研究者データベース

研究者情報

マスター

アカウント(マスター)

  • 氏名

    村田 史郎(ムラタ シロウ), ムラタ シロウ

所属(マスター)

  • 獣医学研究院 獣医学部門 病原制御学分野

所属(マスター)

  • 獣医学研究院 獣医学部門 病原制御学分野

独自項目

syllabus

  • 2020, 免疫学特論, Advanced Lecture on Immunology, 博士後期課程, 獣医学院
  • 2020, 免疫学特論, Advanced Lecture on Immunology, 博士後期課程, 国際感染症学院
  • 2020, 伝染病学実習, Practice in Infectious Diseases, 学士課程, 獣医学部, 細菌、ウイルス、原虫、同定、診断
  • 2020, 獣医疫学演習, Seminar in Veterinary Epidemiology, 学士課程, 獣医学部, 疫学研究手法、原因論、リスク分析、統計学
  • 2020, 動物衛生学実習, Practice in Animal Hygiene, 学士課程, 獣医学部, 飼養衛生、衛生管理、疾病予防、ワクチン、消毒、アニマルウェルフェア
  • 2020, 草地飼料学, Grassland Science and Animal Feeding, 学士課程, 獣医学部, 飼料資源 試料評価 飼料調製 放牧 草地からの家畜生産

timetable

  • 博士後期課程, 獣医学研究科, 2020, 獣医科学基礎科目B 免疫学特論
  • 学士課程, 獣医学部, 2020, 伝染病学実習
  • 学士課程, 獣医学部, 2020, 動物衛生学実習
  • 学士課程, 獣医学部, 2020, 獣医疫学演習
  • 学士課程, 獣医学部, 2020, 草地飼料学
  • 学士課程, 獣医学部, 2020, アドバンスト演習

researchmap

プロフィール情報

学位

  • 博士(獣医学)(北海道大学)

プロフィール情報

  • 村田, ムラタ
  • 史郎, シロウ
  • ID各種

    201301059666482919

業績リスト

研究キーワード

  • 腫瘍   ウイルス学   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

経歴

  • 2010年03月 - 現在 北海道大学大学院獣医学研究科 助教
  • 2008年04月 - 2010年02月 農林水産省動物検疫所

学歴

  •         - 2008年03月   北海道大学 大学院獣医学研究科
  •         - 2004年03月   北海道大学 獣医学部

論文

  • Sotaro Fujisawa, Shiro Murata, Masaki Takehara, Julia Aoyama, Ayu Morita, Masayoshi Isezaki, Shwe Yee Win, Takuma Ariizumi, Takumi Sato, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Osamu Ichii, Satoru Konnai, Kazuhiko Ohashi
    Vaccine 2021年09月08日 
    The poultry red mite (Dermanyssus gallinae; PRM) is a blood-sucking ectoparasite of chickens that is a threat to poultry farming worldwide and significantly reduces productivity in the egg-laying industry. Chemical acaricides that are widely used in poultry farms for the prevention of PRMs are frequently ineffective due to the emergence of acaricide-resistant PRMs. Therefore, alternative control methods are needed, and vaccination is a promising strategy for controlling PRMs. A novel adipocyte-plasma membrane-associated protein-like molecule (Dg-APMAP) is highly expressed in blood-fed PRMs according to a previous RNA sequencing analysis. Here, we attempted to identify the full sequence of Dg-APMAP, study its expression in different life stages of PRMs, and evaluate its potential as a vaccine antigen. Dg-APMAP mRNA was expressed in the midgut and ovaries, and in all life stages regardless of feeding states. Importantly, in vitro feeding of PRMs with plasma derived from chickens immunized with the recombinant protein of the extracellular region of Dg-APMAP significantly reduced their survival rate in nymphs and adults, which require blood meals. Our data suggest that the host immune responses induced by vaccination with Dg-APMAP could be an effective strategy to reduce the suffering caused by PRMs in the poultry industry.
  • Otgontuya Ganbaatar, Satoru Konnai, Tomohiro Okagawa, Yutaro Nojima, Naoya Maekawa, Yoshiki Ichikawa, Atsushi Kobayashi, Tomoyuki Shibahara, Yojiro Yanagawa, Hidetoshi Higuchi, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Immunity, inflammation and disease 2021年08月20日 
    INTRODUCTION: Chronic infections lead to the functional exhaustion of T cells. Exhausted T cells are phenotypically differentiated by the surface expression of the immunoinhibitory receptor, such as programmed death-1 (PD-1). The inhibitory signal is produced by the interaction between PD-1 and its PD-ligand 1 (PD-L1) and impairs the effector functions of T cells. However, the expression dynamics of PD-L1 and the immunological functions of the PD-1/PD-L1 pathway in chronic diseases of pigs are still poorly understood. In this study, we first analyzed the expression of PD-L1 in various chronic infections in pigs, and then evaluated the immune activation by the blocking assay targeting the swine PD-1/PD-L1 pathway. METHODS: In the initial experiments, anti-bovine PD-L1 monoclonal antibodies (mAbs) were tested for cross-reactivity with swine PD-L1. Subsequently, immunohistochemical analysis was conducted using the anti-PD-L1 mAb. Finally, we assessed the immune activation of swine peripheral blood mononuclear cells (PBMCs) by the blockade with anti-PD-L1 mAb. RESULTS: Several anti-PD-L1 mAbs tested recognized swine PD-L1-expressing cells. The binding of swine PD-L1 protein to swine PD-1 was inhibited by some of these cross-reactive mAbs. In addition, immunohistochemical analysis revealed that PD-L1 was expressed at the site of infection in chronic infections of pigs. The PD-L1 blockade increased the production of interleukin-2 from swine PBMCs. CONCLUSIONS: These findings suggest that the PD-1/PD-L1 pathway could be also involved in immunosuppression in chronic infections in pigs. This study provides a new perspective on therapeutic strategies for chronic diseases in pigs by targeting immunosuppressive pathways.
  • Fumi Kayasaki, Tomohiro Okagawa, Satoru Konnai, Junko Kohara, Yamato Sajiki, Kei Watari, Otgontuya Ganbaatar, Shinya Goto, Hayato Nakamura, Honami Shimakura, Erina Minato, Atsushi Kobayashi, Manabu Kubota, Nobuhiro Terasaki, Akira Takeda, Haruka Noda, Mitsuru Honma, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi
    Veterinary microbiology 254 108976 - 108976 2021年03月 
    Diarrhea is a major cause of death in calves and this is linked directly to economic loss in the cattle industry. Fermented milk replacer (FMR) has been used widely in clinical settings for calf feeding to improve its health and growth. However, the protective efficacy of FMR on calf diarrhea remains unclear. In this study, we verified the preventive effects of FMR feeding on calf diarrhea using an experimental infection model of bovine rotavirus (BRV) in newborn calves and a field study in dairy farms with calf diarrhea. In addition, we evaluated the protective efficacy of lactic acid bacteria-supplemented milk replacer (LAB-MR) in an experimental infection model. In the experimental infection, calves fed FMR or high-concentrated LAB-MR had diarrhea, but the water content of feces was lower and more stable than that of calves fed normal milk replacer. The amount of milk intake also decreased temporarily, but recovered immediately in the FMR- and LAB-MR-fed calves. As compared with the control calves, FMR- or LAB-MR-fed calves showed less severe or reduced histopathological lesions of enteritis in the intestinal mucosa. In a field study using dairy calves, FMR feeding significantly reduced the incidence of enteritis, mortality from enteritis, duration of a series of treatment for enteritis, number of consultations, and cost of medical care for the disease. These results suggest that feeding milk replacer-based probiotics to calves reduces the severity of diarrhea and tissue damage to the intestinal tract caused by BRV infection and provides significant clinical benefits to the prevention and treatment of calf diarrhea.
  • Yamato Sajiki, Satoru Konnai, Reiko Nagata, Satoko Kawaji, Hayato Nakamura, Sotaro Fujisawa, Tomohiro Okagawa, Naoya Maekawa, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Yasuyuki Mori, Kazuhiko Ohashi
    The Journal of veterinary medical science 83 2 162 - 166 2021年02月25日 
    Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteritis of ruminants. Previous studies have shown that programmed death-ligand 1 (PD-L1) is associated with the disease progression, and PD-L1 blockade activates MAP-specific Th1 responses in vitro. Here, we performed anti-PD-L1 antibody administration using 2 MAP-infected cattle at the late subclinical stage of infection. After administration, bacterial shedding was reduced or maintained at a low level. Additionally, MAP-specific Th1 cytokine production was upregulated, and CD69 expression was increased in T cells. Collectively, the treatment has a potential as a novel control method against Johne's disease.
  • Naoya Maekawa, Satoru Konnai, Maki Nishimura, Yumiko Kagawa, Satoshi Takagi, Kenji Hosoya, Hiroshi Ohta, Sangho Kim, Tomohiro Okagawa, Yusuke Izumi, Tatsuya Deguchi, Yukinari Kato, Satoshi Yamamoto, Keiichi Yamamoto, Mikihiro Toda, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    NPJ precision oncology 5 1 10 - 10 2021年02月12日 
    Immunotherapy targeting programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) represents promising treatments for human cancers. Our previous studies demonstrated PD-L1 overexpression in some canine cancers, and suggested the therapeutic potential of a canine chimeric anti-PD-L1 monoclonal antibody (c4G12). However, such evidence is scarce, limiting the clinical application in dogs. In the present report, canine PD-L1 expression was assessed in various cancer types, using a new anti-PD-L1 mAb, 6C11-3A11, and the safety and efficacy of c4G12 were explored in 29 dogs with pulmonary metastatic oral malignant melanoma (OMM). PD-L1 expression was detected in most canine malignant cancers including OMM, and survival was significantly longer in the c4G12 treatment group (median 143 days) when compared to a historical control group (n = 15, median 54 days). In dogs with measurable disease (n = 13), one dog (7.7%) experienced a complete response. Treatment-related adverse events of any grade were observed in 15 dogs (51.7%). Here we show that PD-L1 is a promising target for cancer immunotherapy in dogs, and dogs could be a useful large animal model for human cancer research.
  • Takuma Ariizumi, Shiro Murata, Sotaro Fujisawa, Masayoshi Isezaki, Naoya Maekawa, Tomohiro Okagawa, Takumi Sato, Eiji Oishi, Akira Taneno, Satoru Konnai, Kazuhiko Ohashi
    The Journal of veterinary medical science 2021年02月11日 
    Poultry red mites (PRMs, Dermanyssus gallinae) are harmful ectoparasites that affect farmed chickens and cause serious economic losses in the poultry industry worldwide. Acaricides are used for PRM control; however, some PRMs have developed acaricide-resistant properties, which have indicated the need for different approaches for PRM control. Therefore, it is necessary to elucidate the biological status of PRMs to develop alternative PRM control strategies. Quantitative polymerase chain reaction (qPCR) allows analysis of the biological status at the transcript level. However, reference genes are preferable for accurate comparison of expression level changes given the large variation in the quality of the PRM samples collected in each farm. This study aimed to identify candidate reference genes with stable expression levels in the different blood feeding states and life stages of PRMs. First, we selected candidates based on the following criteria: sufficient expression intensity and no significant expression difference between fed and starved states. We selected and characterized seven candidate reference genes. Among them, we evaluated the gene expression stability between the starved and fed states using RefFinder; moreover, we compared their expression levels in each life-stage and identified two reference genes, Elongation factor 1-alpha (ELF1A)-like and apolipophorins-like. Finally, we evaluated the utility of the candidates as reference genes, and the use of ELF1A-like and apolipophorins-like successfully normalized ATP synthase subunit g -like gene expression. Thus, ELF1A-like and apolipophorins-like could be suitable reference genes in PRMs.
  • Yamato Sajiki, Satoru Konnai, Yoshinori Ikenaka, Kevin Christian Montecillo Gulay, Atsushi Kobayashi, Luís Fernando Parizi, Benvindo Capela João, Kei Watari, Sotaro Fujisawa, Tomohiro Okagawa, Naoya Maekawa, Carlos Logullo, Itabajara da Silva Vaz Jr, Shiro Murata, Kazuhiko Ohashi
    Scientific reports 11 1 1063 - 1063 2021年01月13日 
    The tick Rhipicephalus microplus is a harmful parasite of cattle that causes considerable economic losses to the cattle breeding industry. Although R. microplus saliva (Rm-saliva) contains several immunosuppressants, any association between Rm-saliva and the expression of immunoinhibitory molecules, such as programmed death (PD)-1 and PD-ligand 1 (PD-L1), has not been described. In this study, flow cytometric analyses revealed that Rm-saliva upregulated PD-1 expression in T cells and PD-L1 expression in CD14+ and CD11c+ cells in cattle. Additionally, Rm-saliva decreased CD69 expression in T cells and Th1 cytokine production from peripheral blood mononuclear cells. Furthermore, PD-L1 blockade increased IFN-γ production in the presence of Rm-saliva, suggesting that Rm-saliva suppresses Th1 responses via the PD-1/PD-L1 pathway. To reveal the upregulation mechanism of PD-1/PD-L1 by Rm-saliva, we analyzed the function of prostaglandin E2 (PGE2), which is known as an inducer of PD-L1 expression, in Rm-saliva. We found that Rm-saliva contained a high concentration of PGE2, and PGE2 treatment induced PD-L1 expression in CD14+ cells in vitro. Immunohistochemical analyses revealed that PGE2 and PD-L1 expression was upregulated in tick-attached skin in cattle. These data suggest that PGE2 in Rm-saliva has the potential to induce the expression of immunoinhibitory molecules in host immune cells.
  • Hiroto Takeuchi, Satoru Konnai, Naoya Maekawa, Satoshi Takagi, Hiroshi Ohta, Noboru Sasaki, Sangho Kim, Tomohiro Okagawa, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Frontiers in veterinary science 8 656715 - 656715 2021年 
    Cancer cells can evade host immune systems via multiple mechanisms. Transforming growth factor beta 1 (TGF-β1) is an immunosuppressive cytokine that induces regulatory T cell (Tregs) differentiation and is involved in immune evasion mechanisms in cancer. The inhibition of the TGF-β1 signaling pathway can suppress cancer progression and metastasis through the modulation of anticancer immune responses. However, to best of our knowledge, no implementation of treatments targeting TGF-β1 has been reported in dog cancers. This study aimed to examine whether TGF-β1 is upregulated in canine cancers. We measured TGF-β1 concentrations in culture supernatants of canine melanoma cell lines and in serum samples from dogs with oral malignant melanoma. TGF-β1 production was observed in several cell lines, and serum TGF-β1 levels were elevated in dogs with oral malignant melanoma. Interestingly, the addition of recombinant TGF-β1 to canine peripheral blood mononuclear cell cultures decreased Th1 cytokine production and increased differentiation of CD4+CD25+Foxp3+ lymphocytes, suggesting that TGF-β1 is immunosuppressive in canine immune systems. We developed a decoy receptor for TGF-β, namely TGF-βRII-Ig, by identifying an open reading frame of the canine TGFBR2 gene. TGF-βRII-Ig was prepared as a recombinant fusion protein of the extracellular region of canine TGF-βRII and the Fc region of canine IgG-B. As expected, TGF-βRII-Ig bound to TGF-β1. In the presence of TGF-β1, the treatment with TGF-βRII-Ig increased Th1 cytokine production and decreased the differentiation of CD4+CD25+Foxp3+ lymphocytes. Our results suggest that TGF-βRII-Ig competitively inhibits the immunosuppressive effects of TGF-β1 and thereby activates immune responses. This study demonstrated the potential of TGF-βRII-Ig as a novel biologic for canine melanoma.
  • Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Hayato Nakamura, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Developmental and comparative immunology 114 103847 - 103847 2021年01月 
    Bovine leukemia virus (BLV) infection is a bovine chronic infection caused by BLV, a member of the genus Deltaretrovirus. In this study, we examined the immunomodulatory effects of GS-9620, a toll-like receptor (TLR) 7 agonist, in cattle (Bos taurus) and its therapeutic potential for treating BLV infection. GS-9620 induced cytokine production in peripheral blood mononuclear cells (PBMCs) as well as CD80 expression in CD11c+ cells and increased CD69 and interferon (IFN)-γ expressions in T cells. Removing CD11c+ cells from PBMCs decreased CD69 expression in T cells in the presence of GS-9620. These results suggest that TLR7 agonism promotes T-cell activation via CD11c+ cells. Analyses using PBMCs from BLV-infected cattle revealed that TLR7 expression in CD11c+ cells was upregulated during late-stage BLV infection. Furthermore, GS-9620 increased IFN-γ and TNF-α production and inhibited syncytium formation in vitro, suggesting that GS-9620 may be used to treat BLV infection.
  • Shiro Murata, Ayaka Taniguchi, Masayoshi Isezaki, Sotaro Fujisawa, Eishi Sakai, Akira Taneno, Osamu Ichii, Takuya Ito, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi
    Parasite (Paris, France) 28 9 - 9 2021年 
    Poultry red mites (PRMs, Dermanyssus gallinae) are ectoparasites that negatively affect farmed chickens, leading to serious economic losses worldwide. Acaricides have been used to control PRMs in poultry houses. However, some PRMs have developed resistance to acaricides, and therefore different approaches are required to manage the problems caused by PRMs. Vaccination of chickens is one of the methods being considered to reduce the number of PRMs in poultry houses. In a previous study, a cysteine protease, Deg-CPR-1, was identified as a candidate vaccine against PRMs distributed in Europe. In this study, we investigated the characteristics of Deg-CPR-1. A phylogenetic analysis revealed that Deg-CPR-1 is closely related to the digestive cysteine proteases of other mite species, and it was classified into a cluster different from that of chicken cathepsins. Deg-CPR-1 of PRMs in Japan has an amino acid substitution compared with that of PRMs in Europe, but it showed efficacy as a vaccine, consistent with previous findings. Deg-CPR-1 exhibited cathepsin L-like enzyme activity. In addition, the Deg-CPR-1 mRNA was expressed in the midgut and in all stages of PRMs that feed on blood. These results imply that Deg-CPR-1 in the midgut may have important functions in physiological processes, and the inhibition of its expression may contribute to the efficacy of a Deg-CPR-1-based vaccine. Further research is required to fully understand the mechanisms of vaccine efficacy.
  • Yamato Sajiki, Satoru Konnai, Zimeng Cai, Kensuke Takada, Tomohiro Okagawa, Naoya Maekawa, Sotaro Fujisawa, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    ImmunoHorizons 4 12 837 - 850 2020年12月21日 
    Combination treatment approaches are increasingly considered to overcome resistance to immunotherapy targeting immunoinhibitory molecules such as programmed death (PD)-1 and PD-ligand 1 (PD-L1). Previous studies have demonstrated that the therapeutic efficacy of anti-PD-L1 Abs is enhanced by combination treatment with cyclooxygenase-2 inhibitors, through downregulation of the immunosuppressive eicosanoid PGE2, although the underlying mechanism remains unclear. In this study, we show that serum PGE2 levels are upregulated after anti-PD-L1 Ab administration in a bovine model of immunotherapy and that PGE2 directly inhibits T cell activation via its receptor E prostanoid (EP) 4. Additionally, anti-PD-L1 Ab induces TNF-α production and TNF-α blockade reduces PGE2 production in the presence of anti-PD-L1 Ab, suggesting that anti-PD-L1 Ab-induced TNF-α impairs T cell activation by PGE2 upregulation. Our studies examining the therapeutic potential of the dual blockade of PD-L1 and EP4 in bovine and murine immune cells reveal that the dual blockade of PD-L1 and EP4 significantly enhances Th1 cytokine production in vitro. Finally, we show that the dual blockade decreases tumor volume and prolongs survival in mice inoculated with the murine lymphoma cell line EG7. Altogether, these results suggest that TNF-α induced by anti-PD-L1 Ab treatment is associated with T cell dysfunction via PGE2/EP4 pathway and that the dual blockade of PD-L1 and EP4 should be considered as a novel immunotherapy for cancer.
  • Shwe Yee Win, Hla Myet Chel, Myint Myint Hmoon, Lat Lat Htun, Saw Bawm, Mar Mar Win, Shiro Murata, Nariaki Nonaka, Ryo Nakao, Ken Katakura
    Acta tropica 212 105719 - 105719 2020年12月 
    Village chicken production, a traditional, small-scale, and extensive backyard poultry industry, has been profitable for local farmers in Myanmar. However, there is scanty information available concerning the infection of these chickens with avian pathogens, including haemoprotozoan parasites. In the present study, we provide the first report of microscopic detection and molecular identification of Leucocytozoon and Plasmodium parasites from seven different areas of Myanmar. Leucocytozoon gametocytes were detected in 17.6% (81/461) of the blood smears from village chickens. The nested polymerase chain reaction (PCR) for targeting Leucocytozoon mitochondrial cytochrome b (cyt b) genes had a 17.6% positive rate. Although the positive rate of nested PCR targeting Plasmodium/Haemoproteus cyt b was 34.3%, the PCR protocol was observed to possibly amplify DNA of a certain species of Leucocytozoon. There were no obvious clinical signs in the infected birds. Statistical analysis of the microscopic detection and PCR detection rates using the age and sex of birds as internal factors revealed that the statistical significances differed according to the study area. The sequencing of 32 PCR products obtained from each study area revealed infection by Leucocytozoon caulleryi in three birds, Leucocytozoon sabrazesi in two birds, Leucocytozoon schoutedeni in two birds, Leucocytozoon sp. in eighteen birds, and Plasmodium juxtanucleare in seven birds; however, Haemoproteus infection was not detected. While L. sabrazesi was detected in chickens from the central region of Myanmar, the other haemosporidians were detected in those from different areas. In the haplotype analysis, we detected 17 haemosporidian cyt b haplotypes, including two for L. caulleryi, one for L. sabrazesi, two for L. schoutedeni, nine for Leucocytozoon sp., and three for P. juxtanucleare. Phylogenetic analysis of the cyt b haplotypes revealed a considerably close genetic relationship among chicken haemosporidians detected in Myanmar, Thailand, and Malaysia. These results indicate that well-recognized widespread species of chicken Leucocytozoon and Plasmodium are distributed nationwide in Myanmar, providing new insights into the ecosystem and control strategies of haemosporidian parasites in domesticated chickens in Myanmar.
  • Andelé M Conradie, Luca D Bertzbach, Jakob Trimpert, Joseph N Patria, Shiro Murata, Mark S Parcells, Benedikt B Kaufer
    PLoS pathogens 16 12 e1009104  2020年12月 
    Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek's disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection.
  • Zhiyuan Yang, Shiro Murata, Sotaro Fujisawa, Masaki Takehara, Ken Katakura, Myint Myint Hmoon, Shwe Yee Win, Saw Bawm, Satoru Konnai, Kazuhiko Ohashi
    BMC veterinary research 16 1 453 - 453 2020年11月23日 
    BACKGROUND: Avian infectious laryngotracheitis (ILT) is a highly contagious viral disease that causes severe economic losses to the poultry industry worldwide. In Southeast Asian countries, including Myanmar, poultry farming is a major industry. Although it is known that infectious respiratory pathogens, including infectious laryngotracheitis virus (ILTV), are a major threat to poultry farms, there are no data currently available on the epidemiology of ILTV in Myanmar. Therefore, in this study, we conducted a molecular detection of ILTV in 20 poultry farms in Myanmar. RESULTS: Of the 57 tested oropharyngeal swabs, 10 were positive for ILTV by polymerase chain reaction of a 647 bp region of the thymidine kinase (TK) gene, giving a prevalence of ILTV of 17.5% (10/57). Further sequencing analysis of infected cell protein 4 (ICP4) gene and glycoprotein B, G, and J (gB, gG, and gJ) genes indicated that these isolates were field strains. Phylogenetic analysis revealed that the Myanmar strains clustered together in a single branch and were closely related to other reference strains isolated from Asian countries. CONCLUSIONS: This study demonstrated the presence of ILTV in poultry farms in Myanmar. The genetic characterization analysis performed provides the fundamental data for epidemiological studies that monitor circulating strains of ILTV in Myanmar.
  • Shiro Murata, Yuka Machida, Masayoshi Isezaki, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi
    Virology journal 17 1 186 - 186 2020年11月23日 
    BACKGROUND: Marek's disease virus (MDV) causes malignant lymphomas in chickens (Marek's disease, MD). MD is currently controlled by vaccination; however, MDV strains have a tendency to develop increased virulence. Distinct diversity and point mutations are present in the Meq proteins, the oncoproteins of MDV, suggesting that changes in protein function induced by amino acid substitutions might affect MDV virulence. We previously reported that recent MDV isolates in Japan display distinct mutations in Meq proteins from those observed in traditional MDV isolates in Japan, but similar to those in MDV strains isolated from other countries. METHODS: To further investigate the genetic characteristics in Japanese field strains, we sequenced the whole genome of an MDV strain that was successfully isolated from a chicken with MD in Japan. A phylogenetic analysis of the meq gene was also performed. RESULTS: Phylogenetic analysis revealed that the Meq proteins in most of the Japanese isolates were similar to those of Chinese and European strains, and the genomic sequence of the Japanese strain was classified into the Eurasian cluster. Comparison of coding region sequences among the Japanese strain and MDV strains from other countries revealed that the genetic characteristics of the Japanese strain were similar to those of Chinese and European strains. CONCLUSIONS: The MDV strains distributed in Asian and European countries including Japan seem to be genetically closer to each other than to MDV strains from North America. These findings indicate that the genetic diversities of MDV strains that emerged may have been dependent on the different vaccination-based control approaches.
  • Naftaly Wang'ombe Githaka, Satoru Konnai, Masayoshi Isezaki, Shinya Goto, Marina Amaral Xavier, Sotaro Fujisawa, Shinji Yamada, Tomohiro Okagawa, Naoya Maekawa, Carlos Logullo, Itabajara da Silva Vaz Jr, Shiro Murata, Kazuhiko Ohashi
    Ticks and tick-borne diseases 11 6 101547 - 101547 2020年11月 
    Ferritin 2 (FER2) is an iron storage protein, which has been shown to be critical for iron homeostasis during blood feeding and reproduction in ticks and is therefore suitable as a component for anti-tick vaccines. In this study, we identified the FER2 of Ixodes persulcatus, a major vector for zoonotic diseases such as Lyme borreliosis and tick-borne relapsing fever in Japan, and investigated its functions. Ixodes persulcatus-derived ferritin 2 (Ip-FER2) showed concentration-dependent iron-binding ability and high amino acid conservation, consistent with FER2s of other tick species. Vaccines containing the recombinant Ip-FER2 elicited a significant reduction of the engorgement weight of adult I. persulcatus. Interestingly, the reduction of engorgement weight was also observed in Ixodes ovatus, a sympatric species of I. persulcatus. In silico analyses of FER2 sequences of I. persulcatus and other ticks showed a greater similarity with I. scapularis and I. ricinus and lesser similarity with Hyalomma anatolicum, Haemaphysalis longicornis, Rhipicephalus microplus, and R. appendiculatus. Moreover, it was observed that the tick FER2 sequences possess conserved regions within the primary structures, and in silico epitope mapping analysis revealed that antigenic regions were also conserved, particularly among Ixodes spp ticks. In conclusion, the data support further protective tick vaccination applications using the Ip-FER2 antigens identified herein.
  • Sotaro Fujisawa, Shiro Murata, Masayoshi Isezaki, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi
    Parasitology international 78 102156 - 102156 2020年10月 [査読有り][通常論文]
  • Yamato Sajiki, Satoru Konnai, Akie Ochi, Tomohiro Okagawa, Naftaly Githaka, Masayoshi Isezaki, Shinji Yamada, Takuya Ito, Shuji Ando, Hiroki Kawabata, Carlos Logullo, Itabajara da Silva Vaz Jr, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi
    Ticks and tick-borne diseases 11 2 101332 - 101332 2020年03月 [査読有り][通常論文]
     
