Yoshifumi Nishikawa

Obihiro University of Agriculture and Veterinary Medicine, Obibiro, Hokkaidō, Japan

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Publications (160)363.93 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans.
    Journal of Veterinary Medical Science 03/2015; DOI:10.1292/jvms.15-0082 · 0.88 Impact Factor
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    ABSTRACT: The apical complex of Toxoplasma gondii enables it to invade virtually all nucleated cells in warm-blooded animals, including humans, making it a parasite of global importance. Anti-T. gondii cellular defense mechanisms depend largely on interferon (IFN)-γ production by immune cells. However, the molecular mechanism of IFN-β-mediated defense remains largely unclear. Here, mouse peritoneal macrophages and murine embryonic fibroblasts (MEFs) primed with recombinant IFN-β and IFN-γ showed different pathways of activation. Treatment of these cells with IFN-β or IFN-γ inhibited T. gondii (Type II PLK strain) growth. Priming macrophages with IFN-β had no effect on inflammatory cytokine expression, inducible nitric oxide synthase or indoleamine 2,3-dioxygenase, nor did it have an effect on their metabolites, nitric oxide and kynurenine, respectively. In contrast, IFN-γ stimulation was characterized by classical macrophage activation and T. gondii elimination. IFN-β activation recruited the immunity-related GTPase M1 (IRGM1) to the parasitophorous vacuole in the macrophages and MEFs. Anti-toxoplasma activities induced by IFN-β were significantly reduced after IRGM1 knockdown in murine macrophages and in IRGM1 deficient MEFs. Thus, this study unravels an alternative pathway of macrophage activation by IFN-β and provides a mechanistic explanation for the contribution of IRGM1 induced by IFN-β to the elimination of T. gondii.
    Cellular Microbiology 01/2015; DOI:10.1111/cmi.12423 · 4.82 Impact Factor
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    ABSTRACT: Neospora caninum is a protozoan parasite that causes neurological disorders in dogs and cattle. It can cause nonsuppurative meningoencephalitis and a variety of neuronal symptoms are observed, particularly in dogs. However, the pathogenic mechanism, including the relationship between the parasite distribution and the clinical signs, is unclear. In this study, to understand the pathogenic mechanism of neosporosis, parasite distribution and lesions were assessed in the brain of mice infected with N. caninum (strain Nc-1). Host gene expression was also analyzed with RNA sequencing (RNA-Seq). The histopathological lesions in the frontal lobe and the medulla oblongata were significantly more severe in symptomatic mice than in asymptomatic mice, although no association between the severity of the lesions and parasite numbers was found. In infected mice, the expression of 772 mouse brain genes was upregulated. A GOstat analysis predicted that the upregulated genes were involved in the host immune response. Genes whose expression correlated positively and negatively with parasite numbers were involved in the host immune response, and neuronal morphogenesis and lipid metabolic processes, respectively. These results suggest that changes in the gene expression profile associated with neuronal functions as well as immune responses can contribute to the pathogenesis in N. caninum-infected animals.
    Scientific Reports 01/2015; 5:7936. DOI:10.1038/srep07936 · 5.08 Impact Factor
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    ABSTRACT: Background Neospora caninum, a Toxoplasma gondii-like obligate intracellular parasite, causes abortion in cattle and neurological signs in canines. To understand neosporosis better, studies on host cell migration and host immune responses during the early phase of infection are important. Although the C-C chemokine receptor 5 (CCR5) plays a crucial role in immune cell migration, the role played by it in protective immunity against N. caninum is poorly understood.MethodsCCR5¿/¿ mice were used to investigate their sensitivity levels to N. caninum infection and their ability to activate immune cells against this parasite.ResultsIncreased mortality and neurological impairment were observed in the N. caninum-infected CCR5¿/¿ mice. In comparison with wild-type mice, CCR5¿/¿ mice experienced poor migration of dendritic cells and natural killer T cells to the site of infection. Dendritic cells in an in vitro culture from CCR5¿/¿ mice could not be activated upon infection with N. caninum. Furthermore, higher levels of IFN-¿ and CCL5 expression, which are associated with brain tissue damage, were observed in the brain tissue of CCR5¿/¿ mice during the acute phase of the infection, while there was no significant difference in the parasite load between the wild-type and CCR5¿/¿ animals. Additionally, a primary microglia culture from CCR5¿/¿ mice showed lower levels of IL-6 and IL-12 production against N. caninum parasites.Conclusions Our findings show that migration and activation of immune cells via CCR5 is required for controlling N. caninum parasites during the early phase of the infection.
