100% lethality, regardless of the infectious dose. In addition, CBA-Pk-1(slc) mice showed decreased levels of parasitemia when compared to CBA-Pk-1(+) mice, in groups given 10, 10(3) or 10(5) parasitized erythrocytes. These results indicate that similar to PK deficiency in rodents, PK deficiency in mice affects the in vivo growth of B. rodhaini and protects the mice from lethal babesiosis.
[Show abstract][Hide abstract] ABSTRACT: We have examined the seroprevalence of BDV in wild Japanese macaques (Macaca fuscata) in the peninsula (Chiba prefecture), Japan. Serum samples from macaques were examined by the ELISA, Western blot and immunofluorescence assays to detect the presence of serum antibodies that react specifically to BDV antigens. Among 49 investigated individuals, 6 (12.2%) showed positive reaction to BDV antigens. RT-PCR studies detected BDV sequences in brain tissue of one case among four seropositive cases examined. Sequence analysis revealed a high degree of genetic conservation between BDV sequences derived from Japanese macaques and those documented for other animal species. Nevertheless, phylogenetic analysis revealed unique differences between macaque and other species derived BDV sequences.
[Show abstract][Hide abstract] ABSTRACT: A significant number of patients are diagnosed with "fevers of unknown origin" (FUO) in Shimane Prefecture in Japan where tick-borne diseases are endemic. We conducted molecular surveys for Babesia microti, Ehrlichia species, and Candidatus Neoehrlichia mikurensis in 62 FUO cases and 62 wild rodents from Shimane Prefecture, Japan. PCR using primers specific for the Babesia 18S small-subunit rRNA (rDNA) gene and Anaplasmataceae groESL amplified products from 45% (28/62) and 25.8% (16/62) of captured mice, respectively. Of the 28 18S rDNA PCR positives, 23 and five samples were positive for Hobetsu- and Kobe-type B. microti, respectively. In contrast, of the 16 groESL PCR positives, eight, one and seven samples were positive for Ehrlichia muris, Ehrlichia sp. HF565 and Candidatus N. mikurensis, respectively. Inoculation of selected blood samples into Golden Syrian hamsters indicated the presence of Hobetsu- and Kobe-type B. microti in four and one sample, respectively. Isolation of the latter strain was considered important as previous studies suggested that the distribution of this type was so far confined to Awaji Island in Hyogo Prefecture, where the first case of transfusion-associated human babesiosis originated. DNA samples from 62 FUO human cases tested negative for B. microti 18S rDNA gene, Anaplasmataceae groESL gene, Rickettsia japonica 17K genus-common antigen gene and Orientia tsutsugamushi 56K antigen gene by PCRs. We also conducted seroepidemiological surveys on 62 human sera collected in Shimane Prefecture from the FUO patients who were suspected of carrying tick-borne diseases. However, indirect immunofluorescent antibody tests using B. microti- and E. muris-infected cells detected IgG against E. muris in only a single positive sample. This study demonstrates the presence of several potentially important tick-borne pathogens in Shimane Prefecture and suggests the need for further study on the causative agents of FUOs.
Microbiology and Immunology 02/2007; 51(4):359-67. DOI:10.1111/j.1348-0421.2007.tb03923.x · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Six Eurasian red squirrels (Sciurus vulgaris orientis), victims of road traffic found during 2002 and 2004 near the Noppro Forest Park in Ebetsu, Hokkaido, Japan, were examined for the presence of Babesia parasites. Three of the six squirrels exhibited positive signals by nested PCRs targeting both the 18S rRNA and beta-tubulin genes. Three squirrels proved to be infected with a B. microti-like parasite as evidenced by sequencing the amplified DNAs and by the morphology of the intraerythrocytic parasites. Genotypically, however, the parasite appeared to be of a new type, as it was clearly distinguishable from any of the known types that have previously been reported in various wild animals. This is the first report showing molecular evidence for the presence of B. microti-like parasites in Sciuridae.
Journal of Veterinary Medical Science 08/2006; 68(7):643-6. DOI:10.1292/jvms.68.643 · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Babesia rodhaini is a rodent hemoparasite closely related to B. microti, the major causative agent of human babesiosis. We tested the infectivity of B. rodhaini for human erythrocytes by using the SCID mouse model in which the circulating erythrocytes were replaced with those of humans. Initially, parasites grew very poorly in the mouse model, but a variant capable of propagating in human erythrocytes emerged after an adaptation period of three weeks. In an attempt to identify parasite proteins involved in the alteration of host cell preference, an expression cDNA library of B. rodhaini was constructed and screened with immune mouse sera. Although we were able to obtain three merozoite surface protein (MSP) genes, sequences of these genes from both the parental strain and human erythrocyte-adapted substrain were identical. Our results suggest that B. rodhaini has potential ability to infect human erythrocytes, but development of this ability may not be brought about by an amino acid change in MSPs.
Journal of Veterinary Medical Science 10/2005; 67(9):901-7. DOI:10.1292/jvms.67.901 · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Raccoons (Procyon lotor), which have recently become feral in Japan, were examined for the presence of Babesia microti-like parasites. Out of 372 raccoons captured in the west-central part of Hokkaido, 24 animals with splenomegaly were selected and tested by nested PCR targeting the babesial 18S rRNA gene. B. microti-like parasites were detected in two of the 24 individuals, and their DNA sequences were identical to that of the B. microti-like parasite reported from raccoons in the United States, suggesting that the parasites were probably imported into Japan and that the life cycle of the parasite has already been established in the country. The potential risk of this B. microti-like parasite spreading among dogs and foxes in Japan will need to be carefully monitored, as parasitization by phylogenetically very close parasites has been reported from such animals.
Journal of Veterinary Medical Science 09/2005; 67(8):825-7. DOI:10.1292/jvms.67.825 · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The spleen plays an important role in cellular immunity leading to parasite elimination, especially in hemo-protozoan parasite infection. In the present study, we quantified the serum IFN-gamma in splenectomized (SP) and non-splenectomized (NSP) cows infected with Theirelia sergenti (TS) to investigate whether the spleen plays a role in the Th1-type cytokine responses in cows following parasite infection. A transient increase in IFN-gamma was observed in the early stages of infection in the NSP cows, and the cows did not develop parasitemia. In contrast, the SP cows showed no IFN-gamma response at the early stage of infection, and the cows developed parasitemia following anemia. The NSP cows produced IgG1 and IgG2 antibodies to TS-specific, whereas the SP cows showed increases only in IgG1. Increased PBMC proliferation by cocanavalin A stimulation was observed concomitant with the IFN-gamma response. Pretreatment with IFN-gamma suppressed the propagation of TS in the SP cattle. These results suggest that the spleen plays an important role in the resistance to TS infection, leading to a Th1-type immune response. IFN-gamma might contribute to the activation of immunocytes and play an important role in the immune response to resist TS proliferation.