    Tick saliva contains immunosuppressants which are important to obtain a blood meal and enhance the infectivity of tick-borne pathogens. In Japan, Ixodes persulcatus is a major vector for Lyme borreliosis pathogens, such as Borrelia garinii, as well as for those causing relapsing fever, such as B. miyamotoi. To date, little information is available on bioactive salivary molecules, produced by this tick. Thus, in this study, we identified two proteins, I. persulcatus derived sialostatin L1 (Ip-sL1) and sL2 (Ip-sL2), as orthologs of I. scapularis derived sL1 and sL2. cDNA clones of Ip-sL1 and Ip-sL2 shared a high identity with sequences of sL1 and sL2 isolated from the salivary glands of I. scapularis. Semi-quantitative PCR revealed that Ip-sL1 and Ip-sL2 were expressed in the salivary glands throughout the life of the tick. In addition, Ip-sL1 and Ip-sL2 were expressed even before the ticks started feeding, and their expression continued during blood feeding. Recombinant Ip-sL1 and Ip-sL2 were developed to characterize the proteins via biological and immunological analyses. These analyses revealed that both Ip-sL1 and Ip-sL2 had inhibitory effects on cathepsins L and S. Ip-sL1 and Ip-sL2 inhibited the production of IP-10, TNFα, and IL-6 by LPS-stimulated bone-marrow-derived dendritic cells (BMDCs). Additionally, Ip-sL1 significantly impaired BMDC maturation. Taken together, these results suggest that Ip-sL1 and Ip-sL2 confer immunosuppressive functions and appear to be involved in the transmission of pathogens by suppressing host immune responses, such as cytokine production and dendritic cell maturation. Therefore, further studies are warranted to investigate the immunosuppressive functions of Ip-sL1 and Ip-sL2 in detail to clarify their involvement in pathogen transmission via I. persulcatus.
  • Otgontuya Ganbaatar, Satoru Konnai, Tomohiro Okagawa, Yutaro Nojima, Naoya Maekawa, Erina Minato, Atsushi Kobayashi, Ryo Ando, Nobuya Sasaki, Daisuke Miyakoshi, Osamu Ichii, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    PloS one 15 11 e0234218  2020年 
    Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on lymphocytes. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. In our previous studies, we have developed anti-bovine PD-L1 monoclonal antibodies (mAbs) and reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Furthermore, we found that blocking antibodies that target PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway for chronic equine diseases, including tumors, remains unclear. In this study, we identified cDNA sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 mAbs against EqPD-L1 using in vitro assays. In addition, we evaluated the expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). The amino acid sequences of EqPD-1 and EqPD-L1 share a considerable identity and similarity with homologs from non-primate species. Two clones of the anti-bovine PD-L1 mAbs recognized EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine PD-1/PD-L1. Of note, immunohistochemistry confirmed the PD-L1 expression in EMM tumor tissues. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These findings showed that our anti-PD-L1 mAbs would be useful for analyzing the equine PD-1/PD-L1 pathway. Further research is warranted to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horses.
  • Yamato Sajiki, Satoru Konnai, Shinya Goto, Tomohiro Okagawa, Kosuke Ohira, Honami Shimakura, Naoya Maekawa, Satoshi Gondaira, Hidetoshi Higuchi, Motoshi Tajima, Yuki Hirano, Junko Kohara, Shiro Murata, Kazuhiko Ohashi
    Frontiers in veterinary science 7 609443 - 609443 2020年 
    Regulatory T cells (Tregs) regulate immune responses and maintain host immune homeostasis. Tregs contribute to the disease progression of several chronic infections by oversuppressing immune responses via the secretion of immunosuppressive cytokines, such as transforming growth factor (TGF)-β and interleukin-10. In the present study, we examined the association of Tregs with Mycoplasma bovis infection, in which immunosuppression is frequently observed. Compared with uninfected cattle, the percentage of Tregs, CD4+CD25highFoxp3+ T cells, was increased in M. bovis-infected cattle. Additionally, the plasma of M. bovis-infected cattle contained the high concentrations of TGF-β1, and M. bovis infection induced TGF-β1 production from bovine immune cells in in vitro cultures. Finally, we analyzed the immunosuppressive effects of TGF-β1 on bovine immune cells. Treatment with TGF-β1 significantly decreased the expression of CD69, an activation marker, in T cells, and Th1 cytokine production in vitro. These results suggest that the increase in Tregs and TGF-β1 secretion could be one of the immunosuppressive mechanisms and that lead to increased susceptibility to other infections in terms of exacerbation of disease during M. bovis infection.
  • Hiroto Takeuchi, Satoru Konnai, Naoya Maekawa, Erina Minato, Yoshiki Ichikawa, Atsushi Kobayashi, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi
    Frontiers in veterinary science 7 330 - 330 2020年 [査読有り][通常論文]
     