    Parasites & Vectors 01/2015; 8(1):5. DOI:10.1186/PREACCEPT-8340837541429534 · 3.25 Impact Factor
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    ABSTRACT: The previous release of our Full-parasites database (http://fullmal.hgc.jp/) brought enhanced functionality, an expanded full-length cDNA content, and new RNA-Seq datasets from several important apicomplexan parasites. The 2015 update witnesses the major shift in the databases content with focus on diverse transcriptomes of the apicomplexan parasites. The content of the database was substantially enriched with transcriptome information for new apicomplexan parasites. The latest version covers a total of 17 species, with addition of our newly generated RNA-Seq data of a total of 909 150 388 tags. Moreover, we have generated and included two novel and unique datasets, which represent diverse nature of transcriptomes in individual parasites in vivo and in vitro. One is the data collected from 116 Indonesian patients infected with Plasmodium falciparum. The other is a series of transcriptome data collected from a total of 38 single cells of P. falciparum cultured in vitro. We believe that with the recent advances our database becomes an even better resource and a unique platform in the analysis of apicomplexan parasites and their interaction with their hosts. To adequately reflect the recent modifications and the current content we have changed the database name to DB-AT—DataBase of Apicomplexa Transcriptomes.
    Nucleic Acids Research 01/2015; DOI:10.1093/nar/gku1240 · 8.81 Impact Factor
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    ABSTRACT: Infectious diarrhea is the most frequent cause of morbidity and mortality in neonatal calves. Cryptosporidium parvum is one of the main pathogens associated with calf diarrhea. Although diarrhea is a symptom of infection with various pathogens, investigations to detect the types of pathogens have never been performed in Japan. This study investigated the prevalence of four major diarrhea-causing pathogens in calves: C. parvum, rotavirus, coronavirus, and enterotoxigenic Escherichia coli (E. coli K99). Commercial immunochromatography testing of all four pathogens and molecular analysis of C. parvum with diarrhea in calves from southernmost Okinawa and northernmost Hokkaido, Japan, were conducted. The frequencies of C. parvum, rotavirus, coronavirus, and E. coli (K99) in Okinawa were 50%, 28%, 2.3%, and 4.7%, respectively. Watery fecal stools were significantly correlated with C. parvum (p<0.05). In oocyst calculations for C. parvum, no significant difference was observed between the single-infection cases and the mixed-infection cases with rotavirus. Interestingly, molecular analyses targeting small subunit ribosomal RNA as well as glycoprotein 60 (GP60) genes revealed that the C. parvum nucleotide sequences from the two prefectures were identical, indicating that C. parvum with a uniform characteristic is distributed throughout Japan. GP60 subtyping analysis identified C. parvum from Okinawa and Hokkaido as belonging to the IIaA15G2R1 subtype, a known zoonotic subtype. Hence, control of cryptosporidiosis is important not only for pre-weaned calves, but also for human health. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
    Parasitology International 12/2014; 64(2). DOI:10.1016/j.parint.2014.11.007 · 2.11 Impact Factor
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    ABSTRACT: The previous release of our Full-parasites database (http://fullmal.hgc.jp/) brought enhanced functionality, an expanded full-length cDNA content, and new RNA-Seq datasets from several important apicomplexan parasites. The 2015 update witnesses the major shift in the databases content with focus on diverse transcriptomes of the apicomplexan parasites. The content of the database was substantially enriched with transcriptome information for new apicomplexan parasites. The latest version covers a total of 17 species, with addition of our newly generated RNA-Seq data of a total of 909 150 388 tags. Moreover, we have generated and included two novel and unique datasets, which represent diverse nature of transcriptomes in individual parasites in vivo and in vitro. One is the data collected from 116 Indonesian patients infected with Plasmodium falciparum. The other is a series of transcriptome data collected from a total of 38 single cells of P. falciparum cultured in vitro. We believe that with the recent advances our database becomes an even better resource and a unique platform in the analysis of apicomplexan parasites and their interaction with their hosts. To adequately reflect the recent modifications and the current content we have changed the database name to DB-AT-DataBase of Apicomplexa Transcriptomes. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Nucleic Acids Research 11/2014; · 8.81 Impact Factor
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    ABSTRACT: Background: The design and development of an effective malaria vaccine against the pre-erythrocytic and erythrocytic-stages of infection present a great challenge. Methods: In the present study, protective efficacy of oligomannose-coated liposome (OML)-entrapped merozoite and sporozoite antigens against Plasmodium berghei challenge infection in BALB/c mice was evaluated. Results: Subcutaneous immunization with truncated merozoite surface protein 1 entrapped with OML (OML-PbMSP1) prolonged survival, but failed to protect the mice from erythrocytic-stage infection, despite the antigen-specific antibody responses induced by the immunization regimen. In contrast, immunization with circumsporozoite protein entrapped with OML (OML-PbCSP) elicited antigen-specific humoral and cellular responses, which correlated with substantial protection against sporozoite challenge infections. Conclusions: The current results represent the use of an oligomannose-coated liposome-based vaccine against pre-erythrocytic and erythrocytic stages malaria infection. This approach may offer a new vaccination strategy against malaria infection.
