研究者データベース

今内 覚(コンナイ サトル)
獣医学研究院 獣医学部門 病原制御学分野
准教授

基本情報

所属

  • 獣医学研究院 獣医学部門 病原制御学分野

職名

  • 准教授

学位

  • 獣医学博士

ホームページURL

J-Global ID

研究キーワード

  • 感染免疫   臨床免疫   腫瘍免疫   ウイルス学   感染症学   マダニ   節足動物媒介性疾患   

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

職歴

  • 2018年07月 - 現在 北海道大学大学院獣医学研究院 先端創薬分野 分野長・准教授
  • 2017年04月 - 現在 北海道大学大学院獣医学研究院 病原制御学分野 准教授
  • 2008年12月 - 2017年03月 北海道大学大学院獣医学研究科 動物疾病制御学講座 准教授
  • 2007年04月 - 2008年11月 北海道大学大学院獣医学研究科 動物疾病制御学講座 助教
  • 2004年12月 - 2007年03月 北海道大学大学院獣医学研究科 動物疾病制御学講座 助手

学歴

  • 2003年03月 -   北海道大学大学院獣医学研究科博士課程単位取得退学

所属学協会

  • 日本ウイルス学会   大動物臨床研究会   日本家畜臨床感染症研究会   動物サイトカイン研究会   日本獣医寄生虫学会   動物用ワクチン-バイオ医薬品研究会   北海道獣医師会   日本獣医師会   日本獣医学会   

研究活動情報

論文

  • Transcriptome dynamics of blood-fed and starved poultry red mites, Dermanyssus gallinae
    Fujisawa S, Murata S, Isezaki M, Oishi E, Taneno A, Maekawa N, Okagawa T, Konnai S, Ohashi K
    Parasitol Int. 102156 - 102156 2020年06月 [査読有り][通常論文]
  • Rhipicephalus microplus cystatin as a potential cross-protective tick vaccine against Rhipicephalus appendiculatus
    Parizi L, Rangel C, Sabadina G, Saggin B, Kiio I, Xavier M, Matos R, Camargo-Mathias M, Seixas A, Konnai S, Ohashi K, Githaka N, da Silva Vaz Jr I
    Tick Tick born Dise 11 3 101378  2020年05月 [査読有り][通常論文]
  • Immunosuppressive Effects of Sialostatins L and L2 Isolated from the Taiga Tick Ixodes persulcatus Schulze
    Sajiki Y, Konnai S, Ochi A, Okagawa T, Githaka N, Isezaki M, Yamada S, Ito T, Ando S, Kawabata H, Da Silva Vaz Jr I, Logullo C, Maekawa N, Murata S, Ohashi K
    Ticks Tick born Dis. 11 2 101332.  2020年03月 [査読有り][通常論文]
  • Satoshi Gondaira, Koji Nishi, Takahiro Tanaka, Takashi Yamamoto, Takanori Nebu, Reina Watanabe, Satoru Konnai, Tomohito Hayashi, Yoshio Kiku, Mariko Okamoto, Kazuya Matsuda, Masateru Koiwa, Hidetomo Iwano, Hajime Nagahata, Hidetoshi Higuchi
    Infection and immunity 88 3 e00521 - 19 2020年02月20日 [査読有り][通常論文]
     
    Mycoplasma bovis is a destructive pathogen that causes large economic losses in rearing cattle for beef and dairy worldwide. M. bovis causes suppression of and evades the host immune response; however, the mechanisms of host immune function involved in M. bovis mastitis have not been elucidated. The purpose of this study was to elucidate the characteristics of the bovine immune response to mycoplasmal mastitis. We evaluated the responsiveness of the bovine mammary gland following infusion of M. bovis Somatic cell counts and bacterial counts in milk from the infected quarter were increased. However, the proliferation of peripheral blood mononuclear cells (blood MNCs) and mononuclear cells isolated from M. bovis-stimulated mammary lymph nodes (lymph node MNCs) did not differ from that in the unstimulated cells. Transcriptome analysis revealed that the mRNA levels of innate immune system-related genes in blood MNCs, complement factor D (CFD), ficolin 1 (FCN1), and tumor necrosis factor superfamily member 13 (TNFSF13) decreased following intramammary infusion of M. bovis The mRNA levels of immune exhaustion-related genes, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) of milk mononuclear cells (milk MNCs) in the infected quarter were increased compared with those before infusion. Increase in immune exhaustion-related gene expression and decrease in innate immune response-related genes of MNCs in quarters from cows were newly characterized by M. bovis-induced mastitis. These results suggested that M. bovis-induced mastitis affected the immune function of bovine MNCs, which is associated with prolonged duration of infection with M. bovis.
  • Expression Analysis of Canine CMTM6 and CMTM4 as Potential Regulators of the PD-L1 Protein in Canine Cancers
    Takeuchi H, Konnai S, Maekawa N, Minato E, Ichikawa Y, Kobayashi A, Okegawa T, Murata S, Ohashi K.
    Front Vet Sci in press  2020年 [査読有り][通常論文]
  • 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
    Front. Vet. Sci. 7 12 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 in press  2020年01月 [査読有り][通常論文]
  • Junko Takei, Shinji Yamada, Satoru Konnai, Tsuyoshi Ishinazaka, Michito Shimozuru, Mika K Kaneko, Yukinari Kato
    Monoclonal antibodies in immunodiagnosis and immunotherapy 38 6 282 - 284 2019年12月 [査読有り][通常論文]
     
    Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.
  • Epitope Mapping of Anti-Bear Podoplanin Monoclonal Antibody PMab-247
    Kato Y, Takei J, Furusawa Y, Sayama Y, Sano M, Konnai S, Kobayashi A, Harada H, Takahashi M, Suzuki H, Yamada S, Kaneko MK
    Monoclon Antib Immunodiagn Immunother. 38 5 230 - 233 2019年09月 [査読有り][通常論文]
  • Immune inhibitory function of bovine CTLA-4 and the effects of its blockade in IFN-γ production
    Watari K, Konnai S, Maekawa N, Okagawa T, Suzuki Y, Murata S, Ohashi K
    BMC Vet Res 15 1 380  2019年09月 [査読有り][通常論文]
  • PMab-247 Detects Bear Podoplanin in Immunohistochemical Analysis
    Takei J, Furusawa Y, Yamada S, Nakamura T, Sayama Y, Sano M, Konnai S, Kobayashi A, Harada H, Kaneko MK, Kato Y
    Monoclon Antib Immunodiagn Immunother. 38 4 171 - 174 2019年08月 [査読有り][通常論文]
  • Prostaglandin E2 induced immune exhaustion and enhancement of anti-viral effects by anti-PD-L1 antibody combined with COX-2 inhibitor in bovine leukemia virus infection
    Sajiki Y, Konnai S, Okagawa T, Nishimori A, Maekawa N, Goto S, Watari K, Minato E, Kobayashi A, Kohara J, Yamada S, Kaneko M, Kato Y, Takahashi H, Terasaki N, Takeda A, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K
    J. Immunol 203 5 1313 - 1324 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]
  • Shinji Yamada, Shunsuke Itai, Takuro Nakamura, Junko Takei, Masato Sano, Satoru Konnai, Atsushi Kobayashi, Shotaro Nakagun, Yoshiyasu Kobayashi, Mika K Kaneko, Yukinari Kato
    Monoclonal antibodies in immunodiagnosis and immunotherapy 38 3 104 - 107 2019年06月 [査読有り][通常論文]
     
    Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolar cells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for immunohistochemical analyses for cetacean tissues.
  • Haematophagous mites on poultry farms in the Republic of the Union of Myanmar
    Takehara M, Murata S, Katakura K, Fujisawa S, Hmoon MM, Win SY, Bawn 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]
  • Carbohydrate Metabolic Compensation Coupled to High Tolerance to Oxidative Stress in Ticks
    Noce BD, Uhl M, Machado J, Waltero C, Abreu L, Silva R, Fonseca R, Barros C, Sabadin G, Konnai S, Silva Vaz Jr. I, Ohashi K, Logullo C
    Sci Rep. 9 4753  2019年03月 [査読有り][通常論文]
  • Effects of bovine tumor necrosis factor alpha decoy receptors on cell death and inflammatory cytokine kinetics: Potential for bovine inflammation therapy
    Fujisawa S, Konnai S, Okagawa T, Maekawa N, Tanaka A, Suzuki Y, Murata S, Ohashi K
    BMC Vet Res. 15 1 68  2019年02月 [査読有り][通常論文]
  • Anti-Horse Podoplanin Monoclonal Antibody PMab-219 is Useful for Detecting Lymphatic Endothelial Cells by Immunohistochemical Analysis
    Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, Kaneko MK
    Monoclon Antib Immunodiagn Immunother. 37 6 272 - 274 2018年12月 [査読有り][通常論文]
  • Detection of Alpaca Podoplanin by Immunohistochemistry Using the Antibovine Podoplanin Monoclonal Antibody PMab-44
    Kato Y, Yamada S, Itai S, Konnai S, Kobayashi A, Kaneko MK
    Monoclon Antib Immunodiagn Immunother. 37 6 269 - 271 2018年12月 [査読有り][通常論文]
  • Immunohistochemical Detection of Sheep Podoplanin Using an Antibovine Podoplanin Monoclonal Antibody PMab-44
    Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, Kaneko MK
    Monoclon Antib Immunodiagn Immunother. 37 6 265 - 268 2018年12月 [査読有り][通常論文]
  • The mouse-canine chimeric anti-dog podoplanin antibody P38B exerts antitumor activity in mouse xenograft models
    Kato Y, Ohishi T, Kawada M, Maekawa N, Konnai S, Itai S, Yamada S, Kaneko MK
    Biochem Biophys Rep. 17 23 - 26 2018年11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]
  • Cattle with a low bovine leukemia virus proviral load are rarely an infectious source
    Mekata H, Yamamoto M, Hayashi T, Kirino Y, Sekiguchi S, Konnai S, Horii Y, Norimine J
    Jpn J Vet Res. 66 3 157 - 163 2018年08月 [査読有り][通常論文]
  • Cooperation of PD-1 and LAG-3 in the exhaustion of CD4+ and CD8+ T cells during bovine leukemia virus infection
    Okagawa T, Konnai S, Nishimori A, Maekawa N, Goto S, Ikebuchi R, Kohara J, Suzuki Y, Yamada S, Kato Y, Murata S, Ohashi K
    Vet Res. 49 1 50  2018年06月 [査読有り][通常論文]
  • Epitope mapping of anti-mouse podoplanin monoclonal antibody PMab-1
    Yamada S, Itai S, Kaneko MK, Konnai S, Kato Y
    Biochem Biophys Rep. 15 52 - 56 2018年06月 [査読有り][通常論文]
  • Evaluation of costimulatory molecules in dogs with B cell high grade lymphoma
    Tagawa M, Kurashima C, Takagi S, Maekawa N, Konnai S, Shimbo G, Matsumoto K, Inokuma H, Kawamoto K, Miyahara K
    PLoS One 13 7 e0201222  2018年06月 [査読有り][通常論文]
  • Prostaglandin E2 Induction Suppresses the Th1 Immune Responses in Cattle with Johne's Disease
    Sajiki Y, Konnai S, Okagawa T, Nishimori A, Maekawa N, Goto S, Ikebuchi R, Nagata R, Kawaji S, Kagawa Y, Yamada S, Kato Y, Nakajima C, Suzuki Y, Murata S, Mori Y, Ohashi K
    Infect Immun. 86 5 e00910-17  2018年04月 [査読有り][通常論文]
  • Identification of an Atypical Enzootic Bovine Leukosis in Japan by Using a Novel Classification of Bovine Leukemia Based on Immunophenotypic Analysis
    Nishimori A, Konnai S, Okagawa T, Maekawa N, Goto S, Ikebuchi R, Nakahara A, Chiba Y, Ikeda M, Murata S, Ohashi K
    Clin Vaccine Immunol. 24 12 e00001-18  2018年04月 [査読有り][通常論文]
  • Maekawa N, Konnai S, Balbin MM, Mingala CN, Gicana KRB, Bernando FAEM, Murata S, Ohashi K
    Ticks Tick Borne Dis. 9 2 266 - 269 2018年02月 [査読有り][通常論文]
  • Mekata H, Yamamoto M, Kirino Y, Sekiguchi S, Konnai S, Horii Y, Norimine J
    J Vet Med Sci. 80 2 316 - 319 2018年02月 [査読有り][通常論文]
  • Adriana Seixas, Maria Fernanda Alzugaray, Lucas Tirloni, Luis Fernando Parizi, Antonio Frederico Michel Pinto, Naftaly Wang'ombe Githaka, Satoru Konnai, Kazuhiko Ohashi, John R. Yates, Carlos Termignoni, Itabajara da Silva Vaz
    TICKS AND TICK-BORNE DISEASES 9 1 72 - 81 2018年01月 [査読有り][通常論文]
     
    The vitellogenin receptor (VgR), which belongs to the low-density lipoprotein receptors (LDLR) family, regulates the absorption of yolk protein accumulated in developing oocytes during oogenesis. In the present study, the full sequence of Rhipicephalus microplus VgR (RmVgR) and the partial sequence of Rhipicephalus appendiculatus VgR (RaVgR) ORF were determined and cloned. The RmVgR amino acid sequence contains the five highly conserved structural motifs characteristic of LDLR superfamily members, the same overall structure as observed in other species. Phylogenetic analysis separated VgRs in two major groups, corresponding to receptors from acarines and insects. Consistent with observations from other arthropods, RmVgR was specifically expressed in the ovarian tissue and its peak of expression occurs in females that are detaching from the host. Silencing with RmVgR dsRNA reduced VgR expression, which resulted in reduced fertility, evidenced by a decrease in the number of larvae. The present study confirms RmVgR is a specific receptor involved in yolk protein uptake and oocyte maturation in R. microplus, playing an important role in tick reproduction.
  • Ochirkhuu N, Konnai S, Odbileg R, Murata S, Ohashi K
    J Vet Med Sci. 79 12 2040 - 2042 2017年12月 [査読有り][通常論文]
  • Expression of Cat Podoplanin in Feline Squamous Cell Carcinomas
    Itai S, Yamada S, Kaneko MK, Harada H, Kagawa Y, Konnai S, Kato Y
    Monoclon Antib Immunodiagn Immunother. 36 6 243 - 250 2017年12月 [査読有り][通常論文]
  • Sajiki Y, Konnai S, Nishimori A, Okagawa T, Maekawa N, Goto S, Nagano M, Kohara J, Kitano N, Takahashi T, Tajima M, Mekata H, Horii Y, Murata S, Ohashi K
    J Vet Med Sci. 79 12 2036 - 2039 2017年12月 [査読有り][通常論文]
  • 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.
  • PMab-52: Specific and Sensitive Monoclonal Antibody Against Cat Podoplanin for Immunohistochemistry
    Yamada S, Itai S, Nakamura T, Yanaka M, Saidoh N, Chang YW, Handa S, Harada H, Kagawa Y, Ichii O, Konnai S, Kaneko MK, Kato Y
    Monoclon Antib Immunodiagn Immunother. 36 5 224 - 230 2017年10月 [査読有り][通常論文]
  • 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
    Immun Inflamm Dis. 5 3 355 - 363 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.
  • A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma
    Maekawa N, Konnai S, Takagi S, Kagawa Y, Okagawa T, Nishimori A, Ikebuchi R, Izumi Y, Deguchi T, Nakajima C, Kato Y, Yamamoto K, Uemura H, Suzuki Y, Murata S, Ohashi K
    Sci Rep. 7 1 8951  2017年08月 [査読有り][通常論文]
  • Characterization of the Anti-Bovine Podoplanin Monoclonal Antibody PMab-44
    Yamada S, Honma R, Kaneko MK, Nakamura T, Yanaka M, Saidoh N, Takagi M, Konnai S, Kato Y
    Monoclon Antib Immunodiagn Immunother. 36 3 129 - 134 2017年06月 [査読有り][通常論文]
  • 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.
  • Shanemae M. Rivera, Ryan Bismark C. Padiernos, Evaristo A. Abella, Satoru Konnai, Claro N. Mingala
    JAPANESE JOURNAL OF VETERINARY RESEARCH 65 2 65 - 74 2017年05月 [査読有り][通常論文]
     
