研究者データベース

大橋 和彦(オオハシ カズヒコ)
獣医学研究院 獣医学部門 病原制御学分野
教授

基本情報

所属

  • 獣医学研究院 獣医学部門 病原制御学分野

職名

  • 教授

学位

  • Ph. D.(コーネル大学(アメリカ合衆国))
  • 獣医学修士(北海道大学)

J-Global ID

研究キーワード

  • 細胞生物学   感染免疫   

研究分野

  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学

職歴

  • 2007年 - 2008年 北海道大学大学院獣医学研究科 動物疾病制御学講座 准教授
  • 2008年 - 北海道大学大学院獣医学研究科 動物疾病制御学講座 教授
  • 2008年 - Professor
  • 2001年 - 2007年 北海道大学大学院獣医学研究科 動物疾病制御学講座 助教授
  • 2001年 - 2007年 Associate Professor
  • 1995年 - 2000年 北海道大学大学院獣医学研究科 動物疾病制御学講座 助手
  • 1995年 - 2000年 Research Associate
  • 1993年 - 1995年 北海道大学獣医学部 家畜伝染病学講座 助手
  • 1993年 - 1995年 Research Associate
  • 1985年 - 1989年 三楽(現メルシャン)株式会社中央研究所 生物評価室 研究員
  • 1985年 - 1989年 Researcher

学歴

  •         - 1993年   コーネル大学大学院   獣医学研究科
  •         - 1993年   Graduate School of Veterinary Medicine, Cornell University
  •         - 1985年   北海道大学   獣医学研究科   予防治療学
  •         - 1985年   北海道大学
  •         - 1983年   北海道大学   獣医学部
  •         - 1983年   北海道大学

所属学協会

  • 日本ウイルス学会   日本獣医学会   日本癌学会   日本免疫学会   

研究活動情報

その他活動・業績

  • Satoru Konnai, Hideto Nishikado, Shinji Yamada, Saiki Imamura, Takuya Ito, Misao Onuma, Shiro Murata, Kazuhiko Ohashi EXPERIMENTAL PARASITOLOGY 127 (2) 467 -474 2011年02月 [査読無し][通常論文]
     
    Lipocalins have been known for their several biological activities in blood-sucking arthropods. Recently, the identification and characterization of lipocalins from Ixodes ricinus (LIRs) have been reported and functions of lipocalins are well documented. In this study, we have characterized four Ixodes persulcatus lipocalins that were discovered while analyzing I. persulcatus tick salivary gland EST library. We show that the four I. persulcatus lipocalins, here after named LIPERs (lipocalin from I. persulcatus) are 28.8-94.4% identical to LIRs from I. ricinus. Reverse transcriptase-PCR analysis revealed that lipocalin genes were expressed specifically in the salivary glands throughout life cycle stages of the ticks and were up-regulated by blood feeding. The specific expressions were also confirmed by Western blotting analysis. Furthermore, to investigate whether native lipocalins are secreted into the host during tick feeding, the reactivity of anti-serum raised against saliva of adult ticks to recombinant lipocalins was tested by Western blotting. The lipocalins are potentially secreted into the host during tick feeding as revealed by specific reactivity of recombinant lipocalins with mouse antibodies to I. persulcatus tick saliva. Preliminary vaccination of mice with recombinant lipocalins elicited that period to reach engorgement was significantly delayed and the engorgement weight was significantly reduced as compared to the control. Further elucidation of the biological functions of LIPERs are required to fully understand the pathways involved in the modulation of host immune responses. (C) 2010 Elsevier Inc. All rights, reserved.
  • Veterinary Microbiology 1 2011年 [査読無し][通常論文]
  • Luis Fernando Parizi, Kiyoko Uemura Utiumi, Saiki Imamura, Misao Onuma, Kazuhiko Ohashi, Aoi Masuda, Itabajara da Silva Vaz EXPERIMENTAL PARASITOLOGY 127 (1) 113 -118 2011年01月 [査読無し][通常論文]
     
    Recombinant Glutathione S-transferase of Haemaphysalis longicornis (rGST-Hl) was expressed in Escherichia coli, purified by affinity chromatography and used in the immunization of cattle. Western blot analysis showed positive antibody response in cattle immunized with rGST-Hl. The tests also showed that immunized bovine sera recognize native Rhipicephalus micro plus proteins in different tissue extracts. Furthermore, the vaccine potential of rGST-Hl was investigated against infestation of Hereford cattle by R. microplus. Vaccination of cattle with rGST-Hl conferred partial cross-protection immunity against R. microplus. Considering the effect on number of engorged ticks, egg laying capacity and egg fertility, the overall efficacy of vaccination was of 57%, as compared with control group. (C) 2010 Elsevier Inc. All rights reserved.
  • Claro N. Mingala, Satoru Konnai, Ryoyo Ikebuchi, Kazuhiko Ohashi COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 34 (1) 55 -63 2011年01月 [査読無し][通常論文]
     
    Characterization of CTLA-4, PD-1 and PDL-1 genes from swamp and riverine type water buffaloes was done by molecular cloning, sequencing and phylogenetic analysis. The cloned cDNA of CTLA-4, PD-1 and PDL-1 contained an open reading frame of 666, 849 and 870 nucleotides, encoding a polypeptide of 221, 282 and 298 amino acids, respectively. Nucleotide sequence homology of both CTLA-4 and PDL-1 had 99.8% in swamp and riverine type, which gives the identical polypeptide. Meanwhile, PD-1 genes of swamp and riverine type water buffaloes had 99.2% of homology in nucleotide sequence, which has substitution of two amino acid residues. The hexapeptide motif, phosphatidylinositol 3'-kinase and potential glycosylation sites were conserved within the tribe Bovinae. Phylogenetic analysis confirmed the degree of relationship between the bubaline species and justify the distinctness of each breeds by the bootstrap value generated. (C) 2010 Elsevier Ltd. All rights reserved.
  • A. Mori, S. Konnai, S. Yamada, A. Hidano, Y. Murase, T. Ito, A. Takano, H. Kawabata, M. Onuma, K. Ohashi INSECT MOLECULAR BIOLOGY 19 (3) 359 -365 2010年06月 [査読無し][通常論文]
     
    Salp15, a 15-kDa tick salivary gland protein, is known for several suppressive activities against host immunity and critical functions for the transmission of Lyme borrelia in Ixodes scapularis and Ixodes ricinus, the major vectors found in North America and Western Europe. Salp15 inhibits the activation of cluster of differentiation (CD)4+T-cells through the repression of T-cell receptor (TCR)-triggered calcium fluxes and interleukin (IL)-2 production. Furthermore, Salp15 adheres to the spirochaeta and specifically interacts with its outer surface protein C. The binding of Salp15 to Borrelia burgdorferi protects it from antibody-mediated killing in vitro. The aim of this study is to identify the Salp15 genes in Ixodes persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan. Two cDNA clones encoding the Salp15-like sequence were obtained from salivary glands of fed female ticks. These genes encode 135- and 132-amino acid proteins, designated Salp15 Iper-1 and Salp15 Iper-2, respectively, both having signal peptide sequences and predicted to be secretory proteins. Salp15 Iper-1 and -2 showed 51.8 and 68.2% similarity to I. scapularis Salp15, respectively. Reverse transcriptase PCR analysis showed that Salp15 Iper genes were expressed specifically in the salivary glands throughout life cycle stages of the ticks and were up-regulated by blood feeding. In the I. persulcatus-derived sequences, the C-terminal part, which is the binding domain to the CD4 molecule of T-cells in I. scapularis Salp15, was well conserved. In the future, it will be necessary to analyse immunosuppressive functions of I. persulcatus Salp15 and their interaction with Borrelia spp. in Japan.
  • Ryoyo Ikebuchi, Satoru Konnai, Yuji Sunden, Misao Onuma, Kazuhiko Ohashi MICROBIOLOGY AND IMMUNOLOGY 54 (5) 291 -298 2010年05月 [査読無し][通常論文]
     
    Recent work has shown that PD-1, an immune inhibitory receptor, is involved in mechanisms for down-regulating immune responses during tumor progression or chronic viral infection. However, in the case of bovine diseases, there have been no reports on this molecule due to lack of information about bovine PD-1. In this study, we performed identification and preliminary characterization of the bovine PD-1 gene in two breeds of cattle. We cloned full cDNA sequences encoding for PD-1 from both Holstein-Friesian and Japanese Black breeds, and found that both of the genes encoded a 282-amino acid protein, which had a signal sequence, transmembrane domain and an immunoreceptor tyrosine-based inhibitory motif. This bovine PD-1 showed 72.9% and 65.6% homology to human and mouse PD-1, respectively, both of which have been well characterized and documented. Quantitative real-time PCR analysis showed that bovine PD-1 is expressed predominantly in T-cells (such as CD4+ and CD8+ cells) and among PBMCs, and is strongly upregulated on T-cell stimulation via ConA. A limited number of cattle were tested yet, as expected, the degree of PD-1 mRNA expression in CD4+ and CD8+ T-cells was greater in cattle with bovine leukemia virus-induced lymphoma than in uninfected cattle. Further studies to characterize the functions of bovine PD-1 are therefore warranted, in order to elucidate the mechanism of the immunosuppression associated with progression of several diseases and therapy in cattle.
  • Hirohisa Mekata, Satoru Konnai, William H. Witola, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi INFECTION GENETICS AND EVOLUTION 9 (6) 1301 -1305 2009年12月 [査読無し][通常論文]
     
    In South American countries, trypanosomiasis as a result of Trypanosoma evansi and Trypanosoma vivax infections causes significant economic losses in livestock. The objectives of this study were to characterize the epidemiology of bovine trypanosomiasis in South America and to draw a comparison between South American and Asian T. evansi isolates based on the polymorphisms in their transferrin receptor encoding gene 6. We assessed the prevalence rates of T. evansi and T vivax infections in cattle in different regions of Peru and Bolivia using the polymerase chain reaction (PCR) and found that, in Lima and Pucallpa in the Republic of Peru, T. evansi infection rates were 5.8% (6/104) and 2.5% (5/195), respectively, while in Santa Cruz, Republic of Bolivia, the infection rate for T. evansi was 11.5% (59/510). The prevalence rates of T. vivax in Lima and Santa Cruz were 3.8% (4/104) and 0.9% (5/510), respectively. In T. evansi, uptake of host transferrin is mediated by a receptor derived from the two expression site-associated genes 6 and 7 (ESAG6 and ESAG7). We previously showed that the ESAG6 depicts genetic diversity among different isolates of T. evansi in Asia. In this study, we cloned and sequenced the ESAG6 genes from T evansi isolates from South America, and found, in addition to some of the previously observed variants, 20 novel variants of ESAG6 genes which could be categorized into three new clades among the various isolates. To conclude, the results obtained in this study suggest that T evansi isolates from South America are more diverse than the Asian isolates. (C) 2009 Elsevier B.V. All rights reserved.
  • Shinji Yamada, Satoru Konnai, Saiki Imamura, Martin Simuunza, Mwelwa Chembensofu, Amos Chota, Andrew Nambota, Misao Onuma, Kazuhiko Ohashi VETERINARY JOURNAL 182 (2) 352 -355 2009年11月 [査読無し][通常論文]
     
    To ascertain the infection rate for tick-borne pathogens in Zambia, an epidemiological survey of Theileria parva, Babesia bigemina and Anaplasma marginale in traditionally managed Sanga cattle was conducted using PCR. Of the 71 native Zambian cattle, 28 (39.4%) were positive for T parva, 16 (22.5%) for B. bigemina and 34 (47.9%) for A. marginale. The mixed infection rate in cattle was 8.5% (6/71), 16.9% (12/71), 7.0% (5/71) and 2.8% (2/71) for T. parval B. bigemina, T. parval A. marginale, B. bigeminal A. marginale and T. parval B. bigeminal A. marginale, respectively. To predict the risk for transmission of tick-borne pathogens from ticks to cattle, a total of 74 Rhipicephalus appendiculatus ticks were collected from a location where cattle had been found positive for T parva. Of the ticks collected, 10 (13.5%) were found to be PCR-positive for T parva. The results suggest that the infection rate for tick-borne pathogens was relatively high in Sanga cattle and that adult R. appendiculatus ticks were highly infected with T parva. (C) 2008 Elsevier Ltd. All rights reserved.
  • Luis Fernando Parizi, Herbert Rech, Carlos Alexandre Sanchez Ferreira, Saiki Imamura, Kazuhiko Ohashi, Misao Onuma, Aoi Masuda, Itabajara da Silva Vaz VETERINARY PARASITOLOGY 164 (2-4) 282 -290 2009年10月 [査読無し][通常論文]
     
    The ticks Rhipicephalus (Boophilus) microplus and Haemaphysalis longicornis are blood-sucking ectoparasites of bovines, causing serious damages to the livestock production. The main control method for these ticks is based on acaricides. However, the use of vaccines has been studied as a promising control strategy. Calreticulin (CRT) is a multifunctional, predominantly intracellular protein present in almost all cells of animals. The secretion of CRT during feeding might be linked to the modulation of the parasite-host interaction. In the present study, recombinant CRTs of R. microplus (rBmCRT) and H. longicornis (rHlCRT) were expressed in Escherichia coli and purified by ion exchange chromatography and used for the immunization of bovines and mouse. ELISA demonstrated that both rCRTs are recognized by the sera of immunized bovines. In silica, despite the difference in amino acid sequences, antigenic index analysis of HlCRT and BmCRT using the Jameson-Wolf algorithm indicated that both proteins were very similar in antigenicity index, although six different epitopes between the tick CRTs have been inferred. These data were corroborated by competitive ELISA analyses, which suggest the presence of different epitopes within the proteins. Western blot analyses showed that anti-rBmCRT and anti-rHlCRT bovine sera also recognized the native proteins in larvae extracts and, moreover, sera of bovines immunized with saliva and extract of salivary glands recognized both recombinant CRTs. Thus, mouse and bovine immune system recognized rCRTs, resulting in the production of antibodies with similar specificity for both recombinant proteins, although different epitopes could be distinguished between rBmCRT and rHlCRT. (C) 2009 Elsevier B.V. All rights reserved.
  • Shinji Yamada, Satoru Konnai, Saiki Imamura, Takuya Ito, Misao Onuma, Kazuhiko Ohashi VACCINE 27 (43) 5989 -5997 2009年10月 [査読無し][通常論文]
     
    Male tick-derived voraxin alpha and voraxin beta, a pair of testicular proteins, are transferred to female via copulation to stimulate female blood feeding in the tick Amblyomma hebraeum (A. hebraeum). Immunized animals with recombinant (r-)voraxin alpha and voraxin beta have been shown as highly resistant to the tick infestation. In this study, we describe the cloning and characterization of voraxin alpha homologue from the tick Rhipicephalus appendiculatus (R. appendiculatus), the major vector for East Coast fever in Eastern Africa. The sequence analysis of the R. appendiculatus voraxin alpha indicated that the deduced amino acid sequence had high similarity with voraxin alpha of the tick A. hebraeum and Dermacentor variabilis, suggesting that voraxin alpha is conserved in different tick genera. Quantitative RT-PCR and Western blotting analysis showed that male voraxin alpha was predominantly expressed in testis and its expression was induced by blood feeding. X appendiculatus voraxina was not secreted into the host during tick feeding and was detected in mated female hemolymph as measured by Western blotting. Preliminary vaccination of rabbits with r-voraxin alpha elicited the humoral immunity and conferred protective immunity against female ticks, resulting in the reduced fed weight. These results suggest that r-voraxin alpha could be a good candidate as anti-tick vaccine. (C) 2009 Elsevier Ltd. All rights reserved.
  • Claro N. Mingala, Satoru Konnai, Motoshi Tajima, Misao Onuma, Kazuhiko Ohashi JOURNAL OF BASIC MICROBIOLOGY 49 (5) 495 -500 2009年10月 [査読無し][通常論文]
     
    Characterization of bovine viral diarrhea virus (BVDV) isolates has been focused of several studies this last decade. Until now lots of new strains are being unfolded maybe due to the viral fast mutation ability. As we focused our research on water buffalo immunology, we were able to identify a probable new BVDV isolates. RNA was extracted from water buffalo blood in the Philippines. The extracted RNA was reverse-transcribed and synthesized cDNA. Oligonucleotide primers from the viral E2 region were used to amplify the target viral gene and later purified, cloned and sequenced. The E2 region with 420 bp nucleotides long was compared with existing published sequences in the GenBank. Based on the constructed phylogenetic tree, the isolated strain showed to be a BVDV type 1b along with Osloss and CP7 strains. Further classification of the new isolates was done within the BVDV type 1b1 group, which was compared with other strains in the sub-group. The analysis revealed that Lamspringe/738, KE9 and 2543/87 were the closest with 92% homology. Additional study is being done to further qualify and quantify the extent of the existence of this new BVDV isolates in water buffalo in the Philippines. This is the first report of BVDV in the Philippines and first concerning BVDV in Philippine water buffalo.
  • Claro N. Mingala, Satoru Konnai, Fe A. Venturina, Misao Onuma, Kazubliko Ohashi RESEARCH IN VETERINARY SCIENCE 87 (2) 213 -217 2009年10月 [査読無し][通常論文]
     
    This study describes the quantification of cytokine expression of vaccinated water buffaloes with FMD inactivated vaccine. Using real-time PCR quantification assay, expression of Th1 (IL-2, IL-12p40, IFN gamma); Th2 (IL-4, IL-10) and inflammatory (IL-6, TNF alpha) cytokines were quantified weekly for the entire three-week duration of the experiment. It was noted that IFN gamma, IL-10 and TNF alpha had peaked on week three post-vaccination while the remaining cytokines peaked on the second week and decreased by the third week. The counteraction between IFN gamma and IL-4 was noted as well as the possible suppressive action of IL-10 to that of IL-2 and IL-12, which is a common phenomenon between Th1 and Th2 cytokines. Synergy between TNFa and IL-6 was also observed. These findings suggest that within the immune system of water buffalo there is a dynamic cell-mediated and humoral interaction in response to immunogen. This assessment of the cytokine expressions is vital for the study of water buffalo disease progression and concurring protective immune responses. (C) 2009 Elsevier Ltd. All rights reserved.
  • Y. Saito, S. Konnai, S. Yamada, S. Imamura, H. Nishikado, T. Ito, M. Onuma, K. Ohashi INSECT MOLECULAR BIOLOGY 18 (4) 531 -539 2009年08月 [査読無し][通常論文]
     
    Ixodes persulcatus is the primary vector for human tick-borne diseases in Japan. A cDNA library was constructed from whole body homogenates of fed nymphs of I. persulcatus. From this library, one cDNA encoding defensin-like antimicrobial peptide was identified. The amino-acid sequence showed high similarity to those of the defensins of other ticks and arthropods. I. persulcatus defensin mRNA transcripts were detected at all life cycle stages of fed ticks and found to be predominantly expressed in the midguts of adult female ticks, but not in the salivary glands, a finding corroborated by Western blotting analysis. To investigate the function of I. persulcatus defensin, we examined its antibacterial activity by evaluation of growth of several bacterial strains in the presence of the synthetic peptide. The defensin from I. persulcatus markedly inhibited the growth of Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis and Corynebacterium renale, but not Gram-negative bacteria except Escherichia coli O157. In conclusion, these results suggest that I. persulcatus defensin may be playing a significant role in the defence against microbes from bloodmeals.
  • Saiki Imamura, Itabajara da Silva Vaz, Satoru Konnai, Shinji Yamada, Chie Nakajima, Misao Onuma, Kazuhiko Ohashi EXPERIMENTAL AND APPLIED ACAROLOGY 48 (4) 345 -358 2009年08月 [査読無し][通常論文]
     
    We report the cloning, expression and characterization of an Haemaphysalis longicornis metalloprotease (named HLMP1). The gene encodes a predicted 550 aminoacid protein with similarity to metalloproteases of the reprolysin family. The protein sequence contains a signal sequence, the zinc-binding motif (HEXXHXXGXXH) common to metalloproteases and a cysteine-rich region. Reverse transcription-PCR expression analysis indicates the presence of mRNA in the salivary gland of larva, nymph and adult ticks. Rabbit repeatedly infested with H. longicornis recognized rHLMP1, suggesting that the immune-response against HLMP1 is naturally induced through the feeding of ticks. Vaccination of rabbit with rHLMP1 produced protective immunity against ticks, resulting in 15.6 and 14.6% mortality in nymph and adult ticks, respectively. This work provides information to understand the tick's defense system, and offers new insights to develop strategies to block this defense system with an anti-tick vaccine based on a metalloprotease.
  • Satoru Konnai, Hirohisa Mekata, Claro N. Mingala, Nancy S. Abes, Charito A. Gutierrez, Jesus Rommel V. Herrera, Alan P. Dargantes, William H. Witola, Libertado C. Cruz, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi INFECTION GENETICS AND EVOLUTION 9 (4) 449 -452 2009年07月 [査読無し][通常論文]
     