    Malaria Journal 11/2014; 13(1):426. DOI:10.1186/1475-2875-13-426 · 3.49 Impact Factor
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    ABSTRACT: In the present study, we examined the contributions of macrophages to the outcome of infection with Babesia microti, the etiological agent of human and rodent babesiosis, in BALB/c mice. Mice were treated with clodronate liposome at different time courses of B. microti infection in order to deplete the macrophages. Notably, a depletion of host macrophages at the early and acute phases of infection caused a significant elevation of parasitemia associated with remarkable mortality in the mice. The depletion of macrophages at the resolving and latent phases of infection resulted in an immediate and temporal exacerbation of parasitemia coupled with mortality in mice. Reconstituting clodronate liposome-treated mice at the acute phase of infection with macrophages from naïve mice resulted in a slight reduction in parasitemia with improved survival, as compared to mice that received the drug alone. These results indicate that macrophages play a crucial role in the control of and resistance to B. microti infection in mice. Moreover, analyses of host immune responses revealed that macrophage-depleted mice diminished their production of Th1 cell-cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Furthermore, depletion of macrophage at different time courses exaggerated the pathogenesis of the infection in deficient IFN-γ(-)/(-) and severe combined immunodeficiency (SCID) mice. Collectively, our data provides important clues about the role of macrophages in the resistance and control of B. microti and implies that the severity of the infection in immunocompromised patients might be due to impairment of the macrophages' function.
    Infection and Immunity 10/2014; 83(1). DOI:10.1128/IAI.02128-14 · 4.16 Impact Factor
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    ABSTRACT: Babesia microti is an emerging zoonotic protozoan organism that causes "malaria-like" symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD(+)) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection.
    Drug Target Insights 07/2014; 8:31-8. DOI:10.4137/DTI.S16504
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    Fumiaki Ihara, Yoshifumi Nishikawa
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    ABSTRACT: Background Lacking enzymes for sterol synthesis, the intracellular protozoan Toxoplasma gondii scavenges cholesterol from host cells to multiply. T. gondii has a complex life cycle consisting of two asexual stages; the proliferative stage (tachyzoite), and the latent stage characterized by tissue cysts (bradyzoite). In vitro, bradyzoite development can be induced by mimicking host immune response stressors through treatment with IFN-γ, heat shock, nitric oxide, and high pH. However, the extent to which host nutrients contribute to stage conversion in T. gondii is unknown. In this study, we examined the impact of host cholesterol levels on stage conversion in this parasite. Methods Growth of T. gondii tachyzoites (ME49 strain) was investigated in Chinese hamster ovary (CHO) cells using various concentrations of low-density lipoprotein (LDL), oleic acid, or glucose. Squalestatin, which is an inhibitor of squalene synthase and is, therefore, an inhibitor of sterol synthesis, was used to treat the CHO cells. Tachyzoite to bradyzoite conversion rates were analyzed by indirect fluorescent antibody tests. Results Parasite growth was significantly enhanced by addition of exogenous LDL, whereas no such enhancement occurred with oleic acids or glucose. In ME49, growth inhibition from squalestatin treatment was not obvious. Although growth of the RH strain was unaffected by squalestatin in the presence of lipoprotein, in its absence growth of this strain was suppressed. The frequency of BAG1-positive vacuoles in ME49 increased under lipoprotein-free conditions. However, addition of exogenous LDL did not increase tachyzoite to bradyzoite conversion in this strain. Furthermore, treatment with squalestatin did not enhance stage conversion. Conclusion Our results suggest that LDL-derived cholesterol levels play a crucial role in bradyzoite conversion in T. gondii.