    Cancer is one of the most significant causes of death in dogs. Antibody drugs targeting the PD-1/PD-L1 axis represent a promising immunotherapy for both human and canine cancers. However, the regulation mechanisms of PD-L1 expression in canine cancers require further investigation to better understand the resistance mechanisms to anti-PD-L1 therapy. Recent reports have shown that CMTM6 and CMTM4 are critical regulators of PD-L1 protein expression in human cancer cells. By preventing PD-L1 from lysosome-mediated degradation, CMTM6 maintains PD-L1 expression on the cell surface. However, the literature has not reported on CMTM6 and CMTM4 in dogs, and their functions are completely unknown. To reveal a regulation mechanism of PD-L1 in canine cancers, this study firstly identified the gene sequences of CMTM6 and CMTM4. Then, the expression analysis of these proteins was performed by immunohistochemistry. Furthermore, the functions of CMTM6 and CMTM4 in regulating PD-L1 expression were examined by gene knockdown of CMTM6 and CMTM4. Canine CMTM6 and CMTM4 displayed high amino acid sequence identities compared with those of humans and mice. An immunohistochemical analysis using cross-reactive antibodies revealed that canine malignant melanoma and osteosarcoma express CMTM6, CMTM4, and PD-L1 simultaneously. Gene knockdown of CMTM6 and CMTM4 with RNA interference significantly reduced the cell surface expression of PD-L1 in a canine cell line. These results suggest that CMTM6 and CMTM4 are regulators of PD-L1 expression in canine cancers and could serve as potential therapeutic targets to enhance antitumor immunity.
  • Goto S, Konnai S, Hirano Y, Kohara J, Okagawa T, Maekawa N, Sajiki Y, Watari K, Minato E, Kobayashi A, Gondaira S, Higuchi H, Koiwa M, Tajima M, Taguchi E, Uemura R, Yamada S, Kaneko M, Kato Y, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K
    Front Vet Sci 7 12 - 12 2020年 [査読有り][通常論文]
     
    Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E2 (PGE2) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE2 in immune dysfunction and the relationship between PGE2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE2 and the PD-1/PD-L1 pathway.
  • Clinical efficacy of the combined treatment of anti-PD-L1 rat-bovine chimeric antibody with a COX-2 inhibitor in calves infected with Mycoplasma bovis
    Goto S, Konnai S, Hirano Y, Kohara J, Okagawa T, Maekawa N, Sajiki Y, Watari K, Minato E, Kobayashi A, Gondaira S, Higuchi H, Koiwa M, Tajima M, Taguchi E, Ishida M, Uemura R, Yamada S, Kaneko M, Kato Y, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K
    Jpn J Vet Res 2020年 [査読有り][通常論文]
  • Watari K, Konnai S, Maekawa N, Okagawa T, Suzuki Y, Murata S, Ohashi K
    BMC Vet Res 15 1 380 - 380 2019年10月29日 [査読有り][通常論文]
     
    BACKGROUND: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is known as an immune inhibitory receptor that is expressed on activated effector T cells and regulatory T cells. When CTLA-4 binds to CD80 or CD86, immunoinhibitory signals are transmitted to retain a homeostasis of the immune response. Recent studies have reported that CTLA-4 is upregulated in chronic infections and malignant neoplasms, contributing to host immune dysfunction. On the other hand, the blockade of CTLA-4 and CD80 or CD86 binding by antibody restores the immune response against these diseases. In a previous report, we indicated that the expression of CTLA-4 was closely associated with disease progression in cattle infected with the bovine leukemia virus (BLV). In this study, we established an anti-bovine CTLA-4 antibody to confirm its immune enhancing effect. RESULTS: Bovine CTLA-4-Ig binds to bovine CD80 and CD86 expressing cells. Additionally, CD80 and CD86 bind to CTLA-4 expressing cells in an expression-dependent manner. Bovine CTLA-4-Ig significantly inhibited interferon-gamma (IFN-γ) production from bovine peripheral blood mononuclear cells (PBMCs) activated by Staphylococcus enterotoxin B (SEB). An established specific monoclonal antibody (mAb) for bovine CTLA-4 specifically recognized only with bovine CTLA-4, not CD28, and the antibody blocked the binding of CTLA-4-Ig to both CD80 and CD86 in a dose-dependent manner. The bovine CTLA-4 mAb significantly restored the inhibited IFN-γ production from the CTLA-4-Ig treated PBMCs. In addition, the CTLA-4 mAb significantly enhanced IFN-γ production from CTLA-4 expressing PBMCs activated by SEB. Finally, we examined whether a CTLA-4 blockade by CTLA-4 mAb could restore the immune reaction during chronic infection; the blockade assay was performed using PBMCs from BLV-infected cattle. The CTLA-4 blockade enhanced IFN-γ production from the PBMCs in response to BLV-antigens. CONCLUSIONS: Collectively, these results suggest that anti-bovine CTLA-4 antibody can reactivate lymphocyte functions and could be applied for a new therapy against refractory chronic diseases. Further investigation is required for future clinical applications.
  • Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Naoya Maekawa, Shinya Goto, Kei Watari, Erina Minato, Atsushi Kobayashi, Junko Kohara, Shinji Yamada, Mika K Kaneko, Yukinari Kato, Hirofumi Takahashi, Nobuhiro Terasaki, Akira Takeda, Keiichi Yamamoto, Mikihiro Toda, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Journal of immunology (Baltimore, Md. : 1950) 203 5 1313 - 1324 2019年09月01日 [査読有り][通常論文]
     
    Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.
  • 森田 鮎, 竹原 昌生, 村田 史郎, 伊勢崎 政美, 藤澤 宗太郎, 種子野 章, 酒井 英史, 宇野 有紀子, 小川 遼, 市居 修, 前川 直也, 岡川 朋弘, 今内 覚, 大橋 和彦
    日本獣医学会学術集会講演要旨集 162回 412 - 412 (公社)日本獣医学会 2019年08月 [査読有り][通常論文]
  • Vector transmission of bovine leukemia virus during summer season in Northern Hokkaido
    Inagaki H, Konnai S, Kaburagi H, Murota H, Takabatake N, Watari K, Okagawa T, Maekawa N, Murata S, Ohashi K
    Jpn. J. Vet. Res 67 3 235 - 239 2019年08月 [査読有り][通常論文]
  • Fujisawa S, Murata S, Takehara M, Katakura K, Hmoon MM, Win SY, Ohashi K
    BMC veterinary research 15 1 261  2019年07月 [査読有り][通常論文]
  • Takehara M, Murata S, Katakura K, Fujisawa S, Hmoon MM, Win SY, Bawm S, Htun LL, Aung YH, Win MM, Isezaki M, Maekawa N, Okagawa T, Konnai S, Ohashi K
    Heliyon 5 4 e01544  2019年04月 [査読有り][通常論文]
  • Fujisawa S, Konnai S, Okagawa T, Maekawa N, Tanaka A, Suzuki Y, Murata S, Ohashi K
    BMC veterinary research 15 1 68  2019年02月 [査読有り][通常論文]
  • Identification of immuno-inhibitory molecules in Mongolian native cattle and yak
    Ochirkhuu N, Konnai S, Odbileg R, Okagawa T, Maekawa N, Murata S, Ohashi K
    Jpn J Vet Res 66 3 177 - 192 2018年08月 [査読有り][通常論文]
  • Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Junko Kohara, Yasuhiko Suzuki, Shinji Yamada, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
    Veterinary research 49 1 50 - 50 2018年06月19日 [査読有り][通常論文]
     
    Bovine leukemia virus (BLV) is a retrovirus that infects B cells in cattle and causes bovine leukosis after a long latent period. Progressive exhaustion of T cell functions is considered to facilitate disease progression of BLV infection. Programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) are immunoinhibitory receptors that contribute to T-cell exhaustion caused by BLV infection in cattle. However, it is unclear whether the cooperation of PD-1 and LAG-3 accelerates disease progression of BLV infection. In this study, multi-color flow cytometric analyses of PD-1- and LAG-3-expressing T cells were performed in BLV-infected cattle at different stages of the disease. The frequencies of PD-1+LAG-3+ heavily exhausted T cells among CD4+ and CD8+ T cells was higher in the blood of cattle with B-cell lymphoma over that of BLV-uninfected and BLV-infected cattle without lymphoma. In addition, blockade assays of peripheral blood mononuclear cells were performed to examine whether inhibition of the interactions between PD-1 and LAG-3 and their ligands by blocking antibodies could restore T-cell function during BLV infection. Single or dual blockade of the PD-1 and LAG-3 pathways reactivated the production of Th1 cytokines, interferon-γ and tumor necrosis factor-α, from BLV-specific T cells of the infected cattle. Taken together, these results indicate that PD-1 and LAG-3 cooperatively mediate the functional exhaustion of CD4+ and CD8+ T cells and are associated with the development of B-cell lymphoma in BLV-infected cattle.
  • Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Reiko Nagata, Satoko Kawaji, Yumiko Kagawa, Shinji Yamada, Yukinari Kato, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Yasuyuki Mori, Kazuhiko Ohashi
    Infection and immunity 86 5 2018年05月 [査読有り][通常論文]
     
    Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, is a bovine chronic infection that is endemic in Japan and many other countries. The expression of immunoinhibitory molecules is upregulated in cattle with Johne's disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E2 (PGE2) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE2 were performed to investigate the immunosuppressive effect of PGE2 during Johne's disease. In vitro PGE2 treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 and programmed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) from healthy cattle. PGE2 was upregulated in sera and intestinal lesions of cattle with Johne's disease. In vitro stimulation with Johnin purified protein derivative (J-PPD) induced cyclooxygenase-2 (COX-2) transcription, PGE2 production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with M. avium subsp. paratuberculosis Therefore, Johnin-specific Th1 responses could be limited by the PGE2 pathway in cattle. In contrast, downregulation of PGE2 with a COX-2 inhibitor promoted J-PPD-stimulated CD8+ T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade induced J-PPD-stimulated CD8+ T-cell proliferation and interferon gamma production in vitro Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies enhanced J-PPD-stimulated CD8+ T-cell proliferation in vitro, suggesting that the blockade of both pathways is a potential therapeutic strategy to control Johne's disease. The effects of COX-2 inhibition warrant further study as a novel treatment of Johne's disease.
  • 柴田明弘, 村田史郎, 中尾哲也, 井関博
    日本獣医師会雑誌 71 3 135‐139  2018年03月20日 [査読無し][通常論文]
  • Yamato Sajiki, Satoru Konnai, Asami Nishimori, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Masashi Nagano, Junko Kohara, Nana Kitano, Toshihiko Takahashi, Motoshi Tajima, Hirohisa Mekata, Yoichiro Horii, Shiro Murata, Kazuhiko Ohashi
    The Journal of veterinary medical science 79 12 2036 - 2039 2017年12月22日 [査読有り][通常論文]
     
    Enzootic bovine leukemia is caused by the bovine leukemia virus (BLV). BLV is transmitted vertically or horizontally through the transfer of infected cells via direct contact, through milk, insect bites and contaminated iatrogenic procedures. However, we lacked direct evidence of intrauterine infection. The purpose of this study was to confirm intrauterine BLV infection in two pregnant dams with high viral load by cesarean delivery. BLV was detected in cord and placental blood, and the BLV in the newborns showed 100% nucleotide identity with the BLV-env sequence from the dams. Notably, a newborn was seropositive for BLV but had no colostral antibodies. In this study, we presented a direct evidence of intrauterine BLV transmission in pregnant dam with a high proviral load. These results could aid the development of BLV control measures targeting viral load.
  • Tumenjargal Sharav, Satoru Konnai, Nyamsuren Ochirkhuu, Erdene T. S. Ochir, Hirohisa Mekata, Yoshihiro Sakoda, Takashi Umemura, Shiro Murata, Tungalag Chultemdorj, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 79 11 1884 - 1888 2017年11月 [査読有り][通常論文]
     
    The genetic characterization and actual prevalence of EIAV in Mongolian horse in the disease endemic region is currently unknown. Here, 11 of 776 horse serum samples from four Mongolian provinces tested positive on agar gel immunodiffusion test. Genomic DNA extracted from all seropositive samples was subjected to nested PCR assay. Among these, three samples tested positive with nested PCR assay and were identified by sequencing analysis based on long termination repeat and tat gene of the virus. Two of the three sequences were identical, with 94.0% identity with the third. These two independent Mongolian EIAV sequences were retained functional motifs, with no dramatic changes but some variability in the U5 region; they were clustered with genotypes from European countries but not with those from China, U.S.A., or Japan.
  • Goto S, Konnai S, Okagawa T, Nishimori A, Maekawa N, Gondaira S, Higuchi H, Koiwa M, Tajima M, Kohara J, Ogasawara S, Kato Y, Suzuki Y, Murata S, Ohashi K
    Immunity, inflammation and disease 5 3 355 - 363 2017年09月 [査読有り][通常論文]
     
    INTRODUCTION: Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway in immunosuppression in bovine mycoplasmosis. METHODS: In the initial experiments, we used enzyme-linked immunosorbent assay to measure interferon-γ (IFN-γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis. RESULTS: Expectedly, IFN-γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD-1+ CD4+ and PD-L1+ CD14+ cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD-1+ CD4+ and PD-1+ CD8+ T cells were negatively correlated with IFN-γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD-1/PD-L1 pathway in vitro by anti-bovine PD-1- and anti-bovine PD-L1 antibodies significantly upregulated the production of IFN-γ from anti-mycoplasma-specific cells. CONCLUSIONS: These results suggest that the PD-1/PD-L1 pathway could be involved in immune exhaustion of bovine mycoplasma-specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways.
  • Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Ayako Nakahara, Yuzumi Chiba, Masaho Ikeda, Shiro Murata, Kazuhiko Ohashi
    CLINICAL AND VACCINE IMMUNOLOGY 24 9 2017年09月 [査読有り][通常論文]
     
    Bovine leukemia is classified into two types: enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). EBL is caused by infection with bovine leukemia virus (BLV), which induces persistent lymphocytosis and B-cell lymphoma in cattle after a long latent period. Although it has been demonstrated that BLVassociated lymphoma occurs predominantly in adult cattle of > 3 to 5 years, suspicious cases of EBL onset in juvenile cattle were recently reported in Japan. To investigate the current status of bovine leukemia in Japan, we performed immunophenotypic analysis of samples from 50 cattle that were clinically diagnosed as having bovine leukemia. We classified the samples into five groups on the basis of the analysis and found two different types of EBL: classic EBL (cEBL), which has the familiar phenotype commonly known as EBL, and polyclonal EBL (pEBL), which exhibited neoplastic proliferation of polyclonal B cells. Moreover, there were several atypical EBL cases even in cEBL, including an early onset of EBL in juvenile cattle. A comparison of the cell marker expressions among cEBL, pEBL, and B-cell-type SBL (B-SBL) revealed characteristic patterns in B-cell leukemia, and these patterns could be clearly differentiated from those of healthy phenotypes, whereas it was difficult to discriminate between cEBL, pEBL, and B-SBL only by the expression patterns of cell markers. This study identified novel characteristics of bovine leukemia that should contribute to a better understanding of the mechanism underlying tumor development in BLV infection.
  • Maekawa N, Konnai S, Balbin MM, Mingala CN, Gicana KRB, Bernando FAEM, Murata S, Ohashi K
    Ticks and tick-borne diseases 2017年09月 [査読有り][通常論文]
  • Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Shiro Murata, Kazuhiko Ohashi
    VECTOR-BORNE AND ZOONOTIC DISEASES 17 8 539 - 549 2017年08月 [査読有り][通常論文]
     