    Trypanosoma evansi (T. evansi) causes the disease called Surra in domestic animals, which is of great economic importance in South Asian countries. In order to improve the diagnosis of Surra, we endeavored to develop a real-time PCR assay for the detection and quantification of parasites in water buffaloes using specific primers for the T. evansi Rode Trypanozoon antigen type (RoTat) 1.2 Variable Surface Glycoprotein (VSG) gene, which is a known diverse DNA region in trypanosomes. The quantitative detection limit of the assay was 10(2) trypanosomes per mL of blood, and the identity of the amplicon was confirmed in all assays by melting curve analysis. To evaluate the clinical applicability of this procedure, detection and estimation of parasitemia in blood samples obtained from water buffaloes and horses were conducted. T evansi was detected in 17/607 (2.8%) blood samples, with parasitemia levels ranging from >10(1) to 10(7) parasites per mL of blood. Interestingly, out of the 17 PCR positive animals, 3 had previously received trypanocidal treatment and 1 had abortion history. These data indicate that real-time PCR for the estimation of putative parasitemia levels is a quantitatively and objectively applicable technique for clinical diagnosis of Surra, and could help to understand disease stage and risk of transmission of T. evansi. (C) 2009 Elsevier B.V. All rights reserved.
  • Claro N. Mingala, Satoru Konnai, Libertado C. Cruz, Misao Onuma, Kazuhiko Ohashi CYTOKINE 46 (2) 273 -282 2009年05月 [査読無し][通常論文]
     
    This moleculo-epidemiological and immunological study through cytokine response assessment was done to know the dynamics of cytokines in the initiation, persistence and association to physiological changes of a particular pathogen in water buffaloes. This is important to understand the magnitude and behavior of disease progression. Water buffalo blood samples gathered from different places in the Philippines revealed a 9.4%. 27.6%, 10.3% and 4.4% prevalence of bovine viral diarrhea virus (BVDV), bovine leukemia virus (BLV), Anaplasma marginale and Babesia bigemina infection, respectively. This was the first surveillance study of BVDV and BLV in the country. Furthermore, cytokine expression of these naturally infected animals was also quantified. BVDV-infected animals had up-regulated expressions of TNF alpha, IL-2 and IL-4; and down-regulated expressions of IFN gamma and IL-12p40 while BLV positive animals had an up-regulated IL-4 and IL-6, and highly expressed IL-10 and IL-12p40 with unchanged IFN gamma expression. Meanwhile, animals infected with A. marginale had all interleukins and IFN gamma up-regulated with significant expression of IL-10 and IL-12p40 similar to the BLV positive animals. Since it was also observed that swamp-type buffaloes were more disease tolerant than riverine-type buffaloes based on the gathered infection rate of each examined pathogen, further assessment was done focusing on the two vital cytokines, IFN gamma and TNF alpha. We quantified IFN gamma and TNF alpha expressions in ConA-stimulated PBMC from both swamp and riverine buffaloes by real-time PCR. Cytokine expression from ConA-stimulated PBMC revealed that both IFN gamma and TNF alpha were more highly expressed in swamp than in riverine buffalo. To further examine the probable cause of expression differences, the proximal promoter region of these two cytokines were sequenced for the presence of nucleotide polymorphism followed by luciferase assay to analyze the effect of these polymorphisms in gene transcription. A single nucleotide polymorphism was found in the IFN gamma (-299) while eight polymorphisms in the TNF alpha promoter (-541, -553, -562, -596, -609, -655, -659, -688). Luciferase assay showed that both IFN gamma promoter and TNF alpha promoter in swamp-type water buffalo had higher transcription activity compared to riverine-type water buffalo. These findings confirm that IFN gamma and TNF alpha transcriptions in these animals were highly affected by the disparity in the cytokine promoter region. This suggests that disease tolerance or susceptibility of these buffaloes could be due to the differences in their relative cytokine transcription and may relate to pathogen-host specific pathogenesis. (C) 2009 Elsevier Ltd. All rights reserved.
  • Rika Kano, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi Journal of Veterinary Medical Science 71 (5) 603 -610 2009年05月 [査読無し][通常論文]
     
    Marek's disease (MD) is a commercially important disease of chickens caused by MD virus (MDV). Although avirulent MDV strains have been used for vaccination to prevent MD outbreaks, the protective mechanism of the vaccine has not been elucidated. In this Study, a comprehensive transcriptional analysis using microarray wits conducted in MDV-infected chickens with and without vaccination at 7 and 21 days post-infection (dpi). The data Suggested that the expression of T cell receptor (TCR) 1-related genes was up-regulated in vaccinated-challenged compared to unvaccinated-challenged chickens during the latent phase of infection. Consistently, this induction was confirmed by quantitative PCR. Flow cytometric analysis revealed that most of TCR1(+) cells expressed CD8 alpha chain brightly. The number of this subpopulation was significantly and specifically increased in vaccinated-challenged chickens at 21 dpi compared to univaccinated-challenged chickens, though it was not the major Population in spleen of chickens. The number of CD8 alpha(high) TCR2(+) cells, the major subpopulation of chicken CD8 alpha(high) cells, was increased in vaccinated chickens with or without challenge compared to univaccinated control chickens. These data suggested that both CD8 alpha(high) TCR1(+) and CD8 alpha(high) TCR2(+) cells could be induced by the vaccination. It is also possible that CD8 alpha(high) TCR1(+) cells might be primed by the vaccination and specifically induced by the challenge with virulent strain of MDV during the latent phase of infection. Thus, CD8 alpha(high) TCR1(+) cell Population is probably one of the key factors involved in the protective mechanism induced by a vaccine strain, CV1988.
  • Carlos Logullo, William H. Witola, Caroline Andrade, Leonardo Abreu, Josiana Gomes, Itabajara da Silva Vaz, Saiki Imamura, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma VETERINARY PARASITOLOGY 161 (3-4) 261 -269 2009年05月 [査読無し][通常論文]
     
    Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism in mammals. GSK-3 belongs to a highly conserved family of serine/threonine protein kinases, whose members are involved in hormonal regulation, nuclear signaling, and cell fate determination in higher eukaryotes. We have cloned and characterized the RmGSK-3 gene from Rhipicephalus (Boophilus) microplus tick embryos. DNA and protein sequence analysis depicted high similarity to the corresponding enzyme, from both vertebrate and invertebrate animals. In addition, the mRNA transcription profile identified during embryogenesis was analyzed. We observed that the RmGSK-3 mRNA rapidly decreases from the 1st to 3rd day of development, and increases from the 3rd to 15th day. After the 15th day of development, we observed a near 50% reduction in RmGSK-3 mRNA transcription in comparison to the 1st day. We detected the GSK-3 P isoform in egg homogenates throughout embryogenesis using Western blot analysis. RmGSK-3 mRNA was present in fat body, midgut and ovary from partially and fully engorged adult female ticks. The highest mRNA level was observed in ovaries from both developmental stages and in first-day eggs. Furthermore, RmGSK-3 activity correlated with glycogen content variation. Finally, kinase activity in egg homogenates was inhibited by the specific inhibitor, SB-216763. These data suggest that RmGSK-3 beta may be involved in glycogen metabolism regulation during R. microplus embryogenesis. (c) 2009 Elsevier B.V. All rights reserved.
  • KANO Rika, KONNAI Satoru, ONUMA Misao, OHASHI Kazuhiko Microbiology and Immunology 53 (4) 224 -232 2009年04月 [査読無し][通常論文]
     
    Marek's disease has been controlled by vaccination with avirulent strains of MDV. However, the protection mechanism following vaccination is not fully understood. In this study immune responses of PBMC and splenocytes derived from vaccinated chickens challenged with virulent MDV were examined using real-time PCR and ELISA. Higher levels of IFN-gamma induction were observed in chickens vaccinated during the latent phase of infection with virulent MDV than in similarly challenged, unvaccinated chickens. Furthermore, the mean expression of IFNGR2 and IFN regulatory factor-3 mRNAs was significantly higher in vaccinated than in unvaccinated chickens. These results show that IFN-gamma could be one of the important factors in prevention of MD by vaccination and is effective during the latent phase of the infection.
  • Satoru Konnai, Chie Nakajima, Saiki Imamura, Shinji Yamada, Hideto Nishikado, Michi Kodama, Misao Onuma, Kazuhiko Ohashi IMMUNOLOGY 126 (2) 209 -219 2009年02月 [査読無し][通常論文]
     
    Previously, a putative immunosuppressant-coding gene was identified from a complementary DNA library derived from the salivary glands of partially-fed Haemaphysalis longicornis. Using real-time polymerase chain reaction, the gene was shown to be predominantly expressed during blood feeding with the site of expression being mainly in the salivary glands; this was confirmed by Western blotting analysis. To investigate the function of this novel protein, in this study, we examined the proliferative responses of bovine mononuclear cells and murine splenic cells as well as the expression of profiles of several cytokines in these cells in the presence of the recombinant protein (H. longicornis-derived 36 000 molecular weight protein: rHL-p36). The addition of rHL-p36 at the beginning of the 72 hr cultivation period clearly inhibited proliferation of several mitogen-stimulated cells in a dose-dependent manner, with concomitantly significant down-regulation of messenger RNA levels for interleukin-2. The inhibitory response could be abrogated by blockage of HL-p36 with antibody, suggesting the direct involvement of rHL-p36 in the cell proliferation. Furthermore, the proliferative response of splenocytes isolated from rHL-p36-inoculated mice was significantly lower than for those from control mice, suggesting that rHL-p36 could also directly suppress immune responses in vivo. Interestingly, microarray analysis of the splenocytes showed that the expression of several immunomodulating genes was down-regulated by rHL-p36 inoculation. In conclusion, these results suggest that HL-p36 is an immunosuppressor that might play an important role in the modulation of host immune responses.
  • Shinji Yamada, Satoru Konnai, Saiki Imamura, Martin Simuunza, Mwelwa Chembensofu, Amos Chota, Andrew Nambota, Misao Onuma, Kazuhiko Ohashi JOURNAL OF VETERINARY MEDICAL SCIENCE 71 (1) 49 -54 2009年01月 [査読無し][通常論文]
     
    Theileria parva (T. parva) causes a highly serious bovine disease called Fast Coast fever (ECF), which is characterized by pyrexia, dyspnea and cachexia and is of great economic importance in African Countries. We hypothesize that the clinical symptoms of ECF could he explained by a cytokine dysregulation. In this study, we investigated the relationship between T parva DNA load and expression levels of cytokine mRNAs in leukocytes from experimentally infected calves by quantitative PCR. The p104 gene, which encodes the T parva 104 kDa microneme-rhoptry protein, was detected in cattle blood from day 10 after T. parvo-infected tick infestation, and the protozoan DNA load was increased together with severity of disease. The mRNA expressions of pro-inflammatory cytokines, such as interleukin (IL)-1 beta and IL-6, were up-regulated with protozoan DNA load increasing. In addition, the level of a type-2 cytokine (IL-10) transcript was also increased during the acute phase. In contrast, the down-regulation or no detectable levels of the expression of type-I cytokines, such as IL-2 and interferon (IFN)-gamma were observed in T. parva-infected animals. Thus. Our observations indicated that high protozoan load and resulting intense inflammatory responses might be involved in the severity of clinical signs observed in T. parva-infection.
  • Shinji Yamada, Yuko Ito, Saiki Imamura, Satoru Konnai, Takuya Ito, Misao Onuma, Kazuhiko Ohashi EXPERIMENTAL PARASITOLOGY 120 (4) 337 -342 2008年12月 [査読無し][通常論文]
     
    A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks. (C) 2008 Elsevier Inc. All rights reserved.
  • Hirohisa Mekata, Satoru Konnai, Martin Simuunza, Mwelwa Chembensofu, Rika Kano, William H. Witola, Mwase E. Tembo, Harrison Chitambo, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi JOURNAL OF VETERINARY MEDICAL SCIENCE 70 (9) 923 -928 2008年09月 [査読無し][通常論文]
     
    The prevalence of trypanosome infections in tsetse flies, Glossina pallidipes, collected front Chiawa and Chakwenga in Zambia with endemic trypanosomosis was assessed by polymerase chain reaction (PCR). Out of the 550 G. pallidipes, 58 (10.5%) flies were found to harbor trypanosome DNA. Infection rates of tsetse with Trypanosoma vivax universal, Trypanosoma congolense savannah, T congolense forest and T. congolense kilifi were 4.2% (23/550), 4.7% (26/550), 1.1% (6/550) and 1.6% (9/550), respectively. To determine the mammalian hosts of T. congolense and T. vivax infections from the tsetse flies, mammalian mitochondrion DNA of blood meal in these flies were analyzed by PCR and subsequent gene sequence analysis of the amplicons. Sequence analysis showed the presence of cytochrome b gene (cyt b) of 7 different mammalian species such as human, elephant, buffalo, goat, warthog, greater kudu and cattle. Goats which were main livestock in these areas were further examined to know the extent of its contribution ill spreading the infection. We examined the prevalence of trypanosome infections in the domestic goat Population in 6 settlements in Chiawa alone. Of the 86 goats sampled, 4 (4.6%), 5 (5.8%), 4 (4.6%) and 4 (4.6%) were positive for T. vivax universal, T. congolense savannah, forest and kilifi, respectively. These findings showed that the host-source of trypanosome infections in vector fly give a vital information about spread of infection. The result of this study will certainly contribute in elucidating more the epidemiology of trypanosomosis.
  • Takehiro Murao, Yoshitaka Omata, Rika Kano, Shiro Murata, Tsukasa Okada, Satoru Konnai, Mitsuhiko Asakawa, Kazuhiko Ohashi, Misao Onuma JOURNAL OF PARASITOLOGY 94 (4) 830 -833 2008年08月 [査読無し][通常論文]
     
    Antibodies to Toxoplasma gondii were assayed by ELISA in 22 experimentally inoculated domestic ducks. In addition a serological assay was carried out at Obihiro, Hokkaido, Japan, in 2004 and 2005, on 221 wild ducks of 5 species: Anas platyrhynchus (n = 111); A. poecilorhyncha (n = 27); A. acute (n = 58); A.penelope (n = 16); and A. crecca (n = 9). Assays were also conducted using sera from 197 wild geese of 2 species, i.e., Anser albifrons (n = 162) and Ans. fabalis (n = 35). Birds were collected between 2003 and 2005 from 3 different areas: Lake Miyajima-numa, Hokkaido, Japan, regions around Anadyr city of Chukotka antonomous okrug, and Lake Makobetukoa, Kamchatka oblast, Russia. The ELISA cutoff value (OD) was >= 0.395 based on results from uninfected ducks; the final dilution ratio recognized as positive was represented by the end titer. The end titer in the experimentally induced ducks ranged from 1:400 to 1:3,200. Antibodies to T. gondii were found in 49 of the 221 wild duck samples from Japan: A. platyrhynchus (22/74); A. poecilorhyncha (2/15); and A. poecilorhyncha (5/12). Thirty-two of 197 wild goose samples were seropositive, i.e., Ans. albifrons (7/51) in 2004 and (11/72) in 2005 in Miyajima-numa, Japan and 9/39 in Chukotka. Russia as well as in Ans. fabalis (5/35) in Kamchatka, for which the end titer ranged from 1:100 to 1:3,200. In immunoblotting, the A.platyrhynchus samples showed specific IgG antibody binding to several antigens in the T. gondii lane, i.e., at 30 and 43 kDa, but not in the Neospora caninum lane. No specific bands were noted in samples for which antibody activity was not detected. These results suggest that wild waterfowl inhabiting Hokkaido, Chukotka, and Kamchatka may be exposed to T. gondii.
  • Satoru Konnai, Hirohisa Mekata, Raadan Odbileg, Martin Simuunza, Mwelwa Chembensof, William Harold Witola, Mwase Enala Tembo, Harrison Chitambo, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi VECTOR-BORNE AND ZOONOTIC DISEASES 8 (4) 565 -573 2008年08月 [査読無し][通常論文]
     
    The prevalence of trypanosome infections in tsetse flies in the Chiawa area of Lower Zambezi in Zambia, with endemic trypanosomosis, was determined by a polymerase chain reaction (PCR) method that allowed the detection of trypanosome DNA and determination of the type of animal host fed on by the tsetse fly Glossina pallidipes, using tsetse-derived DNA extracts as templates. Ninety G. pallidipes (82 females and 8 males; 18.3%) of the 492 flies captured by baited biconical traps tested positive for the presence of Trypanosoma brucei species genomic DNA. Of the 90 T. brucei-positive flies, 47 (52.2%) also tested positive for vertebrate mitochondrial DNA. Sequence analysis of the vertebrate mitochondrial DNA amplicons established that they originated from 8 different vertebrate species, namely, human (Hoino sapiens), African elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), waterbuck (Kobus ellipsipymnus), roan antelope (Hippotragus equinus), greater kudu (Tragelaphus strepsiceros), warthog (Phacochoerus africanus), and goat (Capra hircus). Furthermore, to investigate the prevalence of trypanosome infections in domestic goats in the same area where trypanosomes had been detected in tsetse files, a total of 86 goats were randomly selected from 6 different herds. Among the selected goats, 36 (41.9%) were found to be positive for T. brucei species. This combined detection method would be an ideal approach not only for mass screening for infection prevalence in tsetse populations, but also for the prediction of natural reservoirs in areas endemic for trypanosomosis.
  • Saiki Imamura, Satoru Konnai, Itabajara da Silva Vaz Junior, Shinji Yamada, Chie Nakajima, Yuko Ito, Tomoko Tajima, Jun Yasuda, Martin Simuunza, Misao Onuma, Kazuhiko Ohashi JAPANESE JOURNAL OF VETERINARY RESEARCH 56 (2) 85 -98 2008年08月 [査読無し][通常論文]
     
    Rhipicephalus appendiculatus serpin-3 (RAS-3), R. appendiculatus serpin-4 (RAS-4) and a 36-kDa immuno-dominant protein of R. appendiculatus (RIM36) were reported as candidate antigens for the anti-tick vaccine to control ixodid ticks. In the present study, we generated recombinant proteins of RAS-3 (rRAS-3), RAS-4 (rRAS-4) and RIM36 (rRIM36), and assessed their potency as an anti-tick cocktail vaccine in cattle model. RT-PCR analysis showed that RAS-3, RAS-4 and RIM36 transcripts were detected in both adult male and female ticks during feeding. Immunization of cattle with the combination of rRAS-3, rRAS-4 and rRIM36 had raised antibodies against all recombinants and anti-sera had reacted with the molecules from the tick salivary gland extract. Tick infestation challenge demonstrated protective immunity against female ticks, resulting in mortality rates of 39.5 and 12.8 % for the vaccinated and control groups, respectively. Moreover, the mortality rate of Theileria parva-infected female ticks was 48.5 and 10.8 % in the vaccinated and control group, respectively. In order to evaluate the levels of pathogen transmission capacity by T parva-infected ticks fed on immunized cattle, the occurrence of T parva in the bovine parotid lymph node and peripheral blood was also determined and quantified by real-time PCR. Although the infection with T parva could not be protected by the vaccine, the occurrence of pathogen in peripheral blood was delayed 1 to 2 days after the infestation challenge in vaccinated group. These results suggest that this cocktail vaccine plays a role in the prevention of tick infestation.
  • Hitoshi Hatai, Kenji Ochiai, Mariko Murakami, Syunsuke Imanishi, Yukiko Tomioka, Takeshi Toyoda, Kazuhiko Ohashi, Takashi Umemura JOURNAL OF VETERINARY MEDICAL SCIENCE 70 (5) 469 -474 2008年05月 [査読無し][通常論文]
     
    Fowl glioma-inducing virus (FGV), which belongs to subgroup A of avian leukosis virus (ALV), is tumorigenic in the nervous system. In a zoological garden in Japan, approximately 40% of chickens, including Japanese fowls, were infected with FGV. Because this zoological garden plays a role as a major supplier of Japanese fowl for other zoological gardens, FGV infection is suspected to have spread among ornamental chickens. In this study, the prevalence of the disease was examined in a total of 129 chickens in three other zoological gardens by nested polymerase chain reaction (PCR), reverse transcription nested PCR and enzyme-linked immunosorbent assay. Twenty-six to 56 percent of the fowls in each of the examined gardens were positive by nested PCR. The phylogenetic analysis revealed that the 3' untranslated region, including the specific sequence of FGV, of the 14 isolated ALVs showed high sequence identity and a close relationship with FGV. In addition, the env gene of the isolates frequently showed mutations and deletions of nucleotides. These results suggest that FGV is prevalent among ornamental chickens kept in zoological gardens in Japan.
  • Satoru Konnai, Claro N. Mingala, Misako Sato, Nancy S. Abes, Fe A. Venturina, Charito A. Gutierrez, Takafumi Sano, Yoshitaka Omata, Libertado C. Cruz, Misao Onuma, Kazuhiko Ohashi ACTA TROPICA 105 (3) 269 -273 2008年03月 [査読無し][通常論文]
     