    The present study was conducted to characterize LAG-3 of swamp- and riverine-type water buffaloes by DNA sequencing, homology and phylogenetic analysis. Bubaline LAG-3 sequence contained an open reading frame of 1551 nucleotide, encoding a polypeptide of 516 amino acids. Nucleotide and amino acid sequence homology of LAG-3 revealed 76-96% and 61-94% identity in water buffalo to that of other mammals, respectively. LAG-3 protein sequence of water buffalo contained four extracellular domains, a transmembrane domain and different conserved regions. There were three N-glycosylation sites, two sequence motifs: 'RGD' and 'WXC' motif and five cysteine residues located at different positions of extracellular region. Likewise, the possible serine phosphorylation site and the 'KTGELE' inhibitory motif were found in the intracellular region of bubaline LAG-3. However, one highly conserved cysteine residue in mammalian LAG-3 was replaced by tyrosine in both swamp- and riverine-type water buffaloes. Phylogenetic analysis generated high bootstrap value between the two types of water buffalo which further confirmed the degree of relationship between bubaline species. This was the first report that describe the genetic characteristic of LAG-3 in swamp- and riverine-type water bufffaloes.
  • Girja S. Pandey, Edgar Simulundu, Danstan Mwiinga, Kenny L. Samui, Aaron S. Mweene, Masahiro Kajihara, Alfred Mangani, Racheal Mwenda, Joseph Ndebe, Satoru Konnai, Ayato Takada
    ARCHIVES OF VIROLOGY 162 4 1051 - 1056 2017年04月 [査読有り][通常論文]
     
    Bovine leukemia virus (BLV) causes enzootic bovine leucosis (EBL) and is responsible for substantial economic losses in cattle globally. However, information in Africa on the disease is limited. Here, based on clinical, hematological, pathological and molecular analyses, two clinical cases of EBL were confirmed in a dairy cattle herd in Zambia. In contrast, proviral DNA was detected by PCR in five apparently healthy cows from the same herd, suggesting subclinical BLV infection. Phylogenetic analysis of the env gene showed that the identified BLV clustered with Eurasian genotype 4 strains. This is the first report of confirmed EBL in Zambia.
  • Development of mPMab-1, a Mouse-Rat Chimeric Antibody Against Mouse Podoplanin
    Yamada S, Kaneko MK, Nakamura T, Ichii O, Konnai S, Kato Y
    Monoclon Antib Immunodiagn Immunother. 36 2 77 - 79 2017年04月 [査読有り][通常論文]
  • 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.
  • 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][招待有り]
     
    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.
  • Evenilton P. Costa, Arnoldo R. Facanha, Criscila S. Cruz, Jhenifer N. Silva, Josias A. Machado, Gabriel M. Carvalho, Mariana R. Fernandes, Renato Martins, Eldo Campos, Nelilma C. Romeiro, Naftaly W. Githaka, Satoru Konnai, Kazuhiko Ohashi, Itabajara S. Vaz, Carlos Logullo
    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1861 1 2922 - 2933 2017年01月 [査読有り][通常論文]
     
    Background: Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase from R. microplus embryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status. Methods: In this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface. Results: Cysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PPi), probably by inducing disulfide bridge formation. Conclusions: Our results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface. General significance: This study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status. (C) 2016 Elsevier B.V. All rights reserved.
  • Carolina K. Rangel, Luis F. Parizi, Gabriela A. Sabadin, Evenilton P. Costa, Nelilma C. Romeiro, Masayoshi Isezaki, Naftaly W. Githaka, Adriana Seixas, Carlos Logullo, Satoru Konnai, Kazuhiko Ohashi, Itabajara da Silva Vaz
    TICKS AND TICK-BORNE DISEASES 8 3 432 - 441 2017年 [査読有り][通常論文]
     
    Cystatins are cysteine peptidase inhibitors that in ticks mediate processes such as blood feeding and digestion. The ixodid tick Ixodes persulcatus is endemic to the Eurasia, where it is the principal vector of Lyme borreliosis. To date, no I. persulcatus cystatin has been characterized. In the present work, we describe three novel cystatins from I. persulcatus, named JpIpcys2a, JpIpcys2b and JpIpcys2c. In addition, the potential of tick cystatins as cross-protective antigens was evaluated by vaccination of hamsters using BrBmcys2c, a cystatin from Rhipicephalus microplus, against I. persulcatus infestation. Sequence analysis showed that motifs that are characteristic of cystatins type 2 are fully conserved in JpIpcys2b, while mutations are present in both JpIpcys2a and JpIpcys2c. Protein-protein docking simulations further revealed that JpIpcys2a, JpIpcys2b and JpIpcys2c showed conserved binding sites to human cathepsins L, all of them covering the active site cleft. Cystatin transcripts were detected in different I. persulcatus tissues and instars, showing their ubiquitous expression during I. persulcatus development. Serological analysis showed that although hamsters immunized with BrBmcys2c developed a humoral immune response, this response was not adequate to protect against a heterologous challenge with I. persulcatus adult ticks. The lack of cross-protection provided by BrBmcys2c immunization is perhaps linked to the fact that cystatins cluster into multigene protein families that are expressed differentially and exhibit functional redundancy. How to target such small proteins that are secreted in low quantities remains a challenge in the development of suitable anti-tick vaccine antigens. (C) 2017 Elsevier GmbH. All rights reserved.
  • Podoplanin Expression in Canine Melanoma
    Ogasawara S, Honma R, Kaneko MK, Fujii Y, Kagawa Y, Konnai S, Kato Y
    Monoclon Antib Immunodiagn Immunother. 35 6 304 - 306 2016年12月 [査読有り][通常論文]
  • 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.
  • Kaneko MK, Honma R, Ogasawara S, Fujii Y, Nakamura T, Saidoh N, Takagi M, Kagawa Y, Konnai S, Kato Y
    Monoclon Antib Immunodiagn Immunother. 35 5 263 - 266 2016年10月 [査読有り][通常論文]
  • 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.
  • Specific Detection of Dog Podoplanin Expressed in Renal Glomerulus by a Novel Monoclonal Antibody PMab-38 in Immunohistochemistry
    Honma R, Kaneko MK, Ogasawara S, Fujii Y, Konnai S, Takagi M, Kato Y
    Monoclon Antib Immunodiagn Immunother. 35 4 212 - 6 2016年08月 [査読有り][通常論文]
  • PMab-44 Detects Bovine Podoplanin in Immunohistochemistry
    Honma R, Ogasawara S, Kaneko MK, Fujii Y, Oki H, Nakamura T, Takagi M, Konnai S, Kato Y
    Monoclon Antib Immunodiagn Immunother. 2016年06月 [査読有り][通常論文]
  • 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.
  • Honma R, Fujii Y, Ogasawara S, Oki H, Konnai S, Kagawa Y, Takagi M, Kaneko MK, Kato Y
    Monoclon Antib Immunodiagn Immunother. 35 2 65 - 72 2016年04月 [査読有り][通常論文]
  • Michihito Tagawa, Naoya Maekawa, Satoru Konnai, Satoshi Takagi
    PLOS ONE 11 2 e0150030  2016年02月 [査読有り][通常論文]
     
    Histiocytic sarcoma is a rapidly progressive and fatal neoplastic disease in dogs. It is unclear whether costimulatory molecules, including CD28, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and programmed death-1 (PD-1), are expressed on peripheral blood lymphocytes (PBLs) of canine patients with histiocytic sarcoma. The objective of this study was to evaluate the expression of CD28, CTLA-4, and PD-1 molecules on PBLs of patients with histiocytic sarcoma, patients with other tumors, and healthy controls. Twenty-six dogs were included in the study, with eight, ten, and eight dogs in the histiocytic sarcoma, other tumor, and healthy control groups, respectively. PBLs and serum were prospectively obtained from patients diagnosed histopathologically with histiocytic sarcoma, other tumors and healthy controls. The surface expression of CTLA-4, CD28, and PD-1 on T lymphocytes was examined using flow cytometric analysis. Serum samples were frozen at -30 degrees C until serum interferon-gamma (IFN-gamma) was measured by enzyme-linked immunosorbent assay. The expression level of CTLA-4 on CD4+ lymphocytes was significantly higher in the histiocytic sarcoma group than in the control group. The expression of CTLA-4 on CD8+ lymphocytes was significantly higher in the histiocytic sarcoma group than in the other two groups. In addition, the expression of PD-1 on CD8+ lymphocytes was significantly higher in the histiocytic sarcoma group than in the control group. However, no significant differences in CD28 expressions and serum IFN-gamma levels were observed. The present results provided evidence showing that the expression levels of CTLA-4 on both CD4+ and CD8+ lymphocytes and PD-1 on CD8+ lymphocytes in peripheral blood obtained from dogs with histiocytic sarcoma were upregulated. The overexpressions of CTLA 4 and PD-1 suggested that antitumor immunity may be suppressed in dogs with histiocytic sarcoma.
  • 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.
  • Bovine leukemia virus reduces anti-viral cytokine activities and NK cytotoxicity by inducing TGF-β secretion from regulatory T cells
    Ohira K, Nakahara A, Konnai S, Okagawa T, Nishimori A, Maekawa N, Ikebuchi R, Kohara J, Murata S, Ohashi K
    Immun Inflamm Dis. 4 1 52 - 63 2016年01月 [査読有り][通常論文]
  • P. L. H. Duran, R. B. C. Padiernos, E. A. Abella, S. Konnai, C. N. Mingala
    INTERNATIONAL JOURNAL OF IMMUNOGENETICS 42 6 469 - 478 2015年12月 [査読有り][通常論文]
     
    Molecular characterization of T-cell immunoglobulin mucin domain-3 (TIM-3) and Galectin-9 (GAL-9) genes of swamp-and riverine-type water buffaloes was conducted to compare these genes with other species; determine the unique characteristic specific in water buffalo; and provide baseline information for the assessment of disease progression in buffalo species. TIM-3 and GAL-9 genes were amplified, purified, sequenced and characterized. The sequence result of TIM-3 in both types of water buffaloes contained 843 nucleotides encoding to 280 amino acids while GAL-9 of swamp-type and riverine-type water buffaloes contained 1023 and 972 nucleotides encoding to 340 and 323 amino acids, respectively. Meanwhile, the nucleotide and amino sequence of TIM-3 in water buffalo were 83-98% and 94-97% identical with other artiodactyl species, respectively. On the other hand, GAL-9 nucleotide and amino acid sequence in water buffalo were 85-98% and 76-96% identical with other artiodactyl species. The tyrosine-kinase phosphorylation motif and potential glycosylation sites were conserved within the tribe Bovinae. It is imperative to have further studies in the assessment of the role of these genes in disease progression in water buffalo during chronic infection. The study is the first report that describes the genetic characteristic of TIM-3 and GAL-9 genes in water buffalo.
  • 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.
  • Hokuto Nakata, Shouta M. M. Nakayama, Yoshinori Ikenaka, Hazuki Mizukawa, Chihiro Ishii, Yared B. Yohannes, Satoru Konnai, Wageh Sobhy Darwish, Mayumi Ishizuka
    ENVIRONMENTAL POLLUTION 205 8 - 15 2015年10月 [査読有り][通常論文]
     
    Nairobi city in Kenya produces 2000 tons/day of garbage, and most of it is dumped onto the Dandora dumping site, home to a quarter-million residents. This study was conducted (1) to assess the contamination levels of nine metals and a metalloid (arsenic) in the blood of pigs, goats, sheep and cattle from Dandora, and (2) to identify a possible source of lead (Pb) pollution. Cadmium (Cd, 0.17-435 mu g/kg, dry-wt) and Pb (90-2710 mu g/kg) levels in blood were generally high, suggesting human exposure to Cd through livestock consumption and Pb poisoning among pigs (2600 mu g/kg) and cattle (354 mu g/kg). Results of Pb isotope ratios indicated that the major exposure route might differ among species. Our results also suggested a possibility that the residents in Dandora have been exposed to the metals through livestock consumption. (C) 2015 Elsevier Ltd. All rights reserved.
  • Bovine Immunoinhibitory Receptors Contribute to Suppression of Mycobacterium avium subsp. paratuberculosis-Specific T-Cell Responses
    Okagawa T, Konnai S, Nishimori A, Ikebuchi R, Mizorogi S, Nagata R, Kawaji S, Tanaka S, Kagawa Y, Murata S, Mori Y, Ohashi K
    Infect Immun. 84 1 77 - 89 2015年10月 [査読有り][通常論文]
  • Hirohisa Mekata, Satoshi Sekiguchi, Satoru Konnai, Yumi Kirino, Yoichiro Horii, Junzo Norimine
    JOURNAL OF VETERINARY MEDICAL SCIENCE 77 9 1115 - 1120 2015年09月 [査読有り][通常論文]
     
    Horizontal transmission is recognized as a major infection route for bovine leukemia virus (BLV), and cattle with high viral loads are considered to be a major infectious source in a herd. However, a correlation between viral loads and the risk of infection has been insufficient to use as a foundation for BLV control strategies. In this report, we examined the epidemiology of BLV infection and the infectious source in a local area. In 2013-2014, BLV infection was investigated in 1,823 cattle from 117 farms in two adjacent districts, Miyazaki, Japan. Seropositive samples for BLV were detected with 88 cattle and in 14 farms. Phylogenetic analysis revealed that 94% of the isolates clustered into genotype I and the remaining isolate into genotype III. Among genotype I, genetically distinct strains were spread at each farm, and cattle infected with less than 3 copies/100 cells did not transmit BLV to other cattle for more than thirty months. This is the first report of concrete data of viral load in relation to viral horizontal transmission under the field condition. The data facilitate farmers and veterinarians understanding the status of BLV infected cattle. This research contributes to BLV infection control and the development of effective BLV eradication programs.
  • 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.
  • Helga Gomes, Jorge Moraes, Naftaly Githaka, Renato Martins, Masayoshi Isezaki, Itabajara da Silva Vaz, Carlos Logullo, Satoru Konnai, Kazuhiko Ohashi
    VETERINARY PARASITOLOGY 211 3-4 266 - 273 2015年07月 [査読有り][通常論文]
     
    Among arthropods, ticks lead as vectors of animal diseases and rank second to mosquitoes in transmitting human pathogens. Cyclin-dependent kinases (CDK) participate in cell cycle control in eukaryotes. CDKs are serine/threonine protein kinases and these catalytic subunits are activated or inactivated at specific stages of the cell cycle. To determine the potential of using CDKs as anti-tick vaccine antigens, hamsters were immunized with recombinant lxodes persulcatus CDK10, followed by a homologous tick challenge. Though it was not exactly unexpected, IpCDK10 vaccination significantly impaired tick blood feeding and fecundity, which manifested as low engorgement weights, poor oviposition, and a reduction in 80% of hatching rates. These findings may underpin the development of more efficacious anti-tick vaccines based on the targeting of cell cycle control proteins. (C) 2015 Elsevier B.V. All rights reserved.
  • Kakinuma Sei-Ichi, Izawa Tomohiro, Matsuda Kei-Ichi, Konnai Satoru, Maeda Yosuke, Ohtsuka Hiromichi
    ACTA VETERINARIA-BEOGRAD 65 2 287 - 296 2015年06月 [査読有り][通常論文]
     
    IFN-tau is a type I interferon, and it is known to be non-virus inducible in ruminants. IFN-tau reduced syncytium formation by PBMC obtained from BLV infected cattle in vitro. In order to clarify the effects of IFN-tau on cellular immune function in Japanese Black (JB) cattle with bovine leukemia virus (BLV) infection, immune related factors of peripheral blood mononuclear cells (PBMC) were analyzed using IFN-tau as a stimulator. Thirty-two JB cattle were used in this investigation, and these cattle were divided into three groups: cattle with enzootic bovine leucosis (EBL) (EBL Group, N=7), clinically healthy cattle with BLV infection (Carrier Group, N=13), and clinically healthy cattle without BLV infection (non-Carrier Group, N=12). A number of mRNA expressions of interleukin-12 and interferon (IFN)-gamma as immune cell activating cytokines, perforin and granulysin as cytotoxic factors, and myxovirus resistance protein (MX)-1 and MX-2 as anti-virus factors of PBMC were analyzed after culturing cells with phytohemagglutinin (PHA) or IFN-tau. The basal mRNA levels of perforin and granulysin in the Carrier Group were significantly higher than those in the non-Carrier Group. Also, significantly higher basal mRNA levels of MX-1 and MX-2 in the EBL Group were detected compared with the non-Carrier Group. The mRNA expressions of perforin and granulysin in PBMC stimulated with PHA were higher in the Carrier Group than those in the non-Carrier Group. There were significantly higher mRNA levels of MX-1 and MX-2 in PBMC stimulated with IFN-tau in the EBL Group compared with those in the non-Carrier Group. These results suggest an enhanced sensitivity of anti-virus reaction in PBMC by IFN-tau treatment in JB cattle with EBL.
  • Yusuke Murase, Satoru Konnai, Shinji Yamada, Naftaly Githaka, Masayoshi Isezaki, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Siro Murata, Kazuhko Ohashi
    INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 60 59 - 67 2015年05月 [査読有り][通常論文]
     