    Anaplasma species are obligate intracellular rickettsial pathogens that cause great economic loss to the animal industry. Few studies on Anaplasma infections in Mongolian livestock have been conducted. This study examined the prevalence of Anaplasma marginale, Anaplasma ovis, Anaplasma phagocytophilum, and Anaplasma bovis by polymerase chain reaction assay in 928 blood samples collected from native cattle and dairy cattle (Bos taurus), yaks (Bos grunniens), sheep (Ovis aries), and goats (Capra aegagrus hircus) in four provinces of Ulaanbaatar city in Mongolia. We genetically characterized positive samples through sequencing analysis based on the heat-shock protein groEL, major surface protein 4 (msp4), and 16S rRNA genes. Only A. ovis was detected in Mongolian livestock (cattle, yaks, sheep, and goats), with 413 animals (44.5%) positive for groEL and 308 animals (33.2%) positive for msp4 genes. In the phylogenetic tree, we separated A. ovis sequences into two distinct clusters based on the groEL gene. One cluster comprised sequences derived mainly from sheep and goats, which was similar to that in A. ovis isolates from other countries. The other divergent cluster comprised sequences derived from cattle and yaks and appeared to be newly branched from that in previously published single isolates in Mongolian cattle. In addition, the msp4 gene of A. ovis using same and different samples with groEL gene of the pathogen demonstrated that all sequences derived from all animal species, except for three sequences derived from cattle and yak, were clustered together, and were identical or similar to those in isolates from other countries. We used 16S rRNA gene sequences to investigate the genetically divergent A. ovis and identified high homology of 99.3-100%. However, the sequences derived from cattle did not match those derived from sheep and goats. The results of this study on the prevalence and molecular characterization of A. ovis in Mongolian livestock can facilitate the control of infectious diseases in livestock.
  • Naoya Maekawa, Satoru Konnai, Satoshi Takagi, Yumiko Kagawa, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Tatsuya Deguchi, Chie Nakajima, Yukinari Kato, Keiichi Yamamoto, Hidetoshi Uemura, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    SCIENTIFIC REPORTS 7 1 8951  2017年08月 [査読有り][通常論文]
     
    Immunotherapy targeting immune checkpoint molecules, programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), using therapeutic antibodies has been widely used for some human malignancies in the last 5 years. A costimulatory receptor, PD-1, is expressed on T cells and suppresses effector functions when it binds to its ligand, PD-L1. Aberrant PD-L1 expression is reported in various human cancers and is considered an immune escape mechanism. Antibodies blocking the PD-1/PD-L1 axis induce antitumour responses in patients with malignant melanoma and other cancers. In dogs, no such clinical studies have been performed to date because of the lack of therapeutic antibodies that can be used in dogs. In this study, the immunomodulatory effects of c4G12, a canine-chimerised anti-PD-L1 monoclonal antibody, were evaluated in vitro, demonstrating significantly enhanced cytokine production and proliferation of dog peripheral blood mononuclear cells. A pilot clinical study was performed on seven dogs with oral malignant melanoma (OMM) and two with undifferentiated sarcoma. Objective antitumour responses were observed in one dog with OMM (14.3%, 1/7) and one with undifferentiated sarcoma (50.0%, 1/2) when c4G12 was given at 2 or 5 mg/kg, every 2 weeks. c4G12 could be a safe and effective treatment option for canine cancers.
  • Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Chie Nakajima, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    FRONTIERS IN IMMUNOLOGY 8 650  2017年06月 [査読有り][通常論文]
     
    Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1)/PD-ligand 1 (PD-L1), is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat-bovine chimeric monoclonal antibody 5D2 (Boch5D2) was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV). Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4(+) T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.
  • Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Yamato Sajiki, Yasuhiko Suzuki, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
    PLOS ONE 12 4 e0174916  2017年04月 [査読有り][通常論文]
     
    Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM(+) B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4(+) T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application.
  • Increase of cells expressing PD-1 and PD-L1 and enhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine mycoplasmosis.
    Goto, S, Konnai, S, Okagawa, T, Nishimori, A, Maekawa, N, Gondaira, S, Higuchi, H, Koiwa, M, Tajima, M, Kohara, J, Ogasawara, S, Kato, Y, Suzuki, Y, Murata, S, Ohashi, K
    Immunity, Inflammation and Disease 10 1 - 9 2017年 [査読有り][通常論文]
  • Yuka Machida, Shiro Murata, Ayumi Matsuyama-Kato, Masayoshi Isezaki, Akira Taneno, Eishi Sakai, Satoru Konnai, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 79 1 115 - 122 2017年01月 [査読有り][通常論文]
     
    Gallid herpesvirus 2 (GaHV-2) causes malignant lymphomas in chickens (Marek's disease, MD). Although MD is controlled through vaccination efforts, field isolates of GaHV-2 have increased in virulence worldwide and even cause MD in vaccinated chickens. GaHV-2 strains are classified into four categories (mild, virulent, very virulent and very virulent +) based on the virulence exhibited in experimental infection in unvaccinated or MD-vaccinated susceptible chickens. Although MD cases are sporadically reported in Japan, the recent field strains of GaHV-2 in Japan have not been characterized. During isolation of recent field strains by using primary chicken kidney cell cultures, a method classically used for GaHV-2 isolation, vaccine strains were simultaneously isolated. Therefore, it is necessary to separate vaccine strains to characterize the virulence and pathogenicity of the GaHV-2 strains currently distributed in Japan. In this study, we prepared cell suspensions from the spleens of MD-symptomatic chickens, inoculated day old -chicks and isolated GaHV-2 strains by primary chicken kidney cell cultures at 2-3 weeks post inoculation. The isolated strains were passaged several times on chicken embryo fibroblast cells, and PCR analysis revealed that the isolated strains were not contaminated with vaccine strains. Moreover, the contaminant vaccine strains were completely removed by the purification of plaques observed in chicken kidney cells. These procedures are necessary to isolate GaHV-2 field strains from vaccine strains in order to carry out future studies to characterize these strains and glean insights into GaHV-2 virulence and pathogenicity.
  • Satoru Konnai, Shiro Murata, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 79 1 1 - 5 2017年01月 [査読有り][通常論文]
     
    Recently, dysfunction of antigen-specific T cells is well documented as T-cell exhaustion and has been defined by the loss of effector functions during chronic infections and cancer in human. The exhausted T cells are characterized phenotypically by the surface expression of immunoinhibitory receptors, such as programmed death 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) and cytotoxic T-lymphocyte antigen 4 (CTLA-4). However, there is still a fundamental lack of knowledge about the immunoinhibitory receptors in the fields of veterinary medicine. In particular, very little is known about mechanism of T cell dysfunction in chronic infection in cattle. Recent our studies have revealed that immunoinhibitory molecules including PD-1/programmed death-ligand 1 (PD-L1) play critical roles in immune exhaustion and disease progression in case of bovine leukemia virus (BLV) infection, Johne's disease and bovine anaplasmosis. This review includes some recent data from us.
  • Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Shiro Murata, Kazuhiko Ohashi
    Journal of Veterinary Medical Science 79 12 2040 - 2042 2017年 [査読有り][通常論文]
     
    Sheep-associated malignant catarrhal fever (SA-MCF), caused by ovine gammaherpesvirus-2 (OvHV-2), is a fatal disease in all ruminants. The epidemiological survey and molecular characterization of OvHV-2 in Mongolian livestock were performed. Of 928 blood samples, 14 were positive for OvHV-2 in sheep and native cattle from Tsenkher County and in sheep from Lun County. Phylogenetic analyses revealed that the tegument gene of OvHV-2 sequences from Mongolian animals is identical to that in animals from Egypt, India, and Turkey, and is 98.0% similar to that in animals from Germany and Brazil. To our knowledge, this is the first confirmed report of OvHV-2 in Mongolian livestock, and could provide useful information for controlling SA-MCF.
  • Tomohiro Okagawa, Satoru Konnai, James R. Deringer, Massaro W. Ueti, Glen A. Scoles, Shiro Murata, Kazuhiko Ohashi, Wendy C. Brown
    INFECTION AND IMMUNITY 84 10 2779 - 2790 2016年10月 [査読有り][通常論文]
     
    The CD4(+) T-cell response is central for the control of Anaplasma marginale infection in cattle. However, the infection induces a functional exhaustion of antigen-specific CD4(+) T cells in cattle immunized with A. marginale outer membrane proteins or purified outer membranes (OMs), which presumably facilitates the persistence of this rickettsia. In the present study, we hypothesize that T-cell exhaustion following infection is induced by the upregulation of immunoinhibitory receptors on T cells, such as programmed death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3). OM-specific T-cell responses and the kinetics of PD-1-positive (PD-1(+)) LAG-3(+) exhausted T cells were monitored in A. marginale-challenged cattle previously immunized with OMs. Consistent with data from previous studies, OM-specific proliferation of peripheral blood mononuclear cells (PBMCs) and interferon gamma (IFN-gamma) production were significantly suppressed in challenged animals by 5 weeks postinfection (wpi). In addition, bacteremia and anemia also peaked in these animals at 5 wpi. Flow cytometric analysis revealed that the percentage of PD-1(+) LAG-3(+) T cells in the CD4(+), CD8(+), and delta gamma T-cell populations gradually increased and also peaked at 5 wpi. A large increase in the percentage of LAG-3(+) gamma delta T cells was also observed. Importantly, in vitro, the combined blockade of the PD-1 and LAG-3 pathways partially restored OM-specific PBMC proliferation and IFN-gamma production at 5 wpi. Taken together, these results indicate that coexpression of PD-1 and LAG-3 on T cells contributes to the rapid exhaustion of A. marginale-specific T cells following infection and that these immunoinhibitory receptors regulate T-cell responses during bovine anaplasmosis.
  • Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Battogtokh Odzaya, Shura Gansukh, Shiro Murata, Kazuhiko Ohashi
    ARCHIVES OF VIROLOGY 161 8 2279 - 2283 2016年08月 [査読有り][通常論文]
     
    Bovine viral diarrhea virus (BVDV) is classified into two species, namely, Bovine viral diarrhea virus 1 and Bovine viral diarrhea virus 2, and affects cattle worldwide, resulting in significant economic loss. The prevalence of BVDV-1 and BVDV-2 infections and its genotypes in Mongolian animals has not been studied. In this study, we surveyed BVDV infection in dairy cattle and yaks from Bornuur and Bulgan counties by RT-PCR, and the average infection rate in the sampling sites was 15.8 % and 20.0 %, respectively. In addition, molecular features of the 5'-UTR region of the BVDV genome in Mongolian cattle and yaks were identified as belonging to the subtypes BVDV-1a and BVDV-2a, respectively. Determining the prevalence, geographical distribution, and molecular diversity of BVDV-1 and BVDV-2 in various host species in Mongolia is important for further studies and process control programs.
  • Naoya Maekawa, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Satoshi Takagi, Yumiko Kagawa, Chie Nakajima, Yasuhiko Suzuki, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
    PLOS ONE 11 6 e0157176  2016年06月 [査読有り][通常論文]
     
    Spontaneous cancers are common diseases in dogs. Among these, some malignant cancers such as oral melanoma, osteosarcoma, hemangiosarcoma, and mast cell tumor are often recognized as clinical problems because, despite their high frequencies, current treatments for these cancers may not always achieve satisfying outcomes. The absence of effective systemic therapies against these cancers leads researchers to investigate novel therapeutic modalities, including immunotherapy. Programmed death 1 (PD-1) is a costimulatory receptor with immunosuppressive function. When it binds its ligands, PD-ligand 1 (PD-L1) or PD-L2, PD-1 on T cells negatively regulates activating signals from the T cell receptor, resulting in the inhibition of the effector function of cytotoxic T lymphocytes. Aberrant PD-L1 expression has been reported in many human cancers and is considered an immune escape mechanism for cancers. In clinical trials, anti-PD-1 or anti-PD-L1 antibodies induced tumor regression for several malignancies, including advanced melanoma, non-small cell lung carcinoma, and renal cell carcinoma. In this study, to assess the potential of the PD-1/PD-L1 axis as a novel therapeutic target for canine cancer immunotherapy, immunohistochemical analysis of PD-L1 expression in various malignant cancers of dogs was performed. Here, we show that dog oral melanoma, osteosarcoma, hemangiosarcoma, mast cell tumor, mammary adenocarcinoma, and prostate adenocarcinoma expressed PD-L1, whereas some other types of cancer did not. In addition, PD-1 was highly expressed on tumor-infiltrating lymphocytes obtained from oral melanoma, showing that lymphocytes in this cancer type might have been functionally exhausted. These results strongly encourage the clinical application of PD-1/PD-L1 inhibitors as novel therapeutic agents against these cancers in dogs.
  • Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Ayako Nakahara, Shiro Murata, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 5 791 - 796 2016年05月 [査読有り][通常論文]
     
    Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective therapy or vaccine; thus, the diagnosis and elimination of BLV-infected cattle are the most effective method to eradicate the infection. Clinical veterinarians need a simpler and more rapid method of diagnosing infection, because both nested polymerase chain reaction (PCR) and real-time PCR are labor intensive, time-consuming, and require specialized molecular biology techniques and expensive equipment. In this study, we describe a novel PCR method for amplifying the BLV provirus from whole blood, thus eliminating the need for DNA extraction. Although the sensitivity of PCR directly from whole blood (PCR-DB) samples as measured in bovine blood containing BLV-infected cell lines was lower than that of nested PCR, the PCR-DB technique showed high specificity and reproducibility. Among 225 clinical samples, 49 samples were positive by nested PCR, and 37 samples were positive by PCR-DB. There were no false positive samples; thus, PCR-DB sensitivity and specificity were 75.51% and 100%, respectively. However, the provirus loads of the samples detected by nested PCR and not PCR-DB were quite low. Moreover, PCR-DB also stably amplified the BLV provirus from tumor tissue samples. PCR-DB method exhibited good reproducibility and excellent specificity and is suitable for screening of thousands of cattle, thus serving as a viable alternative to nested PCR and real-time PCR.
  • Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Asami Nishimori, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi
    ARCHIVES OF VIROLOGY 161 4 985 - 991 2016年04月 [査読有り][通常論文]
     
    Epidemiological studies have indicated that bovine leukemia virus (BLV) infection is globally distributed. However, no information regarding the disease and genetic diversity of the virus in the cattle of Mongolia is currently available. In this study, the prevalence of BLV was assessed using PCR, and the genetic diversity was analyzed through DNA sequencing. Of the 517 samples tested, 20 positives were identified. Phylogenetic analysis showed that six, one, and four isolates were classified into genotype 4, 7, and 1, respectively. Most isolates were clustered with isolates from Eastern Europe and Russia. This study is the first to investigate the BLV genotype in Mongolia.
  • Ohira K, Nakahara A, Konnai S, Okagawa T, Nishimori A, Maekawa N, Ikebuchi R, Kohara J, Murata S, Ohashi K
    Immunity, inflammation and disease 4 1 52 - 63 2016年03月 [査読有り][通常論文]
  • Kochi Toyomane, Satoru Konnai, Ayano Niwa, Naftaly Githaka, Masayoshi Isezaki, Shinji Yamada, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Shiro Murata, Kazuhiko Ohashi
    TICKS AND TICK-BORNE DISEASES 7 1 119 - 125 2016年 [査読有り][通常論文]
     
    Ixodes ricinus immunosuppressor (Iris) is a tick salivary gland protein derived from I. ricinus. In this study, Iris homolog was identified in the salivary glands of Ixodes persulcatus, which is the specific vector of the Lyme disease agent in Japan. The homolog was named Ipis-1. To investigate the function of Ipis-1, we prepared a recombinant Ipis-1 expressed in COS-7 cells as a rabbit IgG Fc-fused protein (Ipis-1-Ig). Cell proliferation assay and IFN-gamma ELISA showed that Ipis-1-Ig inhibits the proliferation and IFN-gamma production of bovine peripheral blood mononuclear cells (PBMCs). Notably, Ipis-1-Ig inhibited the cell proliferation and production of IFN-gamma in bovine PBMCs even when CD14(+) cells were depleted, suggesting that Ipis could directly interact with T cells and inhibit their functions. In conclusion, Ipis could contribute to the establishment of environments suitable for tick blood feeding and pathogen transmission by suppressing the function of immune cells. (C) 2015 Elsevier GmbH. All rights reserved.
  • Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Ryoyo Ikebuchi, Seiko Mizorogi, Reiko Nagata, Satoko Kawaji, Shogo Tanaka, Yumiko Kagawa, Shiro Murata, Yasuyuki Mori, Kazuhiko Ohashi
    INFECTION AND IMMUNITY 84 1 77 - 89 2016年01月 [査読有り][通常論文]
     