    In the Philippines, insufficient consideration has been given to the implementation of systematic control measures against major abortifacient infectious agents in livestock. To elucidate the epidemiology of abortifacient infectious agents in livestock, the prevalence of four abortifacient agents was assessed. Initially, a total of 96 cattle including 17 cows with history of abortion were examined in a herd in Luzon at the request of the farm owner. Six (35.3%) of the 17 aborting cows were found to be serologically positive for Neospora caninum (N. caninum), whereas the seroprevalence in non-aborting cows was 15.9% (10/63). Four of the 6 serologically positive aborting cows were also RT-PCR-positive for bovine viral diarrhea virus (BVDV). Two (12.5%) of the 16 bulls examined were also found to be infected with BVDV, suggesting a putative risk factor of transmission via semen. Based on sequence analysis, the isolates detected belong to BVDV type 1b group. Furthermore, an epidemiological survey of abortifacient infectious agents was conducted with various species of livestock from herds located in Luzon. Out of the 105 water buffalo samples collected, 4 (3.8%) were indicated positive to N. caninum, 2 (1.9%) to Toxoplasma gondii (T gondii) and 2 (1.9%) to Trypanosoma evansi (T evansi). The overall seroprevalence of N. caninum in goat and sheep were 23.6% (21/89) and 26.3% (10/38), respectively. BVDV was not detected in these herds. The findings of this exploratory study indicate a relationship between infection and bovine abortion and that a lager study is required to statistically confirm this relationship. (C) 2007 Elsevier B.V. All rights reserved.
  • Raadan Odbileg, Byambaa Purevtseren, Dorj Gantsetseg, Bazartseren Boldbaatar, Tumurjav Buyannemekh, Zagd Galmandakh, Janchivdorj Erdenebaatar, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi JOURNAL OF VETERINARY MEDICAL SCIENCE 70 (2) 197 -201 2008年02月 [査読無し][通常論文]
     
    In the present study, we determined the levels of cytokines produced by camel (Camelus bactrianus) peripheral blood mononuclear cells (PBMCs) in response to live attenuated Brucella abortus (B. abortus) S19 vaccine. Seven camels were vaccinated with commercial B. abortus S 19 vaccine, and their cytokine responses were determined using a real-time PCR assay. Cytokine responses to B. abortus S 19 were examined at 6 hr, 48 hr and 1, 2 and 3 weeks post-vaccination. Serological tests were performed to further confirm these immune responses. The results revealed that IFN-gamma and IL-6 were upregulated during the first week post-vaccination. Low level expressions of IL-1 alpha, IL-1 beta, TNF-alpha and IL-10 and no expression of IL-2 and IL-4 were observed compared with the control camels. The findings showed that B. abortus stimulates cell-mediated immunity by directly activating camel Thl cells to secrete IFN-gamma. This quantification of cytokine expression in camels is essential for understanding of Camelidae disease development and protective immune responses. This is the first report of in vivo camel cytokine quantification after vaccination.
  • Satoru Konnai, Yoichi Saito, Hideto Nishikado, Shinji Yamada, Saiki Imamura, Akina Mori, Takuya Ito, Misao Onuma, Kazuhiko Ohashi JAPANESE JOURNAL OF VETERINARY RESEARCH 55 (2-3) 85 -92 2008年01月 [査読無し][通常論文]
     
    Ixodes persulcatus Schulze (I. persulcatus) is distributed in Russia and Far East Asia including Japan, and has been implicated as the vector of several human pathogens. In particular, I. persulcatus acts as the only tick vector for human lyme borreliosis in Japan. In order to elucidate the mechanism of transmission of I. persulcatus -borne pathogens, we developed a laboratory colony of I. persulcatus. Ticks were fed on Syrian hamster and engorged ticks that had dropped off the animals were collected and maintained to allow them to molt. Tick rearing was performed in incubator at 20 degrees C with 95% relative humidity and 12-hour light/dark photo-period regimen. We found out that adult females fed for 8 +/- 2 days and had a pre-oviposition period lasting for 7 +/- 2 days. The minimum egg incubation period was 1 month with the hatched larvae feeding for 3 +/- 1 days and molting to nymphs 3-4 months thereafter. Meanwhile, the nymphs fed for 4 +/- 1 days and molted to adult 2-3 months thereafter. For future analysis of gene expression profiles in I. persulcatus, we cloned and sequenced the actin gene (a housekeeping gene), and found that it is 92.7% to 98.6% homologous to the published sequences of related ixodid ticks. This laboratory colony of I. persulcatus will facilitate investigations on the role of tick-derived molecules on the transmission of I. persulcatus -borne pathogens and will be important for identification of potential anti-tick vaccine and acaricide target molecules.
  • Hitoshi Hatai, Kenji Ochiai, Katsue Nagakura, Syunsuke Imanishi, Akihiro Ochi, Rie Kozakura, Masaaki Ono, Masanobu Goryo, Kazuhiko Ohashi, Takashi Umemura AVIAN PATHOLOGY 37 (2) 127 -137 2008年 [査読無し][通常論文]
     
    Fowl glioma is characterized by multiple nodular growth of astrocytes, and fowl glioma-inducing virus belonging to avian leukosis virus has been isolated from Japanese bantam as a causal agent. Subcutaneous neoplasms of the head and neck have been reported in layer chickens since 2003 in Japan, and fowl glioma concurred in these affected layers. In the present study, the histopathology of 240 layers, including 18 layers with subcutaneous neoplasms and 222 layers kept with the affected layers, was performed to clarify the characteristics of fowl glioma in layers. Microscopically, 103 layers showed non-suppurative encephalitis, and four layers had locally extensive proliferation or multiple nodules of astrocytes. Gliomas concurred in 11 layers with subcutaneous neoplasms and occurred independently in three layers. In addition, two layers had locally extensive proliferation of small, round cells in the cerebrum. The fowl glioma-inducing virus genome was not detected in the affected brains by nested polymerase chain reaction. Ten isolates were obtained from the affected brains. By nucleotide sequencing of the env gene, SU coding regions of these isolates were most closely related to myeloblastosis-associated virus-like viruses, but TM regions showed the highest similarity to endogenous viral (ev) loci. The genome of one isolate mainly consisted of ev loci and contained several parts of other avian leukosis/sarcoma viruses. These results show that the causal avian leukosis virus of fowl glioma is not just fowl glioma-inducing virus and that different avian leukosis virus strains having oncogenicity in the central nervous system by recombination are spread in layers in Japan.
  • S. Murata, K.-S. Chang, Y. Yamamoto, T. Okada, S.-I. Lee, S. Konnai, M. Onuma, Y. Osa, M. Asakawa, K. Ohashi ARCHIVES OF VIROLOGY 152 (8) 1523 -1526 2007年08月 [査読無し][通常論文]
     
    Marek's disease (MD) virus (MDV) is known to cause malignant lymphomas in chickens. In 2001, we first reported an MD case in a white-fronted goose (Anser albifrons) in Japan. Therefore, the prevalence of MDV in the wild geese was surveyed by nested PCR using feather-tip samples in Japan and the Far East region of Russia, breeding habitats of geese migrating to Japan. MDV was detected in about 30% of analyzed white-fronted geese. Furthermore, by nucleotide sequence analysis, we confirmed that this MDV shows high homology to very virulent MDV, suggesting that highly virulent MDV is widespread in white-fronted geese migrating between Japan and Far East region of Russia.
  • Tsukasa Okada, Michihiro Takagi, Shiro Murata, Misao Onuma, Kazuhiko Ohashi JOURNAL OF GENERAL VIROLOGY 88 (8) 2111 -2120 2007年08月 [査読無し][通常論文]
     
    In tumour cell lines established from Marek's disease (MID) lymphomas L-meq is consistently expressed. It contains a 180 bp insertion encoding additional copies of the proline-rich repeat in the meq open reading frame and its product may contribute to the maintenance of MID virus (MDV) latency. In this study, we identified a novel spliced form of the meq transcript in MD-derived lymphoblastoid cell lines and in MDV-infected cells. This transcript, termed Delta meq, encodes an N-terminal 98 aa of the Meq protein and lacks part of the basic leucine zipper (bZIP) and transactivation domains. In MID cell lines, transcription of L-meq was significantly downreguiated, while that of the Delta meq transcript was upregulated during apoptosis. These observations were also confirmed at the protein expression level. Reporter assays using meq- and interleukin-2 (IL-2)-promoter-driven luciferase vectors revealed that AMeq suppressed transactivation by L-Meq or Meq in a dose-dependent manner. Immunoprecipitation confirmed that AMeq was associated with L-Meq or Meq physically. These results suggest that AMeq could be involved in 2006 apoptosis in MID cell lines as it works as a negative regulator of L-Meq and Meq by direct interaction.
  • Satoru Konnai, Shinji Yamada, Saiki Imamura, Martin Simuunza, Mwelwa Chembensof, Amos Chota, Andrew Nambota, Kazuhiko Ohashi, Misao Onuma Vector-Borne and Zoonotic Diseases 7 (2) 241 -248 2007年06月 [査読無し][通常論文]
     
    Theileria parva, the agent of East Coast fever (ECF), is transmitted to the host during the blood meal feeding of Rhipicephalus appendiculatus ticks. In order to investigate the relationship between the attachment duration of R. appendiculatus and the transmission of T. parva, infected adult ticks were allowed to attach to naive mice for variable lengths of time. Attached ticks and host animal's back skin biopsies from the tick attachment site were collected daily, starting from 24 hours post-tick attachment, and used for seminested polymerase chain reaction (PCR) detection of T. parva. T. parva-infected ticks started to transmit the parasites from 72 hours post-tick attachment. As expected, the transmission of T. parva from ticks to mouse skin increased with duration of tick attachment. Transmission of the parasites was 77.7%, 100%, 85.5%, and 100% on day 4, 5, 6, and 7 post-tick attachment, respectively, as could be detected from mice skin biopsies taken from T. parva-infected ticks' attachment sites. These results have important implications for our understanding of early events in the transmission of T. parva and would help in the development of effective pharmacologic substances and/or vaccines against ticks. © Mary Ann Liebert, Inc.
  • Claro N. Mingala, Raadan Odbileg, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 30 (2) 119 -131 2007年03月 [査読無し][通常論文]
     
    The current research concerned in the cloning, sequencing and phylogenctic analysis of inflammatory cytokine (IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha) genes from swamp buffalo and two bubaline breeds, CB (cross between swamp and riverine type buffalo) and the Bulgarian Murrah buffalo. Multiple sequence comparison showed a high homology between the bubaline breeds, which ranged from 99.3% to 100.0% similarity, whereas from 98.6% to 99.0% compared to cattle. The phylogenetic analysis had confirmed and justified the degree of relationship between these bubaline species and their distinctness to each other by the bootstrap value (%) generated. These findings were discussed with particular attention to the diversity of the inflammatory cytokine proteins within closely related species. The result of this study concluded that a small difference in the cytokine structures might be the reason behind or has a contributory factor on the previous reports about the existence of disease resistance. However, in-depth study is necessary to further qualify these findings. (c) 2007 Elsevier Ltd. All rights reserved.
  • Journal of Veterinary Diagnostic Investigation 19 471 -478 2007年 [査読無し][通常論文]
  • Tatsufumi Usui, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 115 (1-2) 17 -23 2007年01月 [査読無し][通常論文]
     
    Immunological control of bovine leukemia virus (BLV)-infection has been reported as dependent on the expression balance of types 1 and 2 cytokines. In this report, mRNA expression of interferon (IFN)-gamma and interleukin (IL)-2 (type 1 cytokines), and of IL-4 and IL-10 (type 2 cytokines) were evaluated in concanavalin A-stimulated peripheral blood mononuclear cells (PBMC) from BLV-infected sheep. Despite the same dose of BLV-infection, the extent of viral propagation was markedly different between eight individual sheep by 12 weeks post infection. The virus did not propagate well in three sheep, which showed augmented mRNA expression of IFN-gamma, a strong indicator of cell-mediated immunity, immediately after BLV-infection. Among the other five sheep having more than 2% of BLV-infected cells among PBMC at 12 weeks post infection, four sheep developed B-cell leukemia or lymphoma within 2 years after infection. These observations indicate IFN-gamma expression may play an important role in the protective mechanism against BLV propagation at the early phase of the infection. (c) 2006 Elsevier B.V. All rights reserved.
  • Claro N. Mingala, Raadan Odbileg, Satoru Konnal, Kazuhiko Ohashi, Misao Onuma VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 113 (3-4) 348 -356 2006年10月 [査読無し][通常論文]
     
    Comparative assessment of Th1 and Th2 cytokines of three bubaline breeds namely swamp buffalo, its crossbreed with riverine buffalo (CB), and the improved breed of Bulgarian Murrah buffalo (BMB), was done by molecular cloning, sequencing and phylogenetic analysis. The Th I cytokines analyzed included IL-2, IL-12p35, IL-12p4O, and IFN-gamma while Th2 cytokines included IL-4 and IL-10. Both groups showed strict conservation in the putative secondary structures and amino acid residues within the tribe Bovini, which indicated functional cross-reactivity. Nucleotide sequence homology ranged from 98.6 to 100.0% and was lowest for IL-12p35. With regard to amino acid sequence, the lowest homology was observed in IL-4 with 97.8%. This substitution was mainly due to differences in mRNA splicing. The phylogenetic relationship of the buffalo breeds was analyzed and showed them as a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle and pigs. A deeper knowledge of these cytokine structures will favor understanding of water buffalo immunology and how much it differs from its closest subspecies and other animals. (c) 2006 Elsevier B.V. All rights reserved.
  • Raadan Odbileg, Byambaa Purevtseren, Zayat Batsukh, Satoru Konnaii, Kazuhiko Ohashi, Misao Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 68 (9) 941 -946 2006年09月 [査読無し][通常論文]
     
    The complementary DNAs of the Th1 (IL-2, 12-12p35, and IFN-gamma) and Th2 (IL-4, IL-10 and IL-13) cytokine genes of the bactrian camel (Camelus bactrianus) were cloned, sequenced, and analyzed. IL-2, IL-4, IL-10, IL-12p35, IL-13, and IFN-gamma were found to have 465, 402, 537, 669, 411, and 501 bp length open reading frames with 154, 133, 178, 222, 136, and 166 amino acid encodings, respectively. The homology ranged from 58.8% to 100% between the nucleotide sequences of the camel cytokine genes and the published sequences of other mammalian genes, including the llama, pig, cow, horse, human, and mouse. The cDNA had highest homology with orders Artiodactyla (pigs and cattle) and Perissodactyla (horses), especially to the recently cloned llama sequences.
  • Michihiro Takagi, Kazuhiko Ohashi, Toshifumi Morimura, Chihiro Sugimoto, Misao Onuma Leukemia Research 30 (8) 987 -992 2006年08月 [査読無し][通常論文]
     
    Several kinds of the p53 transcripts in which their open reading frames (ORFs) were truncated (ranging from 101 to 765 bp) were identified in Marek's disease (MD)-derived tumor cell lines as well as avian leukosis- and reticuloendotheliosis-derived ones, detected by nested RT-PCR and subsequent nucleotide sequence analysis. In these ORFs, regions encoding the proline-rich and DNA-binding domains of the p53 protein were frequently deleted, and many of these deletions were found to cause frame shift. Western blot analysis using anti-p53 monoclonal antibodies revealed that multiple p53 isoform proteins with various molecular weights including 45-46, 35 and 28 kDa were expressed in these tumor cell lines, though the p53 protein with a molecular weight of 49 kDa was detected in chicken embryo fibroblasts transformed by the SV40 T antigen as a control. Since no deletions were found in the p53 gene of these MD tumor cell lines, truncations in the p53 ORFs observed in this study might result from alternative splicing of the p53 gene. (c) 2005 Elsevier Ltd. All rights reserved.
  • Tatsufumi Usui, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 112 (3-4) 296 -301 2006年08月 [査読無し][通常論文]
     
    Tumor necrosis factor (TNF)-cL is thought to be one of the cytokines that account for bovine leukemia virus (BLV)-induced B-cell lymphoproliferative disorder, however, information on TNF-alpha expression in B-cells is limited. In this study, the expression of TNF-alpha in IgM(+) B-cells from BLV-infected sheep with or without lymphocytosis was determined. Freshly isolated IgM(+) B-cells from three sheep with lymphocytosis constitutively transcribed TNF-alpha mRNA. Although TNF-alpha mRNA expression in IgM(+) B-cells was transiently up-regulated after cell culture, TNF-alpha mRNA expression was markedly higher in lymphocytotic sheep when compared to that of non-lymphocytotic sheep or uninfected sheep. Expression of membranebound TNF-alpha on IgM(+) B-cells was also augmented in lymphocytotic sheep. TNF-alpha expression in lymphocytotic sheep may support the proliferation of B-cells. (c) 2006 Elsevier B.V. All rights reserved.
  • Satoru Konnai, Saiki Imamura, Chie Nakajima, William Harold Witola, Shinji Yamada, Martin Simuunza, Andrew Nambota, Jun Yasuda, Kazuhiko Ohashi, Misao Onuma ACTA TROPICA 99 (1) 34 -41 2006年08月 [査読無し][通常論文]
     
    In order to investigate the transmission dynamics of Theileria parva (T parva) by the brown ear tick, Rhipicephalus appendiculatus (R. appendiculatus), under experimental conditions, detection of T parva in ticks and cattle was performed by a quantitative real-time PCR assay. A calf inoculated with a T parva mixture became PCR-positive for T parva infection on day 8 post-inoculation, and subsequently, nymphal ticks were introduced and maintained to feed on the infected calf for 6 days. Engorged nymphs were collected daily and allowed to molt into adults, and overall, 70.8% (121/171) of the adult ticks acquired the T parva infection. Furthermore, the T parva infection rate in ticks under field conditions was monitored by real-time PCR in R. appendiculatus ticks collected from a traditionally managed pastoral land of Zambia, on which Sanga breed cattle are traditionally reared and the area has endemic East Coast fever (ECF). A total of 70 cattle were randomly selected in the same area and 67 (95.7%) were found to be serologically positive for R. appendiculatus tick antigen (RIM36). Twenty-nine (43.3%) of the 67 serologically positive cattle were real-time PCR-positive for T parva, although no piroplasms could be detected in the blood smears. Unexpectedly, out of 614 R. appendiculatus nymphal and adult ticks collected by flagging vegetation, 4.1% were positive for T parva DNA. However, since the rate of transmission of T parva from infected cattle to ticks and vice versa and the serological evidence of exposure to R. appendiculatus ticks in naturally exposed cattle were relatively high, it would be wise in such a case to consider vector control as well as vaccination against ECF as control measures. (c) 2006 Elsevier B.V. All rights reserved.
  • Satoru Konnai, Tatsufumi Usui, Manabu Ikeda, Junko Kohara, Toh-ichi Hirata, Kosuke Okada, Kazuhiko Ohashi, Misao Onuma MICROBES AND INFECTION 8 (8) 2163 -2171 2006年07月 [査読無し][通常論文]
     
    In a previous report, we had indicated that in a sheep model, the expression of tumor necrosis factor (TNF)-alpha was closely associated with disease progression in sheep experimentally infected with bovine leukemia virus (BLV). However, individual variabilities are observed in these responses in BLV-infected animals. To attempt to identify genetic factors promoting the progression to BLV-induced lymphoma, we endeavored to determine whether there are any polymorphisms in the TNF-alpha gene among 291 individuals and whether this would affect the level of TNF-alpha expression and concomitant progression of BLV-induced disease or increase in the provirus load in the carriers. We found that the frequency of the TNF-alpha -824G allele, which has been associated with low transcription activity of the promoter/predicted enhancer region of the bovine TNF-alpha gene, was higher in individuals with BLV-induced lymphoma than in asymptomatic carrier individuals. In addition, we observed a tendency for increased BLV-provirus load in cattle with TNF-alpha -824G/G homozygote compared to TNF-alpha -824A/A homozygote or TNF-alpha -824A/G. These data suggest that the observed polymorphism in the promoter region of TNF-alpha gene could at least in part contribute to the progression of lymphoma in BLV-infection. (c) 2006 Elsevier SAS. All rights reserved.
  • A Tsuda, WH Witola, S Konnai, K Ohashi, M Onuma PARASITOLOGY INTERNATIONAL 55 (2) 135 -142 2006年06月 [査読無し][通常論文]
     