    Salp15, a 15-kDa tick salivary gland protein, has several suppressive modes of activity against host immunity and plays a critical role in the transmission of Lyme disease spirochetes in Ixodes scapularis and Ixodes ricinus, major vectors of Lyme disease in North America and Western Europe. Salp15 adheres to Borrelia burgdorferi and specifically interacts with its outer surface protein C (OspC), protecting the spirochete from antibody-mediated cytotoxicity and facilitating infection in the mice. Recently, we identified two Salp15 homologues, IperSalp15-1 and IperSalp15-2, in Ixodes persulcatus, a vector for Lyme disease in Japan. Here we describe the function of IperSalp15 in the transmission of Lyme borreliosis. To investigate the function of IperSalp15, recombinant IperSalp15-1 and IperSalp15-2 were prepared in bacterial and insect cells. Both were identified in the sera of tick-immunized hamsters, indicating that these are secretory proteins in exposed host animals. Solid-phase overlay and indirect fluorescence assays showed that IperSalp15 binds to OspC from B. burgdorferi, Borrelia garinii, and Borrelia afzelii. Importantly, this binding likely protected the spirochete from antibody-mediated cytotoxicity in vitro. In addition, IperSalp15 tended to facilitate infection in mice. Thus, further characterization of tick molecules, including IperSalp15, could lead to the development of new strategies to prevent the transmission of tick-borne diseases. (C) 2015 Elsevier Ltd. All rights reserved.
  • Satoshi Gondaira, Hidetoshi Higuchi, Hidetomo Iwano, Keiichi Nakajima, Kazuhiro Kawai, Shuhei Hashiguchi, Satoru Konnai, Hajime Nagahata
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 165 1-2 45 - 53 2015年05月 [査読有り][通常論文]
     
    Mycoplasma bovis is known as a significant pathogen and cause of large economic losses in beef and dairy calves worldwide. Numerous factors appear to play an important role in the development of disease during infection with M. bovis, e.g., inhibition of immune cell proliferation and induction of lymphocyte apoptosis. However, the mechanisms involved in M. bovis infections have not been explored and remain incompletely understood. We investigated the major cytokine mRNA expression in bovine PBMC stimulated with M. bovis, for comparison, Staphylococcus aureus and Escherichia coli, which are the representative mastitis-causing pathogens. Here we demonstrated that live M. bovis significantly induced tumor necrosis factor alpha (TNF-alpha), interleukin 12p40 (IL-12), and interferon gamma (IFN-gamma) mRNA expression in bovine peripheral blood mononuclear cells (PBMC) at a multiplicity of infection (MOI) of 1000 but not at an MOI of 10 and 100. Live M. bovis at MOIs of 1, 10, and 100 induced significant bovine PBMC proliferative responses compared with unstimulated bovine PBMC. Furthermore, we showed that the cultural supernatant of M. bovis induced a significant increase in TNF-alpha, IL-6, and IL-10 mRNA expression in bovine PBMC. Our results suggest that M. bovis weakly affects the cellular integrity of bovine PBMC and induces clear proliferative responses and associated cytokine production in them. However, large numbers of live M. bovis are required to induce an immune response in bovine PBMC. (C) 2015 Elsevier B.V. All rights reserved.
  • Hirohisa Mekata, Satoshi Sekiguchi, Satoru Konnai, Yumi Kirino, Kazuyuki Honkawa, Nariaki Nonaka, Yoichiro Horii, Junzo Norimine
    VETERINARY RECORD 176 10 254 - + 2015年03月 [査読有り][通常論文]
     
    The perinatal transmission of bovine leukaemia virus (BLV) plays a critical role in the spread and persistence of BLV infection in cattle herds. The purpose of this study was to examine the frequency of perinatal infections in an area in Japan and investigate some risk factors associated with infection. Altogether, 129 calves born to BLV-infected cows in a herd in Japan were tested for infection immediately after birth and again at one month of age using nested PCR. Twenty-four calves (18.6 per cent) were infected with BLV, of which 14 (10.8 per cent) and 10 (7.7 per cent) calves were infected via the transplacental and the birth canal routes, respectively. Maternal viral loads, breed, the presence or absence of assistance during parturition and the number of births per dam were evaluated to investigate risk factors associated with infection. Maternal viral load was significantly correlated with the frequency of perinatal infection, and more than 40 per cent of newborn calves born to dams with high viral loads were infected with BLV. The results of this study could contribute towards developing effective eradication programmes by providing necessary data for replacement of breeding cow in the field.
  • 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.
  • Luis Fernando Parizi, Gabriela Alves Sabadin, Mara Fernanda Alzugaray, Adriana Seixas, Carlos Logullo, Satoru Konnai, Kazuhiko Ohashi, Aoi Masuda, Itabajara da Silva Vaz
    PARASITES & VECTORS 8 122  2015年02月 [査読有り][通常論文]
     
    Background: Cystatins are a group of cysteine protease inhibitors responsible for physiological proteolysis regulation and present in a wide range of organisms. Studies about this class of inhibitors in parasites have contributed to clarify their roles in important physiological processes, like blood digestion and modulation of host immune response during blood feeding. Thus, cystatins are a subject of research on the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to find potential targets in multi-species control methods. However, cystatin functions in ticks remain undetermined, especially in Rhipicephalus microplus and Ixodes ovatus, two species that affect livestock and human health, respectively. Methods: Here we report the inhibitory profile of two R. microplus (BrBmcys2b and BrBmcys2c) and one I. ovatus (JpIocys2a) cystatins to commercial cathepsins B, C, and L. The presence of native cystatins in R. microplus tissues was analyzed using sera against recombinant BrBmcys2b and BrBmcys2c. Also, a peptide from JpIocys2a was synthesized for rabbit immunization, and this serum was used to analyze the cross antigenicity between R. microplus and I. ovatus cystatins. Results: Enzymatic inhibition profile of tick cystatins shows a distinct modulation for cathepsins related to tick blood digestion and evasion of host immune response. Furthermore, BrBmcys2b was detected in saliva and different tissues along tick stages, while BrBmcys2c was detected mainly in gut from partially engorged R. microplus females, demonstrating a distinct pattern of cystatin expression, secretion and traffic between tick tissues. Moreover, phylogenetic analysis suggests that JpIocys2a belongs to the group of tick gut secreted cystatins. Finally, cross-antigenicity assays revealed that antibodies against the JpIocys2a peptide recognize native and recombinant R. microplus cystatins. Conclusion: The presence of these proteins in different tissues and their ability to differentially inhibit cathepsins suggest distinct roles for JpIocys2a, BrBmcys2b, and BrBmcys2c in blood digestion, egg and larvae development, and modulation of host immune response in tick physiology. The cross-antigenicity between native and recombinant cystatins supports further experiments using JpIocys2a, BrBmcys2b, and BrBmcys2c as vaccine antigens.
  • Renato Martins da Silva, Barbara Della Noce, Camila Fernanda Waltero, Evenilton Pessoa Costa, Leonardo Araujo de Abreu, Naftaly Wang'ombe Githaka, Jorge Moraes, Helga Fernandes Gomes, Satoru Konnai, Itabajara da Silva Vaz, Kazuhiko Ohashi, Carlos Logullo
    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 16 1 1821 - 1839 2015年01月 [査読有り][通常論文]
     
    In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells.
  • 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.
  • Abid Ali, Lucas Tirloni, Masayoshi Isezaki, Adriana Seixas, Satoru Konnai, Kazuhiko Ohashi, Itabajara da Silva Vaz Junior, Carlos Termignoni
    EXPERIMENTAL AND APPLIED ACAROLOGY 63 4 559 - 578 2014年08月 [査読有り][通常論文]
     
    Metalloproteases (MPs) have been considered essential for blood feeding and other physiological functions in several hematophagous animals, including ticks. We report the characterization of MP sequences of three important ticks from Asia, Africa and America: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) and R. microplus (BrRm-MPs). Amino acid sequence identity between R. microplus and R. sanguineus MPs ranged from 76 to 100 %, and identities among I. persulcatus, I. ricinus and I. scapularis MP sequences ranged from 88 to 97 %. This high sequence identity and typical functional motifs show that all sequences are MPs. The presence of a zinc binding site, a Met-turn and cysteine rich domain at the C-terminal region indicates that these proteins belong to the reproplysin family of MPs. Differences in amino acid sequences of BrRm-MP1, BrRm-MP2, BrRm-MP4 and BrRm-MP5 (from Porto Alegre strain ticks) were 6, 2, 7 and 5 %, respectively, when compared with sequences deposited in GenBank for the same genes from other R. microplus isolates. Analyses of MPs predicted that they have various highly antigenic regions. Semi-quantitative RT-PCR analysis revealed the presence of transcripts in salivary glands of partially and fully fed female ticks. None of these transcripts were observed in males (except BrRm-MP4) and eggs. These enzymes may be functional components required during tick feeding to manipulate host defenses and support tick hematophagy.
  • A. Hidano, S. Konnai, S. Yamada, N. Githaka, M. Isezaki, H. Higuchi, H. Nagahata, T. Ito, A. Takano, S. Ando, H. Kawabata, S. Murata, K. Ohahsi
    INSECT MOLECULAR BIOLOGY 23 4 466 - 474 2014年08月 [査読有り][通常論文]
     
    Salp16, a 16-kDa tick salivary gland protein, is known to be the molecule involved in the transmission of Anaplasma phagocytophilum, an obligate intracellular pathogen causing zoonotic anaplasmosis, from its mammalian hosts to Ixodes scapularis. Recently, the presence of A. phagocytophilum was documented in Japan and Ixodes persulcatus was identified as one of its vectors. The purpose of this study was to identify Salp16 genes in I. persulcatus and characterize their function. Two cDNA clones encoding the Salp16-like sequences were obtained from the salivary glands of fed female I. persulcatus ticks and designated Salp16 Iper1 and Iper2. Gene expression analyses showed that the Salp16 Iper genes were expressed specifically in the salivary glands and were up-regulated by blood feeding. These proteins attenuated the oxidative burst of activated bovine neutrophils and inhibited their migration induced by the chemoattractant interleukin-8 (IL-8). These results demonstrate that Salp16 Iper proteins contribute to the establishment of blood feeding as an immunosuppressant of neutrophil, an essential factor in innate host immunity. Further examination of the role of Salp16 Iper in the transmission of pathogens, including A. phagocytophilum, will increase our understanding of the tick-host-pathogen interface.
  • 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.
  • Sato, Kozue, Takano, Ai, Konnai, Satoru, Nakao, Minoru, Ito, Takuya, Koyama, Kojiro, Kaneko, Minoru, Ohnishi, Makoto, Kawabata, Hiroki
    EMERGING INFECTIOUS DISEASES 20 8 1391 - 1393 2014年08月 [査読有り][通常論文]
     
    We confirmed infection of 2 patients with Borrelia miyamotoi in Japan by retrospective surveillance of Lyme disease patients and detection of B. miyamotoi DNA in serum samples. One patient also showed seroconversion for antibody against recombinant glycerophosphodiester phosphodiesterase of B. miyamotoi. Indigenous relapsing fever should be considered a health concern in Japan.
  • Takano, Ai, Toyomane, Kochi, Konnai, Satoru, Ohashi, Kazuhiko, Nakao, Minoru, Ito, Takuya, Andoh, Masako, Maeda, Ken, Watarai, Masahisa, Sato, Kozue, Kawabata, Hiroki
    PLOS ONE 9 8 e104532  2014年08月 [査読有り][通常論文]
     
    During 2012-2013, a total of 4325 host-seeking adult ticks belonging to the genus Ixodes were collected from various localities of Hokkaido, the northernmost island of Japan. Tick lysates were subjected to real-time PCR assay to detect borrelial infection. The assay was designed for specific detection of the Relapsing fever spirochete Borrelia miyamotoi and for unspecific detection of Lyme disease-related spirochetes. Overall prevalence of B. miyamotoi was 2% (71/3532) in Ixodes persulcatus, 4.3% (5/117) in Ixodes pavlovskyi and 0.1% (1/676) in Ixodes ovatus. The prevalence in I. persulcatus and I. pavlovskyi ticks were significantly higher than in I. ovatus. Co-infections with Lyme disease-related spirochetes were found in all of the tick species. During this investigation, we obtained 6 isolates of B. miyamotoi from I. persulcatus and I. pavlovskyi by culture in BSK-M medium. Phylogenetic trees of B. miyamotoi inferred from each of 3 housekeeping genes (glpQ, 16S rDNA, and flaB) demonstrated that the Hokkaido isolates were clustered with Russian B. miyamotoi, but were distinguishable from North American and European B. miyamotoi. A multilocus sequence analysis using 8 genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) suggested that all Japanese B. miyamotoi isolates, including past isolates, were genetically clonal, although these were isolated from different tick and vertebrate sources. From these results, B. miyamotoi-infected ticks are widely distributed throughout Hokkaido. Female I. persulcatus are responsible for most human tick-bites, thereby I. persulcatus is likely the most important vector of indigenous relapsing fever from tick bites in Hokkaido.
  • 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.
  • Kyunglee Lee, Ai Takano, Kyle Taylor, Mariko Sashika, Michito Shimozuru, Satoru Konnai, Hiroki Kawabata, Toshio Tsubota
    TICKS AND TICK-BORNE DISEASES 5 6 841 - 847 2014年 [査読有り][通常論文]
     
    A relapsing fever Borrelia sp. similar to Borrelia lonestari (herein referred to as B. lonestari-like) was detected from wild sika deer (Cervus nippon yesoensis) and Haemaphysalis ticks in the eastern part of Hokkaido, Japan. The total prevalence of this Borrelia sp. in tested deer blood samples was 10.6% using conventional PCR and real-time PCR. The prevalence was significantly higher in deer fawns compared to adults (21.9% and 9.4%, respectively). Additionally, there was significant regional difference between our two sampling areas, Shiretoko and Shibetsu with 17% and 2.8% prevalence, respectively. Regional differences were also found in tick species collected from field and on deer. In the Shiretoko region, Haemaphysalis spp. were more abundant than Ixodes spp., while in Shibetsu, Ixodes spp. were more abundant. Using real-time PCR analysis, B. lonestari-like was detected from 2 out of 290 adult Haemaphysalis spp. ticks and 4 out of 76 pools of nymphs. This is the first report of a B. lonestari-like organism in Haemaphysalis spp. ticks, and the first phylogenetic analysis of this B. lonestari-like organism in Asia. Based on our results, Haemaphysalis spp. are the most likely candidates to act as a vector for B. lonestari-like; furthermore, regional variation of B. lonestari-like prevalence in sika deer may be dependent on the population distribution of these ticks. (C) 2014 Elsevier GmbH. All rights reserved.
  • Parizi LF, Githaka NW, Acevedo C, Benavides U, Seixas A, Logullo C, Konnai S, Ohashi K, Masuda A, da Silva Vaz I J
    Ticks Tick Borne Dis. 4 6 492 - 9 2013年12月 [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]
  • Githaka N, Konnai S, Skilton R, Kariuki E, Kanduma E, Murata S, Ohashi K
    Parasitol Int. 62 5 448 - 53 2013年10月 [査読有り][通常論文]
  • Gomes H, Romeiro NC, Braz GR, de Oliveira EA, Rodrigues C, da Fonseca RN, Githaka N, Isezaki M, Konnai S, Ohashi K, da Silva Vaz I Jr, Logullo C, Moraes J
    PLoS One. 8 10 e76128.  2013年10月 [査読有り][通常論文]
  • Suzuki S, Konnai S, Okagawa T, Ikebuchi R, Shirai T, Sunden Y, Mingala CN, Murata S, Ohashi K
    Microbiol Immunol. 57 8 600 - 4 2013年08月 [査読有り][通常論文]
  • Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K
    Vet Res. 44 59  2013年07月 [査読有り][通常論文]
  • 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.
  • Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 6 785 - 790 2013年06月 [査読有り][通常論文]
     