    Parasites & Vectors 05/2014; 7(1):248. DOI:10.1186/1756-3305-7-248 · 3.25 Impact Factor
  • Chisa Abe, Sachi Tanaka, Fumiaki Ihara, Yoshifumi Nishikawa
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    ABSTRACT: Upon Neospora caninum infection, we observed that murine macrophages showed greater activation and increased IL-6, IL-12p40 and IFN-γ production. Many macrophages migrated to the site of infection. Furthermore, macrophage-depleted mice exhibited increased sensitivity to N. caninum infection. This study indicates that macrophages are required for achieving protective immunity against N. caninum.
    Clinical and vaccine Immunology: CVI 05/2014; 21(8). DOI:10.1128/CVI.00082-14 · 2.37 Impact Factor
  • 83rd Annual Meeting of Japanese Society of Parasitology, Ehime University, Japan.; 03/2014
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    ABSTRACT: Toxoplasma gondii hijacks host cells to allow it to disseminate throughout a host animal; however, the migratory machinery involved in this process has not been well characterized. We examined the functional role of T. gondii cyclophilin 18 (TgCyp18) in host cell recruitment using recombinant parasites transfected with TgCyp18. High levels of TgCyp18 enhanced IL-12 production in cysteine-cysteine chemokine receptor 5 (CCR5) knockout mice (CCR5-/-) that had been infected peritoneally with T. gondii. Recruitment of CD11b+ cells to the infection site was enhanced in a CCR5-independent manner. T. gondii spread to several organs, particularly the liver, in a TgCyp18-dependent and CCR5-independent manner. Additionally, CCL5 levels were upregulated in macrophages treated with recombinant protein TgCyp18 and in the peritoneal fluids of the infected CCR5-/- mice. Furthermore, the chemokines involved in macrophage migration, CCL2 and CXCL10, were upregulated in the livers of CCR5-/- mice infected with recombinant parasites that had been transfected with TgCyp18. TgCyp18 may play a crucial role in macrophage migration, and in assisting with transport of T. gondii via CCR5-independent mechanisms. TgCyp18 may also play a role with CCL5 in the migration of macrophages to the site of infection, and with CCL2 and CXCL10 in the transport of T. gondii-infected cells to the liver.
    BMC Microbiology 03/2014; 14(1):76. DOI:10.1186/1471-2180-14-76 · 2.98 Impact Factor
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    ABSTRACT: Abstract The resistance of Babesia parasites to current anti-babesiosis drugs is an issue of major concern. The inosine 5'-monophosphate dehydrogenase (IMPDH) of Babesia gibsoni has been identified and characterized as a molecular drug target in our previous studies. In the present study, inhibitory effects of IMPDH inhibitors (mycophenolate mofetil, mizoribine, ribavirin, 7-nitroindole, and mycophenolic acid) were evaluated in vitro or in vivo. In an inhibition assay of recombinant B. gibsoni IMPDH (BgIMPDH) activity, mycophenolate mofetil was the most potent inhibitor (IC50 = 2.58 ± 1.32 μM) while ribavirin was the least potent. The inhibitory effects of mycophenolate mofetil, mizoribine, ribavirin, and 7-nitroindole on the in vitro growths of B. gibsoni and Babesia bovis were also assessed. The results revealed that mycophenolate mofetil was the most potent inhibitor of the multiplications of both B. gibsoni (IC50 = 0.13 ± 0.05 μM) and B. bovis (IC50 = 0.97 ± 0.49 μM). Ribavirin was also the least potent for both B. gibsoni and B. bovis in vitro. Mycophenolic acid, a metabolite of mycophenolate mofetil, caused an inhibition of Babesia microti in mice with noticeable improvement in hematological parameters of the infected mice (ED50 = 44.15 ± 12.53 mg/kg). Although the report here provide a non-exhaustive view of potential treatment strategy without addressing the potential adverse effect of immune suppression on infections, these results indicated that the IMPDH might be a molecular target of MPA for B. microti. Altogether, we provide a basis for development of antibabesia prodrugs by targeting IMPDH of the parasites in treatment of babesiosis.