    Drug resistance in Trypanosoma brucei causes severe problems for people and domestic animals, but molecular mechanisms of the resistance are not well known. Programmed cell death (PCD) is a fundamental process in both multicellular and unicellular organisms, and it is speculated to be one of the important factors contributing to the emergence of drug resistance. We have previously reported that the expression of TAO appears to play a role in the inhibition of the PCD-like phenomenon development in T brucei. In this study, to ascertain the correlation between the development of the PCD-like phenomenon and the expression of TAO in T. brucei, we genetically engineered T. brucei for conditional overexpression of the TAO gene. TAO over-expressing transgenic T. brucei was refractory to the development of the PCD-like phenomenon compared to the wild-type, indicating that expression of TAO might have a regulatory role on PCD development. Furthermore, the transgenic cells showed resistance to suramin and antrycide. We postulated that intracellular reactive oxygen species (ROS) may be involved in the mechanism of resistance to antrycide because augmentation of ROS in transgenic cells was lower than that in the wild-type cells following treatment with antrycide. These results suggest a possible correlation of PCD to drug resistance in T. brucei. (c) 2006 Elsevier Ireland Ltd. All rights reserved.
  • Michihiro Takagi, Tamami Takeda, Yuki Asada, Chihiro Sugimoto, Misao Onuma, Kazuhiko Ohashi JOURNAL OF VETERINARY MEDICAL SCIENCE 68 (6) 561 -566 2006年06月 [査読無し][通常論文]
     
    To examine the roles of a short form of p53 in the regulation of apoptosis in chicken lymphoblastoid tumor cell lines derived from Marek's disease (MD) and avian leukosis (AL), the expressions of the p53 proteins were analyzed in these cell lines in which apoptosis was chemically induced. In MSB 1-0 derived from MD, the expression of a 40 kDa protein of p53 was decreased and that of a 32 kDa protein, a short form of p53, was increased during apoptosis induced by actinomycin D. In 1104B1 derived from AL, the expressions of 42 and 32 kDa of p53 were increased during the apoptosis. The short form of p53 was undetectable in these cell lines when apoptosis was blocked by the pretreatment with endonuclease inhibitor, Zn2+, protease inhibitors, TPCK and TLCK, or caspase inhibitor, Z-VAD-FMK. In the transcriptional level, the expressions of bcl-2 and 1AP were decreased in these cell lines during actinomycin D-induced apoptosis, but no change was detected in the expression level of p53. These results suggest that, in these chicken tumors, the short form of p53 could play a role in the initiation of apoptosis induced by the chemotherapeutic compound, and that the regulation of its expression may be important for the maintenance of transformation status.
  • C Nakajima, S Imamura, S Konnai, S Yamada, H Nishikado, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 68 (5) 447 -452 2006年05月 [査読無し][通常論文]
     
    A novel thrombin inhibitory protein coding gene was identified from a cDNA library derived from salivary gland of partially-fed Haemaphysalis longicornis (hard tick). The gene encoded a 93-amino acid protein. designated chimadanin. Which had a signal peptide sequence and was predicted to be a secretory protein. It showed no similarity to any other previously identified proteins or conserved domain sequences. The gene was expressed during blood feeding and suggested to be expressed mainly in the salivary gland. The predicted mature region of chimadanin was expressed in Escherichia coli and characteristics of the recombinant chimadanin were determined. The activated partial thromboplastin time and the prothrombin time in sheep plasma were significantly prolonged by chimadanin in a dose dependent manner. Amidolytic activity of thrombin was also inhibited by chimadanin in a dose dependent manner and it suggested that chimadanin was an anticoagulant with thrombin inhibitory activity. This newly identified thrombin inhibitor may play an important role in tick blood feeding.
  • S Imamura, B Namangala, T Tajima, ME Tembo, J Yasuda, K Ohashi, M Onuma VACCINE 24 (13) 2230 -2237 2006年03月 [査読無し][通常論文]
     
    We have previously undertaken preliminary characterization of two Rhipicephalus appendiculutus serine protease inhibitors (RAS-1 and -2) as anti-tick vaccine candidates. In this study, to clarify this hypothesis, we generated and further characterized recombinant RAS-1 and -2 (rRAS-1 and -2) and tested their potency as a cocktail anti-tick vaccine in cattle. RT-PCR analysis showed that RAS-1 and -2mRNA transcripts are expressed during all life cycle stages of ticks, independent of sex. As judged by SDS-PAGE rRAS-1 and -2 migrated as a molecular weight of around 64 and 60 kDa protein, respectively, considering that the expression vector produced a recombinant protein fused with 18-22 kDa TRX protein. RAS-1 and -2 were found not to be secreted into the bite site as determined by the reactivity of anti-tick saliva sera to rRAS-1 and -2, suggesting that both proteins are concealed antigens. Vaccination of cattle with a combination of rRAS-1 and -2 conferred significant protective immunity against ticks, resulting in 61.4% reduction in nymph engorgement rate, and in 28 and 43% increased mortality rate in adult female and male ticks, respectively. This is the first report on an anti-tick vaccine trial using a combination of two different serpins derived from R. appendiculatus, and using cattle as a natural host. (c) 2005 Elsevier Ltd. All rights reserved.
  • S Konnai, T Usui, M Ikeda, J Kohara, T Hirata, K Okada, K Ohashi, M Onuma ARCHIVES OF VIROLOGY 151 (2) 347 -360 2006年02月 [査読無し][通常論文]
     
    We previously reported that tumor necrosis factor alpha (TNF-alpha) was one of the cytokines that contributed to the leukemogenesis caused by bovine leukemia virus (BLV). To determine if the spontaneous cell proliferation observed in the late disease stages, such as persistent lymphocytosis and lymphosarcoma, correlated with the expression level of TNF-alpha, we analyzed the mRNA expression levels for TNF-alpha in spontaneously proliferating PBMCs derived from BLV-infected cattle. The mean mRNA expression level for TNF-alpha was higher in the spontaneously proliferating PBMCs derived from BLV-infected cattle than in non-spontaneously proliferating PBMCs from normal cattle. The TNF-alpha protein level in the PBMCs was determined by flow cytometric analysis, and it was noted that most of the cells expressing membrane-bound TNF-alpha in the spontaneously proliferating cells were CD5 or sIgM(+) -cells. Additionally, in order to determine if this spontaneous proliferation can be blocked by anti-bovine TNF-alpha MAb, the spontaneously proliferating PBMCs from a BLV-infected cattle were cultured in the presence of the MAb. The addition of this MAb at the beginning of the 72 h-cultivation clearly inhibited spontaneous proliferation of cells in a dose-dependent manner, indicating the direct involvement of TNF-alpha in the spontaneous proliferation of PBMCs during the late disease stage. These data suggest that an aberrant expression of TNF-alpha might contribute to the progression of bovine leukosis in animals which develop persistent lymphocytosis of B-cells or B-cell lymphosarcoma.
  • A Tsuda, WH Witola, K Ohashi, M Onuma PARASITOLOGY INTERNATIONAL 54 (4) 243 -251 2005年12月 [査読無し][通常論文]
     
    Trypanosoma brucei rhodesiense is one of the causative agents of African Trypanosomiasis. Programmed cell death (PCD) is fundamental in the development, homeostasis and immune mechanisms of multicellular organisms. It has been shown that, other than occurring in multicellular organisms, the PCD phenomenon also takes place in unicellular organisms. In the present study, we have found that under high-density axenic culture conditions, bloodstream form of T h. rhodesiense depicts a PCD-like phenomenon. We investigated the association of the PCD-like phenomenon with expression of trypanosome alternative oxidase (TAO) under low-temperature stress conditions. We observed that bloodstream form of T b. rhodesiense did not show any PCD but had Lip-regulated expression of TAO. Inhibition of TAO by the addition of ascofranone caused the development of PCD in bloodstream T h. rhodesiense under low-temperature stress, implying that expression of TAO may contribute to the inhibition of PCD. (c) 2005 Elsevier Ireland Ltd. All rights reserved.
  • HM Pham, S Konnai, T Usui, KS Chang, S Murata, M Mase, K Ohashi, M Onuma ARCHIVES OF VIROLOGY 150 (12) 2429 -2438 2005年12月 [査読無し][通常論文]
     
    In order to rapidly detect and differentiate Newcastle disease virus (NDV) isolates, a method based on real-time PCR SYBR Green I melting-curve analysis of the fusion (F) protein gene was developed. The detection limit of real-time PCR was 9 x 10(2). plasmid copies and was 100 times more sensitive than conventional PCR. Thirty eight reference NDV strains were rapidly identified by their distinctive melting temperatures (T(m)s): 89.23 +/- 0.27 degrees C for velogenic strains, 90.17 +/- 0.35 degrees C for pigeon mesogenic strains, 91.25 +/- 0.14 degrees C for two lentogenic strains (B1 and Ishii). No amplification was detected from unrelated RNA samples by this method. This real-time PCR directly detected NDV from infected tissues and eliminated the gel electrophoretic step for analyzing PCR product using ethidium bromide. The total time for a PCR run was less than 1 hour. The results obtained in this study showed that the real-time PCR presented here is a good screening test for the identification of NDV.
  • H Hatai, K Ochiai, Y Tomioka, T Toyoda, K Hayashi, M Anada, M Kato, A Toda, K Ohashi, E Ono, T Kimura, T Umemura AVIAN PATHOLOGY 34 (6) 473 -479 2005年12月 [査読無し][通常論文]
     
    The complete nucleotide sequence of the avian leukosis virus causing so-called fowl glioma has been previously determined. Primers were designed for detection of the fowl glioma-causal virus (FGV) based on the 3' untranslated region of the viral genome. The provirus and viral RNA of FGV were specifically detected in various organs and tissues, including feather pulp, from experimentally infected birds using nested polymerase chain reaction (PCR) and reverse transcription nested PCR. The prevalence of FGV was evaluated in 131 Japanese fowls of a zoological garden in Japan based on the detection of the FGV genome in feather pulp using PCR and the detection of viral antigen in faeces by enzyme-linked immunosorbent assay. FGV proviral DNA was detected in feather pulp of 52 birds (39.7%) by nested PCR. Later, nine dead birds from among the 52 were histologically diagnosed as having fowl glioma and found to have the proviral DNA in the affected brain. These results demonstrated that the PCR-based detection of FGV in feather pulp is useful for epidemiological studies on fowl glioma.
  • M Ozawa, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (12) 1237 -1241 2005年12月 [査読無し][通常論文]
     
    Three individual peptide sequences, EVSHPKVG, WVTTSNQNV, and SGGSNRSP, which have potentials to bind to Newcastle disease virus (NDV), were identified by the biopanning method using phage display technology. The binding specificities of these peptides presented on phages were confirmed by ELISA competition assay using chicken anti-NDV antiserum. The synthetic peptides designed based on these results partially neutralized the infection of NDV in vitro. The peptide-motives identified here have the potential to lead to the identification of novel molecules that inhibit the NDV infection independent of the immune system.
  • WY He, K Ohashi, C Sugimoto, M Onuma EXPERIMENTAL PARASITOLOGY 111 (3) 143 -153 2005年11月 [査読無し][通常論文]
     
    A gene encoding a protein (Tocp1) from Theileria orientalis was isolated from a cDNA library and the deduced amino acid sequence of Tocp1 has 476 amino acids. The primary structure of Tocp1 is similar to eukaryotic thiol proteases (EC 3.4.22.-), but no enzymatic activity was observed with the substitution of essential cysteine at the cysteine active site for glycine. Southern blot analysis showed that multiple genes similar to Tocp1 were present in the parasite genome. Sequence analysis of the genome of the parasite showed that there are at least five different genes similar to Tocp1. Tocpl transcripts were detected in the T. orientalis piroplasma by Northern blot analysis. Western blot analysis showed that Tocp1 was expressed in the piroplasm of T. orientalis. To address the role of Tocpl in the life cycle of T. orientalis, Tocp1 was expressed using pET32 expression system. Binding affinity to haemoglobin was demonstrated by enzyme-linked immunosorbent assay. (c) 2005 Elsevier Inc. All rights reserved.
  • WH Witola, A Tsuda, N Inoue, K Ohashi, M Onuma PARASITOLOGY 131 635 -646 2005年11月 [査読無し][通常論文]
     
    Drug resistance is now a severe and increasing problem in trypanosomes, but molecular details of mechanisms of resistance are only beginning to unveil. There is urgent need to clearly elucidate the different mechanisms of drug resistance in trypanosomes in order to circumvent existing resistance problems and avoid emergence of resistance to the next generation drugs. In this study, we cloned and characterized I novel gene, TeDR40, whose expression is associated with resistance to berenil in Trypanosoma evansi. Expression analysis showed that the gene was at least 1000-fold upregulated in resistant parasites and the encoded protein appeared to have a ubiquitous cellular localization. To investigate the association of TeDR40 with berenil-resistance, we genetically modified wild-type berenil-sensitive T. evansi for inducible overexpression of the TeDR40 gene. Induction of over-expression of TeDR40 in T. evansi led to decreased (P<0(.)01) sensitivity to berenil. Our findings indicate a possible correlation between over-expression of a novel gene, TeDR40, and reduced sensitivity to berenil in an in vitro-cultured clonal line of T. evansi.
  • C Nakajima, ID Vaz, S Imamura, S Konnai, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (11) 1127 -1131 2005年11月 [査読無し][通常論文]
     
    A cDNA library was constructed from salivary glands of partially-fed adult female Haemaphysalis longicornis (hard tick). Randomly selected clones were sequenced and a total of 633 sequences were analyzed by bioinformatic programs. The sequences were grouped into 213 clusters, with each cluster being considered to be composed of mRNAs derived from the same gene or closely related genes. About 36% of the mRNA sequences showed significant similarity to known proteins in the non-redundant protein database by the NCBI blastx program and appeared to be coding for functional predicted proteins, whereas the remaining 64% had no similar sequences. Two thirds of the predicted proteins were annotated as basic cellular proteins (housekeeping proteins). Among the functional predicted protein sequences, other than the housekeeping proteins, several protease inhibitors including anticoagulants, two metalloproteases and a potential immunosuppressive protein could be identified. These proteins may play important roles during tick feeding and could be novel anti-tick vaccine candidates.
  • S Konnai, T Usui, M Ikeda, J Kohara, T Hirata, K Okada, K Ohashi, M Onuma VIROLOGY 339 (2) 239 -248 2005年09月 [査読無し][通常論文]
     
    Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-alpha and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-alpha-induced responses, in this study we examined the TNF-alpha-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-alpha (rTNF-alpha) was significantly higher than those from AL cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5(+) or sIgM(+) cells and these cells showed resistance to TNF-a-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-alpha-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection. (c) 2005 Elsevier Inc. All rights reserved.
  • R Odbileg, S Konnai, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (9) 921 -925 2005年09月 [査読無し][通常論文]
     
    We cloned, sequenced and analyzed the cDNAs encoding Camelidae inflammatory cytokines, including llama (lama glama) interleukin (IL)-1 alpha, IL-1 beta, IL-6, tumor necrosis factor (TNF)-alpha and camel (Camelus bactrianus) IL-6 and TNF-alpha. The similarity levels of the deduced amino acid sequences of IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha from llama (camel) to those from other mammalian species, ranged from 60.7% to 87.7%, 52.8% to 75.3%, 41.4% to 98.6%, and 72.9% to 99.6%, respectively. Phylogenetic analyses based on nucleic acid sequences showed that llama IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha were more closely related to those of camel, pig, cattle, sheep and horse than to those of human, dog, cat, mouse and rat.
  • KS Chang, K Ohashi, SI Lee, M Takagi, M Onuma Japanese Journal of Veterinary Research 53 (1-2) 3 -11 2005年08月 [査読無し][通常論文]
     
    The meq gene was thought to be only detected in Marek's disease virus serotype 1 (MDV 1) including a very virulent strain, Md5, while L-meq, in which a 180-bp sequence is inserted into the meq open reading frame, is found in other strains of MDV I, such as CVI 988/R6. However, both meq and L-meq were previously detected by PCR in chickens infected with MDV I, suggesting that MDV 1 may consists of at least two subpopulations, one with meq, the other with L-meq. To further analyze these subpopulations, we analyzed the time course changes in distribution of these subpopulations among T cell subsets from chickens infected with MDV1. Both meq and L-meq were detected in CD4(+) and CD8(+) T cells infected with strain ME or CVI 988/R6. The shift in MDV subpopulations from one displaying meq to the other displaying L-meq and/or the conversion from meq to L-meq occurred mainly in the CD8(+) T cell subset from Md5-infected chickens. PCR products corresponding to L-meq rather than meq were frequently amplified from the CD8(+) T cell subset from CVI 988/R 6 -infected chickens. These results suggest that a dominant sub-population of MDV 1 changes depending on the T cell subsets, and that L-meq is dominantly present in the CD8(+) T cells which play a role in the clearance of pathogenic agents.
  • K Ohya, T Matsumura, N Itchoda, K Ohashi, M Onuma, C Sugimoto JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 25 (8) 459 -466 2005年08月 [査読無し][通常論文]
     
    Type I interferons (IFN-alpha/beta) were originally thought to be antiviral cytokines, but it has recently been reported that they also play an important role in potentiating innate and adaptive immune responses. Moreover, several studies have shown that the oral administration of type I IFN ameliorates various biologic activities. Here, we studied the ability of orally administered IFN-alpha to protect mice from systemic Listeria monocytogenes infection. Daily oral administration of purified natural IFN-alpha at a concentration of 1000 international units (IU)/20 mu l reduced the bacterial burden in infected organs. We also examined the protective effect of IFN-alpha expressed in transgenic potato plants. A much lower concentration of IFN-alpha (20 IU/20 mu l) in the plant extracts was almost as protective as much higher concentrations of purified natural IFN-alpha. Our observations indicate that transgenic cytokine-expressing plants can be used prophylactically as edible pharmaceuticals to enhance systemic defense responses in humans and animals.
  • WY He, K Ohashi, C Sugimoto, M Tsuji, M Onuma JAPANESE JOURNAL OF VETERINARY RESEARCH 53 (1-2) 27 -35 2005年08月 [査読無し][通常論文]
     
    A cDNA clone encoding a prohibitin-like protein (Toprh) was isolated from a piroplasm cDNA library of Theileria orientalis and its nucleotide sequence was determined. An open reading frame, encoding a polypeptide of 278 amino acid residues, was found in Toprh cDNA sequence. An intron of 89 bp was identified when this cDNA clone was compared with the Toprh gene in the genome of T orientalis. The deduced amino acid sequence of Toprh shares 93.8, 93.1 and 69.1% identities with the prohibitins of T parva (from chromosome 1), T annulata (from chromosome 1), and Plasmodium falciparum, (from chromosome 10), respectively. By Western blot analysis, Toprh was found to be expressed in the piroplasm stage of the parasites.
  • R Odbileg, SI Lee, K Ohashi, M Onuma VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 104 (3-4) 145 -153 2005年04月 [査読無し][通常論文]
     
    This paper describes the cloning and sequence analysis of the cDNAs encoding the T helper (Th) 2 cytokines of llama including interleukin-4 (IL-4), IL-10 and IL-13. The cDNAs encoding for IL-4, IL-10 and IL-13 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-4, IL-10 and IL-13 were found to be 402, 537 and 411 bp in length, with open reading frames encoding 133, 178 or 136 amino acids, respectively. Homology analyses of nucleotide and deduced amino acid sequences of llama IL-4, IL-10 and IL-13 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla (pig, cattle) and Perissodactyla (horse). (c) 2004 Elsevier B.V. All rights reserved.
  • HM Pham, C Nakajima, K Ohashi, M Onuma JOURNAL OF CLINICAL MICROBIOLOGY 43 (4) 1646 -1650 2005年04月 [査読無し][通常論文]
     
    We have evaluated a diagnostic system based on the loop-mediated isothermal amplification (LAMP) assay for the rapid, simple, and sensitive detection of Newcastle disease virus (NDV) directly from culture isolates as well as clinical samples. By using one set of specific primers targeting the fusion protein gene, the LAMP assay rapidly amplified the target gene within 2 h, requiring only a regular laboratory water bath or heat block for reaction. The results obtained from testing the genomes of 38 NDV strains, other different viruses, and clinical samples of experimentally infected chickens showed that LAMP was as sensitive and specific as nested PCR. All LAMP-positive samples were positive by nested PCR. The LAMP assay is faster than nested PCR, cost-effective, and easy to perform. Our results clearly demonstrate that the LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV infection.
  • T Toyoda, K Ochiai, K Ohashi, Y Tomioka, T Kimura, T Umemura VETERINARY PATHOLOGY 42 (2) 176 -183 2005年03月 [査読無し][通常論文]
     