    The ecologies of Lyme disease Borrelia spp. are very specific to location, as they are dependent upon the spirochete species and genotypes, the vectors and the host vertebrates present. In Hokkaido, Japan, where two human pathogenic, Lyme disease Borrelia spp. are present, and human cases are reported annually, the ecologies have been poorly studied. Our goal was to determine whether variation in borrelial infection rates among rodent species sharing an environment, is due to immunological or ecological differences. To this end, we examined the relationships between tick burden and borrelial infection, by including examination of agreement between nested PCR, as a test for infection, and serology, as a test for exposure. We collected 868 rodents, comprised of four species commonly found in Hokkaido, and tested for infection rates with Borrelia spp. using PCR for the borrelial flaB gene, seroprevalence of Borrelia afzelii and Borrelia garinii using ELISA, and attachment of ticks by direct counts. We noted a correlation between differential nymph and larval burdens and the borrelial infection rates found among the four rodent species. Furthermore, there was significant correlation between infection and seroprevalence of B. afzelii and B. garinii (P<0.01), between infection and Ixodes persulcatus nymph burden (P<0.01), and between seroprevalence and I. persulcatus nymph burden (P<0.01). The close agreement among rodent species seroprevalences with infection rates and tick burdens suggest the differences in infection rates of Borrelia spp. may largely be a direct consequence of differential exposure to vectors.
  • 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]
  • 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.
  • Leonardo Araujo de Abreu, Christiano Calixto, Camila Fernanda Waltero, Barbara Pitta Della Noce, Naftaly Wang'ombe Githaka, Adriana Seixas, Luis Fernando Parizi, Satoru Konnai, Itabajara da Silva Vaz, Kazuhiko Ohashi, Carlos Logullo
    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1830 3 2574 - 2582 2013年03月 [査読有り][通常論文]
     
    Background: Tick embryogenesis is a metabolically intensive process developed under tightly controlled conditions and whose components are poorly understood. Methods: In order to characterize the role of AKT (protein kinase B) in glycogen metabolism and cell viability, glycogen determination, identification and cloning of an AKT from Rhipicephalus microplus were carried out, in parallel with experiments using RNA interference (RNAi) and chemical inhibition. Results: A decrease in glycogen content was observed when AKT was chemically inhibited by 10-DEBC treatment, while GSK3 inhibition by alsterpaullone had an opposing effect. RmAKT ORF is 1584-bp long and encodes a polypeptide chain of 60.1 kDa. Phylogenetic and sequence analyses showed significant differences between vertebrate and tick AKTs. Either AKT or GSK3 knocked down cells showed a 70% reduction in target transcript levels, but decrease in AKT also reduced glycogen content, cell viability and altered cell membrane permeability. However, the GSK3 reduction promoted an increase in glycogen content. Additionally, either GSK3 inhibition or gene silencing had a protective effect on BME26 viability after exposure to ultraviolet radiation. R. microplus AKT and GSK3 were widely expressed during embryo development. Taken together, our data support an antagonistic role for AKT and GSK3, and strongly suggest that such a signaling axis is conserved in tick embryos, with AKT located upstream of GSK3. General significance: The AKT/GSK3 axis is conserved in tick in a way that integrates glycogen metabolism and cell survival, and exhibits phylogenic differences that could be important for the development of novel control methods. (C) 2013 Elsevier B.V. All rights reserved.
  • 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).
  • Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota
    VECTOR-BORNE AND ZOONOTIC DISEASES 13 2 92 - 97 2013年02月 [査読有り][通常論文]
     
    To clarify how Borrelia miyamotoi is maintained in the environment in Hokkaido, we examined Ixodes persulcatus for its prevalence among wild rodents and its tick vector by detecting a portion of the borrelial flaB gene in rodent urinary bladder and blood samples, and from whole ticks. We compared B. miyamotoi infection rates to Borrelia garinii and Borrelia afzelii, which are human Lyme disease pathogens also carried by wild rodents, and which are transmitted by the same vector tick. Whereas B. garinii and B. afzelii showed age dependence of infection rates among wild rodents (18.4% and 9.9% among adults and 6.0% and 3.4% among sub-adults, respectively) when looking at urinary bladder samples, B. miyamotoi infection rates were not age dependent for either blood (4.2% among adults, and 7.9% among sub-adults) or urinary bladder samples (1.0% among adults, and 1.7% among sub-adults). Moreover, while B. garinii and B. afzelii infection rates showed increases across months (June, July [p < 0.05] and August [p < 0.01] had higher rates than in May for adult rodents with B. garinii, and July and August had higher rates than in May [p < 0.01] for adult rodents with B. afzelii), B. miyamotoi infection rates did not show significant month dependence. These differences in month and age dependence led us to suspect that B. miyamotoi may not develop persistent infections in wild rodents, as B. garinii and B. afzelii are thought to. Furthermore, we examined the extent of rodent exposure to I. persulcatus nymphs and larvae throughout most of the tick's active season (May through September), and determined that B. miyamotoi infection rates in sub-adult rodents were correlated with larval burden (p < 0.01), suggesting that larvae may be very important in transmission of B. miyamotoi to wild rodents.
  • 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.
  • Luis F. Parizi, Naftaly W. Githaka, Carlos Logullo, Satoru Konnai, Aoi Masuda, Kazuhiko Ohashi, Itabajara da Silva Vaz
    VETERINARY JOURNAL 194 2 158 - 165 2012年11月 [査読有り][通常論文]
     
    As blood-sucking parasites, ticks inflict great damage to animals and humans in many parts of the world. The continued use of chemical acaricides is not sustainable due to increasing tick resistance, growing public concern over drug residues in food and in the environment, and the high cost of developing new acaricides. Therefore, an alternative control strategy is urgently needed. Vaccines against ticks have been shown to be functionally feasible, as highlighted by the success of Bm86 vaccines against Rhipicephalus (Boophilus) microplus and closely related tick species. However, a limited number of tick antigens with cross-protective epitopes have been characterized so far, limiting widespread deployment of the available vaccines, including those derived from Bm86. Therefore, identifying tick antigens with potential broad-spectrum protection against multiple tick species is subject of vigorous research at present. In this paper, progress towards effective anti-tick vaccines is reviewed in the light of emerging data from studies including heterologous tick challenge. Taken together, these studies indicate that the decades-long search for a universal tick vaccine is making progress, with such a vaccine likely to be based on multiple cross-reactive antigens. (c) 2012 Elsevier Ltd. All rights reserved.
  • 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.
  • Yoshinori Ikenaka, Shouta M. M. Nakayama, Taro Muroya, John Yabe, Satoru Konnai, Wageh Sobhy Darwish, Kaampwe Muzandu, Kennedy Choongo, Geoffrey Mainda, Hiroki Teraoka, Takashi Umemura, Mayumi Ishizuka
    ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 31 10 2300 - 2305 2012年10月 [査読有り][通常論文]
     
    The Republic of Zambia is rich in mineral resources, such as zinc (Zn) and lead (Pb), and mining is a key industry in Zambia. A previous study of Pb pollution in Kabwe, one of the main mining areas, found that soil was contaminated with high levels of toxic metals over a substantial area. In the present study, the authors focus on toxic metal pollution in cattle, one of the most important domestic animals in Zambia. Blood samples from cattle in Kabwe and a control area (Lusaka) were tested for toxic metal content. They also measured mRNA expression of metal-responsive proteins and cytokines in white blood cells using real-time reverse transcriptase polymerase chain reaction. In the present in vitro study, The authors cultured peripheral blood mononuclear cells (PBMCs) from cattle, exposing them to Pb acetate for 24?h and analyzing mRNA expression of metal-responsive proteins and selected cytokines. Lead concentrations in cattle blood from Kabwe were significantly greater than those from Lusaka, as were the mRNA expressions of metallothionein-2 (MT-2), tumor necrosis factor-a (TNF-a), interferon-? (IFN-?), interleukin-1 beta (IL-1 beta), IL-6, and inducible nitric oxide synthase (iNOS). The present in vitro study demonstrated that Pb exposure led to an increase in the expressions of MT-2, TNF-a, IL-1 beta, and iNOS, similar to those found in vivo. These results indicate the possibility of immune system modulations in cattle from the Kabwe area. Environ. Toxicol. Chem. 2012; 31: 23002305. (c) 2012 SETAC
  • 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.
  • Effects of interferon-tau on cattle persistently infected with bovine viral diarrhea virus
    Junko Kohara, Yumiko Nishikura, Satoru Konnai, Motoshi Tajima, Misao Onuma
    JAPANESE JOURNAL OF VETERINARY RESEARCH 60 2-3 63 - 70 2012年08月 [査読有り][通常論文]
     
    In this study, the antiviral effects of bovine interferon-tau (boIFN-tau) on bovine viral diarrhea virus (BVDV) were examined in vitro and in vivo. In the in vitro experiments, the replication of cytopathic and non-cytopathic BVDV was inhibited in the bovine cells treated with boIFN-tau. The replication of BVDV was completely suppressed by boIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the effect of boIFN-tau on virus propagation in cattle persistently infected (PI) with non-cytopathic BVDV, boIFN-tau was subcutaneously administered to PI cattle at 10(5) U/kg or 10(6) U/kg body weight 5 times per week for 2 weeks. No physical abnormality such as depression was observed in the cattle during the experiment. The mean BVDV titers in the serum of the PI cattle decreased slightly during the boIFN-tau administration period with the dose of 10(6) U/kg. However, the BVDV titers in the serum returned to the pre-administration level after the final boIFN-tau administration. These results suggest that boIFN-tau demonstrates an anti-BVDV effect, reducing the BVDV level in serum transiently when injected into PI cattle.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Takano, Ai, Sugimori, Chieko, Fujita, Hiromi, Kadosaka, Teruki, Taylor, Kyle R., Tsubota, Toshio, Konnai, Satoru, Tajima, Tomoko, Sato, Kozue, Watanabe, Haruo, Ohnishi, Makoto, Kawabata, Hiroki
    TICKS AND TICK-BORNE DISEASES 3 4 259 - 261 2012年 [査読有り][通常論文]
     
    A novel relapsing fever Borrelia sp. was found in Amblyomma geoemydae in Japan. The novel Borrelia sp. was phylogenetically related to the hard (ixodid) tick-borne relapsing fever Borrelia app. Borrelia miyamotoi and B. lonestari. The novel relapsing fever Borrelia sp. was detected in 39 A. geoemydae (39/274: 14.2%), of which 14(14/274: 5.1%) were co-infected with the novel relapsing fever Borrelia sp. and Borrelia sp. tAG, one of the reptile-associated borreliae. Transstadial transmission of the novel relapsing fever Borrelia sp. occurred in the tick midgut and the salivary glands, although Borrelia sp. tAG was only detected in the tick midgut. The difference of the borrelial niche in molted ticks might be associated with borrelial characterization. (c) 2012 Published by Elsevier GmbH.
  • 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.
  • Ai Takano, Hiromi Fujita, Teruki Kadosaka, Satoru Konnai, Tomoko Tajima, Haruo Watanabe, Makoto Ohnishi, Hiroki Kawabata
    ENVIRONMENTAL MICROBIOLOGY REPORTS 3 5 632 - 637 2011年10月 [査読有り][通常論文]
     
    The genus Borrelia is arthropod-borne infectious agents in vertebrates, and is classified into Lyme disease (LD) Borrelia spp. and Relapsing fever (RF) Borrelia spp. In addition to these Borrelia groups, we recently reported reptile-associated (REP) Borrelia spp. from reptiles and from hard-bodied ticks, which probably transmitted the REP Borrelia spp. In this study, we investigated the presence of REP Borrelia sp. in moulted ticks, and found that trans-stadial transmission of REP Borrelia sp. occurred in the midgut, while it was observed that REP Borrelia sp. entered the salivary gland during blood-feeding. This characteristic is also found in LD Borrelia spp., which are also transmitted by hard-bodied ticks. Although phylogenetic analysis demonstrated that REP Borrelia spp. are similar to RF Borrelia spp., the ecology of the spirochaetes within the vector ticks is different for REP Borrelia spp. and RF Borrelia spp. Elucidation of the evolutionary history of the genus Borrelia and its adaptation to ticks promises to be of great interest to researchers of vector-borne microorganisms.
  • 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.
  • Ai Takano, Minoru Nakao, Toshiyuki Masuzawa, Nobuhiro Takada, Yasuhiro Yano, Fubito Ishiguro, Hiromi Fujita, Takuya Ito, Xiaohang Ma, Yozaburo Oikawa, Fumihiko Kawamori, Kunihiko Kumagai, Toshiyuki Mikami, Nozomu Hanaoka, Shuji Ando, Naoko Honda, Kyle Taylor, Toshio Tsubota, Satoru Konnai, Haruo Watanabe, Makoto Ohnishi, Hiroki Kawabata
    JOURNAL OF CLINICAL MICROBIOLOGY 49 5 2035 - 2039 2011年05月 [査読有り][通常論文]
     
    Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.
  • 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.
  • Claro N. Mingala, Satoru Konnai, Ryoyo Ikebuchi, Kazuhiko Ohashi
    COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 34 1 55 - 63 2011年01月 [査読有り][通常論文]
     
    Characterization of CTLA-4, PD-1 and PDL-1 genes from swamp and riverine type water buffaloes was done by molecular cloning, sequencing and phylogenetic analysis. The cloned cDNA of CTLA-4, PD-1 and PDL-1 contained an open reading frame of 666, 849 and 870 nucleotides, encoding a polypeptide of 221, 282 and 298 amino acids, respectively. Nucleotide sequence homology of both CTLA-4 and PDL-1 had 99.8% in swamp and riverine type, which gives the identical polypeptide. Meanwhile, PD-1 genes of swamp and riverine type water buffaloes had 99.2% of homology in nucleotide sequence, which has substitution of two amino acid residues. The hexapeptide motif, phosphatidylinositol 3'-kinase and potential glycosylation sites were conserved within the tribe Bovinae. Phylogenetic analysis confirmed the degree of relationship between the bubaline species and justify the distinctness of each breeds by the bootstrap value generated. (C) 2010 Elsevier Ltd. All rights reserved.
  • A. Mori, S. Konnai, S. Yamada, A. Hidano, Y. Murase, T. Ito, A. Takano, H. Kawabata, M. Onuma, K. Ohashi
    INSECT MOLECULAR BIOLOGY 19 3 359 - 365 2010年06月 [査読有り][通常論文]
     
    Salp15, a 15-kDa tick salivary gland protein, is known for several suppressive activities against host immunity and critical functions for the transmission of Lyme borrelia in Ixodes scapularis and Ixodes ricinus, the major vectors found in North America and Western Europe. Salp15 inhibits the activation of cluster of differentiation (CD)4+T-cells through the repression of T-cell receptor (TCR)-triggered calcium fluxes and interleukin (IL)-2 production. Furthermore, Salp15 adheres to the spirochaeta and specifically interacts with its outer surface protein C. The binding of Salp15 to Borrelia burgdorferi protects it from antibody-mediated killing in vitro. The aim of this study is to identify the Salp15 genes in Ixodes persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan. Two cDNA clones encoding the Salp15-like sequence were obtained from salivary glands of fed female ticks. These genes encode 135- and 132-amino acid proteins, designated Salp15 Iper-1 and Salp15 Iper-2, respectively, both having signal peptide sequences and predicted to be secretory proteins. Salp15 Iper-1 and -2 showed 51.8 and 68.2% similarity to I. scapularis Salp15, respectively. Reverse transcriptase PCR analysis showed that Salp15 Iper genes were expressed specifically in the salivary glands throughout life cycle stages of the ticks and were up-regulated by blood feeding. In the I. persulcatus-derived sequences, the C-terminal part, which is the binding domain to the CD4 molecule of T-cells in I. scapularis Salp15, was well conserved. In the future, it will be necessary to analyse immunosuppressive functions of I. persulcatus Salp15 and their interaction with Borrelia spp. in Japan.
  • Ryoyo Ikebuchi, Satoru Konnai, Yuji Sunden, Misao Onuma, Kazuhiko Ohashi
    MICROBIOLOGY AND IMMUNOLOGY 54 5 291 - 298 2010年05月 [査読有り][通常論文]
     
    Recent work has shown that PD-1, an immune inhibitory receptor, is involved in mechanisms for down-regulating immune responses during tumor progression or chronic viral infection. However, in the case of bovine diseases, there have been no reports on this molecule due to lack of information about bovine PD-1. In this study, we performed identification and preliminary characterization of the bovine PD-1 gene in two breeds of cattle. We cloned full cDNA sequences encoding for PD-1 from both Holstein-Friesian and Japanese Black breeds, and found that both of the genes encoded a 282-amino acid protein, which had a signal sequence, transmembrane domain and an immunoreceptor tyrosine-based inhibitory motif. This bovine PD-1 showed 72.9% and 65.6% homology to human and mouse PD-1, respectively, both of which have been well characterized and documented. Quantitative real-time PCR analysis showed that bovine PD-1 is expressed predominantly in T-cells (such as CD4+ and CD8+ cells) and among PBMCs, and is strongly upregulated on T-cell stimulation via ConA. A limited number of cattle were tested yet, as expected, the degree of PD-1 mRNA expression in CD4+ and CD8+ T-cells was greater in cattle with bovine leukemia virus-induced lymphoma than in uninfected cattle. Further studies to characterize the functions of bovine PD-1 are therefore warranted, in order to elucidate the mechanism of the immunosuppression associated with progression of several diseases and therapy in cattle.
  • Hirohisa Mekata, Satoru Konnai, William H. Witola, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi
    INFECTION GENETICS AND EVOLUTION 9 6 1301 - 1305 2009年12月 [査読有り][通常論文]
     