    Johne's disease (paratuberculosis) is a chronic enteritis in cattle that is caused by intracellular infection with Mycobacterium avium subsp. paratuberculosis. This infection is characterized by the functional exhaustion of T-cell responses to M. avium subsp. paratuberculosis antigens during late subclinical and clinical stages, presumably facilitating the persistence of this bacterium and the formation of clinical lesions. However, the mechanisms underlying T-cell exhaustion in Johne's disease are poorly understood. Thus, we performed expression and functional analyses of the immunoinhibitory molecules programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) and lymphocyte activation gene 3 (LAG-3)/major histocompatibility complex class II (MHC-II) in M. avium subsp. paratuberculosis-infected cattle during the late subclinical stage. Flow cytometric analyses revealed the upregulation of PD-1 and LAG-3 in T cells in infected animals, which suffered progressive suppression of interferon gamma (IFN-gamma) responses to the M. avium subsp. paratuberculosis antigen. In addition, PD-L1 and MHC-II were expressed on macrophages from infected animals, consistent with PD-1 and LAG-3 pathways contributing to the suppression of IFN-gamma responses during the subclinical stages of M. avium subsp. paratuberculosis infection. Furthermore, dual blockade of PD-L1 and LAG-3 enhanced M. avium subsp. paratuberculosis-specific IFN-gamma responses in blood from infected animals, and in vitro LAG-3 blockade enhanced IFN-gamma production from M. avium subsp. paratuberculosis-specific CD4(+) and CD8(+) T cells. Taken together, the present data indicate that M. avium subsp. paratuberculosis-specific T-cell exhaustion is in part mediated by PD-1/PD-L1 and LAG-3/MHC-II interactions and that LAG-3 is a molecular target for the control of M. avium subsp. paratuberculosis-specific T-cell responses.
  • Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Shiro Murata, Kazuhiko Ohashi
    JAPANESE JOURNAL OF VETERINARY RESEARCH 63 4 191 - 194 2015年11月 [査読有り][通常論文]
     
    Johne's disease is a chronic infection with Mycobacterium avium susp. paratuberculosis (MAP), which causes huge economic losses to cattle industry. The seroprevalence of MAP in cattle of Mongolian was estimated by an ELISA assay using 356 serum samples which were collected from eleven provinces and Ulaanbaatar city. Out of these samples, 3 (0.84%) were found to be seropositive for MAP, originating from Tsenkher sum of Arkhangai province, Murun sum of khuvsgul province, and Bornuur sum of Tuv province in Mongolia. This study represents first conformation of Johne's disease in Mongolian cattle. These findings provide vital information that can be used for the planning and execution of control measures for Johne's disease in the Mongolian cattle industry.
  • Nyamsuren Ochirkhuu, Satoru Konnai, Claro N. Mingala, Tomohiro Okagawa, Marvin Villanueva, Flor Marie Immanuelle R. Pilapil, Shiro Murata, Kazuhiko Ohashi
    VETERINARY PARASITOLOGY 212 3-4 161 - 167 2015年09月 [査読有り][通常論文]
     
    In the Philippines, vector-borne disease is one of the important problems in the livestock industry. To elucidate the epidemiology of vector-borne diseases in cattle on Luzon Island, the Philippines, the prevalence of five protozoan agents was assessed by polymerase chain reaction. Out of the 339 samples, 324 (95.5%), 154 (45.4%), 209 (61.6%), 140 (41.3%), and 2 (0.6%) were positive for Anaplasma marginale, Babesia bigemina, Babesia bovis, Theileria spp., and Trypanosoma evansi infections, respectively. Mixed infections were detected in 290 (85.5%) samples, of which 115 (33.9%) had two pathogens, 144 (42.5%) had three pathogens, and 31 (9.1%) had four kinds of pathogens. 16S rRNA gene was 100% identical in A. marginale compared with the same lineage across the world. B. bovis RAP-1 and B. bigemina AMA-1 genes were identical with 92.27%-100% and 97.07%-100% sequences, respectively, in the database (Asian isolates). MPSP genes of Theileria spp. were 83.51%-100% identical with the one another. Phylogenetic analysis showed that they belong to the groups of T. sergenti and T. buffeli. Positive rates of the tick-borne pathogens were extremely high in this area. These findings provide vital information that can be used for the planning and execution of effective control measures for vector-borne diseases in the Philippine cattle industry. (C) 2015 Elsevier B.V. All rights reserved.
  • Hirohisa Mekata, Shiro Murata, Claro Niegos Mingala, Kazuhiko Ohashi, Satoru Konnai
    JOURNAL OF VETERINARY MEDICAL SCIENCE 77 8 1017 - 1019 2015年08月 [査読有り][通常論文]
     
    Trypanosoma evansi causes wasting disease in many livestock. T. evansi infection gives rise to inflammatory immune responses, which contribute to the development of inflammation-associated tissue injury. We previously reported that regulatory dendritic cells (DCs), which act as potential regulators of inflammation, were activated in infected mice and transfer of regulatory DCs to infected mice prolonged their survival. However, the kinetics of regulatory DCs in cattle, which are natural hosts of T. evansi, remained unclear. In this study, we report that the expressions of CCL8 and IL-10, which promote the development of regulatory DCs, were up-regulated in cattle experimentally infected with T. evansi. This finding is potentially useful for studying the control strategy of T. evansi infection in cattle.
  • Saori Suzuki, Satoru Konnai, Tomohiro Okagawa, Ryoyo Ikebuchi, Asami Nishimori, Junko Kohara, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 163 3-4 115 - 124 2015年02月 [査読有り][通常論文]
     
    Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4(+)CD25(+)Foxp3(+) T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3(+)CD4(+) cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-gamma expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CfLA-4(+) cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4+ T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4(+) and CD25(+) T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4(+) cells in the CD4(+) T cell subpopulation was positively correlated with TGF-beta mRNA expression, suggesting that CD4(+)CTLA-4(+) T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-gamma mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection. (C) 2014 Elsevier B.V. All rights reserved.
  • Murase Y, Konnai S, Yamada S, Githaka N, Isezaki M, Ito T, Takano A, Ando S, Kawabata H, Murata S, Ohashi K
    Insect Biochemistry and Molecular Biology 60 59 - 67 2015年 [査読有り][通常論文]
  • Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    IMMUNOLOGY 142 4 551 - 561 2014年08月 [査読有り][通常論文]
     
    Programmed death-ligand 1 (PD-L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death-1 (PD-1). However, the mechanism of PD-L1-mediated inhibitory signalling after PD-L1 cross-linking by anti-PD-L1 monoclonal antibody (mAb) or PD-1-immunogloblin fusion protein (PD-1-Ig) is still unknown, although it may induce cell death of PD-L1(+) cells required for regular immune reactions. In this study, PD-1-Ig or anti-PD-L1 mAb treatment was tested in cell lines that expressed PD-L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD-L1-mediated cell death. PD-L1 cross-linking by PD-1-Ig or anti-PD-L1 mAb primarily increased the number of dead cells in PD-L1(high) cells, but not in PD-L1(low) cells; these cells were prepared from Cos-7 cells in which bovine PD-L1 expression was induced by transfection. The PD-L1-mediated cell death also occurred in Cos-7 and HeLa cells transfected with vectors only encoding the extracellular region of PD-L1. In bovine lymphocytes, the anti-PD-L1 mAb treatment up-regulated interferon-gamma (IFN-gamma) production, whereas PD-1-Ig treatment decreased this cytokine production and cell proliferation. The IFN-gamma production in B-cell-depleted peripheral blood mononuclear cells was not reduced by PD-1-Ig treatment and the percentages of dead cells in PD-L1(+) B cells were increased by PD-1-Ig treatment, indicating that PD-1-Ig-induced immunosuppression in bovine lymphocytes could be caused by PD-L1-mediated B-cell death. This study provides novel information for the understanding of signalling through PD-L1.
  • Ayumi Matsuyama-Kato, Shiro Murata, Masayoshi Isezaki, Sarah Takasaki, Rika Kano, Satoru Konnai, Kazuhiko Ohashi
    ARCHIVES OF VIROLOGY 159 8 2123 - 2126 2014年08月 [査読有り][通常論文]
     
    PD-L2 is a ligand of the immunoinhibitory receptor PD-1. Here, we report functional and expression analyses of PD-L2 in tumor lesions and spleens from chickens infected with gallid herpesvirus 2 (GaHV-2, Marek's disease virus), which induces malignant lymphomas in chickens. We show that the expression of IFN-gamma protein was decreased in PBMCs and splenocytes co-cultured with PD-L2-expressing cells and that the expression of PD-L2 mRNA was significantly higher in the spleens of infected chickens in the latent phase and in tumor lesions caused by GaHV-2. These results suggest that chicken PD-L2 has an immunoinhibitory function and is involved in the establishment of latency and tumor formation by GaHV-2.
  • Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Ayako Nakahara, Shiro Murata, Kazuhiko Ohashi
    JOURNAL OF GENERAL VIROLOGY 95 Pt 8 1832 - 1842 2014年08月 [査読有り][通常論文]
     
    Bovine leukemia virus (BLV) induces abnormal B-cell proliferation and B-cell lymphoma in cattle, where the BLV provirus is integrated into the host genome. BLV-infected B-cells rarely express viral proteins in vivo, but short-term cultivation augments BLV expression in some, but not all, BLV-infected B-cells. This observation suggests that two subsets, i.e. BLV-silencing cells and BLV-expressing cells, are present among BLV-infected B-cells, although the mechanisms of viral expression have not been determined. In this study, we examined B-cell markers and viral antigen expression in B-cells from BLV-infected cattle to identify markers that may discriminate BLV, expressing cells from BLV-silencing cells. The proportions of IgM(high) B-cells were increased in blood lymphocytes from BLV-infected cattle. IgM(high) B-cells mainly expressed BLV antigens, whereas IgM(low) B-cells did not, although the provirus load was equivalent in both subsets. Several parameters were investigated in these two subsets to characterize their cellular behaviour. Real-time PCR and microarray analyses detected higher expression levels of some proto-oncogenes (e.g. Maf, Jun and Fos) in IgM(low) B-cells than those in IgM(high) B-cells. Moreover, lymphoma cells obtained from the lymph nodes of 14 BLV-infected cattle contained IgM(low) or IgM(-) B-cells but no IgM(high) B-cells. To our knowledge, this is the first study to demonstrate that IgM(high) B-cells mainly comprise BLV-expressing cells, whereas IgM(low) B-cells comprise a high proportion of BLV-silencing B-cells in BLV-infected cattle.
  • Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Chie Nakajima, Yasuhiko Suzuki, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi
    MICROBIOLOGY AND IMMUNOLOGY 58 7 388 - 397 2014年07月 [査読有り][通常論文]
     
    Previous reports from this group have indicated that the immunoinhibitory programmed death (PD)-1 receptor and its ligand, PD-L1, are involved in the mechanism of immune evasion of bovine chronic infection. However, no functional analysis of bovine PD-L2 in cattle has been reported. Thus, in this study, the molecular function of bovine PD-L2 was analyzed in vitro. Recombinant PD-L2 (PD-L2-Ig), which comprises an extracellular domain of bovine PD-L2 fused to the Fc portion of rabbit IgG1, was prepared based on the cloned cDNA sequence for bovine PD-L2. Bovine PD-L2-Ig bound to bovine PD-1-expressing cells and addition of soluble bovine PD-1-Ig clearly inhibited the binding of PD-L2-Ig to membrane PD-1 in a dose-dependent manner. Cell proliferation and IFN-gamma production were significantly enhanced in the presence of PD-L2-Ig in peripheral blood mononuclear cells (PBMCs) from cattle. Moreover, PD-L2-Ig significantly enhanced IFN-gamma production from virus envelope peptides-stimulated PBMCs derived from bovine leukemia virus-infected cattle. Interestingly, PD-L2-Ig-induced IFN-gamma production was further enhanced by treatment with anti-bovine PD-1 antibody. These data suggest potential applications of bovine PD-L2-Ig as a therapy for bovine diseases.
  • Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K
    PloS one 9 6 e98415  2014年06月 [査読有り][通常論文]
     
    Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together induce the "exhausted'' status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells. In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1-expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-gamma production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity in vivo, and its therapeutic potential has to be further discussed.
  • Naftaly Githaka, Satoru Konnai, Richard Bishop, David Odongo, Isaac Lekolool, Edward Kariuki, Francis Gakuya, Lucy Kamau, Masayoshi Isezaki, Shiro Murata, Kazuhiko Ohashi
    VETERINARY PARASITOLOGY 202 3-4 180 - 193 2014年05月 [査読有り][通常論文]
     
    Waterbuck (Kobus defassa), an ungulate species endemic to the Eastern African savannah, is suspected of being a wildlife reservoir for tick-transmitted parasites infective to livestock. Waterbuck is infested by large numbers of Rhipicephalus appendiculatus, the tick vector for Theileria parva, and previous data suggests that the species may be a source of T. parva transmission to cattle. In the present study, a total of 86 cattle and 26 waterbuck blood samples were obtained from Marula, a site in Kenya endemic for East Coast fever (ECF) where the primary wildlife reservoir of T. parva the Cape buffalo (Syncerus coffer) is also common. To investigate for the presence of cattle-infective Theileria parasites, DNA specimens extracted from the blood samples were subjected to two diagnostic assays; a nested PCR based on the p104 gene that is specific for T. parva, and a reverse line blot (RLB) incorporating 13 oligonucleotide probes including all of the Theileria spp. so far described from livestock and wildlife in Kenya. Neither assay provided evidence of T. parva or Theileria sp. (buffalo) infection in the waterbuck DNA samples. By contrast, majority of the cattle samples (67.4%) were positive for T. parva using a nested PCR assay. The RLB assay, including a generic probe for the genus Theileria, indicated that 25/26 (96%) of the waterbuck samples were positive for Theileria, while none of the 11 Theileria species-specific probes hybridized with the waterbuck-derived PCR products. Phylogenetic analysis of 18S ribosomal RNA (18S rRNA) and internal transcribed spacer (ITS) sequences within the RLB-positive waterbuck samples revealed the occurrence of three Theileria genotypes of unknown identity designated A, B and C. Group A clustered with Theileria equi, a pathogenic Theileria species and a causative agent of equine piroplasmosis in domestic equids. However, DNA from this group failed to hybridize with the T. equi oligonucleotide present on the RLB filter probe, suggesting the occurrence of novel taxa in these animals. This was confirmed by DNA sequencing that revealed heterogeneity between the waterbuck isolates and previously reported T. equi genotypes. Group B parasites clustered closely with Theileria luwenshuni, a highly pathogenic parasite of sheep and goats reported from China. Group C was closely related to Theileria ovis, an apparently benign parasite of sheep. Together, these findings provided no evidence that waterbuck plays a role in the transmission of T. parva. However, novel Theileria genotypes detected in this bovid species may be of veterinary importance. (C) 2014 Elsevier B.V. All rights reserved.
  • Hidano A, Konnai S, Yamada S, Githaka N, Isezaki M, Higuchi H, Nagahata H, Ito T, Takano A, Ando S, Kawabata H, Murata S, Ohashi K
    Insect Molecular Biology 23 4 466 - 474 2014年 [査読有り][通常論文]
  • Ooshiro M, Konnai S, Katagiri Y, Afuso M, Arakaki N, Tsuha O, Murata S, Ohashi K
    Vet Rec. 173 21 527  2013年11月 [査読有り][通常論文]
  • Naftaly Githaka, Satoru Konnai, Robert Skilton, Edward Kariuki, Esther Kanduma, Shiro Murata, Kazuhiko Ohashi
    Parasitology International 62 5 448 - 453 2013年10月 [査読有り][通常論文]
     
    Recently, mortalities among giraffes, attributed to infection with unique species of piroplasms were reported in South Africa. Although haemoparasites are known to occur in giraffes of Kenya, the prevalence, genetic diversity and pathogenicity of these parasites have not been investigated.In this study, blood samples from 13 giraffes in Kenya were investigated microscopically and genomic DNA extracted. PCR amplicons of the hyper-variable region 4 (V4) of Theileria spp. small subunit ribosomal RNA (18S rRNA) gene were hybridized to a panel of genus- and species-specific oligonucleotide probes by reverse line blot (RLB). Two newly designed oligonucleotide probes specific for previously identified Theileria spp. of giraffes found single infections in eight of the specimens and mixed infections in the remaining five samples.Partial 18S rRNA genes were successfully amplified from 9 samples and the PCR amplicons were cloned. A total of 28 plasmid clones representing the Kenyan isolates were analyzed in the present study and compared with those of closely-related organisms retrieved from GenBank. In agreement with RLB results, the nucleotide sequence alignment indicated the presence of mixed infections in the giraffes. In addition, sequence alignment with the obtained 18S rRNA gene sequences revealed extensive microheterogeneities within and between isolates, characterized by indels in the V4 regions and point mutations outside this region. Phylogeny with 18S rRNA gene sequences from the detected parasites and those of related organisms places Theileria of giraffes into two major groups, within which are numerous clades that include the isolates reported in South Africa. Collectively, these data suggest the existence of at least two distinct Theileria species among giraffes, and extensive genetic diversity within the two parasite groups. © 2013 Elsevier Ireland Ltd.
  • Saori Suzuki, Satoru Konnai, Tomohiro Okagawa, Ryoyo Ikebuchi, Tatsuya Shirai, Yuji Sunden, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi
    Microbiology and Immunology 57 8 600 - 604 2013年08月 [査読有り][通常論文]
     