    Intraneural perineurioma is an extremely rare condition characterized by perineurial cell proliferation within peripheral nerve (PN) sheaths. In the veterinary field, this entity has been reported only in a dog. We examined multiple enlargements of PNs in 11 chickens (Gallus gallus domesticus) (9 Japanese bantams and 2 specific pathogen-free White Leghorn), which were inoculated with an avian leukosis virus (ALV) causing so-called fowl glioma. All chickens clinically exhibited progressive leg paralysis. Lumbosacral plexus, brachial plexus, and/or spinal ganglion were commonly affected, and these nerves contained a diffuse proliferation of spindle cells arranged concentrically in characteristic onion bulb-like structures surrounded by residual axons and myelin sheaths. The spindle cells were immunohistochemically negative for S-100alpha/beta protein. Electron microscopy revealed that these cells were characterized by short bipolar cytoplasmic processes, occasional cytoplasmic pinocytotic vesicles, and discontinuous basal laminae. These features are consistent with those of intraneural perineurioma. Furthermore, the specific sequence of the ALV was detected in the PN lesions of 8/11 (73%) birds by polymerase chain reaction. These results indicate that the multiple intraneural perineuriomas of chicken may be associated with the ALV-A causing fowl glioma.
  • T Kozasa, M Tajima, Yasutomi, I, K Sano, K Ohashi, M Onuma VETERINARY MICROBIOLOGY 106 (1-2) 41 -47 2005年03月 [査読無し][通常論文]
     
    The prevalence of bovine viral diarrhea virus (BVDV) in dairy herds in Hokkaido, Japan, was estimated by reverse transcription polymerase chain reaction (RT-PCR) using bulk tank milk samples. Sixteen out of 265 dairy herds were identified as BVDV positive, and at least one persistently infected (PI) cattle was recognized in each of the positive herds except for two herds of which, owners did not agree to examine individual cows. The proportion of positive herds with a history of BVDV PI was significantly higher than that with no history of BVDV PI (odds ratio (OR) 4.25, 95% confidence interval (0) 1.471-12.278, p = 0.004). The herds examined for BVDV were divided into two groups, high and low disease incidence groups based on the occurrence of diseases such as diarrhea, pneumonia or abortion in the past I year. The BVDV positive herds in the high disease incidence group were significantly more than that in the low disease incidence group (OR 2.92, CI 1.110-7.683, p = 0.024). It was observed that there were significantly (p = 0.008) more PI calves or heifers in farms of high disease incidence group than in farms of low disease incidence group. These results suggested that bulk tank milk test was available method for the detection of PI animals in dairy herds, and the existence of PI non-lactating cows in herd correlated with the incidence of diseases of the diarrhea or respiratory disorders. (c) 2004 Elsevier B.V. All rights reserved.
  • R Odbileg, S Konnai, T Usui, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (2) 195 -198 2005年02月 [査読無し][通常論文]
     
    We have developed a method by which llama cytokine mRNAs can be quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of llama, reverse transcribed to cDNA, and cytokine profiles for interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF) alpha were quantified by real-time PCR. The expressions of mRNAs of inflammatory cytokines IL-1alpha, IL-1beta, IL-6 and TNFalpha were upregulated upon stimulation with LPS in a dose- and time-dependent manner. Incubation of PBMCs with 100 and 1,000 pg/ml of LPS for 3 to 6 hr resulted in the acceleration of the mRNA levels of inflammatory cytokines. Here, we describe a highly sensitive and reproducible method to quantify the transcription of llama cytokine mRNAs by real-time RT-PCR with the double-stranded DNA-binding dye SYBR Green I.
  • ID Vaz, S Imamura, C Nakajima, FC de Cardoso, CAS Ferreira, G Renard, A Masuda, K Ohashi, N Onuma VETERINARY PARASITOLOGY 127 (2) 147 -155 2005年01月 [査読無し][通常論文]
     
    The nucleotide and deduced amino acid sequences of the actins from ticks, Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus, have been determined. Nucleotide sequence analysis showed open reading frames of 1128-nucleotide-long encoding proteins of 376 amino acids with a predicted molecular weight of 41.82 kDa each. Comparison between the nucleic acid and deduced amino acid sequences as well as structural and phylogenetic analyses of thew genes confirmed the high similarity among actins from ticks in comparison to other species. (C) 2004 Elsevier B.V. All rights reserved.
  • S Imamura, ID Vaz, M Sugino, K Ohashi, M Onuma VACCINE 23 (10) 1301 -1311 2005年01月 [査読無し][通常論文]
     
    The application of anti-tick vaccine has been shown to be the most promising alternative tick control strategy compared to the current use of acaricides that suffer from a number of limitations. The success of this strategy is dependent on the cloning, and characterization of tick molecules involved in the mediation of tick central physiological roles. Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a cDNA with high similarity in the reactive center loop (RCL) to representative serpin, heparin cofactor If. We have named this novel gene as Haemaphysalis longicornis serpin-2 (HLS2). RT-PCR analysis showed that HLS2 mRNA transcripts are not expressed in salivary glands but in hemolymph by feeding ticks. HLS2 was not introduced into the bite site as measured by Western blot analysis. The activated partial thromboplastin time (APTT) and the thrombin inhibitory assay using recombinant HLS2 (rHLS2) demonstrated prolonged coagulation time and inhibition of thrombin activity. These results suggested that HLS2 is present only in hemolymph of the feeding ticks and the function of HLS2 is homeostasis in tick physiological compartment. Vaccination of rabbits with rHLS2 conferred protective immunity against ticks, resulting in 44.6 and 43.0% mortality in nymphal and adult ticks, respectively. These results show that rHLS2 could be an important candidate as a component of a cocktail anti-tick vaccine. (C) 2004 Elsevier Ltd. All rights reserved.
  • WH Witola, N Sarataphan, N Inoue, K Ohashi, M Onuma ACTA TROPICA 93 (1) 63 -73 2005年01月 [査読無し][通常論文]
     
    Bloodstream trypanosomes take up iron needed for their propagation through the transferrin receptor that, in Trypanosoma brucei, is encoded by expression-site-associated genes (ESAGs), ESAG6 and 7 genes located in variant surface glycoprotein expression sites. ESAG6 and 7 genes in different expression sites have been shown to encode transferrin receptors with varying affinities for polymorphic transferrins. T brucei could cope with the different host transferrins by switching between expression sites. ESAG6- and 7-encoded transferrin receptor appear to be present in Trypanosoma evansi but the genes have not yet been characterized. In this study, we cloned and sequenced different members of ESAG6 genes in seven isolates of T evansi from geographically distinct localities in Thailand. We assessed the intra- and inter-species genetic variability in the transferrin receptor gene regions involved in transferrin binding and established that T evansi, like T brucei, has widely diverse ESAG6 genes. In addition, T evansi possess a clade of ESAG6 variants not observed in T brucei and different T evansi strains share at least two conserved variants. We further noted that T evansi possesses all the reported T equiperdum ESAG6 variants as a subset. Our findings depict a correlation between the genetic diversity in the transferrin-binding regions of ESAG6 genes with the broad host range of T evansi and T brucei compared to the narrow host range of Trypanosoma equiperdum. (C) 2004 Elsevier B.V. All rights reserved.
  • HM Pham, KS Chang, M Mase, K Ohashi, M Onuma ARCHIVES OF VIROLOGY 149 (8) 1559 -1569 2004年08月 [査読無し][通常論文]
     
    Nucleocapsid protein (NP) gene of Newcastle disease virus (NDV) strains mainly isolated in Japan from 1930 to 2001 was genetically characterized. By deduced amino acid sequence comparison, the N-terminal region (from 1 to 401 residues) of the NP protein was found to be highly conserved, while the C-terminal region was highly variable among the NDV isolates. A phylogenetic tree construct based on the nucleotide sequence of the complete NP gene revealed that the old (prior to 1970s) and the new (after 1980s) isolates could be classified into two major different groups, i.e., a group comprising virulent strains, and another group composed of avirulent strains. By restriction enzyme analysis using Pst I, none of the virulent strains were cleaved, while avirulent strains were cleaved. The results may be useful for simple primary screening test for differentiating NDV isolates.
  • WH Witola, N Inoue, K Ohashi, M Onuma EXPERIMENTAL PARASITOLOGY 107 (1-2) 47 -57 2004年05月 [査読無し][通常論文]
     
    Drug resistance of trypanosomes is now a problem, but its underlying mechanisms are not fully understood. Cellular uptake of the major trypanocidal drugs is thought to occur through an adenosine transporter. The adenosine transporter-1 gene, TbAT1, encoding a P2-like nucleoside transporter has previously been cloned from Trypanosoma brucei brucei, and when expressed in yeast, it showed very similar substrate specificity to the P2-nucleoside transporter, but could not transport diamidines (pentamidine and diminazene). We have cloned and sequenced a similar gene (TevAT1) from Trypanosoma evansi and found it to have 99.7% identity to the TbAT1 gene. To elucidate the role of the TevAT1 gene on diamidine trypanocidal effect, we genetically engineered T evansi for conditional knock-out of the TevAT1 gene by RNA interference (RNAi). Induction of the RNAi resulted in 10-fold depletion of TevAT1 mRNA, with concomitantly significant resistance to diminazene aceturate (berenil). The induced parasites propagated normally and attained peak cell density at an in vitro concentration of berenil, 5.5-fold higher than the IC100 of the wild-type. TevAT1 knock-out had no effect on the trypanocidal activity of suramin and antrycide, but conferred some resistance to samorin. Our findings validate the significance of the TevAT1 adenosine transporter-1 gene in mediating the trypanocidal effect of diamidines in T evansi. Further, we show for the first time that RNAi gene silencing in T evansi can be induced using plasmids designed for T brucei. We also demonstrate the usefulness of real-time PCR in rapidly quantifying mRNA levels in trypanosomes. (C) 2004 Elsevier Inc. All rights reserved.
  • S Meas, M Nakayama, T Usui, Y Nakazato, J Yasuda, K Ohashi, M Onuma JAPANESE JOURNAL OF VETERINARY RESEARCH 52 (1) 3 -8 2004年05月 [査読無し][通常論文]
     
    We report herein on the first evidence for the presence of bovine immunodeficiency virus (BIV) in Zambia. Serological surveillance of BIV and bovine leukemia virus (BLV) was conducted in traditional cattle herds in Zambia. Out of a total of 262 sera analyzed, 11.4% were found positive for anti-BIV p26 antibodies as determined by Western blot analysis, while 5.0% were positive for anti-BLV gp51 antibodies as detected by immunodiffusion test. Peripheral blood mononuclear cells from BIV seropositive cattle were found to have BIV-provirus DNA, as detected by nested polymerase chain reaction. A nucleotide sequence corresponding to a 298 bp fragment of the BIV pol gene was also analyzed. Amino acid sequences of these Zambian pol gene products showed 98.0 to 100% homology to the American strain BIV R29,97.0 to 99.0% to Japanese BIV isolates, and divergence ranged from 0.0 to 2.0% among Zambian BIV isolates.
  • Y Tomioka, K Ochiai, K Ohashi, E Ono, T Toyoda, T Kimura, T Umemura JOURNAL OF GENERAL VIROLOGY 85 (3) 647 -652 2004年03月 [査読無し][通常論文]
     
    So-called fowl glioma is a retroviral infectious disease caused by avian leukosis virus subgroup A (ALV-A). We determined the complete nucleotide sequence of the virus genome. The full-length sequence was consistent with a genetic organization typical of a replication-competent type C retrovirus lacking viral oncogenes. The coding sequences were well conserved with those of replication-competent viruses, but the 3' noncoding regions including LTR were most related to those of replication-defective sarcoma viruses. The U3 region of the LTR had a few deletions and several point mutations compared to that of other ALVs. The promoter activities of the LTRs of glioma-inducing ALV and ALV-A standard strain, RAV-1, were equivalent in chick embryo fibroblasts (CEF), while that of glioma-inducing ALV was significantly lower than that of RAV-1 in human astrocytic cells. These subtle differences of the promoter activity of the LTR may be related to the induction of glial neoplasm.
  • 日本獣医師会雑誌 57 705 -709 2004年 [査読無し][通常論文]
  • 日本獣医師会雑誌 57 511 -514 2004年 [査読無し][通常論文]
  • Cloning, expression and partial characterization of a Haemaphysalis longicornis and a Rhipicephalus appendiculatus glutathione S-transferases.
    Insect Molecular Biology 13 219 -225 2004年 [査読無し][通常論文]
  • Cloning and Sequence analysis of llama cytokines related to cell-mediated immunity.
    Veterinary Immunology and Immunopathology 102 93 -102 2004年 [査読無し][通常論文]
  • Y Tomioka, K Ochiai, K Ohashi, T Kimura, T Umemura AVIAN PATHOLOGY 32 (6) 617 -624 2003年12月 [査読無し][通常論文]
     
    We have previously isolated an avian leukosis virus (ALV) from a chicken affected with so-called fowl glioma. A resistance-inducing factor test indicated that the isolate was classified into a subgroup A. The distribution and pathogenicity were investigated in C/O specific pathogen free chickens infected in ovo with this virus. Histologically, 11 of 12 (92%) infected birds had non-suppurative encephalitis and three birds (25%) showed the characteristic nodules of fowl glioma at 50 or 100 days of age. Non-suppurative myocarditis with matrix inclusions and atypical myocytes were also noted in nine (75%) of the birds and the ALV antigens were immunohistochemically detected in various general organs as well as the central nervous system and heart. The semi-quantitative determination of the proviral DNA and viral RNA supported the immunohistochemical results and indicated that the virus was likely to replicate especially in myocardial fibres. The isolated ALV failed to induce other neoplastic lesions in this line of chickens within the experimental period of 100 days, despite the broad tissue tropism throughout the body. These results confirmed that this virus was able to induce glioma in embryo-inoculated chickens.
  • T Usui, S Konnai, S Tajima, S Watarai, Y Aida, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (11) 1201 -1205 2003年11月 [査読無し][通常論文]
     
    A DNA vaccination trial was performed on sheep to determine whether vaccination with bovine leukemia virus (BLV) transactivator Tax DNA is effective against BLV infection. Immunization was carried out with cationic liposomes containing the Tax-expressing plasmid DNA and subsequently all sheep were challenged with BLV. BLV titers in peripheral blood mononuclear cell (PBMC) determined by syncytium formation assay and BLV provirus load detected by genomic PCR analysis showed higher levels of virus titers in control sheep than those in Tax-vaccinated sheep. Higher levels of IFN-gamma mRNA expression have been demonstrated in vaccinated sheep after the challenge. These results suggested that Th1 type immune response induced by Tax DNA vaccine inhibited BLV propagation in vaccinated sheep at the early phase of infection.
  • M Nakajima, M Kodama, H Yanase, T Iwanaga, A Mulenga, K Ohashi, M Onuma VETERINARY PARASITOLOGY 115 (4) 355 -363 2003年08月 [査読無し][通常論文]
     
    There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development. (C) 2003 Elsevier B.V. All rights reserved.
  • S Konnai, T Usui, K Ohashi, M Onuma VETERINARY MICROBIOLOGY 94 (4) 283 -294 2003年07月 [査読無し][通常論文]
     
    For a practical need, fast and efficient methods to quantify mRNA expression are expecting. By using real-time reverse transcription polymerase chain reaction (RT-PCR) with the double-stranded DNA-binding dye SYBR Green I as a novel method, cytokine profiles (IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12p40 and IFN-gamma) were analyzed in peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected animals. In aleukemic cattle, IFN-gamma and IL-12p40 mRNA expression was significantly increased compared to those in cattle with persistent lymphocytosis. The similar results were obtained in the case of sheep experimentally infected with BLV. Real-time quantitative PCR technique is an applicable technique for analysis of cytokine profiles in field. (C) 2003 Elsevier Science B.V. All rights reserved.
  • M Sugino, S Imamura, A Mulenga, M Nakajima, A Tsuda, K Ohashi, M Onuma VACCINE 21 (21-22) 2844 -2851 2003年06月 [査読無し][通常論文]
     
    Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a 1350 bp cDNA which encodes a 378 polypeptide with high sequence similarity to several known serpins. We have named this gene as Haemaphysalis longicornis serpin-1 (HLS1). Northern blotting and reverse transcription (RT)-PCR analysis of total RNA from unfed or partially fed whole ticks as well as dissected tick organs revealed that transcription of HLS1 mRNA was induced by blood meal feeding during the slow feeding phase (24-48 h post-attachment) only in the tick midguts. Vaccination of rabbits with recombinant HLS1 (rHLS1) expressed in Escherichia coli resulted. in 43.9 and 11.2% mortality of nymph and adult ticks which were fed on immunized rabbits. Polyclonal rabbit antibodies to tick saliva did not react with rHLS1, suggesting that native HLS1 was not secreted into the host during tick feeding. rHLS1 could be a potential candidate for a cocktail anti-tick vaccine. (C) 2003 Elsevier Science Ltd. All rights reserved.
  • JL Zhou, K Ohashi, M Yamasaki, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (4) 519 -521 2003年04月 [査読無し][通常論文]
     
    For studying protein trafficking in Babesia-infected erythrocyte, we describe the cloning of a Rab5, one of molecular marker for vesicle trafficking in eukaryotic cells, gene homologue in Babesia gibsoni (BgRab5). The full-length cDNA of BgRab5 is 1,020 bp long with an open reading frame encoding a protein of 220 amino acids. The deduced amino acid sequence of BgRab5 contained the highly conserved GTP-binding consensus sequence and shares about 40% homology with that of Rab5 from Plasmodium falciparum, Toxoplasma gondii, Dog, Lotus japonicusor, Oryza sativa. Northern blot analysis showed that the BgRab5 probe hybridized with a 1 kb band in total RNA from parasitized erythrocytes, that was consistent with the size of the BgRab5 full-length cDNA.
  • T Usui, S Meas, S Konnai, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (2) 287 -289 2003年02月 [査読無し][通常論文]
     
    Serological survey of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) infection was conducted in dairy cattle from 10 different regions of Hokkaido, Japan. Among 390 cattle, 11.0% of cattle were BIV-seropositive and 3.3% were BLV-seropositive. Moreover, in two dairy farms, where bovine leukosis has been reported, prevalence of BIV infections were 6.4 and 9.1%, respectively. In contrast, among 150 beef cattle, 16.6% were BIV-seropositive while none was BLV-seropositive. Dual infections with BLV and BIV in dairy cattle were tested by using 107 BLV-seropositive sera, and 20 sera were found BIV-positive (18.7%). These results indicate that BIN infection was widespread in Hokkaido.
  • M Nakajima, H Yanase, T Iwanaga, M Kodama, K Ohashi, M Onuma JAPANESE JOURNAL OF VETERINARY RESEARCH 50 (4) 157 -163 2003年02月 [査読無し][通常論文]
     
    Tick vaccine development plays an important role in current tick control strategies. Previously, we have produced three different isotypes of monoclonal antibodies (mAbs) which recognized a midgut protein of adult Haemaphysalis longicornis. These mAbs, typed as IgG1, 2a, and 2b, reacted with a 76 kDa surface protein of midgut cells. We speculated that the 76 kDa protein may be an unknown antigen for a tick vaccine and the three mAbs may work as probes to clone the protein. In this study, to test whether these three isotypes have anti-tick effects and if so which works more effectively, we conducted passive immunization in BALB/c mice with each of the mAbs, and infested the mice with adult ticks. All isotypes significantly reduced the number of hatched larvae, compared to controls, however, no differences in the magnitude of the reduction were observed among the three.
  • Japanese Journal of Veterinary Research 51 1 -6 2003年 [査読無し][通常論文]
  • KS Chang, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 64 (12) 1097 -1101 2002年12月 [査読無し][通常論文]
     