    In South American countries, trypanosomiasis as a result of Trypanosoma evansi and Trypanosoma vivax infections causes significant economic losses in livestock. The objectives of this study were to characterize the epidemiology of bovine trypanosomiasis in South America and to draw a comparison between South American and Asian T. evansi isolates based on the polymorphisms in their transferrin receptor encoding gene 6. We assessed the prevalence rates of T. evansi and T vivax infections in cattle in different regions of Peru and Bolivia using the polymerase chain reaction (PCR) and found that, in Lima and Pucallpa in the Republic of Peru, T. evansi infection rates were 5.8% (6/104) and 2.5% (5/195), respectively, while in Santa Cruz, Republic of Bolivia, the infection rate for T. evansi was 11.5% (59/510). The prevalence rates of T. vivax in Lima and Santa Cruz were 3.8% (4/104) and 0.9% (5/510), respectively. In T. evansi, uptake of host transferrin is mediated by a receptor derived from the two expression site-associated genes 6 and 7 (ESAG6 and ESAG7). We previously showed that the ESAG6 depicts genetic diversity among different isolates of T. evansi in Asia. In this study, we cloned and sequenced the ESAG6 genes from T evansi isolates from South America, and found, in addition to some of the previously observed variants, 20 novel variants of ESAG6 genes which could be categorized into three new clades among the various isolates. To conclude, the results obtained in this study suggest that T evansi isolates from South America are more diverse than the Asian isolates. (C) 2009 Elsevier B.V. All rights reserved.
  • Shinji Yamada, Satoru Konnai, Saiki Imamura, Martin Simuunza, Mwelwa Chembensofu, Amos Chota, Andrew Nambota, Misao Onuma, Kazuhiko Ohashi
    VETERINARY JOURNAL 182 2 352 - 355 2009年11月 [査読有り][通常論文]
     
    To ascertain the infection rate for tick-borne pathogens in Zambia, an epidemiological survey of Theileria parva, Babesia bigemina and Anaplasma marginale in traditionally managed Sanga cattle was conducted using PCR. Of the 71 native Zambian cattle, 28 (39.4%) were positive for T parva, 16 (22.5%) for B. bigemina and 34 (47.9%) for A. marginale. The mixed infection rate in cattle was 8.5% (6/71), 16.9% (12/71), 7.0% (5/71) and 2.8% (2/71) for T. parval B. bigemina, T. parval A. marginale, B. bigeminal A. marginale and T. parval B. bigeminal A. marginale, respectively. To predict the risk for transmission of tick-borne pathogens from ticks to cattle, a total of 74 Rhipicephalus appendiculatus ticks were collected from a location where cattle had been found positive for T parva. Of the ticks collected, 10 (13.5%) were found to be PCR-positive for T parva. The results suggest that the infection rate for tick-borne pathogens was relatively high in Sanga cattle and that adult R. appendiculatus ticks were highly infected with T parva. (C) 2008 Elsevier Ltd. All rights reserved.
  • Claro N. Mingala, Satoru Konnai, Fe A. Venturina, Misao Onuma, Kazubliko Ohashi
    RESEARCH IN VETERINARY SCIENCE 87 2 213 - 217 2009年10月 [査読有り][通常論文]
     
    This study describes the quantification of cytokine expression of vaccinated water buffaloes with FMD inactivated vaccine. Using real-time PCR quantification assay, expression of Th1 (IL-2, IL-12p40, IFN gamma); Th2 (IL-4, IL-10) and inflammatory (IL-6, TNF alpha) cytokines were quantified weekly for the entire three-week duration of the experiment. It was noted that IFN gamma, IL-10 and TNF alpha had peaked on week three post-vaccination while the remaining cytokines peaked on the second week and decreased by the third week. The counteraction between IFN gamma and IL-4 was noted as well as the possible suppressive action of IL-10 to that of IL-2 and IL-12, which is a common phenomenon between Th1 and Th2 cytokines. Synergy between TNFa and IL-6 was also observed. These findings suggest that within the immune system of water buffalo there is a dynamic cell-mediated and humoral interaction in response to immunogen. This assessment of the cytokine expressions is vital for the study of water buffalo disease progression and concurring protective immune responses. (C) 2009 Elsevier Ltd. All rights reserved.
  • Claro N. Mingala, Satoru Konnai, Motoshi Tajima, Misao Onuma, Kazuhiko Ohashi
    JOURNAL OF BASIC MICROBIOLOGY 49 5 495 - 500 2009年10月 [査読有り][通常論文]
     
    Characterization of bovine viral diarrhea virus (BVDV) isolates has been focused of several studies this last decade. Until now lots of new strains are being unfolded maybe due to the viral fast mutation ability. As we focused our research on water buffalo immunology, we were able to identify a probable new BVDV isolates. RNA was extracted from water buffalo blood in the Philippines. The extracted RNA was reverse-transcribed and synthesized cDNA. Oligonucleotide primers from the viral E2 region were used to amplify the target viral gene and later purified, cloned and sequenced. The E2 region with 420 bp nucleotides long was compared with existing published sequences in the GenBank. Based on the constructed phylogenetic tree, the isolated strain showed to be a BVDV type 1b along with Osloss and CP7 strains. Further classification of the new isolates was done within the BVDV type 1b1 group, which was compared with other strains in the sub-group. The analysis revealed that Lamspringe/738, KE9 and 2543/87 were the closest with 92% homology. Additional study is being done to further qualify and quantify the extent of the existence of this new BVDV isolates in water buffalo in the Philippines. This is the first report of BVDV in the Philippines and first concerning BVDV in Philippine water buffalo.
  • Shinji Yamada, Satoru Konnai, Saiki Imamura, Takuya Ito, Misao Onuma, Kazuhiko Ohashi
    VACCINE 27 43 5989 - 5997 2009年10月 [査読有り][通常論文]
     
    Male tick-derived voraxin alpha and voraxin beta, a pair of testicular proteins, are transferred to female via copulation to stimulate female blood feeding in the tick Amblyomma hebraeum (A. hebraeum). Immunized animals with recombinant (r-)voraxin alpha and voraxin beta have been shown as highly resistant to the tick infestation. In this study, we describe the cloning and characterization of voraxin alpha homologue from the tick Rhipicephalus appendiculatus (R. appendiculatus), the major vector for East Coast fever in Eastern Africa. The sequence analysis of the R. appendiculatus voraxin alpha indicated that the deduced amino acid sequence had high similarity with voraxin alpha of the tick A. hebraeum and Dermacentor variabilis, suggesting that voraxin alpha is conserved in different tick genera. Quantitative RT-PCR and Western blotting analysis showed that male voraxin alpha was predominantly expressed in testis and its expression was induced by blood feeding. X appendiculatus voraxina was not secreted into the host during tick feeding and was detected in mated female hemolymph as measured by Western blotting. Preliminary vaccination of rabbits with r-voraxin alpha elicited the humoral immunity and conferred protective immunity against female ticks, resulting in the reduced fed weight. These results suggest that r-voraxin alpha could be a good candidate as anti-tick vaccine. (C) 2009 Elsevier Ltd. All rights reserved.
  • Saiki Imamura, Itabajara da Silva Vaz, Satoru Konnai, Shinji Yamada, Chie Nakajima, Misao Onuma, Kazuhiko Ohashi
    EXPERIMENTAL AND APPLIED ACAROLOGY 48 4 345 - 358 2009年08月 [査読有り][通常論文]
     
    We report the cloning, expression and characterization of an Haemaphysalis longicornis metalloprotease (named HLMP1). The gene encodes a predicted 550 aminoacid protein with similarity to metalloproteases of the reprolysin family. The protein sequence contains a signal sequence, the zinc-binding motif (HEXXHXXGXXH) common to metalloproteases and a cysteine-rich region. Reverse transcription-PCR expression analysis indicates the presence of mRNA in the salivary gland of larva, nymph and adult ticks. Rabbit repeatedly infested with H. longicornis recognized rHLMP1, suggesting that the immune-response against HLMP1 is naturally induced through the feeding of ticks. Vaccination of rabbit with rHLMP1 produced protective immunity against ticks, resulting in 15.6 and 14.6% mortality in nymph and adult ticks, respectively. This work provides information to understand the tick's defense system, and offers new insights to develop strategies to block this defense system with an anti-tick vaccine based on a metalloprotease.
  • Y. Saito, S. Konnai, S. Yamada, S. Imamura, H. Nishikado, T. Ito, M. Onuma, K. Ohashi
    INSECT MOLECULAR BIOLOGY 18 4 531 - 539 2009年08月 [査読有り][通常論文]
     
    Ixodes persulcatus is the primary vector for human tick-borne diseases in Japan. A cDNA library was constructed from whole body homogenates of fed nymphs of I. persulcatus. From this library, one cDNA encoding defensin-like antimicrobial peptide was identified. The amino-acid sequence showed high similarity to those of the defensins of other ticks and arthropods. I. persulcatus defensin mRNA transcripts were detected at all life cycle stages of fed ticks and found to be predominantly expressed in the midguts of adult female ticks, but not in the salivary glands, a finding corroborated by Western blotting analysis. To investigate the function of I. persulcatus defensin, we examined its antibacterial activity by evaluation of growth of several bacterial strains in the presence of the synthetic peptide. The defensin from I. persulcatus markedly inhibited the growth of Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis and Corynebacterium renale, but not Gram-negative bacteria except Escherichia coli O157. In conclusion, these results suggest that I. persulcatus defensin may be playing a significant role in the defence against microbes from bloodmeals.
  • Satoru Konnai, Hirohisa Mekata, Claro N. Mingala, Nancy S. Abes, Charito A. Gutierrez, Jesus Rommel V. Herrera, Alan P. Dargantes, William H. Witola, Libertado C. Cruz, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi
    INFECTION GENETICS AND EVOLUTION 9 4 449 - 452 2009年07月 [査読有り][通常論文]
     
    Trypanosoma evansi (T. evansi) causes the disease called Surra in domestic animals, which is of great economic importance in South Asian countries. In order to improve the diagnosis of Surra, we endeavored to develop a real-time PCR assay for the detection and quantification of parasites in water buffaloes using specific primers for the T. evansi Rode Trypanozoon antigen type (RoTat) 1.2 Variable Surface Glycoprotein (VSG) gene, which is a known diverse DNA region in trypanosomes. The quantitative detection limit of the assay was 10(2) trypanosomes per mL of blood, and the identity of the amplicon was confirmed in all assays by melting curve analysis. To evaluate the clinical applicability of this procedure, detection and estimation of parasitemia in blood samples obtained from water buffaloes and horses were conducted. T evansi was detected in 17/607 (2.8%) blood samples, with parasitemia levels ranging from >10(1) to 10(7) parasites per mL of blood. Interestingly, out of the 17 PCR positive animals, 3 had previously received trypanocidal treatment and 1 had abortion history. These data indicate that real-time PCR for the estimation of putative parasitemia levels is a quantitatively and objectively applicable technique for clinical diagnosis of Surra, and could help to understand disease stage and risk of transmission of T. evansi. (C) 2009 Elsevier B.V. All rights reserved.
  • Carlos Logullo, William H. Witola, Caroline Andrade, Leonardo Abreu, Josiana Gomes, Itabajara da Silva Vaz, Saiki Imamura, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma
    VETERINARY PARASITOLOGY 161 3-4 261 - 269 2009年05月 [査読有り][通常論文]
     
    Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism in mammals. GSK-3 belongs to a highly conserved family of serine/threonine protein kinases, whose members are involved in hormonal regulation, nuclear signaling, and cell fate determination in higher eukaryotes. We have cloned and characterized the RmGSK-3 gene from Rhipicephalus (Boophilus) microplus tick embryos. DNA and protein sequence analysis depicted high similarity to the corresponding enzyme, from both vertebrate and invertebrate animals. In addition, the mRNA transcription profile identified during embryogenesis was analyzed. We observed that the RmGSK-3 mRNA rapidly decreases from the 1st to 3rd day of development, and increases from the 3rd to 15th day. After the 15th day of development, we observed a near 50% reduction in RmGSK-3 mRNA transcription in comparison to the 1st day. We detected the GSK-3 P isoform in egg homogenates throughout embryogenesis using Western blot analysis. RmGSK-3 mRNA was present in fat body, midgut and ovary from partially and fully engorged adult female ticks. The highest mRNA level was observed in ovaries from both developmental stages and in first-day eggs. Furthermore, RmGSK-3 activity correlated with glycogen content variation. Finally, kinase activity in egg homogenates was inhibited by the specific inhibitor, SB-216763. These data suggest that RmGSK-3 beta may be involved in glycogen metabolism regulation during R. microplus embryogenesis. (c) 2009 Elsevier B.V. All rights reserved.
  • Claro N. Mingala, Satoru Konnai, Libertado C. Cruz, Misao Onuma, Kazuhiko Ohashi
    CYTOKINE 46 2 273 - 282 2009年05月 [査読有り][通常論文]
     
    This moleculo-epidemiological and immunological study through cytokine response assessment was done to know the dynamics of cytokines in the initiation, persistence and association to physiological changes of a particular pathogen in water buffaloes. This is important to understand the magnitude and behavior of disease progression. Water buffalo blood samples gathered from different places in the Philippines revealed a 9.4%. 27.6%, 10.3% and 4.4% prevalence of bovine viral diarrhea virus (BVDV), bovine leukemia virus (BLV), Anaplasma marginale and Babesia bigemina infection, respectively. This was the first surveillance study of BVDV and BLV in the country. Furthermore, cytokine expression of these naturally infected animals was also quantified. BVDV-infected animals had up-regulated expressions of TNF alpha, IL-2 and IL-4; and down-regulated expressions of IFN gamma and IL-12p40 while BLV positive animals had an up-regulated IL-4 and IL-6, and highly expressed IL-10 and IL-12p40 with unchanged IFN gamma expression. Meanwhile, animals infected with A. marginale had all interleukins and IFN gamma up-regulated with significant expression of IL-10 and IL-12p40 similar to the BLV positive animals. Since it was also observed that swamp-type buffaloes were more disease tolerant than riverine-type buffaloes based on the gathered infection rate of each examined pathogen, further assessment was done focusing on the two vital cytokines, IFN gamma and TNF alpha. We quantified IFN gamma and TNF alpha expressions in ConA-stimulated PBMC from both swamp and riverine buffaloes by real-time PCR. Cytokine expression from ConA-stimulated PBMC revealed that both IFN gamma and TNF alpha were more highly expressed in swamp than in riverine buffalo. To further examine the probable cause of expression differences, the proximal promoter region of these two cytokines were sequenced for the presence of nucleotide polymorphism followed by luciferase assay to analyze the effect of these polymorphisms in gene transcription. A single nucleotide polymorphism was found in the IFN gamma (-299) while eight polymorphisms in the TNF alpha promoter (-541, -553, -562, -596, -609, -655, -659, -688). Luciferase assay showed that both IFN gamma promoter and TNF alpha promoter in swamp-type water buffalo had higher transcription activity compared to riverine-type water buffalo. These findings confirm that IFN gamma and TNF alpha transcriptions in these animals were highly affected by the disparity in the cytokine promoter region. This suggests that disease tolerance or susceptibility of these buffaloes could be due to the differences in their relative cytokine transcription and may relate to pathogen-host specific pathogenesis. (C) 2009 Elsevier Ltd. All rights reserved.
  • Rika Kano, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 71 5 603 - 610 2009年05月 [査読有り][通常論文]
     
    Marek's disease (MD) is a commercially important disease of chickens caused by MD virus (MDV). Although avirulent MDV strains have been used for vaccination to prevent MD outbreaks, the protective mechanism of the vaccine has not been elucidated. In this Study, a comprehensive transcriptional analysis using microarray wits conducted in MDV-infected chickens with and without vaccination at 7 and 21 days post-infection (dpi). The data Suggested that the expression of T cell receptor (TCR) 1-related genes was up-regulated in vaccinated-challenged compared to unvaccinated-challenged chickens during the latent phase of infection. Consistently, this induction was confirmed by quantitative PCR. Flow cytometric analysis revealed that most of TCR1(+) cells expressed CD8 alpha chain brightly. The number of this subpopulation was significantly and specifically increased in vaccinated-challenged chickens at 21 dpi compared to univaccinated-challenged chickens, though it was not the major Population in spleen of chickens. The number of CD8 alpha(high) TCR2(+) cells, the major subpopulation of chicken CD8 alpha(high) cells, was increased in vaccinated chickens with or without challenge compared to univaccinated control chickens. These data suggested that both CD8 alpha(high) TCR1(+) and CD8 alpha(high) TCR2(+) cells could be induced by the vaccination. It is also possible that CD8 alpha(high) TCR1(+) cells might be primed by the vaccination and specifically induced by the challenge with virulent strain of MDV during the latent phase of infection. Thus, CD8 alpha(high) TCR1(+) cell Population is probably one of the key factors involved in the protective mechanism induced by a vaccine strain, CV1988.
  • Rika Kano, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi
    MICROBIOLOGY AND IMMUNOLOGY 53 4 224 - 232 2009年04月 [査読有り][通常論文]
     