    In the present study, we monitored Foxp3+ T cells in bovine leukemia virus (BLV)-infected cattle. By flow cytometric analysis, the proportion of Foxp3+CD4+ cells from persistent lymphocytotic cattle was significantly increased compared to control and AL cattle. Interestingly, the proportion of Foxp3+CD4+ cells correlated positively with the increased number of lymphocytes, virus titer and virus load, whereas it inversely correlated with IFN-γ mRNA expression, suggesting that Foxp3+CD4+ T cells in cattle have a potentially immunosuppressive function. Further studies are necessary to elucidate the detailed mechanism behind the increased Treg during BLV infection. © 2013 The Societies and Wiley Publishing Asia Pty Ltd.
  • 免疫抑制受容体PD-1のリガンドPD-L2の機能的特徴と臨床応用研究
    西森朝美, 今内 覚, 池渕良洋, 岡川朋弘, 村田史郎, 大橋和彦
    動物用ワクチンーバイオ医薬品研究会ニュースレター 7 19 - 20 2013年06月 [査読無し][招待有り]
  • Shiro Murata, Tomoyuki Hashiguchi, Yuko Hayashi, Yuki Yamamoto, Ayumi Matsuyama-Kato, Sarah Takasaki, Masayoshi Isezaki, Misao Onuma, Satoru Konnai, Kazuhiko Ohashi
    INFECTION GENETICS AND EVOLUTION 16 137 - 143 2013年06月 [査読有り][通常論文]
     
    Serotype 1 strains of Marek's disease virus (MDV-1) cause malignant lymphomas in chickens (Marek's disease; MD). Although MD has been controlled by vaccination, field isolates of MDV-1 have tended to increase in virulence and cause MD even in vaccinated chickens. Meq, a putative MDV-1 oncoprotein, resembles the Jun/Fos family of basic leucine zipper (bZIP) transcription factors and can regulate the expression of viral and cellular genes as a homodimer or as a heterodimer with a variety of bZIP family proteins. Sequencing analysis of some of the viral genes of various MDV-1 strains revealed a distinct diversity of and point mutations in Meq, which may contribute to changes in the transcriptional activities of Meq and, consequently, to increases in MDV-1 oncogenicity. However, few reports have characterized MDV-1 strains isolated in Japan. In this study, we established the amino acid sequences of MDV-1 field isolates from Japan in order to determine whether they display a distinct diversity of and point mutations in Meq. In addition, we analyzed the transactivation activities of the Meq proteins in order to evaluate whether the observed mutations affect their functions. Japanese MDV-1 isolates displayed the distinct mutations in basic region 2 (BR2) and proline-rich repeats (PRRs) of the Meq proteins as well as some unique mutations. Reporter assays revealed that the amino acid substitutions in BR2 and the PRRs affected the Meq transactivation activity. These results suggest that the distinct mutations are also present in the Meq proteins of MDV-1 isolates from Japan and affect their transactivation activities. (C) 2013 Elsevier B.V. All rights reserved.
  • Suzuki S, Konnai S, Okagawa T, Ikebuchi R, Shirai T, Sunden Y, Mingala CN, Murata S, Ohashi K
    Microbiology and immunology 2013年06月 [査読有り][通常論文]
  • Saiki Imamura, Mari Nakamizo, Michiko Kawanishi, Nao Nakajima, Kinya Yamamoto, Mariko Uchiyama, Fumiya Hirano, Hidetaka Nagai, Mayumi Kijima, Ryoyo Ikebuchi, Hirohisa Mekata, Shiro Murata, Satoru Konnai, Kazuhiko Ohashi
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 153 1-2 153 - 158 2013年05月 [査読有り][通常論文]
     
    In order to analyze bovine immune reactions against the Gram-negative bacterial vaccine, bovine whole-blood culture was used to investigate the pro-inflammatory cytokine responses stimulated with lipopolysaccharides (LPS) extracted from Escherichia colt, Salmonella enterica, Pseudomonas aeruginosa, and Klebsiella pneumoniae. We also examined the interaction between LPS and aluminum hydroxide gel for endotoxin activity and pro-inflammatory cytokine responses of whole bovine blood. Alteration in the mRNA concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and IL-10 in whole-blood culture at 4 h after stimulation with different doses of LPS was observed and determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The mRNA concentrations of TNF-alpha and IL-1 beta changed in a dose-dependent manner and differed depending on the type of LPS. Limulus test revealed that endotoxin activity was remarkably reduced when aluminum hydroxide gel was added to LPS. In contrast, the mRNA concentration of TNF-alpha in whole bovine blood was enhanced by LPS mixed with aluminum hydroxide gel. These results suggest that bovine whole-blood culture can be utilized to detect endotoxin activity of Gram-negative bacterial vaccines. In addition, whole-blood culture offers several advantages, such as ease of performance, few preparation artifacts, and a physiological cell environment, for investigating bovine immune response compared with the Limulus test. (C) 2013 Elsevier B.V. All rights reserved.
  • Hirohisa Mekata, Satoru Konnai, Claro N. Mingala, Nancy S. Abes, Charito A. Gutierrez, Alan P. Dargantes, William H. Witola, Noboru Inoue, Misao Onuma, Shiro Murata, Kazuhiko Ohashi
    PARASITOLOGY RESEARCH 112 4 1513 - 1521 2013年04月 [査読有り][通常論文]
     
    In recent years, the emergence of highly pathogenic Trypanosoma evansi strains in the Philippines has resulted in substantial losses in livestock production. In this study, we isolated T. evansi from infected-water buffaloes in the Philippines and analyzed their virulence using mice and cattle. A total of 10 strains of T. evansi were isolated. Evaluation of the virulence of each strain using mice depicted significant differences among the strains in the prepatent period, the level of parasitemia, and the survival time of the infected animals. In mice infected with the highly pathogenic T. evansi, signs of excessive inflammation such as marked splenomegaly and increase more than 6-fold in the number of leukocytes were observed at 8 days post-infection. To study the virulence of the parasite strains in cattle (which are the common T. evansi hosts in Philippines), cattle were infected with the T. evansi isolates that showed high and low virulence in mice. The rate of parasite growth and the length of the prepatent periods were found to be similar to those observed in mice for the respective strains. The cattle infected with the highly pathogenic strain developed anemia and a marked decrease in leukocyte counts. To determine the cause of the pathological changes, we analyzed the expression levels of inflammatory cytokines and observed up-regulation of tumor necrosis factor-alpha in anemic infected cattle. Our findings suggest that the epidemic of T. evansi in the Philippines is characterized by T. evansi strains with varying virulences from low to very high pathogenicity in cattle.
  • Yusuke Murase, Satoru Konnai, Naftaly Githaka, Arata Hidano, Kyle Taylor, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Toshio Tsubota, Shiro Murata, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 2 215 - 218 2013年02月 [査読有り][通常論文]
     
    In this study, the prevalence of Borrelia infections in Ixodes ticks from a site in Hokkaido, Japan, with confirmed cases of Lyme disease was determined by a PCR method capable of detecting and differentiating between strains of pathogenic Borrelia, with particular emphasis on Borrelia garinii (B. garinii) and Borrelia afzelli (B. afzelli), using tick-derived DNA extracts as template. A total of 338 ticks, inclusive of 284 Ixodes persulcatus (I. persulcatus), were collected by flagging vegetation in mid-spring. Ninety-eight (34.5%) of I. persukatus tested positive for Borrelia species DNA, whereas the overall prevalence of Borrelia species in Ixodes ovatus and Haemaphysalis longicornis ticks was 19.5 and 7.7%, respectively. PCR-RFLP and sequence analysis of Borrelia rrj(5S)-rr/(23S) intergenic spacer DNA amplicons indicated that they originated from three different Borrelia species namely, B. garinii, B. afzelii and B. japonica. Among the I. persulcatus species, which is a known vector of human borreliosis, 86 were mono-infected with B. garinii, 2 ticks were mono-infected with B. afzelii and whereas 12 ticks had dual infections. Most significant, 11 of the I. persukatus ticks were coinfected with Anaplasma phagocytophilum and B. garinii. The difference between the number of obtained and expected co-infections was significant (chi(2)=4.32, P=0.038).
  • Satoru Konnai, Saori Suzuki, Tatsuya Shirai, Ryoyo Ikebuchi, Tomohiro Okagawa, Yuji Sunden, Claro N. Mingala, Misao Onuma, Shiro Murata, Kazuhiko Ohashi
    COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 36 1 63 - 69 2013年01月 [査読有り][通常論文]
     
    An immunoinhibitory receptor, lymphocyte activation gene-3 (LAG-3), which is mainly expressed in T-cells, is involved in the immune evasion of several pathogens causing chronic infections and tumors. However, unlike human or mouse LAG-3, no functional analysis of LAG-3 has been reported in domestic animals. Thus, in this study, bovine LAG-3 expression was analyzed in bovine leukemia virus (BLV)-infected cattle. In persistent lymphocytotic (PL) cattle, the numbers of LAG-3(+)CD4(+) cells and LAG-3(+)CD8(+) cells were conserved whilst the number of MHC class II+ cells was remarkably higher than in the control animals. In contrast, the mean fluorescence intensity (MFI) for LAG-3 on PBMCs from PL cattle was significantly increased compared to control and asymptomatic (AL) cattle. Specifically, the LAG-3 expression level was significantly increased in both CD4(+) and CD8(+) T cells from PL cattle. LAG-3 expression correlated positively with increased numbers of lymphocytes and MHC class II+ cells in infected animals. Preliminary results from PD-L1 and LAG-3 blockade assay revealed that IFN-gamma and IL-2 expressions were significantly up-regulated by addition of anti- PD-L1 and LAG-3 antibodies in PBMCs from PL cattle. These findings suggest that LAG-3 might be involved in the inhibition of T-cell function through its binding and signaling on MHC class II molecule during BLV infection. (c) 2012 Elsevier Ltd. All rights reserved.
  • Saiki Imamura, Satoru Konnai, Shinji Yamada, Luis F. Parizi, Naftaly Githaka, Itabajara da S. Vaz, Shiro Murata, Kazuhiko Ohashi
    TICKS AND TICK-BORNE DISEASES 4 1-2 138 - 144 2013年 [査読有り][通常論文]
     
    Vaccines are among the alternative tick control methods expected to replace at least in part the volumes of chemical acaricides currently used worldwide. However, a vaccination approach depends on a host immune response against proteins that are essential to tick physiology. The cystatin family is a protein class recently investigated to compose an effective antigen in a tick vaccine. In this study, a cDNA from Rhipicephalus appendiculatus with high sequence similarity to cystatins type 2 was identified by random sequencing analysis and called R. appendiculatus cystatin 1 (Ra-cyst-1). DNA sequence analysis showed that the cloned Ra-cyst-1 has a 423-bp open reading frame and codified to a 140-amino acid polypeptide. The putative mature protein consists of 115 amino acid residues with a deduced molecular weight of 12.8 kDa. The highly conserved G (P-I), QxVxG (P-II), and PW (P-III) type 2 cystatins motifs are present in Ra-cyst-1 cDNA. RT-PCR analysis showed that the Ra-cyst-1 gene is expressed in nymph, male, and female midgut following blood feeding, but not in the salivary glands of fed females. In addition, Western blot revealed that recombinant Ra-cyst-1 was not recognized by sera derived from rabbits infested with ticks, suggesting that this cystatin is not secreted into the host during infestation. We hypothesize that Ra-cyst-1 may play a role in the tick feeding process and could be a concealed antigen candidate in further anti-tick vaccination trials. (C) 2012 Elsevier GmbH. All rights reserved.
  • Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Veterinary Research 44 1 59  2013年 [査読有り][通常論文]
     
    Programmed death-1 (PD-1) is a known immunoinhibitory receptor that contributes to immune evasion of various tumor cells and pathogens causing chronic infection, such as bovine leukemia virus (BLV) infection. First, in this study, to establish a method for the expression and functional analysis of bovine PD-1, hybridomas producing monoclonal antibodies (mAb) specific for bovine PD-1 were established. Treatment with these anti-PD-1 mAb enhanced interferon-gamma (IFN-γ) production of bovine peripheral blood mononuclear cells (PBMC). Next, to examine whether PD-1 blockade by anti-PD-1 mAb could upregulate the immune reaction during chronic infection, the expression and functional analysis of PD-1 in PBMC isolated from BLV-infected cattle with or without lymphoma were performed using anti-PD-1 mAb. The frequencies of both PD-1+ CD4+ T cells in blood and lymph node and PD-1 + CD8+ T cells in lymph node were higher in BLV-infected cattle with lymphoma than those without lymphoma or control uninfected cattle. PD-1 blockade enhanced IFN-γ production and proliferation and reduced BLV-gp51 expression and B-cell activation in PBMC from BLV-infected cattle in response to BLV-gp51 peptide mixture. These data show that anti-bovine PD-1 mAb could provide a new therapy to control BLV infection via upregulation of immune response.© 2013 Ikebuchi et al. licensee BioMed Central Ltd.
  • Naftaly Githaka, Satoru Konnai, Edward Kariuki, Esther Kanduma, Shiro Murata, Kazuhiko Ohashi
    ACTA TROPICA 124 1 71 - 78 2012年10月 [査読有り][通常論文]
     
    Piroplasms frequently infect domestic and wild carnivores. At present, there is limited information on the occurrence and molecular identity of these tick-borne parasites in wild felids in Kenya. In 2009, a pair of captive lions (Panthare leo) was diagnosed with suspected babesiosis and mineral deficiency at an animal orphanage on the outskirts of Nairobi, Kenya. Blood smears indicated presences of haemoparasites in the erythrocytes, however, no further investigations were conducted to identify the infecting agent. The animals recovered completely following diet supplementation and treatment with anti-parasite drug. In this report, we extracted and detected parasite DNA from the two lions and seven other asymptomatic feline samples; two leopards (Panthera pardus) and five cheetahs (Acinonyx jubatus). Reverse line blot with probes specific for Babesia spp. of felines indicated the presence of new Babesia species or genotypes in the lions and leopards, and unknown Theileria sp. in the cheetahs. Phylogenetic analyses using partial sequences of 185 ribosomal RNA (185 rRNA) gene showed that the parasite infecting the lions belong to the Babesia canis complex, and the parasite variant detected in the leopards clusters in a clade bearing other Babesia spp. reported in wild felids from Africa. The cheetah isolates falls in the Theileria sensu stricto group. Our findings indicate the occurrence of potentially new species or genotypes of piroplams in all three feline species. (C) 2012 Elsevier B.V. All rights reserved.
  • Tomohiro Okagawa, Satoru Konnai, Hirohisa Mekata, Naftaly Githaka, Saori Suzuki, Edward Kariuki, Francis Gakuya, Esther Kanduma, Tatsuya Shirai, Ryoyo Ikebuchi, Yoshinori Ikenaka, Mayumi Ishizuka, Shiro Murata, Kazuhiko Ohashi
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 148 3-4 373 - 379 2012年08月 [査読有り][通常論文]
     
    Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus coffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo. (c) 2012 Elsevier B.V. All rights reserved.
  • H. Mekata, S. Konnai, C. N. Mingala, N. S. Abes, C. A. Gutierrez, A. P. Dargantes, W. H. Witola, N. Inoue, M. Onuma, S. Murata, K. Ohashi
    PARASITE IMMUNOLOGY 34 6 318 - 329 2012年06月 [査読有り][通常論文]
     