    The meq gene encoding a 339-amino-acid bZIP transactivator protein has been identified as a candidate oncogene of Marek's disease virus serotype I (MDV1). which induces malignant lymphomas in chickens. We have previously reported that, in addition to meq, L-meq, in which a 180-bp sequence is inserted into the region encoding the transactivation domain of meq, is also detected in chickens experimentally infected with MDV. To further analyze the diversity in meq, PCR was performed using a primer set which specifically amplify the proline-rich repeat (PRR) region in the transactivation domain of meq. In CV1988/R6, a vaccine strain of MDV1, and JM, an MDV I strain attenuated by prolonged passage in vitro, a major band of a 0.8 kb corresponding to L-meq as well as a minor band of 0.6 kb corresponding to meq was detected by PCR. Furthermore, extra 0.5- and 0.3-kb bands, corresponding to genes termed as short meq (S-meq). and very short meq (VS-meq), respectively, were also detected. These genes were also detected in MDV-transformed cell lines, MSB1 and MTB1. In Md5, an oncogenic MDV1, attenuated by prolonged passage in vitro, the 0.6-kb meq was consistently detected, and 0.5-kb S-meq was occasionally detected. This diversity in meq was due to the difference in the copy number of the PRR region: L-meq and meq contained 9 and 6 copies of PRR while 4 and 2 copies of PRR were present in S-meq and VS-meq, respectively. Thus, the meq gene is polymorphic in the attenuated MDV1 and the MDV-transformed cell lines, and gene products from different meq genes may have different functions from each other.
  • KS Chang, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 64 (12) 1091 -1095 2002年12月 [査読無し][通常論文]
     
    Meq is one of the candidate oncogenes in the MDV1 genome. We previously reported a difference in the meq open reading frame (ORF) between oncogenic and non-oncogenic MDV1: L-meq. in which a 180-bp sequence is inserted into the meq ORF, is detected in non-oncogenic MDV1. To study the functions of a gene product of L-meq (L-MEQ), transactivation by L-MEQ was analyzed by dual luciferase assay using a reporter gene under the control of long (-1--873 bp) and short (-1--355 bp) meq promoter (LMP and SMP, respectively). LMP showed higher promoter function than SMP. L-MEQ transactivated the expression of the reporter gene, but less than MEQ did. In the presence of SNIP or the cytomegalovirus immediate-early promoter, the same or slightly higher transactivation was observed in cells cotransfected with both meq and L-meq than cells transfected only with meq. However, in the presence of LMP, lower transactivation was observed in cells cotransfected with both meq and L-meq than cells transfected only with meq, suggesting that L-MEQ can be a transrepressor. Replication of a vvMDV1 was enhanced in the cells with meq. Interestingly, however, replication of vvMDV1 was suppressed in the cells with L-meq or with both L-meq and meq, compared to untransfected cells. Thus, L-MEQ could suppress replication of vvMDV1 displaying the meq gene in coinfetced cells.
  • JL Zhou, A Mulenga, M Yamasaki, K Ohashi, Y Maede, M Onuma EXPERIMENTAL PARASITOLOGY 101 (4) 210 -214 2002年08月 [査読無し][通常論文]
     
    Members of the Rab subfamily of GTPases have been implicated as important components in vesicle trafficking in the eukaryotes, individual Rab proteins have a remarkable degree of specific subcellular localization. As a first step towards developing a set of compartment specific probes for studying protein trafficking in Babesia-infected erythrocyte, here we describe the cloning and characterization of Rab6 and Rab11 gene homologues in Babesia gibsoni (BgRab6 and BgRab11). The deduced amino acid sequence of both BgRab6 and BgRab11 contained the highly conserved GTP-binding consensus sequence and C-terminal cysteines. Northern blotting analysis of total RNA hybridized a 1.3 kb band on both BgRab6 and BgRab11 probed blots consistent with the expected size. Using a GTP-binding assay we demonstrated that Escherichia coli expressed recombinant BgRab6 and BgRab11 were able to bind GTP. BgRab6 and BgRab11 represent the first two molecular markers of B. gibsoni.
  • KS Chang, SI Lee, K Ohashi, A Ibrahim, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 64 (5) 413 -417 2002年05月 [査読無し][通常論文]
     
    In the genome of strains of very virulent Marek's disease virus serotype 1 (vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF. termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CVI988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.
  • N Iwata, K Ochiai, K Hayashi, K Ohashi, T Umemura JOURNAL OF VETERINARY MEDICAL SCIENCE 64 (5) 395 -399 2002年05月 [査読無し][通常論文]
     
    C/O specific pathogen-free White Leghorn chickens were intracerebrally inoculated at one day of age with a brain homogenate of Japanese bantams (Gallus gallus domesticus) affected with fowl glioma. Histologically, six of eight inoculated chickens developed nonsuppurative meningoencephalitis in cerebrum and two of them had the characteristic lesions of fowl glioma. Hyperplastic lymphoid foci concomitantly developed in many organs of these birds, especially in the heart. Apart from these lymphoid foci, lymphocytic myocarditis was observed in all inoculated birds. Matrix inclusions were also noted in myocardial cells. Immunohistochemically, avian leukosis virus antigens were detected in reticular cells in the lymphoid foci, mesangial cells of the kidney, smooth muscle cells of the blood vessels, and myocardial cells, Of these tissues, the myocardium of all inoculated birds consistently showed strong reactivity for this antigens. The matrix inclusions were also positive for the antigens. These results suggest that the causal virus of fowl glioma has a high propensity to replicate, especially in myocardium and nonsuppurative myocarditis occurs associated with so-called fowl glioma.
  • N Iwata, K Ochiai, K Hayashi, K Ohashi, T Umemura AVIAN PATHOLOGY 31 (2) 193 -199 2002年04月 [査読無し][通常論文]
     
    So-called fowl glioma is characterized by multiple nodular gliomatous growths associated with disseminated non-suppurative encephalitis. To investigate the possibility of the induction of the gliomatous lesions, chicks of Japanese bantams (Gallus gallus domesticus) and specific pathogen free chickens (C/O strain White Leghorn) were intracerebrally inoculated with a brain homogenate or culture supernatant from a bantam affected with fowl glioma. All bantams and 16 chickens (89%) in the inoculated groups showed non-suppurative encephalitis, and the 18 bantams (82%) and five chickens (28%) developed multiple nodules consisting of aggregations of astrocytes in the cerebrum. These astrocytes had avian leukosis virus (ALV) antigen. By Southern blot analysis, the ALV sequence was detected both in DNA prepared from the brains of the inoculated birds and in DNA from the inoculum. Ultrastructurally, tadpole-shaped particles, approximately 100 nm in diameter, were detected in the concentrated supernatant of the chicken embryo fibroblasts, and budding of the particles was noted. These results substantiated that fowl glioma of the bantams could be transmitted by intracerebral inoculation of the affected tissue and that the causal agent was an unidentified strain of ALV.
  • K Ohya, N Itchoda, K Ohashi, M Onuma, C Sugimoto, T Matsumura JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 22 (3) 371 -378 2002年03月 [査読無し][通常論文]
     
    We report the successful insertion of the cDNA of human tumor necrosis factor-alpha (HuTNF-alpha) into the genome of potato plant species, Solanum tuberosum, using Agrobacterium tumefacience-mediated transformation. HuTNF-alpha is a known and essential cytokine mediating host defense against tumors and infectious diseases and an immunomodulating agent. To enhance the accumulation of foreign gene product expression in plant cells, the molecular design of the constructed HuTNF-alpha is presented. Transcription and translation of TNF-alpha in transformants were confirmed by Northern blot, RT-PCR, ELISA, and Western blot, respectively. Expression of the bioactive HuTNF-alpha in plant cells was confirmed by way of the cytotoxic effect of the extract obtained from the transformants against marine L929 cells. We think that the expression level of HuTNFalpha (15 mug/g potato plant tissue) obtained in the present study may be sufficient to induce responses/effects similar to those generated by TNF-alpha in human milk administered orally. We believe that the TNF-alpha expressed in edible potato plants has tremendous potential for clinical use in the areas of medicine and veterinary science. Its usefulness and applicability, therefore, need to be fully explored.
  • Biological and pathological studies on Marek’s disease virus (strains Md5 and CVI988C/R6) using polymerase chain reaction (PCR).
    1st International Science Conference of Faculty of Veterinary Medicine 1 -13 2002年 [査読無し][通常論文]
  • The L-meq gene detected in chicken cells infected with Marek's disease virus serotype 1.
    1st International Science Conference of Faculty of Veterinary Medicine 21 -26 2002年 [査読無し][通常論文]
  • Japanese Journal of Veterinary Research 50 9 -16 2002年 [査読無し][通常論文]
  • S Meas, T Usui, K Ohashi, C Sugimoto, M Onuma VETERINARY MICROBIOLOGY 84 (3) 275 -282 2002年01月 [査読無し][通常論文]
     
    Vertical transmission of bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) was investigated in five dairy cattle herds in Hokkaido, where 36.1 and 17.0% of cattle were BLV and BIN seropositive, respectively, and 9.9% of dams were co-infected with both BIV and BLV. Twenty six cases of offspring born from dams infected with only BLV (17 cases) or with both BIN and BLV (9 cases) were examined for the presence of BLV and BIV before and after colostrum feeding by polymerase chain reaction (PCR) and syncytium assay. After birth, all calves were separated immediately from their dams. The offspring born from BLV-positive dams were BLV-negative before colostrum feeding, suggesting that no transplacental transmission had occurred. Thereafter, these offspring were fed colostrum or milk from their dams, but still remained BLV-negative. The other offspring born from BLV-positive dams were fed with BLV-negative colostrum, or with pasteurized BLV-positive colostrum. All these calves remained negative for BLV infection, suggesting that in utero transmission of BLV is negligible. In the case of offspring born from darns co-infected with BLV and BIV, calves were BIV-positive before colostrum feeding at I day after the birth, indicating in utero transmission of BIV. After colostrum feeding from their dams, newborn calves became BLV-positive. In addition, one calf was BLV-positive even before colostrum feeding. These results suggest that BIV can be transmitted to offspring in utero. and that BLV can be transmitted through colostrum or milk if dams are infected with both BIV and BLV. (C) 2002 Elsevier Science B.V. All rights reserved.
  • M Inoue, D Van Nguyen, S Meas, K Ohashi, S Sen, C Sugimoto, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 63 (10) 1155 -1157 2001年10月 [査読無し][通常論文]
     
    A survey of Theileria parasite infection in cattle in Cambodia and Vietnam was carried out by using allele-specific polymerase chain reaction. A total of 137 blood samples from draught animals in Cambodia and 40 blood samples from dairy cattle in Vietnam were analyzed. In Cambodia, 69 out of 137(50.4%) samples were PCR-positive containing mainly the Thai and the C type parasites. In Vietnam, 11(27.5%) samples were positive and all were of the Thai type parasite.
  • K Ohya, T Matsumura, K Ohashi, M Onuma, C Sugimoto JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 21 (8) 595 -602 2001年08月 [査読無し][通常論文]
     
    Plant expression systems have advantages over other in vitro expression systems in terms of low production costs and low risk of contamination by animal viruses or bacterial endotoxins. In this study, cDNA encoding two subtypes of human interferon-alpha 2b and 8 (HuIFN-alpha 2b and HuIFN-alpha8) were introduced into potato plants (Solanum tuberosum) using Agrobacterium-mediated transformation. Transcription and translation of the inserted HuIFN-alpha cDNA were confirmed by Northern blot analysis and ELISA, respectively. Bioactivity of the products was assayed by inhibition of vesicular stomatitis virus (VSV) replication on a human amniotic cell line. However, because of the presence of substances in potato tissue extracts that were toxic to animal cells, successful demonstration of IFN bioactivity in the transformants; was achieved only after removal of such substances by dialysis. The maximum level of IFN activity in plant extracts was 560 IU/g of tissue. These results indicated that the HuIFN-alpha gene introduced into the potato plant was correctly translated and transcribed in plant cells. This report for the first time shows that biologically active animal cytokines with potential pharmaceutical applications could be expressed in transgenic potato plants.
  • H Kabeya, A Fukuda, K Ohashi, C Sugimoto, M Onuma VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 81 (1-2) 129 -139 2001年08月 [査読無し][通常論文]
     
    To examine whether tumor necrosis factor alpha (TNF alpha) contributes to the pathogenesis of bovine leukemia virus (BLV) infection, the mRNA expression patterns of TNF alpha and its receptors, type I (TNF RI) and type 2 (TNF R2) were investigated. Sheep inoculated with BLV were divided into two groups: one was BLV-positive and the other BLV-negative based on the detection in peripheral blood mononuclear cells (PBMC). Expression of TNF R1 mRNA was down-regulated in PBMC from the BLV-positive compared to BLV-negative sheep. No difference was shown in the expression levels of TNF R2 mRNA between the two groups, Furthermore, proliferative responses of PBMC in the presence of TNF alpha were observed from the BLV-positive, but not BLV-negative sheep. Membrane-bound TNF alpha (mTNF alpha) is thought to be one of the ligands, inducing B-cell activation. Flow cytometric analysis demonstrated that the number of PBMC that were positive for mTNF alpha expression, was increased in the BLV-positive sheep. Thus, the expression of TNF alpha and its receptors may be closely associated with lymphocytosis induced by BLV. (C) 2001 Elsevier Science B.V, All rights reserved.
  • A Tsuda, A Mulenga, C Sugimoto, M Nakajima, K Ohashi, M Onuma VACCINE 19 (30) 4287 -4296 2001年07月 [査読無し][通常論文]
     
    Immunological control of ticks is currently the only sustainable and practical alternative method to the current use of acaricides which has serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. We used a combination of immuno-screening of an adult tick cDNA library as well as the 3 and 5 rapid amplification of cDNA ends to clone two cDNAs, encoding tick saliva proteins from Haemaphysicalis longicornis. The two cDNAs herein named HL 34 and 35 are 1000 by each and encode polypeptides with 292 and 321 amino acid residues respectively. Northern blotting analysis of total RNA from ticks at different feeding stages revealed that expression of both HL 34 and HL35 mRNAs is induced during the slow feeding phase. We speculate that the functions of both genes are closely associated with blood feeding. Expression analysis by RT-PCR showed that both genes are expressed in other tick organs in addition to salivary glands. Recombinant HL 34 was successfully expressed in Escherichia coli and its suitability as a tick vaccine antigen was analyzed in rabbits. We propose that rHL34 could be a useful component of a cocktail tick vaccine. (C) 2001 Elsevier Science Ltd. All rights reserved.
  • A Mulenga, C Sugimoto, G Ingram, K Ohashi, O Misao INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 31 (8) 817 -825 2001年06月 [査読無し][通常論文]
     
    Host vaccination against tick infestation is at present the most practical and sustainable alternative tick control method to the current acaricide use which has serious limitations. However the success of this approach to control ticks depends upon the identification of target vaccine antigens. Members of the serine proteinase gene family may represent an interesting group of proteins to target as candidate antigens because of their involvement in regulation of many physiological functions and development processes in a wide range of organisms. We used RT-PCR with the 3' and 5' RACE to clone two cDNAs encoding full-length serine proteinases from the hard tick, Haemaphysalis longicornis. RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His(57) and Ser(195) conserved among most known serine proteinase. Gene specific primers designed from nucleotide sequences of the RT-PCR products were used to prime the 3' and 5' RACE. Southern blotting analysis showed that both HLSG-1 and -2 are single copy. The 2 cDNAs, HLSG-1 and -2 are 1.2 and 1.0 kb long in size with open reading frames encoding polypeptides with 37.7 and 31.2 kDa predicted molecular mass respectively. Northern blotting analysis of total RNA from unfed and partially fed whole ticks showed that the expression of mRNAs for both HLSG-1 and -2 was induced by blood feeding. Expression analysis by RT-PCR showed that both HLSG-1 and -2 are expressed in other tick organs in addition to salivary glands and midguts. The 6 serine proteinase consensus cyteine residues are well conseverd in both HLSG-1 and -2. We have discussed our findings with respect to tick vaccine development research. (C) 2001 Elsevier Science Ltd. All rights reserved.
  • SI Lee, K Ohashi, C Sugimoto, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 63 (4) 427 -432 2001年04月 [査読無し][通常論文]
     
    The mechanisms of Marek's disease virus (MDV) entry to host cells have not yet been analyzed. Heparan sulfate (HS) on the cell surface serves as a receptor for several herpesviruses in mammalian species. in this study, we demonstrated that plaque formation by cell-free MDV is inhibited by the addition of soluble heparin to the cell culture. Moreover, pretreatment of susceptible cells, chicken embryo fibroblasts, with heparinase, partially reduced infectivity of the cell-free MDV. From these results, it was suggested that the MDV entry, at least in the case of cell-free MDV, is dependent on the presence of cell surface glycosaminoglycans, principally HS.
  • M Takagi, K Ohashia, T Takeda, Y Asada, Y Wakita, C Sugimoto, M Onuma, J Kawano, R Osawa, A Shimizu CURRENT PROGRESS ON MAREK'S DISEASE RESEARCH 305 -312 2001年 [査読無し][通常論文]
     
    A 122-bp deletion form of the p53 transcript was found in Marek's disease (MD)-derived cell lines, MSB1 and MTB1. To determine whether this deletion is dependent on the cell cycle, the p53 transcripts expressed in three MSB1 subclones, MSB1-O, -clone 18 and -41C, and their gene products were analyzed by RT-PCR, nested-PCR and Western blotting. Throughout the cell cycle and even during apoptosis induced by actinomycin D, the deleted form of the p53 transcript was continuously expressed, and no difference was found in the expression pattern of the deleted form, suggesting that deletion in the p53 transcripts does not depend on the cell cycle. When apoptosis was induced, however, the p53 protein with a smaller size detected by anti-p53 monoclonal antibody was expressed in MSB1-O. Therefore, the translation of the smaller p53 protein from the deleted transcript may play a role in the initiation of apoptosis. In addition, two short forms of the p53 transcript, produced by the deletions in exon 8 and 10, and from exon 8 to 10, were further identified in MSB I subclones by nested-PCR. Thus, several kinds of deleted forms of the p53 transcripts are present in MD tumor cell lines though their roles in the tumorigenesis by MDV remain to be established.
  • Differences in the kinetic changes of lymphocyte populations and cytokine profiles between chickens genetically susceptible and resistant to Marek's disease.
    Current progress on Marek's diease research 295 -301 2001年 [査読無し][通常論文]
  • S Meas, K Ohashi, C Sugimoto, M Onuma ARCHIVES OF VIROLOGY 146 (5) 1037 -1045 2001年 [査読無し][通常論文]
     
    Isolates of bovine immunodeficiency virus (BIV) exhibit a striking genomic diversity, most of which are located in the viral envelope gene. Since this property of the BIV group of viruses may play an important role in the pathobiology of the virus, the surface envelope gene, particularly the conserved (C) 2, hypervariable (V) 1, V2 and C3 regions, of eleven different isolates from different environments with different bovine breeds naturally infected with BIV, including dairy cows in Japan, buffaloes in Pakistan and draught animals in Cambodia, were sequenced. When compared to the nucleotide sequence of American BIV isolates, all Asian BIV field isolates seem to be smaller, several base substitutions were observed in the V1 region, and deletions were also found in the V2 region of env gene in these samples. However, deduced amino acid sequences were not so different among isolates from different bovine breeds, suggesting that bovine susceptibility to BIV infection may not depend upon bovine breed or buffaloes. Moreover, phylogenetic analysis revealed that genotypes were distinct between Asian and American BIV isolates and these results also provide an information on the molecular epidemiology of naturally occurring BIV infection in cattle and buffaloes
  • SI Lee, K Ohashi, M Takagi, KS Chang, C Sugimoto, M Onuma CURRENT PROGRESS ON MAREK'S DISEASE RESEARCH 289 -294 2001年 [査読無し][通常論文]
     
    Serotype I strains of Marek's disease virus (MDV1), except attenuated vaccine strains, are known to cause lymphomas in visceral organs of infected chickens. We have previously found that a 178-bp sequence was inserted to the meq open reading frame (ORF) of strains CVI988/C and CVI988/R6, attenuated non-oncogenic MDV1 strains (9). To further determine if this insertion could be detected in other NMV1 strains, southern blot and nucleotide sequence analyses were done with total cellular DNA from MDV1-infected chicken embryo fibroblasts. The insertion of 178-bp sequence was detected in the meq ORF of strain JM, a mild MDV1 strain, and occurred at the exactly same position as observed in CVI988. This insertion was not detected in strain Md5 which had been passaged 17 times in vitro. The biological significance and function of this long meq gene is not yet elucidated, but this insertion is not CVI988-specific and can occur in oncogenic MDV1 strains.
  • S Meas, K Ohashi, S Tum, M Chhin, K Te, K Miura, C Sugimoto, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 62 (7) 779 -781 2000年07月 [査読無し][通常論文]
     