    Marek's disease has been controlled by vaccination with avirulent strains of MDV. However, the protection mechanism following vaccination is not fully understood. In this study immune responses of PBMC and splenocytes derived from vaccinated chickens challenged with virulent MDV were examined using real-time PCR and ELISA. Higher levels of IFN-gamma induction were observed in chickens vaccinated during the latent phase of infection with virulent MDV than in similarly challenged, unvaccinated chickens. Furthermore, the mean expression of IFNGR2 and IFN regulatory factor-3 mRNAs was significantly higher in vaccinated than in unvaccinated chickens. These results show that IFN-gamma could be one of the important factors in prevention of MD by vaccination and is effective during the latent phase of the infection.
  • Satoru Konnai, Chie Nakajima, Saiki Imamura, Shinji Yamada, Hideto Nishikado, Michi Kodama, Misao Onuma, Kazuhiko Ohashi
    IMMUNOLOGY 126 2 209 - 219 2009年02月 [査読有り][通常論文]
     
    Previously, a putative immunosuppressant-coding gene was identified from a complementary DNA library derived from the salivary glands of partially-fed Haemaphysalis longicornis. Using real-time polymerase chain reaction, the gene was shown to be predominantly expressed during blood feeding with the site of expression being mainly in the salivary glands; this was confirmed by Western blotting analysis. To investigate the function of this novel protein, in this study, we examined the proliferative responses of bovine mononuclear cells and murine splenic cells as well as the expression of profiles of several cytokines in these cells in the presence of the recombinant protein (H. longicornis-derived 36 000 molecular weight protein: rHL-p36). The addition of rHL-p36 at the beginning of the 72 hr cultivation period clearly inhibited proliferation of several mitogen-stimulated cells in a dose-dependent manner, with concomitantly significant down-regulation of messenger RNA levels for interleukin-2. The inhibitory response could be abrogated by blockage of HL-p36 with antibody, suggesting the direct involvement of rHL-p36 in the cell proliferation. Furthermore, the proliferative response of splenocytes isolated from rHL-p36-inoculated mice was significantly lower than for those from control mice, suggesting that rHL-p36 could also directly suppress immune responses in vivo. Interestingly, microarray analysis of the splenocytes showed that the expression of several immunomodulating genes was down-regulated by rHL-p36 inoculation. In conclusion, these results suggest that HL-p36 is an immunosuppressor that might play an important role in the modulation of host immune responses.
  • Shinji Yamada, Satoru Konnai, Saiki Imamura, Martin Simuunza, Mwelwa Chembensofu, Amos Chota, Andrew Nambota, Misao Onuma, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 71 1 49 - 54 2009年01月 [査読有り][通常論文]
     
    Theileria parva (T. parva) causes a highly serious bovine disease called Fast Coast fever (ECF), which is characterized by pyrexia, dyspnea and cachexia and is of great economic importance in African Countries. We hypothesize that the clinical symptoms of ECF could he explained by a cytokine dysregulation. In this study, we investigated the relationship between T parva DNA load and expression levels of cytokine mRNAs in leukocytes from experimentally infected calves by quantitative PCR. The p104 gene, which encodes the T parva 104 kDa microneme-rhoptry protein, was detected in cattle blood from day 10 after T. parvo-infected tick infestation, and the protozoan DNA load was increased together with severity of disease. The mRNA expressions of pro-inflammatory cytokines, such as interleukin (IL)-1 beta and IL-6, were up-regulated with protozoan DNA load increasing. In addition, the level of a type-2 cytokine (IL-10) transcript was also increased during the acute phase. In contrast, the down-regulation or no detectable levels of the expression of type-I cytokines, such as IL-2 and interferon (IFN)-gamma were observed in T. parva-infected animals. Thus. Our observations indicated that high protozoan load and resulting intense inflammatory responses might be involved in the severity of clinical signs observed in T. parva-infection.
  • Shinji Yamada, Yuko Ito, Saiki Imamura, Satoru Konnai, Takuya Ito, Misao Onuma, Kazuhiko Ohashi
    EXPERIMENTAL PARASITOLOGY 120 4 337 - 342 2008年12月 [査読有り][通常論文]
     
    A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks. (C) 2008 Elsevier Inc. All rights reserved.
  • Hirohisa Mekata, Satoru Konnai, Martin Simuunza, Mwelwa Chembensofu, Rika Kano, William H. Witola, Mwase E. Tembo, Harrison Chitambo, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 70 9 923 - 928 2008年09月 [査読無し][通常論文]
     
    The prevalence of trypanosome infections in tsetse flies, Glossina pallidipes, collected front Chiawa and Chakwenga in Zambia with endemic trypanosomosis was assessed by polymerase chain reaction (PCR). Out of the 550 G. pallidipes, 58 (10.5%) flies were found to harbor trypanosome DNA. Infection rates of tsetse with Trypanosoma vivax universal, Trypanosoma congolense savannah, T congolense forest and T. congolense kilifi were 4.2% (23/550), 4.7% (26/550), 1.1% (6/550) and 1.6% (9/550), respectively. To determine the mammalian hosts of T. congolense and T. vivax infections from the tsetse flies, mammalian mitochondrion DNA of blood meal in these flies were analyzed by PCR and subsequent gene sequence analysis of the amplicons. Sequence analysis showed the presence of cytochrome b gene (cyt b) of 7 different mammalian species such as human, elephant, buffalo, goat, warthog, greater kudu and cattle. Goats which were main livestock in these areas were further examined to know the extent of its contribution ill spreading the infection. We examined the prevalence of trypanosome infections in the domestic goat Population in 6 settlements in Chiawa alone. Of the 86 goats sampled, 4 (4.6%), 5 (5.8%), 4 (4.6%) and 4 (4.6%) were positive for T. vivax universal, T. congolense savannah, forest and kilifi, respectively. These findings showed that the host-source of trypanosome infections in vector fly give a vital information about spread of infection. The result of this study will certainly contribute in elucidating more the epidemiology of trypanosomosis.
  • Prevalence and Source of Trypanosome Infections in Field-Captured Vector Flies (Glossina pallidipes) in Southeastern Zambia
    Hirohisa Mekata, Satoru Konnai, Martin Simuunza, Mwelwa Chembensofu, Rika Kano, William H. Witola, Mwase E. Tembo, Harrison Chitambo, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 70 9 923 - 928 2008年09月 [査読有り][通常論文]
     
    The prevalence of trypanosome infections in tsetse flies, Glossina pallidipes, collected front Chiawa and Chakwenga in Zambia with endemic trypanosomosis was assessed by polymerase chain reaction (PCR). Out of the 550 G. pallidipes, 58 (10.5%) flies were found to harbor trypanosome DNA. Infection rates of tsetse with Trypanosoma vivax universal, Trypanosoma congolense savannah, T congolense forest and T. congolense kilifi were 4.2% (23/550), 4.7% (26/550), 1.1% (6/550) and 1.6% (9/550), respectively. To determine the mammalian hosts of T. congolense and T. vivax infections from the tsetse flies, mammalian mitochondrion DNA of blood meal in these flies were analyzed by PCR and subsequent gene sequence analysis of the amplicons. Sequence analysis showed the presence of cytochrome b gene (cyt b) of 7 different mammalian species such as human, elephant, buffalo, goat, warthog, greater kudu and cattle. Goats which were main livestock in these areas were further examined to know the extent of its contribution ill spreading the infection. We examined the prevalence of trypanosome infections in the domestic goat Population in 6 settlements in Chiawa alone. Of the 86 goats sampled, 4 (4.6%), 5 (5.8%), 4 (4.6%) and 4 (4.6%) were positive for T. vivax universal, T. congolense savannah, forest and kilifi, respectively. These findings showed that the host-source of trypanosome infections in vector fly give a vital information about spread of infection. The result of this study will certainly contribute in elucidating more the epidemiology of trypanosomosis.
  • Satoru Konnai, Hirohisa Mekata, Raadan Odbileg, Martin Simuunza, Mwelwa Chembensof, William Harold Witola, Mwase Enala Tembo, Harrison Chitambo, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi
    VECTOR-BORNE AND ZOONOTIC DISEASES 8 4 565 - 573 2008年08月 [査読有り][通常論文]
     
    The prevalence of trypanosome infections in tsetse flies in the Chiawa area of Lower Zambezi in Zambia, with endemic trypanosomosis, was determined by a polymerase chain reaction (PCR) method that allowed the detection of trypanosome DNA and determination of the type of animal host fed on by the tsetse fly Glossina pallidipes, using tsetse-derived DNA extracts as templates. Ninety G. pallidipes (82 females and 8 males; 18.3%) of the 492 flies captured by baited biconical traps tested positive for the presence of Trypanosoma brucei species genomic DNA. Of the 90 T. brucei-positive flies, 47 (52.2%) also tested positive for vertebrate mitochondrial DNA. Sequence analysis of the vertebrate mitochondrial DNA amplicons established that they originated from 8 different vertebrate species, namely, human (Hoino sapiens), African elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), waterbuck (Kobus ellipsipymnus), roan antelope (Hippotragus equinus), greater kudu (Tragelaphus strepsiceros), warthog (Phacochoerus africanus), and goat (Capra hircus). Furthermore, to investigate the prevalence of trypanosome infections in domestic goats in the same area where trypanosomes had been detected in tsetse files, a total of 86 goats were randomly selected from 6 different herds. Among the selected goats, 36 (41.9%) were found to be positive for T. brucei species. This combined detection method would be an ideal approach not only for mass screening for infection prevalence in tsetse populations, but also for the prediction of natural reservoirs in areas endemic for trypanosomosis.
  • 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.
  • Saiki Imamura, Satoru Konnai, Itabajara da Silva Vaz Junior, Shinji Yamada, Chie Nakajima, Yuko Ito, Tomoko Tajima, Jun Yasuda, Martin Simuunza, Misao Onuma, Kazuhiko Ohashi
    JAPANESE JOURNAL OF VETERINARY RESEARCH 56 2 85 - 98 2008年08月 [査読有り][通常論文]
     
    Rhipicephalus appendiculatus serpin-3 (RAS-3), R. appendiculatus serpin-4 (RAS-4) and a 36-kDa immuno-dominant protein of R. appendiculatus (RIM36) were reported as candidate antigens for the anti-tick vaccine to control ixodid ticks. In the present study, we generated recombinant proteins of RAS-3 (rRAS-3), RAS-4 (rRAS-4) and RIM36 (rRIM36), and assessed their potency as an anti-tick cocktail vaccine in cattle model. RT-PCR analysis showed that RAS-3, RAS-4 and RIM36 transcripts were detected in both adult male and female ticks during feeding. Immunization of cattle with the combination of rRAS-3, rRAS-4 and rRIM36 had raised antibodies against all recombinants and anti-sera had reacted with the molecules from the tick salivary gland extract. Tick infestation challenge demonstrated protective immunity against female ticks, resulting in mortality rates of 39.5 and 12.8 % for the vaccinated and control groups, respectively. Moreover, the mortality rate of Theileria parva-infected female ticks was 48.5 and 10.8 % in the vaccinated and control group, respectively. In order to evaluate the levels of pathogen transmission capacity by T parva-infected ticks fed on immunized cattle, the occurrence of T parva in the bovine parotid lymph node and peripheral blood was also determined and quantified by real-time PCR. Although the infection with T parva could not be protected by the vaccine, the occurrence of pathogen in peripheral blood was delayed 1 to 2 days after the infestation challenge in vaccinated group. These results suggest that this cocktail vaccine plays a role in the prevention of tick infestation.
  • Satoru Konnai, Claro N. Mingala, Misako Sato, Nancy S. Abes, Fe A. Venturina, Charito A. Gutierrez, Takafumi Sano, Yoshitaka Omata, Libertado C. Cruz, Misao Onuma, Kazuhiko Ohashi
    ACTA TROPICA 105 3 269 - 273 2008年03月 [査読有り][通常論文]
     
    In the Philippines, insufficient consideration has been given to the implementation of systematic control measures against major abortifacient infectious agents in livestock. To elucidate the epidemiology of abortifacient infectious agents in livestock, the prevalence of four abortifacient agents was assessed. Initially, a total of 96 cattle including 17 cows with history of abortion were examined in a herd in Luzon at the request of the farm owner. Six (35.3%) of the 17 aborting cows were found to be serologically positive for Neospora caninum (N. caninum), whereas the seroprevalence in non-aborting cows was 15.9% (10/63). Four of the 6 serologically positive aborting cows were also RT-PCR-positive for bovine viral diarrhea virus (BVDV). Two (12.5%) of the 16 bulls examined were also found to be infected with BVDV, suggesting a putative risk factor of transmission via semen. Based on sequence analysis, the isolates detected belong to BVDV type 1b group. Furthermore, an epidemiological survey of abortifacient infectious agents was conducted with various species of livestock from herds located in Luzon. Out of the 105 water buffalo samples collected, 4 (3.8%) were indicated positive to N. caninum, 2 (1.9%) to Toxoplasma gondii (T gondii) and 2 (1.9%) to Trypanosoma evansi (T evansi). The overall seroprevalence of N. caninum in goat and sheep were 23.6% (21/89) and 26.3% (10/38), respectively. BVDV was not detected in these herds. The findings of this exploratory study indicate a relationship between infection and bovine abortion and that a lager study is required to statistically confirm this relationship. (C) 2007 Elsevier B.V. All rights reserved.
  • Raadan Odbileg, Byambaa Purevtseren, Dorj Gantsetseg, Bazartseren Boldbaatar, Tumurjav Buyannemekh, Zagd Galmandakh, Janchivdorj Erdenebaatar, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 70 2 197 - 201 2008年02月 [査読有り][通常論文]
     
    In the present study, we determined the levels of cytokines produced by camel (Camelus bactrianus) peripheral blood mononuclear cells (PBMCs) in response to live attenuated Brucella abortus (B. abortus) S19 vaccine. Seven camels were vaccinated with commercial B. abortus S 19 vaccine, and their cytokine responses were determined using a real-time PCR assay. Cytokine responses to B. abortus S 19 were examined at 6 hr, 48 hr and 1, 2 and 3 weeks post-vaccination. Serological tests were performed to further confirm these immune responses. The results revealed that IFN-gamma and IL-6 were upregulated during the first week post-vaccination. Low level expressions of IL-1 alpha, IL-1 beta, TNF-alpha and IL-10 and no expression of IL-2 and IL-4 were observed compared with the control camels. The findings showed that B. abortus stimulates cell-mediated immunity by directly activating camel Thl cells to secrete IFN-gamma. This quantification of cytokine expression in camels is essential for understanding of Camelidae disease development and protective immune responses. This is the first report of in vivo camel cytokine quantification after vaccination.
  • Establishment of a laboratory colony of taiga tick Ixodes persulcatus for tick-borne pathogen transmission studies
    Satoru Konnai, Yoichi Saito, Hideto Nishikado, Shinji Yamada, Saiki Imamura, Akina Mori, Takuya Ito, Misao Onuma, Kazuhiko Ohashi
    JAPANESE JOURNAL OF VETERINARY RESEARCH 55 2-3 85 - 92 2008年01月 [査読有り][通常論文]
     