    Trypanosoma evansi (T.similar to evansi) causes a wasting disease in almost all mammals. Trypanosoma evansi infection gives rise to the inflammatory responses that contribute to the development of inflammation-associated tissue injury. To determine what kinds of inflammatory molecules play roles in the pathogenicity of T.similar to evansi infection, polymerase chain reaction array analysis was performed on samples from the infected and uninfected mice. The inflammatory cytokine and chemokine storm, caused mainly by macrophages, was observed. On the other hand, the expression levels of Ccl8 and Il10 in splenocytes were also markedly increased. These results suggested an augmentation in the number and activity of regulatory dendritic cells (DCs). Therefore, the kinetics of regulatory DCs in T.similar to evansi-infected mice were investigated. During T.similar to evansi infection, the regulatory DCs became prevalent, with reducing the amount of inflammatory DCs. Interestingly, when the regulatory DCs were implanted into T.similar to evansi-infected mice, the survival was prolonged, and the expression levels of inflammatory molecules were suppressed. Taken together, these results showed that a subset of regulatory DCs acted as a potential regulator of the inflammatory responses.
  • Shiro Murata, Yuko Hayashi, Ayumi Kato, Masayoshi Isezaki, Sarah Takasaki, Misao Onuma, Yuichi Osa, Mitsuhiko Asakawa, Satoru Konnai, Kazuhiko Ohashi
    VETERINARY JOURNAL 192 3 538 - 540 2012年06月 [査読有り][通常論文]
     
    Marek's disease virus serotype 1 (MDV-1) strains cause malignant lymphoma in chickens. MDV-1 has been previously reported to be widespread in white-fronted geese (Anser albifrons); however, the prevalence of MDV-1 in other wild birds has not been determined. In this study, we investigated the prevalence of MDV-1 in various wild birds in Hokkaido, Japan. The MDV-1 genome was widespread in geese and ducks, but was not detected in other birds. MDV-1 was detected in both sedentary and migratory species. These results suggest that, in Japan. MDV-1 is widespread in wild goose and duck populations, and that resident ducks may be significant carriers and reservoirs of MDV-1. (C) 2011 Elsevier Ltd. All rights reserved.
  • Tomohiro Okagawa, Satoru Konnai, Ryoyo Ikebuchi, Saori Suzuki, Tatsuya Shirai, Yuji Sunden, Misao Onuma, Shiro Murata, Kazuhiko Ohashi
    VETERINARY RESEARCH 43 45  2012年05月 [査読有り][通常論文]
     
    The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4(+) and CD8(+) cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4(+) and CD8(+) cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-gamma and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-gamma mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.
  • Ayumi Matsuyama-Kato, Shiro Murata, Masayoshi Isezaki, Rika Kano, Sara Takasaki, Osamu Ichii, Satoru Konnai, Kazuhiko Ohashi
    VIROLOGY JOURNAL 9 94  2012年05月 [査読有り][通常論文]
     
    Background: An immunoinhibitory receptor, programmed death-1 (PD-1), and its ligand, programmed death-ligand 1 (PD-L1), are involved in immune evasion mechanisms for several pathogens causing chronic infections and for neoplastic diseases. However, little has been reported for the functions of these molecules in chickens. Thus, in this study, their expressions and roles were analyzed in chickens infected with Marek's disease virus (MDV), which induces immunosuppression in infected chickens. Results: A chicken T cell line, Lee1, which constitutively produces IFN-gamma was co-cultured with DF-1 cells, which is a spontaneously immortalized chicken fibroblast cell line, transiently expressing PD-L1, and the IFN-gamma expression level was analyzed in the cell line by real-time RT-PCR. The IFN-gamma expression was significantly decreased in Lee1 cells co-cultured with DF-1 cells expressing PD-L1. The expression level of PD-1 was increased in chickens at the early cytolytic phase of the MDV infection, while the PD-L1 expression level was increased at the latent phase. In addition, the expression levels of PD-1 and PD-L1 were increased at tumor lesions found in MDV-challenged chickens. The expressions levels of PD-1 and PD-L1 were also increased in the spleens and tumors derived from MDV-infected chickens in the field. Conclusions: We demonstrated that the chicken PD-1/PD-L1 pathway has immunoinhibitory functions, and PD-1 may be involved in MD pathogenesis at the early cytolytic phase of the MDV infection, whereas PD-L1 could contribute to the establishment and maintenance of MDV latency. We also observed the increased expressions of PD-1 and PD-L1 in tumors from MDV-infected chickens, suggesting that tumor cells transformed by MDV highly express PD-1 and PD-L1 and thereby could evade from immune responses of the host.
  • S. Suzuki, S. Konnai, T. Okagawa, N. W. Githaka, E. Kariuki, F. Gakuya, E. Kanduma, T. Shirai, R. Ikebuchi, Y. Ikenaka, M. Ishizuka, S. Murata, K. Ohashi
    INTERNATIONAL JOURNAL OF IMMUNOGENETICS 39 2 170 - 182 2012年04月 [査読有り][通常論文]
     
    The African buffalo (Syncerus caffer) has been implicated as the reservoir of several bovine infectious agents. However, there is insufficient information on the protective immune responses in the African buffalo, particularly in infected animals. In this study, we analysed Th1 cytokines IL-2 and IFN-?, and Th2 cytokines IL-4 and IL-10. The cloned cDNA of IL-2, IL-4, IL-10 and IFN-? contained an open reading frame of 468, 501, 408 and 540 nucleotides, encoding polypeptides of 155, 166, 135 and 179 amino acids, respectively. Nucleotide sequence homology of IL-2, IFN-? and IL-4 was more than 98% between the African buffalo and cattle, which resulted in identical polypeptides. Meanwhile, IL-10 gene of African buffalo and cattle had 95% homology in nucleotide sequence, corresponding to thirteen amino acid residues substitution. Cysteine residues and potential glycosylation sites were conserved within the family Bovinae. Phylogenetic analyses including cytokines of the African buffalo placed them within a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle, water buffalo, sheep, goat, pig and artiodactyl wildlife. A deeper understanding of the structure of these cytokines will shed light on their protective role in the disease-resistant African buffalo in comparison with other closely related species.
  • Satoru Konnai, Shinji Yamada, Saiki Imamura, Hideto Nishikado, Naftaly Githaka, Takuya Ito, Ai Takano, Hiroki Kawabata, Shiro Murata, Kazuhiko Ohashi
    TICKS AND TICK-BORNE DISEASES 3 2 75 - 77 2012年 [査読有り][通常論文]
     
    The tick receptor for outer surface protein A (TROSPA) is an Ixodes scapularis (I. scapularis) receptor for Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease in North America. The blockade of TROSPA has been shown to reduce B. burgdorferi adherence to the I. scapularis gut in vivo. Thus. TROSPA is one of the potential targets for the development of vector-antigen-based vaccines to prevent the transmission of B. burgdorferi. The aim of this study is to identify the TROSPA gene in I. persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan. The cDNA clone encoding the TROSPA-like sequence with 483 nucleotides was obtained from whole-body homogenates of fed nymphs of I. persulcatus. The putative amino acid sequence of I. persulcatus TROSPA was 88.2% and 87.8% identical to that of I. scapularis and I. ricinus, respectively. This finding will facilitate investigations on the role of I. persulcatus TROSPA and its interaction with Borrelia spp. and will have important implications on endeavors to develop a tick vaccine. (C) 2012 Elsevier GmbH. All rights reserved.
  • Tatsuya Shirai, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Saori Suzuki, Yuji Sunden, Misao Onuma, Shiro Murata, Kazuhiko Ohashi
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 144 3-4 462 - 467 2011年12月 [査読有り][通常論文]
     
    Lymphocyte activation gene-3 (LAG-3), a major histocompatibility complex (MHC) class II binding CD4 homologue has recently been shown as one of the mechanisms for down-regulating immune responses during chronic disease progression. For the first time, we cloned LAG-3 from two breeds of cattle (Holstein and Japanese Black), and analyzed its expression levels in cattle infected with bovine leukemia virus (BLV), a chronic viral infection that leads to immuno-suppression. The cloned cDNA of bovine LAG-3 have an open reading frame of 1551 nucleotides, encoding a polypeptide of 515 amino acids in length. Similar to the swine LAG-3, the bovine LAG-3 protein sequence consisted of four extracellular domains, a transmembrane domain and an inhibitory motif, KTGELE. We found that the bovine LAG-3 mRNA transcripts were expressed predominantly on T-cells such as CD4(+) and CD8(+) cells, among peripheral blood mononuclear cells. In subsequent expression analysis, LAG-3 mRNA expression on CD4(+) T-cells from BLV-infected cattle was upregulated compared to that in normal cattle. Comparable results were obtained with CD8(+) T-cells from cattle infected with BLV. We further observed strong upregualtion of MHC class II molecule, the ligand for LAG-3 in BLV-infected cattle. These findings indicate an important role for inhibitory receptor molecules such as LAG-3 in chronic bovine infections and future studies will elucidate the specific role of LAG-3 in bovine diseases. (C) 2011 Elsevier B.V. All rights reserved.
  • Ryoyo Ikebuchi, Satoru Konnai, Tatsuya Shirai, Yuji Sunden, Shiro Murata, Misao Onuma, Kazuhiko Ohashi
    VETERINARY RESEARCH 42 103  2011年09月 [査読有り][通常論文]
     
    The inhibitory receptor programmed death-1 (PD-1) and its ligand, programmed death-ligand 1 (PD-L1) are involved in immune evasion mechanisms for several pathogens causing chronic infections. Blockade of the PD-1/PD-L1 pathway restores anti-virus immune responses, with concomitant reduction in viral load. In a previous report, we showed that, in bovine leukemia virus (BLV) infection, the expression of bovine PD-1 is closely associated with disease progression. However, the functions of bovine PD-L1 are still unknown. To investigate the role of PD-L1 in BLV infection, we identified the bovine PD-L1 gene, and examined PD-L1 expression in BLV-infected cattle in comparison with uninfected cattle. The deduced amino acid sequence of bovine PD-L1 shows high homology to the human and mouse PD-L1. The proportion of PD-L1 positive cells, especially among B cells, was upregulated in cattle with the late stage of the disease compared to cattle at the aleukemic infection stage or uninfected cattle. The proportion of PD-L1 positive cells correlated positively with prediction markers for the progression of the disease such as leukocyte number, virus load and virus titer whilst on the contrary, it inversely correlated with the degree of interferon-gamma expression. Blockade of the PD-1/PD-L1 pathway in vitro by PD-L1-specific antibody upregulated the production of interleukin-2 and interferon-gamma, and correspondingly, downregulated the BLV provirus load and the proportion of BLV-gp51 expressing cells. These data suggest that PD-L1 induces immunoinhibition in disease progressed cattle during chronic BLV infection. Therefore, PD-L1 would be a potential target for developing immunotherapies against BLV infection.
  • Shiro Murata, Tsukasa Okada, Rika Kano, Yuko Hayashi, Tomoyuki Hashiguchi, Misao Onuma, Satoru Konnai, Kazuhiko Ohashi
    VIRUS GENES 43 1 66 - 71 2011年08月 [査読有り][通常論文]
     
    Marek's disease virus (MDV) is an oncogenic herpesvirus that causes malignant lymphomas in chickens. Recent field isolates of MDV have tended to exhibit increasing virulence, and MDV strains are currently classified into four categories based on their relative virulence. Meq, a putative MDV oncoprotein, resembles the Jun/Fos family of basic leucine zipper (bZIP) transcription factors and can regulate the expression of viral and cellular genes as a homodimer or as a heterodimer with a variety of bZIP family proteins. MDV isolates display distinct diversity and point mutations in Meq, which may contribute to changes in the transcriptional activities of Meq and subsequently, to observed increases in MDV oncogenicity. In this study, we introduced mutations into the meq gene and used dual luciferase reporter assays to analyze the transcriptional activities of the resulting Meq proteins to determine whether distinct mutations in Meq could be responsible for differences in transcriptional activity among MDV strains. A proline-to-alanine substitution at position 217, the second position of one of the proline direct repeats in the transactivation domain, enhanced the transactivation activity of Meq. In addition, we found that two substitutions at positions 283 and 320 affected transactivation activity. These results suggest that the distinct diversity of and point mutations in the Meq proteins are responsible for differences in transactivation activity among MDV strains.
  • Yusuke Murase, Satoru Konnai, Arata Hidano, Naftali W. Githaka, Takuya Ito, Ai Takano, Hiroki Kawabata, Manabu Ato, Tomoko Tajima, Motoshi Tajima, Misao Onuma, Shiro Murata, Kazuhiko Ohashi
    VETERINARY MICROBIOLOGY 149 3-4 504 - 507 2011年05月 [査読有り][通常論文]
     
    The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmaposis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals. Crown Copyright (C) 2010 Published by Elsevier B.V. All rights reserved.
  • 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 [査読有り][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.
  • 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 [査読有り][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.
  • Shiro Murata, Kyung-Soo Chang, Sung-Il Lee, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi
    JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION 19 5 471 - 478 2007年09月 [査読有り][通常論文]
     
    For the easy survey of Marek's disease virus (MDV), feather tip-derived DNA from MDV-infected chickens can be used because feather tips are easy to collect and feather follicle epithelium is known to be the only site of productive replication of cell-free MDV. To develop a diagnostic method to differentiate highly virulent strains of MDV from the attenuated MDV vaccine strain, CV1988, which is widely used, nested polymerase chain reaction (PCR) was performed to detect a segment of the meq gene in feather tip samples of chickens experimentally infected with MDV. In chickens infected with Md5, a strain of oncogenic MDV, the meq gene was consistently detected, whereas the L-meq gene, in which a 180-base pair (180-bp) sequence is inserted into the meq gene, was detected in CV1988-infected chickens. Moreover, the meq gene was mainly detected even in chickens co-infected with both Md5 and CV1988. These results suggest that this method is appropriate for the surveillance of the highly virulent MDV infection in the field.
  • 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 [査読有り][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 [査読有り][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.
  • 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 [査読有り][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.