    Journal of Parasitology 02/2014; 100(4). DOI:10.1645/13-278.1 · 1.26 Impact Factor
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    ABSTRACT: Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of mammals and birds, causing toxoplasmosis. Several types of vaccines against T. gondii have been developed, but these have limitations in terms of their safety and inadequate efficacy. T. gondii profilin (TgPF) is a potential immunodominant antigen for a candidate vaccine. In this study, we encapsulated TgPF in oligomannose-coated liposomes (OMLs) to evaluate the immune response induced by this vaccine. C57BL/6 mice were immunized with TgPF-OML three times at 14-day intervals and challenged with T. gondii. TgPF-OML increased the survival of the mice and reduced the parasite burden in their brains after T. gondii infection. Immunization with TgPF-OML also induced TgPF-specific interferon-γ production and IgG antibodies in mice. Our results demonstrate that OML-encapsulated TgPF triggers strong humoral and cellular responses against T. gondii, and that TgPF-OML is a candidate vaccine that warrants further development.
    Vaccine 02/2014; 32(16). DOI:10.1016/j.vaccine.2014.01.095 · 3.49 Impact Factor
  • Journal of parasitic diseases 01/2014; DOI:10.1007/s12639-014-0530-7
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    ABSTRACT: A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite's growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.
    PLoS ONE 12/2013; 8(12):e83305. DOI:10.1371/journal.pone.0083305 · 3.53 Impact Factor
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    ABSTRACT: A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (BgGARP) of Babesia gibsoni. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibodies against BgGARP when compared with the control groups. The antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. Two weeks after the booster immunization with a recombinant vaccinia virus expressing BgGARP, the dogs were challenged with B. gibsoni parasite. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia. These results indicated that this type of DNA/rvv prime-boost immunization approach may have use against B. gibsoni infection in dogs.
    Acta Parasitologica 12/2013; 58(4):619-23. DOI:10.2478/s11686-013-0183-1 · 0.97 Impact Factor
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    ABSTRACT: The emergence of drug resistance and adverse side effects of current bovine babesiosis treatment suggest that the search of new drug targets and development of safer and effective compounds are required. This study focuses on dihydroorotate dehydrogenase (DHODH), the fourth enzyme of pyrimidine biosynthesis pathway as a potential drug target for bovine babesiosis. Recombinant Babesia bovis DHODH protein (rBboDHODH) was produced in Escherichia coli and used for characterization and measurement of enzymatic activity. Furthermore, the effects of DHODH inhibitors were evaluated in vitro. The recombinant B. bovis DHODH histidine fusion protein (rBboDHODH) had 42.4-kDa molecular weight and exhibited a specific activity of 475.7 ± 245 Unit/mg, a Km= 276.2 µM for L-dihydroorotate and a Km= 94.41 µM for decylubiquinone. A 44-kDa band of native BboDHODH was detected by Western blot analysis and found in parasites mitochondria using a confocal microscope. Among DHODH inhibitors, atovaquone (ATV) and leflunomide (LFN) significantly inhibited the activity of rBboDHODH as well as the growth of B. bovis in vitro. The half maximal inhibitory concentration (IC50) of ATV and LFN was 2.38 ± 0.53 nM and 52.41 ± 11.47 µM, respectively. These results suggest that BboDHODH might be a novel target for development of new drug for treatment of B. bovis infection.
    Journal of Veterinary Medical Science 11/2013; 76(3). DOI:10.1292/jvms.13-0419 · 0.88 Impact Factor

Publication Stats

2k Citations
363.93 Total Impact Points

Institutions

  • 2001–2015
    • Obihiro University of Agriculture and Veterinary Medicine
      • National Research Center for Protozoan Diseases
      Obibiro, Hokkaidō, Japan
    • Chiang Mai University
      Amphoe Muang Chiang Mai, Chiang Mai, Thailand
  • 2009
    • National Institute of Advanced Industrial Science and Technology
      • Research Center for Medical Glycoscience
      Tsukuba, Ibaraki, Japan
  • 2004
    • Yale-New Haven Hospital
      New Haven, Connecticut, United States
  • 1998–2002
    • The University of Tokyo
      • Department of Global Agricultural Sciences
      Edo, Tōkyō, Japan