    Ixodes persulcatus Schulze (I. persulcatus) is distributed in Russia and Far East Asia including Japan, and has been implicated as the vector of several human pathogens. In particular, I. persulcatus acts as the only tick vector for human lyme borreliosis in Japan. In order to elucidate the mechanism of transmission of I. persulcatus -borne pathogens, we developed a laboratory colony of I. persulcatus. Ticks were fed on Syrian hamster and engorged ticks that had dropped off the animals were collected and maintained to allow them to molt. Tick rearing was performed in incubator at 20 degrees C with 95% relative humidity and 12-hour light/dark photo-period regimen. We found out that adult females fed for 8 +/- 2 days and had a pre-oviposition period lasting for 7 +/- 2 days. The minimum egg incubation period was 1 month with the hatched larvae feeding for 3 +/- 1 days and molting to nymphs 3-4 months thereafter. Meanwhile, the nymphs fed for 4 +/- 1 days and molted to adult 2-3 months thereafter. For future analysis of gene expression profiles in I. persulcatus, we cloned and sequenced the actin gene (a housekeeping gene), and found that it is 92.7% to 98.6% homologous to the published sequences of related ixodid ticks. This laboratory colony of I. persulcatus will facilitate investigations on the role of tick-derived molecules on the transmission of I. persulcatus -borne pathogens and will be important for identification of potential anti-tick vaccine and acaricide target molecules.
  • 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.
  • Konnai S, Yamada S, Imamura S, Simuunza M, Chembensof M, Chota A, Nambota A, Ohashi K, Onuma M
    Vector Borne Zoonotic Dis. 7 2 241 - 8 2007年07月 [査読有り][通常論文]
  • Claro N. Mingala, Raadan Odbileg, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma
    COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 30 2 119 - 131 2007年03月 [査読有り][通常論文]
     
    The current research concerned in the cloning, sequencing and phylogenctic analysis of inflammatory cytokine (IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha) genes from swamp buffalo and two bubaline breeds, CB (cross between swamp and riverine type buffalo) and the Bulgarian Murrah buffalo. Multiple sequence comparison showed a high homology between the bubaline breeds, which ranged from 99.3% to 100.0% similarity, whereas from 98.6% to 99.0% compared to cattle. The phylogenetic analysis had confirmed and justified the degree of relationship between these bubaline species and their distinctness to each other by the bootstrap value (%) generated. These findings were discussed with particular attention to the diversity of the inflammatory cytokine proteins within closely related species. The result of this study concluded that a small difference in the cytokine structures might be the reason behind or has a contributory factor on the previous reports about the existence of disease resistance. However, in-depth study is necessary to further qualify these findings. (c) 2007 Elsevier Ltd. All rights reserved.
  • Tatsufumi Usui, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 115 1-2 17 - 23 2007年01月 [査読有り][通常論文]
     
    Immunological control of bovine leukemia virus (BLV)-infection has been reported as dependent on the expression balance of types 1 and 2 cytokines. In this report, mRNA expression of interferon (IFN)-gamma and interleukin (IL)-2 (type 1 cytokines), and of IL-4 and IL-10 (type 2 cytokines) were evaluated in concanavalin A-stimulated peripheral blood mononuclear cells (PBMC) from BLV-infected sheep. Despite the same dose of BLV-infection, the extent of viral propagation was markedly different between eight individual sheep by 12 weeks post infection. The virus did not propagate well in three sheep, which showed augmented mRNA expression of IFN-gamma, a strong indicator of cell-mediated immunity, immediately after BLV-infection. Among the other five sheep having more than 2% of BLV-infected cells among PBMC at 12 weeks post infection, four sheep developed B-cell leukemia or lymphoma within 2 years after infection. These observations indicate IFN-gamma expression may play an important role in the protective mechanism against BLV propagation at the early phase of the infection. (c) 2006 Elsevier B.V. All rights reserved.
  • Claro N. Mingala, Raadan Odbileg, Satoru Konnal, Kazuhiko Ohashi, Misao Onuma
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 113 3-4 348 - 356 2006年10月 [査読有り][通常論文]
     
    Comparative assessment of Th1 and Th2 cytokines of three bubaline breeds namely swamp buffalo, its crossbreed with riverine buffalo (CB), and the improved breed of Bulgarian Murrah buffalo (BMB), was done by molecular cloning, sequencing and phylogenetic analysis. The Th I cytokines analyzed included IL-2, IL-12p35, IL-12p4O, and IFN-gamma while Th2 cytokines included IL-4 and IL-10. Both groups showed strict conservation in the putative secondary structures and amino acid residues within the tribe Bovini, which indicated functional cross-reactivity. Nucleotide sequence homology ranged from 98.6 to 100.0% and was lowest for IL-12p35. With regard to amino acid sequence, the lowest homology was observed in IL-4 with 97.8%. This substitution was mainly due to differences in mRNA splicing. The phylogenetic relationship of the buffalo breeds was analyzed and showed them as a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle and pigs. A deeper knowledge of these cytokine structures will favor understanding of water buffalo immunology and how much it differs from its closest subspecies and other animals. (c) 2006 Elsevier B.V. All rights reserved.
  • Raadan Odbileg, Byambaa Purevtseren, Zayat Batsukh, Satoru Konnaii, Kazuhiko Ohashi, Misao Onuma
    JOURNAL OF VETERINARY MEDICAL SCIENCE 68 9 941 - 946 2006年09月 [査読有り][通常論文]
     
    The complementary DNAs of the Th1 (IL-2, 12-12p35, and IFN-gamma) and Th2 (IL-4, IL-10 and IL-13) cytokine genes of the bactrian camel (Camelus bactrianus) were cloned, sequenced, and analyzed. IL-2, IL-4, IL-10, IL-12p35, IL-13, and IFN-gamma were found to have 465, 402, 537, 669, 411, and 501 bp length open reading frames with 154, 133, 178, 222, 136, and 166 amino acid encodings, respectively. The homology ranged from 58.8% to 100% between the nucleotide sequences of the camel cytokine genes and the published sequences of other mammalian genes, including the llama, pig, cow, horse, human, and mouse. The cDNA had highest homology with orders Artiodactyla (pigs and cattle) and Perissodactyla (horses), especially to the recently cloned llama sequences.
  • Tatsufumi Usui, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 112 3-4 296 - 301 2006年08月 [査読有り][通常論文]
     
    Tumor necrosis factor (TNF)-cL is thought to be one of the cytokines that account for bovine leukemia virus (BLV)-induced B-cell lymphoproliferative disorder, however, information on TNF-alpha expression in B-cells is limited. In this study, the expression of TNF-alpha in IgM(+) B-cells from BLV-infected sheep with or without lymphocytosis was determined. Freshly isolated IgM(+) B-cells from three sheep with lymphocytosis constitutively transcribed TNF-alpha mRNA. Although TNF-alpha mRNA expression in IgM(+) B-cells was transiently up-regulated after cell culture, TNF-alpha mRNA expression was markedly higher in lymphocytotic sheep when compared to that of non-lymphocytotic sheep or uninfected sheep. Expression of membranebound TNF-alpha on IgM(+) B-cells was also augmented in lymphocytotic sheep. TNF-alpha expression in lymphocytotic sheep may support the proliferation of B-cells. (c) 2006 Elsevier B.V. All rights reserved.
  • Satoru Konnai, Saiki Imamura, Chie Nakajima, William Harold Witola, Shinji Yamada, Martin Simuunza, Andrew Nambota, Jun Yasuda, Kazuhiko Ohashi, Misao Onuma
    ACTA TROPICA 99 1 34 - 41 2006年08月 [査読有り][通常論文]
     
    In order to investigate the transmission dynamics of Theileria parva (T parva) by the brown ear tick, Rhipicephalus appendiculatus (R. appendiculatus), under experimental conditions, detection of T parva in ticks and cattle was performed by a quantitative real-time PCR assay. A calf inoculated with a T parva mixture became PCR-positive for T parva infection on day 8 post-inoculation, and subsequently, nymphal ticks were introduced and maintained to feed on the infected calf for 6 days. Engorged nymphs were collected daily and allowed to molt into adults, and overall, 70.8% (121/171) of the adult ticks acquired the T parva infection. Furthermore, the T parva infection rate in ticks under field conditions was monitored by real-time PCR in R. appendiculatus ticks collected from a traditionally managed pastoral land of Zambia, on which Sanga breed cattle are traditionally reared and the area has endemic East Coast fever (ECF). A total of 70 cattle were randomly selected in the same area and 67 (95.7%) were found to be serologically positive for R. appendiculatus tick antigen (RIM36). Twenty-nine (43.3%) of the 67 serologically positive cattle were real-time PCR-positive for T parva, although no piroplasms could be detected in the blood smears. Unexpectedly, out of 614 R. appendiculatus nymphal and adult ticks collected by flagging vegetation, 4.1% were positive for T parva DNA. However, since the rate of transmission of T parva from infected cattle to ticks and vice versa and the serological evidence of exposure to R. appendiculatus ticks in naturally exposed cattle were relatively high, it would be wise in such a case to consider vector control as well as vaccination against ECF as control measures. (c) 2006 Elsevier B.V. All rights reserved.
  • Satoru Konnai, Tatsufumi Usui, Manabu Ikeda, Junko Kohara, Toh-ichi Hirata, Kosuke Okada, Kazuhiko Ohashi, Misao Onuma
    MICROBES AND INFECTION 8 8 2163 - 2171 2006年07月 [査読有り][通常論文]
     
    In a previous report, we had indicated that in a sheep model, the expression of tumor necrosis factor (TNF)-alpha was closely associated with disease progression in sheep experimentally infected with bovine leukemia virus (BLV). However, individual variabilities are observed in these responses in BLV-infected animals. To attempt to identify genetic factors promoting the progression to BLV-induced lymphoma, we endeavored to determine whether there are any polymorphisms in the TNF-alpha gene among 291 individuals and whether this would affect the level of TNF-alpha expression and concomitant progression of BLV-induced disease or increase in the provirus load in the carriers. We found that the frequency of the TNF-alpha -824G allele, which has been associated with low transcription activity of the promoter/predicted enhancer region of the bovine TNF-alpha gene, was higher in individuals with BLV-induced lymphoma than in asymptomatic carrier individuals. In addition, we observed a tendency for increased BLV-provirus load in cattle with TNF-alpha -824G/G homozygote compared to TNF-alpha -824A/A homozygote or TNF-alpha -824A/G. These data suggest that the observed polymorphism in the promoter region of TNF-alpha gene could at least in part contribute to the progression of lymphoma in BLV-infection. (c) 2006 Elsevier SAS. All rights reserved.
  • A Tsuda, WH Witola, S Konnai, K Ohashi, M Onuma
    PARASITOLOGY INTERNATIONAL 55 2 135 - 142 2006年06月 [査読有り][通常論文]
     
    Drug resistance in Trypanosoma brucei causes severe problems for people and domestic animals, but molecular mechanisms of the resistance are not well known. Programmed cell death (PCD) is a fundamental process in both multicellular and unicellular organisms, and it is speculated to be one of the important factors contributing to the emergence of drug resistance. We have previously reported that the expression of TAO appears to play a role in the inhibition of the PCD-like phenomenon development in T brucei. In this study, to ascertain the correlation between the development of the PCD-like phenomenon and the expression of TAO in T. brucei, we genetically engineered T. brucei for conditional overexpression of the TAO gene. TAO over-expressing transgenic T. brucei was refractory to the development of the PCD-like phenomenon compared to the wild-type, indicating that expression of TAO might have a regulatory role on PCD development. Furthermore, the transgenic cells showed resistance to suramin and antrycide. We postulated that intracellular reactive oxygen species (ROS) may be involved in the mechanism of resistance to antrycide because augmentation of ROS in transgenic cells was lower than that in the wild-type cells following treatment with antrycide. These results suggest a possible correlation of PCD to drug resistance in T. brucei. (c) 2006 Elsevier Ireland Ltd. All rights reserved.
  • C Nakajima, S Imamura, S Konnai, S Yamada, H Nishikado, K Ohashi, M Onuma
    JOURNAL OF VETERINARY MEDICAL SCIENCE 68 5 447 - 452 2006年05月 [査読有り][通常論文]
     
    A novel thrombin inhibitory protein coding gene was identified from a cDNA library derived from salivary gland of partially-fed Haemaphysalis longicornis (hard tick). The gene encoded a 93-amino acid protein. designated chimadanin. Which had a signal peptide sequence and was predicted to be a secretory protein. It showed no similarity to any other previously identified proteins or conserved domain sequences. The gene was expressed during blood feeding and suggested to be expressed mainly in the salivary gland. The predicted mature region of chimadanin was expressed in Escherichia coli and characteristics of the recombinant chimadanin were determined. The activated partial thromboplastin time and the prothrombin time in sheep plasma were significantly prolonged by chimadanin in a dose dependent manner. Amidolytic activity of thrombin was also inhibited by chimadanin in a dose dependent manner and it suggested that chimadanin was an anticoagulant with thrombin inhibitory activity. This newly identified thrombin inhibitor may play an important role in tick blood feeding.
  • Experimental transmission of Bovine leukemia virus in cattle via rectal palpation
    Junko Kohara, Satoru Konnai, Misao Onuma
    JAPANESE JOURNAL OF VETERINARY RESEARCH 54 1 25 - 30 2006年05月 [査読有り][通常論文]
     
    We examined whether Bovine leukemia virus (BLV) was transmitted by rectal palpation using a common sleeve between a BLV-infected cow and BLV-negative steers. Three of four steers developed antibodies against BLV as determined by agar-gel immunodiffusion (AGID) test between 7 to 10 weeks after the first rectal palpation using common sleeves from BLV-infected cow. In the steers, BLV proviral DNA were detected by PCR I to 5 weeks earlier than detection of the antibodies by the AGID test. Our experiments demonstrated that rectal palpation is a potential cause of BLV spread in herds and that detection of BLV proviral DNA in cattle by PCR is useful screening test for early diagnosis of BLV infection.
  • S Konnai, T Usui, M Ikeda, J Kohara, T Hirata, K Okada, K Ohashi, M Onuma
    ARCHIVES OF VIROLOGY 151 2 347 - 360 2006年02月 [査読有り][通常論文]
     
    We previously reported that tumor necrosis factor alpha (TNF-alpha) was one of the cytokines that contributed to the leukemogenesis caused by bovine leukemia virus (BLV). To determine if the spontaneous cell proliferation observed in the late disease stages, such as persistent lymphocytosis and lymphosarcoma, correlated with the expression level of TNF-alpha, we analyzed the mRNA expression levels for TNF-alpha in spontaneously proliferating PBMCs derived from BLV-infected cattle. The mean mRNA expression level for TNF-alpha was higher in the spontaneously proliferating PBMCs derived from BLV-infected cattle than in non-spontaneously proliferating PBMCs from normal cattle. The TNF-alpha protein level in the PBMCs was determined by flow cytometric analysis, and it was noted that most of the cells expressing membrane-bound TNF-alpha in the spontaneously proliferating cells were CD5 or sIgM(+) -cells. Additionally, in order to determine if this spontaneous proliferation can be blocked by anti-bovine TNF-alpha MAb, the spontaneously proliferating PBMCs from a BLV-infected cattle were cultured in the presence of the MAb. The addition of this MAb at the beginning of the 72 h-cultivation clearly inhibited spontaneous proliferation of cells in a dose-dependent manner, indicating the direct involvement of TNF-alpha in the spontaneous proliferation of PBMCs during the late disease stage. These data suggest that an aberrant expression of TNF-alpha might contribute to the progression of bovine leukosis in animals which develop persistent lymphocytosis of B-cells or B-cell lymphosarcoma.
  • 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.
  • K Okada, N Nakae, K Kuramochi, S Yin, M Ikeda, S Takami, T Hirata, M Goryo, S Numakunai, S Takeshima, M Takahashi, S Tajima, S Konnai, M Onuma, Y Aida
    JOURNAL OF VETERINARY MEDICAL SCIENCE 67 12 1231 - 1235 2005年12月 [査読有り][通常論文]
     
    Sheep were inoculated with high tax coded pBLV-IF (H group, Nos.1-5) of bovine leukemia virus (BLV), wild tax coded pBLV-IF (W group, Nos. 6-11), or control plasmid (C group, Nos. 12-14). During the observation period (4 to 46 months), 5 of 5 cases in H group and 3 of 6 cases (Nos. 6, 7, 9) in W group became positive tor gp51. Only 1 case in H group became leukemic, and one case each of H and W groups developed lymphoma. In No. 3, lesions Were found in multiple organs including the lymph nodes, gastrointestinal tract following abomasum, and heart. In No. 6, lesions of lymphoma were found only in the jejunum, and heart. Morphologically, small to middle-sized lymphocytic neoplastic (NP) cells were found in both cases, but lymphoblastic NP cells were found only in No. 3. By immunohistochemical examination, the phenotypes of NP dwells were determined as CD1(-), CD4(-), CD5(-)-, CD8 alpha(-), sIgM(+), lambda light chain(+), B-B4(+), MHC class II+ in both case. The results of this study indicate that inoculation of pBLV-IF can induce lymphocytic and lymphoblastic leukemia/lymphoma in sheep. Additionally, it is suggested that the expression rate of tax gene is not associated with the development of leukemia/lymphoma in sheep experimentally inoculated with pBLV-IF.
  • C Nakajima, ID Vaz, S Imamura, S Konnai, K Ohashi, M Onuma
    JOURNAL OF VETERINARY MEDICAL SCIENCE 67 11 1127 - 1131 2005年11月 [査読有り][通常論文]
     