MISC

書籍等出版物

  • 感染症の生態学
    長雄一, 大橋和彦, 村田史郎 (担当:共著範囲:第20章 鳥インフルエンザ)
    共立出版 2016年03月
  • Ducks: Habitat, Behavior, and Diseases
    Asakawa, M, Nakade, T, Murata, S, Ohashi, K, Osa, Y, Taniyama, H (担当:共著範囲:Recent viral disease of Japanese Anatid with a fatal case of Marek’s disease in an endangered species, white-fronted goose (Anser albifrons).)
    Nova Science Publishers 2013年

講演・口頭発表等

  • Transcriptome analysis in blood-fed and starved poultry red mites, Dermanyssus gallinae  [通常講演]
    Fujisawa, S, Murata, S, Takehara, M, Isezaki, M, Ogawa, R, Uno, Y, Taneno, A, Okagawa, T, Maekawa, N, Konnai, S, Ohashi, K
    The 4th International Symposium on Parasite Infections in Poultry, Vieena 2019年06月 ポスター発表
  • Cysteine protease and ferritin 2 as vaccine antigens to control poultry red mites, Dermanyssus gallinae  [通常講演]
    S. Murata, M. Isezaki, A. Taniguchi, S. Fujisawa, A. Taneno, E. Sakai, Y. Uno, R. Ogawa, O. Ichii, T. Ito, M. Takehara, A. Morita, N. Maekawa, T. Okagawa, S. Konnai, K. Ohashi
    The 4th International Symposium on Parasite Infections in Poultry, Vienna 2019年06月 口頭発表(一般)
  • マレック病に関する最近の知見について ―日本に分布するマレック病ウイルスの性状  [招待講演]
    村田史郎, 今内覚, 大橋和彦
    鶏病研究会佐賀県支部鶏病技術研修会 2019年06月 口頭発表(招待・特別)
  • 抗ワクモワクチン開発に向けた試み  [招待講演]
    村田 史郎
    第284回鶏病事例検討会 2018年09月 口頭発表(招待・特別)
  • Molecular detection of Marek’s disease virus in poultry farms in Myanmar  [通常講演]
    Shiro Murata, Masaki Takehara, Ken Katakura, Myint Myint Hmoon, Shwe Yee Win, Masayoshi Isezaki, Satoru Konnai, Kazuhiko Ohashi
    The 12th International Symposium on Marek's Disease and Avian Herpesviruses 2018年07月 ポスター発表
  • Enhanced immune responses after vaccination with a gallid alphaherpesvirus 2 (Marek’s disease virus, MDV) expressing PD-1  [通常講演]
    Shiro Murata, Benedikt B Kaufer, Nikolaus Osterrieder, Masayoshi Isezaki, Satoru Konnai, Kazuhiko Ohashi
    42nd International Herpesvirus Workshop 2017年07月 ポスター発表
  • The Role of Meq Mutations on Marek’s Disease Virus Pathogenesis  [通常講演]
    Nirajan Bhandari, Phaedra Tavlarides-Hontz, Upendra Katneni, Benedikt B. Kaufer, Shiro Murata, Mark S. Parcells
    The 11th International Symposium on Marek's Disease and Avian herpesviruses 2016年07月 口頭発表(一般)
  • Sequence analysis of a Marek’s disease virus strain isolated in Japan  [通常講演]
    Shiro Murata, Yuka Machida, Masayoshi Isezaki, Satoru Konnai, Kazuhiko Ohashi
    The 11th International Symposium on Marek's Disease and Avian herpesviruses 2016年07月 ポスター発表
  • Expression analysis of programmed death ligand 2 in tumors caused by the avian oncovirus Marek’s disease virus  [通常講演]
    Shiro Murata, Ayumi Matsuyama-Kato, Masayoshi Isezaki, Satoru Konnai, Kazuhiko Ohashi
    The 39th Annual International Herpesvirus Workshop 2014年07月 ポスター発表
  • Characterization of Meq proteins from field isolates of Marek’s disease virus in Japan  [通常講演]
    Shiro Murata, Tomoyuki Hashiguchi, Yuko Hayashi, Yuki Yamamoto, Ayumi Kato, Sarah Takasaki, Masayoshi Isezaki, Satoru Konnai, Kazuhiko Ohashi
    The 9th International Symposium on Marek’s disease and Avian Herpesviruses 2012年06月 口頭発表(一般)
  • Characterization of immunoinhibitory factors PD-1/PD-L1 in chickens infected with Marek’s disease virus  [通常講演]
    Ayumi Matsuyama-Kato, Shiro Murata, Masayoshi Isezaki, Rika Kano, Sarah Takasaki, Osamu Ichii, Satoru Konnai, Kazuhiko Ohashi
    The 9th International Symposium on Marek’s disease and Avian Herpesviruses 2012年06月 口頭発表(一般)
  • Amino acid substitutions or insertion in the Meq proteins could affect their transactivation and transformation abilities  [通常講演]
    Shiro Murata, Tomoyuki Hashiguchi, Tsukasa Okada, Rika Kano, Misao Onuma, Satoru Konnai, Kazuhiko Ohashi
    International Union of Microbiological Societies 2011 Congress 2011年09月 口頭発表(一般)
  • Regulation of the interferon gamma expression by an oncoprotein of Marek’s disease virus in wild waterfowls and chickens  [通常講演]
    Ayako Matsubara, Shiro Murata, Rika Kano, Tomoyuki Hashiguchi, Masayoshi Isezaki, Ayumi Kato, Misao Onuma, Satoru Konnai, Kazuhiko Ohashi
    The 9th International Veterinary Immunology Symposium 2010年08月 口頭発表(一般)
  • Analysis of transcriptional activities by the Meq proteins with highly virulent Marek’s disease virus strains  [通常講演]
    Shiro MURATA, Tsukasa OKADA, Rika KANO, Yuko HAYASHI, Satoru KONNAI, Misao ONUMA, Kazuhiko OHASHI
    4th International Workshop of the Molecular Pathogenesis of Marek's disease Virus 2006年08月 口頭発表(一般)

所属学協会

  • 日本獣医学会   

共同研究・競争的資金等の研究課題

  • 腸管細胞膜タンパク質を標的としたウイルスベクターの応用による抗ワクモワクチン開発
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2020年07月 -2023年03月 
    代表者 : 村田 史郎
  • 鶏の外部寄生虫を種横断的に防除する”ユニバーサル”ワクチン候補分子の探索
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 大橋 和彦, 村田 史郎, 今内 覚
  • 伴侶動物における腫瘍横断的治療法の新規実用化のための実証研究
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 今内 覚, 村田 史郎, 大橋 和彦
     
    免疫チェックポイントに係る免疫抑制因子を標的とした特異的に腫瘍を排除する抗体医薬品を開発し、難治性の悪性腫瘍に罹ったイヌに対する臨床応用研究を行った。ポメラニアン、12歳、去勢雄。CT検査において右上顎犬歯尾側より発生する腫瘤性の病変を認め、内側咽頭後リンパ節は両側ともに腫大していた。原発巣に対して外科的切除を行ったが、病理検査によりマージン部に悪性黒色腫細胞の残存を認めた。残存病変に対する放射線照射を計画していたが、プランニングCTで口腔内に再発病変を認めた。そして放射線治療終了後の定期健診 (初回照射から6週目) におけるCT検査にて、再発巣の増大と肺転移の出現を認めた(Stage IVに進行)。その後、抗PD-L1抗体(5 mg/kg、2週間に1度)による免疫療法を開始した。1クール(3回投与、6週間)終了時に効果判定としてCT検査を行った。その結果、免疫療法開始前に長径10.9 mmだった口腔内の再発巣が、CT検査において画像上完全に消失した。同様に、複数あった肺転移巣においても縮小・消失が認められた。肺において微小な病変が残存していることが疑われたため、総合的には部分奏功と判定した。免疫療法開始以前に放射線治療を行っているため、口腔の再発病変の消失は放射線治療の効果によるとも考えられるが、照射終了後のCT検査において明らかな縮小が認められていないことから抗PD-L1抗体による治療効果の寄与が大きいと考えられた。このことから、抗PD-L1抗体は肺転移のあるイヌ悪性黒色腫に対する有効な治療法となりうると考えられた。
  • ワクチン抗原を分泌する組換えマレック病ウイルスを用いたワクモ防除法の開発
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 村田 史郎, 今内 覚, 大橋 和彦
     
    日本を含め多くの国々の養鶏場において、ワクモによる被害は深刻な問題となっている。ワクモ対策に貢献するため、殺虫剤に変わる新たな手法として、ワクモ防除用ワクチンの開発を目指して研究を行っている。これまでに複数のワクチン抗原候補を同定しており、その有用性を評価してきた。加えて、ワクチン接種時にかかる負担を軽減させるため、ワクチン抗原遺伝子を挿入したウイルスベクターの応用についても、組換えマレック病弱毒生ワクチンを用いて検討を行ってきた。本研究では、これまでに作製したワクチン抗原遺伝子挿入組換えマレック病弱毒生ワクチンの効果増大を目指し、従来型ワクチンに改良を加え、抗原分泌型の弱毒生ワクチンの作製とその効果検証を行うことを目的とした。 本年度は、ワクチン抗原分泌型のマレック病弱毒生ワクチンゲノムの作製を行った。本研究では、マレック病ウイルス強毒株であるRB1B株のmeq遺伝子とワクチン抗原遺伝子を置換させたウイルスゲノムプラスミドを用いた。meq遺伝子はマレック病ウイルスの病態形成に必須であるため、ワクチン抗原遺伝子の挿入に伴い、meq遺伝子が欠損することとなり、マレック病ウイルスの病原性も欠失する。ワクチン抗原分泌型とするため、ウイルスゲノム中のワクチン抗原遺伝子上流に分泌シグナルを挿入し、改良型のマレック病弱毒生ワクチンゲノムプラスミドを作製した。さらに抗原の細胞外分泌時に宿主細胞への影響を低減させるため、ワクチン抗原のタンパク質機能を失活させる点変異をワクチン抗原遺伝子中に挿入した。以上より、ワクチン抗原分泌型の弱毒マレック病生ワクチンゲノムプラスミドが完成した。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 大橋 和彦, 村田 史郎, 今内 覚
     
    マレック病ウイルス(MDV)は、鶏に悪性リンパ腫を主徴とするマレック病(MD)を引き起こし、養鶏業に大きな被害をもたらしてきたが、現在では生ワクチンの開発・実用化により効果的に予防されている。しかし野外においては、MDVの病原性が増強する傾向にあり、ワクチン接種鶏でMDが発生するワクチンブレークが問題となっている。しかしMDVの野外における病原性進化の分子機構については未だ不明のままである。これまでの我々の研究で、国内のワクチンブレーク鶏から分離されたMDVでは、全ゲノム解析により、米国で報告されているmeq遺伝子の多型も存在するが、従来報告されていない多型や他のウイルス遺伝子の異常も検出されている。そこで本研究では、近年日本国内の養鶏等から検出される MDVの分子生物学的性状を従来の分離株と比較して、これまで報告されていない新規の病原性進化機構を明らかにすることとした。 前年度の解析で、国内で分離されたMDV株が米国の強毒株686株とは異なる病原性を示すことが示されたので、今年度はこの国内分離株の全ゲノム解析を行い、より詳細な比較解析を行った。その結果、病態発現に重要なvIL-8遺伝子やウイルス複製に関わる因子、ウイルスの構造蛋白等にアミノ酸置換を伴う相違が多く検出されたが、これらの箇所は中国や欧州由来のMDV株に見られた配列とは一致していた。そしてウイルス構造蛋白のひとつであるUL36に関してC末端の反復配列数に差が見られ、病原性との相関が疑われた。今後より詳細な機能解析をする必要がある。 また、アジア諸国に分布するMDVの疫学調査・性状を解析を行う目的でミャンマーでの分子疫学調査を実施して、いくつかの農場でMDV遺伝子(meq遺伝子)が検出されたが、現時点で強毒株と思われるものは検出されなかった。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 大橋 和彦, 村田 史郎, 今内 覚
     
    世界各地で養鶏業に大きな被害をもたらす鶏の外部寄生虫であるワクモ(Dermanyssus gallinae)の新規防除法として抗ワクモワクチンを開発するために、世界各地でワクモ材料を採取し、ワクチン候補分子として同定したワクモ遺伝子群について多型解析を実施した。そして、同定した分子群について、ワクモのin vitro吸血システムや吸血攻撃試験でその有効性を検証した結果、同定した分子群を用いた抗ワクモワクチンはワクモに対する防御効果を示した。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2016年04月 -2018年03月 
    代表者 : 村田 史郎
     
    ワクモ(鳥類の外部寄生虫)による吸血被害は、世界中の養鶏場で深刻な問題となっている。殺虫剤に変わる新たなワクモ防除法として、ワクチンによる防除法開発に取り組んでいる。本研究では、使用しやすいワクチンの作製を目指し、ウイルスベクターの応用を検討した。ウイルスはほぼ全ての鶏に接種されるマレック病弱毒生ワクチンを用いて、ワクチン抗原遺伝子が挿入された組換えウイルスを作製した。組換えウイルス感染細胞では、抗原タンパク質の発現が認められており、新たなワクモ防除用ワクチンとして期待される。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2016年04月 -2018年03月 
    代表者 : 大橋 和彦, 村田 史郎, 今内 覚
     
    養鶏業に多大な被害をもたらしているワクモに対する新規ワクチンの開発を目指して、その候補抗原としてワクモ中腸に発現する分子の探索を行い、3種の分子を同定し、これら分子がワクモの中腸や卵巣で発現していることを確認した。また既に我々が同定した候補抗原であるカテプシンD様タンパク質について解析した結果、この分子が発育ステージ等に関わらず恒常的に発現し、吸血鶏由来の血清に反応しないことから非暴露型抗原であることが示された。今回同定した分子のより詳細な性状解析を行い、ワクチン抗原として有用性を評価していく必要がある。
  • 日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2014年04月 -2018年03月 
    代表者 : 今内 覚, 村田 史郎, 大橋 和彦
     
    牛白血病ウイルス(BLV)は、日本を含む多くの国で高い陽性率が報告されている。本研究では、牛白血病清浄化ガイドラインの構築を目的としてBLV感染牛における種々の伝播リスクについて解析を行った。その結果、リンパ球増多症を呈するウシ、ウイルス量が多いウシおよび発症牛が水平感染源になっていることを確認した。さらに感染ウイルス量が多い妊娠牛から帝王切開術により子牛を摘出し、子牛のへの直接的な垂直感染を証明した。本成果は、ウイルス量が高い母牛からの産子の感染検査は、清浄化対策において極めて重要となり、陽性牛から陽性牛が生まれると農場での清浄化は極めて難しく、子牛への感染防御は重要であることを示した。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 大橋 和彦, 村田 史郎, 今内 覚
     
    鶏に悪性リンパ腫を主徴とするマレック病(MD)を引き起こすマレック病ウイルス(MDV)の野外における病原性進化の分子機構を解明するために、国内でワクチン接種したにも関わらずMDが発生するワクチンブレーク鶏からMDVを単離して、その分子生物学的性状及びウイルス学的性状を解析した。その結果、分離したMDVは、病原性に重要なウイルス遺伝子に新規の多型が同定され、鶏に病原性を示したが、全ゲノムの系統樹解析の結果、米国等で従来報告されていたものとは異なる分子機構で病原性進化している可能性が示唆された。
  • 日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2013年04月 -2017年03月 
    代表者 : 今内 覚, 村田 史郎, 大橋 和彦, 川治 聡子
     
    家畜の悪性伝染病では免疫抑制が認められるが、その機序については明らかになっていなかった。本研究では、海外で甚大な経済的被害を与えているアナプラズマ症、東海岸熱、スーラ病、牛結核、ヨーネ病、牛白血病、ウマ伝染性貧血などの悪性伝染病の免疫抑制機序について解析を行った。その結果、Programmed death 1 (PD-1)やProgrammed death-ligand 1 (PD-L1)などの免疫抑制因子(免疫チェックポイント因子)が病態に深く関与することが明らかとなった。また、抗体などで免疫抑制因子を阻害すると、抗病原体免疫が回復することを明らかにした。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2013年04月 -2015年03月 
    代表者 : 村田 史郎
     
    近年マレック病ウイルスは、野外において病原性の増強が報告されている。そこで、より防御効果の高い新たなワクチン開発を行うために、免疫抑制因子であるPD1とPD-L1の機構に着目した。分泌型PD-1遺伝子を挿入した組換え生ワクチンを作製し、宿主が持つPD1/PD-L1による免疫抑制能を競合的に緩和させ、より強い細胞性免疫が誘導できるワクチン開発を行った。その結果、既存のワクチンと比較して、新規組換えワクチンを接種した鶏由来の末梢血単核球は、MDV抗原に対する高いインターフェロンγ産生能を示し、本組換えワクチンがより効果的な細胞性免疫応答を誘導する能力を持つことが示唆された。
  • 日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2011年11月 -2015年03月 
    代表者 : 今内 覚, 大橋 和彦, 村田 史郎, 田島 誉士
     
    増加する牛白血病ウイルス(BLV)による地方病牛白血病は、現在、日本の畜産生産上深刻な問題となっている。本研究ではBLVに新規感染した牛の感染源と媒介昆虫対策による感染阻止効果について検討した。新規感染した牛のBLV遺伝子(env遺伝子)を解析した結果、BLV陽性牛群内でリンパ球増多症を呈するウイルス量が多い牛の遺伝子と一致し、BLVの感染伝播においてハイリスク要因であることが示唆された。一方、吸血昆虫対策を実施した結果、新規感染は認められなかった。このことから、高ウイルス保有牛は感染源になりやすいこと、および昆虫対策は感染伝播阻止に有効であることが示された。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2011年04月 -2014年03月 
    代表者 : 大橋 和彦, 今内 覚, 村田 史郎, 村田 史郎
     
    Programmed death 1 (PD-1)レセプターとProgrammed death ligands (PD-L1およびPD-L2)は、難治性感染症や腫瘍において、病原体や腫瘍細胞の免疫抑制機構に役割を果たすことが知られている。本研究により、鶏においても、他の動物と同様に、PD-1およびPD-L1およびPD-L2が免疫抑制能を有することが示された。そしてマレック病ウイルス感染鶏では、PD-1およびPD-L1およびPD-L2が発現しており、特に腫瘍病変部でPD-1およびPD-L2が腫瘍の免疫回避に関与して、マレック病の病態進行に重要な役割を果たすことが示唆された。
  • 秋山記念生命科学振興財団研究助成
    秋山記念生命科学振興財団:
    研究期間 : 2010年 -2010年 
    代表者 : 村田史郎


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