    Since bovine immunodeficiency virus (BIV), known as bovine lentivirus, has been detected in dairy and beef cattle in various countries around the world, a prevalence study of antibodies to BIV and bovine leukemia virus (BLV) was conducted in draught animals in five provinces in Cambodia, where protozoan parasite infections were suspected in some animals. To clarify the status of draught animals including Haryana, Brahman, mixed-breed, local breed cattle and muscle water buffaloes, a total of 544 cattle and 42 buffaloes were tested, and 26.3 and 16.7%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting. There were 5.3% positive for anti-BLV antibodies detected by immunodiffusion test among the cattle, but no reactors among buffaloes and no dual infection for both BIV and BLV was determined in this study. Peripheral blood mononuclear cells from BIV-seropositive cattle were found to have BIV-provirus DNA, as detected by polymerase chain reaction and subsequent Southern blot hybridization. This is the First evidence for the presence of BIV and BLV infections in draught animals in tropical countries such as Cambodia. This wide distribution of BIV suggests its association with problems in animal health as reported worldwide, and that a primary BIV infection can predispose death of affected animals by other aggressive pathogens or stresses.
  • A Mulenga, C Sugimoto, K Ohashi, M Onuma BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE 1501 (2-3) 219 -226 2000年06月 [査読無し][通常論文]
     
    An immunogenic 84 kDa protein was isolated and purified from whole tick extracts of Haemaphysalis longicornis larvae by a combination of ion exchange, reverse phase and hydrophobic interaction chromatographies. The protein, when injected intradermally into rabbits exposed to repeated tick feeding, induces an immediate cutaneous hypersensitivity reaction. It has been purified to homogeneity as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis and silver staining. Amino acid sequences for two peptides derived from proteolytic cleavage of p84 were scanned against known proteins on the SWISS-PROT database. A 7 residue motif, ISGWGNT present in one of the two peptides appeared conserved in both vertebrate and invertebrate trypsin-like serine proteinases, while another 7 amino acid motif, HVPAGQI present in the second peptide showed homology to an Escherichia coli ATP-binding protein. We have discussed our findings in relation to isolation and characterization of target antigens for tick vaccine candidates. (C) 2000 Elsevier Science B.V. All rights reserved.
  • S Meas, J Seto, C Sugimoto, M Bakhsh, M Riaz, T Sato, K Naeem, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 62 (3) 329 -331 2000年03月 [査読無し][通常論文]
     
    A survey of antibodies to bovine immunodeficiency virus (BIV) known as bovine lentivirus and bovine leukemia virus (BLV) was conducted with samples from water buffalo and cattle populations in Pakistan. A total of 370 water buffaloes and 76 cattle were tested, and 10.3% and 15.8%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting, while 0.8% of water buffaloes and no cattle were positive for anti-BLV antibodies determined by immunodiffusion test. BIV-seropositive water buffaloes and cattle were found to have BIV proviral DNA in the peripheral blood mononuclear cells determined by nested polymerase chain reaction. This is the first report of BIV infections in water buffaloes.
  • SI Lee, M Takagi, K Ohashi, C Sugimoto, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 62 (3) 287 -292 2000年03月 [査読無し][通常論文]
     
    Serotype 1 strains of Marek's disease virus (MDV1), except attenuated vaccine strains, are known to cause lymphomas in visceral organs of infected chickens. To know additional genetic differences between oncogenic and nononcogenic MDV1, polymerase chain reaction (PCR) was performed to amplify the meq gene of the viral genome. In addition to the 1,062-bp band including the native meq open reading frame (ORF), a 1.2-kb band was amplified from the DNA sample prepared from chick embryo fibroblast infected with an attenuated strain, CVI988, but not with oncogenic strains. Sequence analysis of the 1.2-kb band showed that a 178-bp sequence was inserted to the meg ORF of CVI988. This ORF could encode for the Meq protein with a different transactivator domain. Southern blot analysis also confirmed the insertion of the 178-bp sequence in the meg ORF of CVI988. This insertion of 178-bp sequence may explain the reason why CVI988 is not oncogenic.
  • Differences in the kinetic changes of lymphocyte populations and cytokine profiles between chickens genetically susceptible and resistant to Marek's disease.
    Proceedings of the International Veterinary Cytokine and Vaccine Conference 318 -321 2000年 [査読無し][通常論文]
  • Involovement of tumor necrosis factor a in the pathogenesis of bovine leukemia virus infection.
    Proceedings of the International Veterinary Cytokine and Vaccine Conference 155 -158 2000年 [査読無し][通常論文]
  • Expression of biologically active molecules in transgenic potatoes.
    Proceedings of the International Veterinary Cytokine and Vaccine Conference 116 -119 2000年 [査読無し][通常論文]
  • Tick saliva proteins interacting with the host immune response factors.
    Proceedings of the International Veterinary Cytokine and Vaccine Conference 93 -96 2000年 [査読無し][通常論文]
  • KO Cho, K Ohashi, M Onuma VETERINARY PATHOLOGY 36 (4) 314 -320 1999年07月 [査読無し][通常論文]
     
    Skin lymphomas induced in 11 specific-pathogen-free chickens by inoculation at 1 day of age with Marek's disease virus (MDV) were biopsied weekly and examined by electron microscopy and immunohistochemistry. In the sequentially biopsied lymphomas, immature MDV particles (abortive replication) were found only in the nuclei of necrotic lymphoblasts within necrotizing neoplasms. The necrotizing lymphomas were observed in two of the 11 experimental birds and were associated with prominent vascular endothelial cell injury, including fibrinoid necrosis of blood vessels. Nonnecrotizing lymphomas biopsied sequentially from the 11 experimental birds did not contain virus particles of any kind in the lymphoblasts and had no distinct vascular lesions. Immunohistochemically, MDV early antigen (pp38), but not late antigens (glycoproteins B and C), was detected only in the necrotizing lymphomas. These findings indicate that abortive MDV replication mainly occurred in necrotic lymphoblasts, which might have been induced by ischemia.
  • K Ochiai, K Ohashi, T Mukai, T Kimura, T Umemura, C Itakura VETERINARY RECORD 145 (3) 79 -81 1999年07月 [査読無し][通常論文]
  • H Kabeya, K Ohashi, N Oyunbileg, Y Nagaoka, Y Aida, C Sugimoto, Y Yokomizo, M Onuma VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 68 (2-4) 255 -265 1999年05月 [査読無し][通常論文]
     
    Protective immune responses were analyzed in eight sheep vaccinated with BLV envelope peptides and experimentally infected with bovine-leukemia virus (BLV). Five of eight peptide-immunized sheep showed a high T-cell proliferative response to the BLV peptides and all of these were protected from the infection. The other three peptide-immunized sheep showed no T-cell proliferative responses to any BLV antigens similar to control sheep, though they also exhibited resistance to BLV challenge. To investigate other mechanisms which suppress BLV expansion in these non-responding sheep, we measured the levels of the cytokine expressions before, and after, BLV challenge using competitive reverse-transcriptase polymerase chain-reaction systems. It was revealed that the expression of tumor necrosis factor alpha (TNF alpha) was higher in BLV-resistant sheep than in BLV-susceptible sheep. Thus, TNF alpha expression rather than specific T-cell activity may play an important role in the protective mechanism against BLV infection, at least during the primary viremia phase. (C) 1999 Elsevier Science B.V. All rights reserved.
  • H Kabeya, K Ohashi, C Sugimoto, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 61 (5) 475 -480 1999年05月 [査読無し][通常論文]
     
    To study the immunomodulative activity caused by bovine leukemia virus envelope (BLV Env) peptide, sheep were immunized with two kinds of Th-epitope peptides, peptide 98 (BLV Env 98-117), and 61 (BLV Env 61-78). Four of eight immunized sheep showed specific proliferative responses against both of the peptide stimulations. To characterize the cells responding to the peptides, peptide-specific cells were established From the responding sheep by the continuous stimulation of peripheral blood mononuclear cells (PBMCs) with either peptide 98 or 61 in vitro. The peptide 98-specific cells consisted of CD4-positive cells, whereas the peptide 61-specific cells consisted of CD8-positive cells and MHC class II-positive cells. In addition, cytokine profile analysis indicated that the peptide 98-stimulated cells expressed IFN-gamma but not IL-10, although the peptide 61-stimulated cells expressed IL-10 but not IFN-gamma. These results show that BLV envelope peptides 98 and 61 can modulate immune responses of sheep lymphocytes in different ways and may contribute to the pathogenesis of BLV infection.
  • A Mulenga, C Sugimoto, G Ingram, K Ohashi, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 61 (5) 497 -502 1999年05月 [査読無し][通常論文]
     
    Immunological protection of mammalian hosts against tick infestation has been proposed as the most sustainable alternative tick control method to the current use of acaricides which has several limitations. The success of this method is dependent on the identification of key molecules for use as tick vaccine antigens. Proteolytic enzymes are involved in a wide range of cellular processes in eukaryotes such as development regulation and nutrition, thus they can be considered as good target antigens for a tick vaccine. In the present study we used primers designed based on the consensus amino acid motifs flanking the conserved active sites C-25 and N-175 present in all papain-like cysteine proteinases to amplify by polymerase chain reaction, sequence and characterize two Haemaphysalis longicornis tick cysteine proteinase genes. Based on the nucleotide and deduced amino acid sequences, both genes were identified as members of the cysteine proteinase gene family by presence in their sequences of consensus motifs flanking the conserved active sites C-25, H-150 and N-175 that are present in all papain-like cysteine proteinases. Both genes are about 1.2 kb in size and show high sequence homology predominantly to invertebrate cathepsin L-like cysteine proteinases.
  • KO Cho, S Meas, NY Park, YH Kim, YK Lim, D Endoh, SI Lee, K Ohashi, C Sugimoto, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 61 (5) 549 -551 1999年05月 [査読無し][通常論文]
     
    Infection of bovine immunodeficiency virus (BIV), a lentivirus, is thought to sporadically occur throughout the world, but seroepidemiological surveys concerning the incidence of BIV are limited and have not been undertaken in Korea. A total of 266 sera from different twenty dairy (Holstein) and twenty-six Korean native beef (Hanwoo) farms of the south-western part of Korea was analyzed for the presence of anti-BIV antibodies by Western blotting. Thirty five percent and 33% of dairy and beef cattle, respectively, were BIV-seropositive. By nested polymerase chain reaction, it was confirmed that these seropositive cows had provirus in the peripheral blood mononuclear cells. To demonstrate the correlation with BIV and bovine leukemia virus (BLV) infection, these sera were also analyzed for anti-BLV antibodies by immunodiffusion test, resulting in high prevalence of BLV infection but relatively a few dual infections. We report herein the first serological detection of antibodies to BIV in Korea.
  • A Mulenga, C Sugimoto, Y Sako, K Ohashi, A Musoke, M Shubash, M Onuma INFECTION AND IMMUNITY 67 (4) 1652 -1658 1999年04月 [査読無し][通常論文]
     
    The use of tick vaccines in mammalian hosts has been shown to be the most promising alternative tick control method to current use of acaricides, which suffers from a number of limitations. However, the success of this method is dependent on the identification, cloning, and in vitro expression of tick molecules involved in the mediation of key physiological roles with respect to the biological success of a tick as a vector and pest. We have sequenced and characterized a Haemaphysalis longicornis tick salivary gland-associated cDNA coding for a 29-kDa extracellular matrix-like protein. This protein is expressed in both unfed and fed immature and mature H. longicornis ticks. The predicted amino acid sequence of p29 shows high homology to sequences of some known extracellular matrix like-proteins with the structural conservation similar to all known collagen proteins. Immunization with the recombinant p29 conferred a significant protective immunity in rabbits, resulting in reduced engorgement weight for adult ticks and up to 40 and 56% mortality in larvae and nymphs that fed on the immunized rabbits. We speculate that this protein is associated with formation of tick cement, a chemical compound that enables the tick to remain attached to the host, and suggest a role for p29 as a candidate tick vaccine molecule for the control of ticks. We have discussed our findings with respect to the search of tick molecules for vaccine candidates.
  • K Ohashi, T Morimura, M Takagi, SI Lee, KO Cho, H Takahashi, Y Maeda, C Sugimoto, M Onuma ACTA VIROLOGICA 43 (2-3) 128 -132 1999年04月 [査読無し][通常論文]
     
    To characterize the molecular events involved in both apoptosis and transformation process induced by Marek's disease virus (MDV), the expressions of the bcl-2 and bcl-x genes, ones of the dominant apoptosis-regulating genes, in Marek's disease (MD) tumor cell lines and cells prepared from MDV-infected chickens were analyzed. The expression of bcl-2 was down-regulated in both CD4(+) and CD8(+) T cells prepared from MDV-infected chickens at 3 weeks p.i. No bcl-2 transcript was detected in MD tumor-derived MSB1 and MTB1 cell lines, which had been established from primary MD tumors. On the other hand, the bcl-xL transcript whose product can also inhibit apoptosis was expressed in cell lines derived from MD. By the treatment with phorbol 12-myristate 13-acetate (PMA) and ionomycin, normal CD4(+) T cells were induced to express bcl-xS which can promote apoptosis, while bcl-xL was constitutively expressed in MD cell lines. Our results suggest that bcl-xL rather than bcl-2 might play an important role in the transformation process by MDV.
  • H Kabeya, K Ohashi, C Sugimoto, M Onuma VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 68 (1) 39 -48 1999年03月 [査読無し][通常論文]
     
    Immunomodulatory activity of two bovine leukaemia virus envelope (BLVEnv) derived peptides were examined in BALB/c mice. One is a peptide homologous to CKS-17 which is known as a 17-amino acid peptide derived from p15E of feline leukaemia virus (CKS-17/BLV), and the other is an 18-amino acid synthetic peptide of BLV Env 61-78 (pep61). Priming with CKS-17/BLV in vitro, as well as CKS-17, significantly suppressed the mitogen-induced proliferative responses of spleen cells in naive BALB/c mice. In addition, priming of spleen cells with pep61 in vitro and in vivo resulted in suppression of lipopolysaccaride-induced B-cell proliferative response. This suppression was partially due to the basic amino acid sequence in the peptide because if the pep61-derived peptide lacking Arg was used, this inhibitory activity was partially restored. In contrast, pep61 enhanced both concanavalin A-stimulated proliferative response and IL-2 production. These findings showed that pep61 may contribute to the modification of the host immune responses in the course of BLV infection. (C) 1999 Elsevier Science B.V. ALI rights reserved.
  • T Morimura, KO Cho, Y Kudo, Y Hiramoto, K Ohashi, M Hattori, C Sugimoto, M Onuma ARCHIVES OF VIROLOGY 144 (9) 1809 -1818 1999年 [査読無し][通常論文]
     
    By vaccination with an attenuated Marek's disease virus (MDV), strain CVI988, chickens ape protected from the development of T cell lymphoma caused by an oncogenic MDV. To clarify the role of T lymphocyte subsets in the protection mechanisms of this vaccine, vaccinated chickens were depleted of T cell subsets by neonatal thymectomy and injections of monoclonal antibodies specific to chicken CD4 and CD8 molecules, and then challenged with an oncogenic MDV, strain Md5. The MDV titers rescued from CD8(+) T cells, which are the main targets for latent infection and subsequent transformation by MDV, was much higher in the CD8-deficient vaccinated chickens than in untreated vaccinated chickens at the early stage of the latent phase. However, the neonatal vaccination prevented lymphoma formation by strain Md5 even in either CD4(+) or CD8(+) T cell-depleted chickens. These results suggest that specific CD8(+) T cell responses induced by the MD vaccine play a crucial role in the prevention of MDV infection during the latent phase, but may not be essential for the prevention of lymphoma formation by an oncogenic MDV.
  • SI Lee, K Ohashi, T Morimura, C Sugimoto, M Onuma ARCHIVES OF VIROLOGY 144 (1) 45 -54 1999年 [査読無し][通常論文]
     
    To know the effect of Marek's disease (MD) vaccines, we analyzed the distribution of MD virus (MDV) among T cell subsets from chickens vaccinated or non-vaccinated with MD vaccine and subsequently challenged with a virulent MDV. The challenged MDV was reisolated preferentially from CD4(+) T cells, and the average titers of challenged MDV rescued were significantly lower in vaccinated chickens compared to that of non-vaccinated chickens. In addition, it was also shown that different serotypes of MDV, CVI988 and SB-1, have remarkable difference in recovery rates of viruses from CD4(+) and CD8(+) T cells, though both CVI988 and SB-1 can reduce the infection rates of virulent MDV to splenocytes.
  • S Meas, H Kabeya, S Yoshihara, K Ohashi, S Matsuki, Y Mikami, C Sugimoto, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 60 (11) 1195 -1202 1998年11月 [査読無し][通常論文]
     
    A seroprevalence study of bovine lentivirus, known as bovine immunodeficiency virus (BIV), was conducted in 12 different dairy herds in Hokkaido, where some herds were a high prevalence of bovine leukemia virus (BLV) infection. Amongst 611 cattle, 28.6% of cattle were BLV-seropositive, and 11.7% of cattle were seropositive for BIV, while 4.2% of cattle were seropositive for both BIV and BLV. For the isolation of BIV, 19 samples of peripheral blood mononuclear cells (PBMC) and one sample of milk-derived leukocytes were prepared from BIV-seropositive cows. These PBMC and leukocyte preparations were then co-cultivated with cc81 cells, a cat cell line transformed by mouse sarcoma virus. BIV was isolated from 17 PBMC and one milk-derived leukocyte samples. The isolated viruses showed slow replication and syncytia formation. Major core antigen, p26 from these isolates were reacted with anti-BIV (American isolate R-29) serum. In addition, proviral DNA was detected in blood and milk samples by nested polymerase chain reaction and subsequent Southern blot hybridization. Nucleotide sequence analysis of the amplified pol gene products showed its 99.0 to 99.7% homology to that of BIV R-29. These results indicate that the Japanese BIV isolates appear to be antigenically and genetically similar to the American R-29. Since BIV was isolated from milk samples, BIV could possibly be transmitted through milk. This is the first report of BIV isolation in Japan.
  • M Takagi, K Ohashi, T Morimura, C Sugimoto, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 60 (8) 923 -929 1998年08月 [査読無し][通常論文]
     
    To determine whether there is any abnormalities of the p53 gene in chicken lymphoblastoid tumor cell lines derived from Mareks disease (MD), lymphoid leukosis, reticuloendotheliosis, and field tumors, some portions of p53 cDNA corresponding to core and C-terminal domains (nucleotide positions 277-1104 in the p53 open reading frame (ORF)) were sequenced. Several mutations were identified in both cell lines and field tumors. However, none of these mutations is localized at the "hot spot", which has been reported as the site for transformation-activating mutations. Moreover, partial cDNA clones with a 122-bp deletion in the p53 ORF were identified in two cell lines, MSB1 and MTB1 derived from MD tumors. Southern blot analysis showed that no deletion occurred in the genome of p53 in MSB1, indicating that deletion occurred at the transcriptional level. This deletion could cause a frame shift of the encoding p53 protein, possibly resulting in the generation of a functionally different p53 protein. However, we confirmed that p53 mRNA without deletion is also present in each of these cell lines. These mutations of the p53 gene and deletion in the p53 transcript may be ones of molecular changes specific to the transformation induced by MD virus.
  • KO Cho, NY Park, D Endoh, K Ohashi, C Sugimoto, C Itakura, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 60 (7) 843 -847 1998年07月 [査読無し][通常論文]
     
    Cytological changes of feather pulp lesions (FPL) sampled chronologically from. the same specific-pathogen free chickens inoculated with Marek's disease virus serotype 1 (MDV) were examined, comparing with their histological changes. The birds having Marek's disease (MD) lymphomas or nerve lesions exhibited the characteristic lesion changes on the cytological smears of FPL; the initial non-suppurative inflammatory to the late lymphomatous FPL. The birds having neither the MD visceral lymphomas nor the nerve lesions manifested only non-suppurative inflammatory FPL on the cytological smears throughout the experimental periods. Histological evaluation of FPL sampled from the same birds confirmed as above mentioned cytological results. From these results, the cytological evaluation of FPL proved to be an effective diagnostic and prognostic tool in foreseeing MD incidence.
  • N Sakakibara, H Kabeya, K Ohashi, C Sugimoto, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 60 (5) 599 -605 1998年05月 [査読無し][通常論文]
     
    The immunogenicity of the bovine leukemia virus (BLV) transactivator protein (tax) was studied by mapping its B-cell and T-cell epitopes. Peptides (18 to 20-mer) overlapping by 10 amino acids, spanning whole amino acid sequence of BLVtax were synthesized. Reconbinant BLV tax protein was used to immunize two different strains of mice, C57BL/6 and BALB/c. B-cell and T-cell epitopes of recombinant BLVtax protein was determined by screening all the 30 synthetic peptides, against immune serum in ELISA for antibody reactivity, and against immune spleen cells in lymphocyte proliferation assay for T-cell stimulation. Peptides with amino acids at position 111-130 and 131-150 were T-cell epitopes for C57BL/6 and BALB/c mice immune cells, respectively. B-cell epitope was mapped to amino acid sequence at 261-280 in both strains of mice. These results imply that BLVtax protein contains some of BLV-immunodominant epitopes and this information may be applied for designing an effective peptide vaccine capable of inducing neutralizing antibodies as well as cellular immunity.
  • T Tasaki, A Nakamura, S Itoh, K Ohashi, Y Yamamoto, M Masuda, H Iwata, A Kazusaka, T Kamataki, S Fujita JOURNAL OF BIOCHEMISTRY 123 (1) 162 -168 1998年01月 [査読無し][通常論文]
     
    Dog CYP2D15 was expressed in Sf9 cells with a recombinant baculovirus. Infection of Sf9 insect cells with a recombinant dog CYP2D15-virus resulted in the expression of a protein which cross-reacted with a polyclonal antibody against a dog CYP2D15-specific peptide, The difference spectrum of CO-complex of reduced P450 of the infected cell microsomes had a maximal absorbance at 449 nm, The specific content of P450 was calculated to be 0.56 nmol/mg of Sf9 cell microsomal protein, Although the expressed dog CYP2D15 showed high catalytic activity for the hydroxylations of bunitrolol and imipramine at low substrate concentration (10 mu M), the catalytic activity for that of debrisoquine (50 mu M) was extremely low as compared with that of CYP2D from other species, Dog liver microsomes also showed bunitrolol and imipramine hydroxylase activities, but not debrisoquine hydroxylase activity at the same substrate concentrations, In addition, the expressed CYP2D showed high catalytic activity for imipramine N-demethylation, Thus, our study reveals that the expressed dog CYP2D15 engages in high catalytic activity and has a unique substrate specificity from other CYP2D subfamilies, Western blot analysis suggested that the dog CYP2D15 contents were less than 4% of the total liver P450 content, assuming that 100% of expressed CYP2D15 incorporated heme.
  • Apoptosis in peripheral CD4+ T cells and thymocytes by Marek's disease virus-infection.
    Leukemia 11 Supplement 3 206 -208 1997年 [査読無し][通常論文]
  • RW Renshaw, C Soine, T Weinkle, PH OConnell, K Ohashi, S Watson, B Lucio, S Harrington, KA Schat JOURNAL OF VIROLOGY 70 (12) 8872 -8878 1996年12月 [査読無し][通常論文]
     
    Chicken infectious anemia virus (CIAV) is a unique infectious agent with an amino acid composition that has been found to be remarkably conserved even in isolates from different parts of the world, We have characterized field isolates of CIAV which vary significantly in terms of their abilities to replicate in culture, demonstrating a biological difference between isolates, Two sublines of MDCC-MSB1 cells that differ in their abilities to support CIAV mere identified. In the MSB1(S) subline the CIA-1 isolate of CIAV was found to be less cytopathogenic than the prototype Cux-1(C) isolate; the MSB1(L) subline, which supports Cux-1(C) replication, was found to be nonpermissive for CIA-1. Alignments of the VP1 sequences of previously examined isolates with those of the field isolates CIA-I and L-028 and the culture-adapted ConnB isolate revealed a previously unreported hypervariable region spanning amino acid positions 139 to 151. Chimeras of Cux-1 (C) and CIA-1 mere constructed to examine the potential for this region to affect cytopathogenicity, Transfer of a 316-bp region of Cus-1(C) open reading frame 1 into CIA-1 produced a virus with a cytopathogenic profile typical of Cux-1(C), indicating that one or both of the amino acid differences at positions 139 and 144 affect the rate of replication or the spread of infection. Transfection experiments with additional chimeras indicated that the inability of CIA-1 to replicate in MSB1(L) cells is mediated by a larger region of the genome which contains the hypervariable region in addition to upstream amino acid differences, Analysis of chimeras excluding the entire region of open reading frame 1 suggested the presence of a secondary mediator in the progression of infection in culture that R as localized to a region containing a single nucleotide difference which results in amino acid differences in both VP2 (V-153) and the nuclear localization signal of VP3 (C-118). Immunofluorescence assays indicated an increased cytoplasmic distribution of VP3 and a general lack of VP3-associated apoptatic bodies in infections of CIA-1 and chimeras containing V-153 or C-118, as opposed to a primarily nuclear distribution and association with well-formed apoptotic bodies in Cux-1(C)-infected cells.
  • N Hirai, H Kabeya, K Ohashi, C Sugimoto, M Onuma JAPANESE JOURNAL OF VETERINARY RESEARCH 44 (3) 153 -163 1996年12月 [査読無し][通常論文]
     
    Experimental bovine immunodeficiency-like virus (BIV)-infection and mixed infection of BIV and bovine leukemia Virus (BLV) were performed on sheep. BIV proviral DNA and anti-BIV antibodies were persistently detected in all BIV-inoculated sheep. A slight increase in lymphocyte counts was observed in BIV-infected sheep, but the percentages of CD4(+) and CD8(+) cells in sheep peripheral blood mononuclear cells (PBMCs) were not significantly changed. A transient decrease in lymphocyte blastogenic response to concanavalin A was observed in two of three BIV-infected sheep at 3-6 months after inoculation. From 6 months after BLV-inoculation to sheep which were previously infected with BIV, the numbers of lymphocytes expressing a tumor-associated antigen (TAA) of bovine leukosis were increased compared to those of a sheep inoculated with BLV alone. The BLV titers in PBMCs and the antibody titers against BLV from sheep infected with both BIV and BLV were higher than those of a sheep inoculated with BLV alone.
  • H Kabeya, K Ohashi, K Ohishi, C Sugimoto, H Amanuma, M Onuma VACCINE 14 (12) 1118 -1122 1996年08月 [査読無し][通常論文]
     
    Protective effects of the gp51 of bovine leukaemia virus (BLV) expressed by a recombinant baculovirus (rgp51) and synthetic multiple antigenic peptides (MAP) of T-helper, T-cytotoxic, and B-cell epitopes of gp51 were investigated against BLV challenge. Two and three sheep were immunized with rgp51 and a mixture of peptides with Freund's complete adjuvant, respectively. BLV was detected from all the immunized sheep at 2 weeks and showed peak levels at 4 weeks after the challenge. However, in two sheep immunized with the mixed peptides, the titer of BLV gradually decreased and one sheep eliminated BLV completely at 28 weeks after the challenge. These two sheep showed higher lymphocyte proliferative responses against the immunized peptides than the other sheep, One of the sheep also showed the specific cytotoxic lymphocyte activity against the BLVgp51-expressing target in vitro. These results suggest the possibility of the peptide vaccine for elimination of BLV in carrier animals in vivo. Copyright (C) 1996 Elsevier Science Ltd.
  • N Hirai, H Kabeya, K Ohashi, C Sugimoto, M Onuma JOURNAL OF VETERINARY MEDICAL SCIENCE 58 (5) 455 -457 1996年05月 [査読無し][通常論文]
     
    Serological survey of bovine immunodeficiency-like virus (BIV) infection was performed in cattle of 3 different farms in Hokkaido, where a relatively high seroprevalence was recorded for bovine leukemia virus (BLV). About a half of 120 cattle tested were seropositive for BLV, while 7.5% of the cattle were seropositive for BIV. Though increased numbers of leukocytes were frequently observed in BLV-seropositive cows, no such changes were observed in BIV-positive but BLV-negative cows. No correlation was demonstrated between BIV- and BLV-seroprevalence of the cattle.
  • Y Hiramoto, SI Lee, T Morimura, K Ohashi, C Sugimoto, M Onuma CURRENT RESEARCH ON MAREK'S DISEASE 130 -135 1996年 [査読無し][通常論文]
     
    Marek's disease (Mn) lymphoma formation can be prevented by the vaccination of a live attenuated XID virus (MDV). We analyzed the effect of MD vaccination on the distribution of MDV among T cell subsets. Chickens vaccinated with CVI988 at one day of age were challenged with Md5 at 5 days of age. Non-vaccinated chickens were also challenged with Md5 at the same day. At 7, 14, 21 and 28 days after the Md5 challenge, MDV was re-isolated from CD4(+) and CD8(+) T cell subsets, and virus titers were compared each other. The fractionation of T cell subsets was achieved by positive immune-magnetic selection using monoclonal antibodies to either CD4 or CD8. The average titer of rescued MDV from vaccinated chickens was significantly power compared to that from nonvaccinated chickens. In addition, the titer of MDV isolated from CD4(+) T cells was consistently higher than that from CD8+ T cells, and these titers were decreased during the experimental period. Only very low titers of MDV were isolated from both CD4 and CD8(+) T cell subsets in vaccinated chickens at 21 and 28 days after challenge. These results suggest that CD4(+) T cell subset is preferentially infected with MDV, and that the infection to CD4(+) as well as CD8(+) T cells was suppressed by vaccination.
  • T Morimura, K Ohashi, M Hattori, C Sugimoto, M Onuma Current Research on Marek's Disease 335 -340 1996年 [査読無し][通常論文]
     
    We investigated whether T cell-immunosuppression induced by Marek's disease virus (MDV)-infection is due to apoptosis. DNA fragmentation analysis showed that CD4(+) but not CD8(+) T cells underwent apoptosis in infected chickens. Furthermore, splenic CD4(+) and CD8(+) T cells in infected chickens did not respond to the stimulation with phorbol 12-myristate 13-acetate plus ionomycin in vitro. This low response of T cells from MDV-infected chickens was at least partially due to apoptotic cell death. Thus, apoptosis observed in MDV-infected chickens may at least in part contribute to the immunosuppression induced by MDV.
  • MORIMURA T, OHASHI K, KON Y, HATTORI M, SUGIMOTO C, ONUMA M Archives of Virology 141 (11) 2243 -2249 1996年 [査読無し][通常論文]
  • H Yamamoto, M Hattori, K Ohashi, C Sugimoto, M Onuma VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 49 (4) 375 -386 1996年01月 [査読無し][通常論文]
     
    The function of CD4(+) T cells in antibody production was examined by using T cell subset-depleted chickens. CD4- and CD8-depleted chickens, established by the combination of thymectomy and injection of T cell subset-specific monoclonal antibodies, were immunized with sheep red blood cells (SRBC). Titers of anti-SRBC antibody produced in CD4-depleted chickens were lower than those in control chickens, while no difference in the antibody production was observed between CD8-depleted and control chickens. In chickens depleted of both CD4(+) and CD8(+) T cells, the recovery of T cells in the periphery was demonstrated starting 3 weeks after T cell depletion. Those T cells recovered in the periphery predominantly expressed CD4 molecules. Although low titers of antibody against SRBC were detected in chickens depleted of both CD4(+) and CD8(+) T cells, an increase of anti-SRBC antibody production was coincidentally observed with the recovery of CD4(+) T cells in the periphery. These results suggest that CD4(+) T cells could differentiate in extrathymic environments in chickens, and have a helper function in antibody production similar to that of intrathymic T cells. These extrathymic T cells, however, showed a lower proliferative response to concanavalin A than intrathymic T cells, suggesting that these extrathymic T cells may have some properties distinct from intrathymic T cells.
  • M Takagi, K Ohashi, T Morimura, C Sugimoto, M Onuma CURRENT RESEARCH ON MAREK'S DISEASE 251 -256 1996年 [査読無し][通常論文]
     
    To determine whether there is any abnormalities of the p53 gene in chicken lymphoblastoid tumor cell lines (derived from Marek's disease (MD), lymphoid leukosis and reticuloendotheliosis tumors) and field tumors, some portions of p53 cDNA corresponding to domains IT to V were sequenced. Several mutations were Identified in both cell lines and field tumors. However, none of these mutations is localized at the "hot spot", which has been regarded as the site for transformation-activating mutations. Moreover, the 122-bp deletion including exon eight of the p53 gene were shown in three cell lines; MSB1, MTB1 and HP1 derived from MD tumors. This deletion could cause a frame shift of the encoding p53 protein, possibly resulting in the generation of a functionally different p53 protein. However, we confirmed that p53 mRNA without deletion is also present in each of these cell lines. In addition, mutations of the: p53 gene were identified in these cell lines. Thus, mutations of the p53 gene and deletion in the p53 transcript may be induced by MD virus, Alternatively, this deletion may result from alternative splicing which may occur in the cell cycle-dependent manner.
  • H YAMAMOTO, M HATTORI, K OHASHI, C SUGIMOTO, M ONUMA JOURNAL OF VETERINARY MEDICAL SCIENCE 57 (5) 945 -946 1995年10月 [査読無し][通常論文]
     
    In chickens inoculated with a Marek's disease (MD) vaccine and subsequently with virulent MD virus (MDV), CD4(+) T cell population was drastically decreased following a transient increase at 21 days after hatching (16 days after MDV infection). To elucidate the immune response after the decrease of CD4(+) T cell population, the antibody production against sheep red blood cells (SRBC) was examined in these chickens. Chickens challenged with a virulent MDV after MD vaccination produced lower titers of anti-SRBC antibody than untreated control chickens. Antibody production against SRBC was also lowered in vaccinated chickens or chickens challenged with a virulent MDV.
  • Immunomodulation of peripheral T cells in chickens induced by Marek's disease virus infection: Involvement in immunosuppression.
    Journal of General Virology 76 2979 -2985 1995年 [査読無し][通常論文]
  • Possible mechanisms of immuno-suppression in chickens induced by Marek’s disease virus.
    日仏獣医学雑誌 6 89 -99 1995年 [査読無し][通常論文]
  • K OHASHI, PH OCONNELL, KA SCHAT VIROLOGY 199 (2) 275 -283 1994年03月 [査読無し][通常論文]
     
    A cDNA library was constructed from poly(A)(+) RNA fractions obtained from a Marek's disease (MD) lymphoblastoid cell line, MDCC-CU41, in which viral gene expression is very limited. Three independent groups (1, 2, and 3) of MD virus (MDV)-specific clones were obtained, which were mapped in the inverted repeat region of the BamHI-A fragment of the MDV genome. Northern blot analysis showed that probes prepared from these cDNA clones hybridized with several transcripts of different sizes in poly(A)(+) RNA of MDCC-CU41, although the amounts of these transcripts were relatively small compared to those in MDV lytically infected cells. Moreover, a small open reading frame, which can encode a 94-amino-acid protein, was identified in the A41 cDNA clone (Group 3). By RNase protection assays, the 1.2-kb Group 3 transcriptional unit has been defined. In indirect immunofluorescent antibody assays, antiserum against the bacterially expressed fusion protein, glutathione S-transferase-A41, reacted specifically with the cytoplasmic regions of MDV (strain RB1B)-infected chick kidney cells. However, MDCC-CU41 did not contain a detectable level of the protein determined by these methods. (C) 1994 Academic Press, Inc.
  • K OHASHI, WP ZHOU, PH OCONNELL, KA SCHAT JOURNAL OF VIROLOGY 68 (2) 1191 -1195 1994年02月 [査読無し][通常論文]
     
    Two Marek's disease (MD) virus BamHI-L-specific cDNA clones were isolated from a cDNA library constructed from poly(A)(+) RNA fractions of an MD lymphoblastoid cell line, MDCC-CU41 (CU41). These clones were mapped to the region corresponding to the BamHI-Q(2), and -L, regions. These clones hybridized with 2.5-, 0.8-, and 0.6-kb transcripts prepared from CU41. The transcriptional unit of the 0.6-kb transcript was determined by RNase protection assays. An open reading frame encoding a 107-amino-acid polypeptide was identified in the 0.6-kb transcript. Reverse transcriptase-PCR demonstrated the presence of this transcript in both CU41 and a reticuloendotheliosis virus-transformed cell line latently infected with MD virus.
  • cDNA clones derived from the Marek's disease tumor cell line MDCC-CU41.
    Proceedings of 19th World's Poultry Congress 54 -57 1992年 [査読無し][通常論文]
  • M HORIE, K OHASHI, H KODAMA, T MIKAMI INTERNATIONAL JOURNAL OF CANCER 47 (2) 238 -243 1991年01月 [査読無し][通常論文]
     
    The physico-chemical nature of Marek's disease tumor-associated surface antigen (MATSA) on Marek's disease (MD) lymphoblastoid tumor cell line (MDCC-MSBI-clo.18) was examined by the cellular enzyme-linked immunosorbent assay (CELISA) and the sandwich enzyme-linked immunosorbent assay (ELISA) using an anti-MATSA immune serum or a monoclonal antibody (MAb) 2B9 developed against MATSA. Our results indicate that MATSA is a glycoprotein and 2B9 recognizes an antigenic site in the protein moiety of MATSA. MATSA was solubilized from MSBI-clo.18 cells by treatment with 0.5% Nonidet P-40, and purified by affinity chromatography coupling with 2B9 and further by ion exchange chromatography on diethylaminoethylcellulose (IECD). MATSA was eluted with 0.2 to 0.3 M KCl in IECD and the purity of MATSA was increased about 2,500-fold. The purified MATSA was shown to have a molecular weight (M(r)) of 70,000 by SDS-PAGE. The reactivity of purified MATSA with anti-thymus cell serum was examined. MATSA was detectable by anti-thymus cell serum, although 2B9, which was used to purify MATSA from MSBI-clo.18 cells, was not reactive to cells prepared from the thymus. However, MATSA was no longer detectable after the absorption of anti-thymus cell serum by chicken bursa cells. The absorption of anti-thymus cell serum by chicken red blood cells (RBC) had no effect on the reactivity against MATSA. These results suggest that MATSA may be a lymphocyte-specific antigen modified during leukemogenesis by Marek's disease virus (MDV).
  • Cytotoxic activity of spleen cells from chickens with transplantable Marek's disease tumors.
    Proceedings of 3rd International Symposium on Marek's Disease 268 -275 1989年 [査読無し][通常論文]
  • Suppression of NK activity of spleen cells by chicken fetal antigen in a Marek's disease lymphoblastoid cell line (MDCC-MSB1).
    International Journal of Cancer 40 378 -382 1987年 [査読無し][通常論文]
  • K OHASHI, T MIKAMI, T HIGASHIHARA, H KODAMA, H IZAWA CANCER RESEARCH 46 (11) 5858 -5863 1986年11月 [査読無し][通常論文]
  • Detection of cell-surface antigens on MDCC-MSB1 cells using monoclonal antibodies.
    2nd International Symposium on Marek's Disease 214 -235 1985年 [査読無し][通常論文]
  • T HIGASHIHARA, T MIKAMI, K OHASHI, H KODAMA, M ONUMA, H IZAWA JAPANESE JOURNAL OF VETERINARY SCIENCE 46 (5) 649 -658 1984年 [査読無し][通常論文]

受賞

  • 2008年 日本獣医学会賞

共同研究・競争的資金等の研究課題

  • 鳥類由来感染症の疫学的研究
    研究期間 : 2008年
  • Epidemiological study on infectious diseases of wild and domestic birds
    研究期間 : 2008年
  • マレック病ウイルスによる腫瘍化の分子メカニズムの解析、ワクチン作用機序の解明
    研究期間 : 1992年
  • 動物感染症に対する免疫応答の分子機序の解明
    研究期間 : 1992年
  • Characterization of molecular mechanisms of immune responses against infectious diseases in animals
    研究期間 : 1992年

教育活動情報

主要な担当授業

  • 感染症学特別研究Ⅰ
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 英語演習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 免疫学 英語論文
  • 感染症学特別演習
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 基礎免疫学
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 基礎免疫学 獲得免疫 自然免疫 生体防御
  • 人獣共通感染症対策専門特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 研究・臨床セミナー
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
  • 感染症学特別研究ⅡA
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 伝染病学各論(伴侶動物)
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 伝染病、感染症、ワクチン、犬、猫、伴侶動物
  • 感染症学特別研究ⅡB
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医疫学演習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 疫学研究手法、原因論、リスク分析、統計学
  • 獣医科学・感染症学基礎科目 免疫学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 魚病学
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 魚病、魚類、水産、感染症
  • 獣医科学特別研究
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 専門獣医科学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 人獣共通感染症対策専門特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学特論演習
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 免疫学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目 免疫学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 専門獣医科学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 動物愛護福祉論
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
  • アドバンスト演習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部

大学運営

学内役職歴

  • 2017年4月1日 - 2019年3月31日 教育研究評議会評議員
  • 2017年4月1日 - 2019年3月31日 大学院国際感染症学院長
  • 2019年4月1日 - 2021年3月31日 教育研究評議会評議員
  • 2019年4月1日 - 2021年3月31日 大学院国際感染症学院長


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