    A cDNA library was constructed from salivary glands of partially-fed adult female Haemaphysalis longicornis (hard tick). Randomly selected clones were sequenced and a total of 633 sequences were analyzed by bioinformatic programs. The sequences were grouped into 213 clusters, with each cluster being considered to be composed of mRNAs derived from the same gene or closely related genes. About 36% of the mRNA sequences showed significant similarity to known proteins in the non-redundant protein database by the NCBI blastx program and appeared to be coding for functional predicted proteins, whereas the remaining 64% had no similar sequences. Two thirds of the predicted proteins were annotated as basic cellular proteins (housekeeping proteins). Among the functional predicted protein sequences, other than the housekeeping proteins, several protease inhibitors including anticoagulants, two metalloproteases and a potential immunosuppressive protein could be identified. These proteins may play important roles during tick feeding and could be novel anti-tick vaccine candidates.
  • S Konnai, T Usui, M Ikeda, J Kohara, T Hirata, K Okada, K Ohashi, M Onuma
    VIROLOGY 339 2 239 - 248 2005年09月 [査読有り][通常論文]
     
    Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-alpha and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-alpha-induced responses, in this study we examined the TNF-alpha-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-alpha (rTNF-alpha) was significantly higher than those from AL cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5(+) or sIgM(+) cells and these cells showed resistance to TNF-a-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-alpha-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection. (c) 2005 Elsevier Inc. All rights reserved.
  • R Odbileg, S Konnai, K Ohashi, M Onuma
    JOURNAL OF VETERINARY MEDICAL SCIENCE 67 9 921 - 925 2005年09月 [査読有り][通常論文]
     
    We cloned, sequenced and analyzed the cDNAs encoding Camelidae inflammatory cytokines, including llama (lama glama) interleukin (IL)-1 alpha, IL-1 beta, IL-6, tumor necrosis factor (TNF)-alpha and camel (Camelus bactrianus) IL-6 and TNF-alpha. The similarity levels of the deduced amino acid sequences of IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha from llama (camel) to those from other mammalian species, ranged from 60.7% to 87.7%, 52.8% to 75.3%, 41.4% to 98.6%, and 72.9% to 99.6%, respectively. Phylogenetic analyses based on nucleic acid sequences showed that llama IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha were more closely related to those of camel, pig, cattle, sheep and horse than to those of human, dog, cat, mouse and rat.
  • M Ikeda, S Konnai, M Onuma, N Ishiguro, M Goryo, K Okada
    JOURNAL OF VETERINARY MEDICAL SCIENCE 67 4 425 - 432 2005年04月 [査読有り][通常論文]
     
    Tumor necrosis factor-alpha (TNF-α) has been reported to be associated with the progression of lymphoproliferative neoplastic diseases and retroviral infections. Hence we examined immunohistochemically the expression patterns of TNF-receptors (TNF-RI and RII) on lymphoma cells derived from the 29 cases of enzootic bovine leukosis (EBL). Lymphomas obtained in 29 animals with EBL were histopathologically classified into three types: diffuse mixed type (10 cases), diffuse large type (9 cases), and diffuse large cleaved type (10 cases). Immunohistochemically using a monoclonal antibody to a bovine lymphocyte surface antigen, the lymphomas were classified into three phenotypes: B-1a (CD5(+)/CD11b(+)), B-1b (CD5(-)/CD11b(+)) and B-2 (conventional 13) (CD5(-)/CDl lb(-)). Interestingly, the lymphoma cells in all animals expressed TNF-RII, but not TNF-RI. Although, in EBL, lymphoma cells of which the histopathological and immunological property differs has been formed, the expression patterns of TNF-Rs had the universality in all lymphoma cells. TNF-RII which induces cell proliferation, was expressed but TNF-RI, which induces cell apoptosis was not expressed on all lymphoma cells, suggesting that TNF-Rs play an important role in the malignant proliferation of B cells and formation of lymphomas in EBL.
  • R Odbileg, S Konnai, T Usui, K Ohashi, M Onuma
    JOURNAL OF VETERINARY MEDICAL SCIENCE 67 2 195 - 198 2005年02月 [査読有り][通常論文]
     
    We have developed a method by which llama cytokine mRNAs can be quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of llama, reverse transcribed to cDNA, and cytokine profiles for interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF) alpha were quantified by real-time PCR. The expressions of mRNAs of inflammatory cytokines IL-1alpha, IL-1beta, IL-6 and TNFalpha were upregulated upon stimulation with LPS in a dose- and time-dependent manner. Incubation of PBMCs with 100 and 1,000 pg/ml of LPS for 3 to 6 hr resulted in the acceleration of the mRNA levels of inflammatory cytokines. Here, we describe a highly sensitive and reproducible method to quantify the transcription of llama cytokine mRNAs by real-time RT-PCR with the double-stranded DNA-binding dye SYBR Green I.
  • Takahashi M, Tajima S, Takeshima SN, Konnai S, Yin SA, Okada K, Davis WC, Aida Y
    Microbes Infect. 6 6 584 - 95 2004年05月 [査読有り][通常論文]
  • T Usui, S Konnai, S Tajima, S Watarai, Y Aida, K Ohashi, M Onuma
    JOURNAL OF VETERINARY MEDICAL SCIENCE 65 11 1201 - 1205 2003年11月 [査読有り][通常論文]
     
    A DNA vaccination trial was performed on sheep to determine whether vaccination with bovine leukemia virus (BLV) transactivator Tax DNA is effective against BLV infection. Immunization was carried out with cationic liposomes containing the Tax-expressing plasmid DNA and subsequently all sheep were challenged with BLV. BLV titers in peripheral blood mononuclear cell (PBMC) determined by syncytium formation assay and BLV provirus load detected by genomic PCR analysis showed higher levels of virus titers in control sheep than those in Tax-vaccinated sheep. Higher levels of IFN-gamma mRNA expression have been demonstrated in vaccinated sheep after the challenge. These results suggested that Th1 type immune response induced by Tax DNA vaccine inhibited BLV propagation in vaccinated sheep at the early phase of infection.
  • S Konnai, Y Nagaoka, S Takesima, M Onuma, Y Aida
    JOURNAL OF DAIRY SCIENCE 86 10 3362 - 3365 2003年10月 [査読有り][通常論文]
     
    The ovine major histocompatibilty complex (Ovar) class II DRB1 second exon was amplified by polymerase chain reaction (PCR) from DNA samples of 52 Suffolk sheep. Polymerase chain reaction products were characterized by the restriction fragment length polymorphism (RFLP) technique using nine restriction enzymes, RsaI, HaeIII, SacI, SacII, DdeI, NciI, Hin1I, EcoRI, and BstNI, yielding 13 types. Sequencing of cloned PCR products identified 16 Ovar-DRB1 alleles. Collectively, all PCR-RFLP patterns exactly matched those predicted from DNA sequences. These findings strongly indicate that the PCR-RFLP method using a combination of nine restriction endonucleases is a very powerful tool in Ovar typing.
  • S Konnai, Y Nagaoka, S Takeshima, M Onuma, Y Aida
    EUROPEAN JOURNAL OF IMMUNOGENETICS 30 4 275 - 282 2003年08月 [査読有り][通常論文]
     
    To investigate the genetic diversity of the sheep MHC (Ovar) class II DRB1 locus, we amplified exon 2 of Ovar-DRB1 alleles by polymerase chain reaction (PCR) and determined the nucleotide sequences of both resultant strands after cloning. In our study of a total of 97 sheep of three breeds, namely, Suffolk, Cheviot and Corriedale, we identified 18 previously published alleles and 17 new alleles. These alleles were 83.4 to 94.1% identical at the nucleotide level and 71.4 to 90.9% identical at the amino acid level to Ovar-DRB1 *0101 . We identified six new alleles in Cheviot sheep and 11 new alleles in Suffolk sheep. Furthermore, we identified 15, 6 and 1 allele in Suffolk, Cheviot and Corriedale sheep, respectively, that have only been found in these breeds to date. Analysis of the frequencies of the various Ovar-DRB1 alleles in each breed indicated that Ovar-DRB1 *0702 was the most frequent allele in Suffolk sheep (23.9%), Ovar-DRB1 *0203 was the most frequent allele in Cheviot sheep (27.5%) and Ovar-DRB1 *0201 was the most frequent allele in Corriedale sheep (25.0%). A comparative analysis of the positions of polymorphic residues in the first extracellular domain of the DRB genes of sheep, humans and mice revealed an extraordinary similarity amongst the positions of polymorphic residues that are associated with the antigen recognition site (ARS). Moreover, the extent of polymorphism seems to be similar in sheep, humans and mice.
  • S Konnai, T Usui, K Ohashi, M Onuma
    VETERINARY MICROBIOLOGY 94 4 283 - 294 2003年07月 [査読有り][通常論文]
     
    For a practical need, fast and efficient methods to quantify mRNA expression are expecting. By using real-time reverse transcription polymerase chain reaction (RT-PCR) with the double-stranded DNA-binding dye SYBR Green I as a novel method, cytokine profiles (IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12p40 and IFN-gamma) were analyzed in peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected animals. In aleukemic cattle, IFN-gamma and IL-12p40 mRNA expression was significantly increased compared to those in cattle with persistent lymphocytosis. The similar results were obtained in the case of sheep experimentally infected with BLV. Real-time quantitative PCR technique is an applicable technique for analysis of cytokine profiles in field. (C) 2003 Elsevier Science B.V. All rights reserved.
  • S Tajima, M Takahashi, S Takeshima, S Konnai, SA Yin, S Watarai, Tana, Y Tanaka, M Onuma, K Okada, Y Aida
    JOURNAL OF VIROLOGY 77 3 1894 - 1903 2003年02月 [査読有り][通常論文]
     
    In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.
  • T Usui, S Meas, S Konnai, K Ohashi, M Onuma
    JOURNAL OF VETERINARY MEDICAL SCIENCE 65 2 287 - 289 2003年02月 [査読有り][通常論文]
     
    Serological survey of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) infection was conducted in dairy cattle from 10 different regions of Hokkaido, Japan. Among 390 cattle, 11.0% of cattle were BIV-seropositive and 3.3% were BLV-seropositive. Moreover, in two dairy farms, where bovine leukosis has been reported, prevalence of BIV infections were 6.4 and 9.1%, respectively. In contrast, among 150 beef cattle, 16.6% were BIV-seropositive while none was BLV-seropositive. Dual infections with BLV and BIV in dairy cattle were tested by using 107 BLV-seropositive sera, and 20 sera were found BIV-positive (18.7%). These results indicate that BIN infection was widespread in Hokkaido.
  • S Konnai, NS Takeshima, S Tajima, SA Yin, K Okada, M Onuma, Y Aida
    MICROBIOLOGY AND IMMUNOLOGY 47 3 223 - 232 2003年 [査読有り][通常論文]
     
    We have reported previously that the alleles of the ovine leukocyte antigen (OLA)-DRB1 gene that encode the Arg-Lys (RK) motif and the Ser-Arg (SR) motif at positions beta(70/71) of the OLA-DRbeta1 domain are associated with resistance and susceptibility, respectively, to development of bovine leukemia virus (BLV)-induced ovine lymphoma. Here, to investigate the different immune response in sheep that carried alleles associated with resistance and susceptible for 30 weeks after infection with BLV, we selected sheep that had the RK/RK or SR/SR genotype among the 52 sheep analyzed by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing of PCR product for the OLA-DRB1 exon 2 and infected them with BLV. Although the number of BLV-infected cells and virus titer had been maintaining low levels throughout the experimental period, the sheep with the RK/RK genotype could induce expansion of CD5-B-cells and rapid production of neutralizing antibody in the early phase of infection. The level of incorporation of [H-3]thymidine by peripheral blood mononuclear cells from the sheep with RK/RK genotype gave a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-helper epitope of the BLV envelope glycoprotein gp51. In contrast, the sheep with SR/SR genotype showed a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-cytotoxic and B-cell epitopes. In such cases, the animals with the RK/RK strongly expressed IFN-gamma, the animals with SR/SR genotype strongly expressed IL-2. To determine the proliferating cells, we tried a blocking assay with monoclonal antibodies such as anti-CD4, -CD8 and -DR molecule. We found that these proliferating cells were MHC-restricted CD4(+) T-cells.
  • Y Kiku, H Matsuzawa, H Ohtsuka, N Terasaki, S Fukuda, S Kon-Nai, M Koiwa, Y Yokomizo, H Sato, TJ Rosol, H Okada, TO Yoshino
    JOURNAL OF VETERINARY MEDICAL SCIENCE 64 8 723 - 726 2002年08月 [査読有り][通常論文]
     
    The effects of chlorpromazine (CPZ), pentoxifylline (PTX) and dexamethasone (DEX) on mRNA expression of lipopolysaccharide (LPS)-induced proinflammatory cytokines were examined in bovine peripheral blood mononuclear cells (PBMCs) in vitro. The expression of inflammatory cytokine mRNAs was analyzed by RT-PCR and Southern blot hybridization in bovine PBMCs. CPZ and DEX decreased the expression of cytokine mRNA (such as interleukin-1beta and tumor necrosis factor-a) after stimulation with LPS in a dose-dependent manner. However, pretreatment with PTX had no inhibitory effect on the mRNA expression of proinflammatory cytokines. These results indicated that pretreatment with CPZ and DEX might be effective to reduce the production of LPS-induced inflammatory cytokines in bovine PBMCs in vitro.
  • 今内 覚, 大塚 浩通, 坂本 哲昭, 中岡 祐司, 菊 住男, 福田 茂夫, 小岩 政照, 高橋 淳吉, 谷山 弘行, 横溝 祐一, 岡田 洋之, 吉野 知男
    The journal of veterinary medical science 63 8 859 - 865 社団法人日本獣医学会 2001年08月25日 [査読有り][通常論文]
     
    一農場で集団発生し, 発熱および下痢を示した搾乳牛のサルモネラ感染症(Salmonella Takoradi) 11例(全例発熱, 下痢)の抹消血について, 末梢血単核球のサブセット, 血清エンドトキシン濃度および炎症性サイトカインの動態を測定し病態との関係を検討した. 発症牛のCD4陽性(CD4^+)T細胞数は健康搾乳牛に比べて著しく低く, CD4^+ T細胞とCD8陽性(CD8^+)T細胞との比が低下していた. これに反して, γδT細胞, MHC IIおよびIgM陽性細胞数は健康搾乳牛より高かった. エンドトキシン血症は一例を除いた罹患牛全例に起こっていたが, 排菌中は非排菌牛に比べてその濃度が高かった. 炎症性サイトカイン中IL-1およびIL-6活性は罹患牛が明らかに高かったが, TNF-αの活性は全ての牛で認められなかった. いっぽう罹患牛の血中IL-2値は健康搾乳牛に比べて低く, IFN-γ値は非排菌牛1例のみが健康搾乳牛に比べて高かった. これらは末梢血単核球におけるサブセットの分布状態との関連が示唆された. 以上の結果は, 牛の急性サルモネラ症では細胞性免疫能が抑制されているが液性免疫能は活性化していることを示唆していた.
  • H Okada, H Ohtsuka, S Kon-Nai, R Kirisawa, Y Yokomizo, T Yoshino, TJ Rosol
    JOURNAL OF VETERINARY MEDICAL SCIENCE 61 1 33 - 35 1999年01月 [査読有り][通常論文]
     
    This investigation was performed to determine the effect of lipopolysaccharide (LPS) on production of interleukin (IL)-1 and IL-6 by bovine mammary epithelial cells in vitro. After confluence, the cells were stimulated with LPS (0.1, 1.0 or 10 mu g/ml) for 4, 8, 24, and 48 hr. LPS increased production of both IL-1 and IL-6 production from mammary cells in a dose dependent manner. The expression of mRNA for IL-1 receptor antagonist (IL-1ra) was demonstrated by reverse transcription-polymerase chain reaction in bovine mammary epithelial cells.

書籍

  • 主要症状を基礎にした牛の臨床3
    今内 覚 他 (担当:分担執筆範囲:トリパノソーマ病、悪性カタル熱)
    デーリィマン社 2020年05月
  • Academic Fantasista 免疫療法で動物を救う
    今内 覚 (担当:分担執筆範囲:免疫療法で動物を救う https://www.hokudai.ac.jp/pr/hokudai_af2018.pdf)
    北海道大学 2019年04月
  • 動物の感染症<第4版>
    今内 覚 (担当:分担執筆範囲:総論:IV感染症の予防と治療: 1. 感染症の予防, 各論 : 11 アデノウイルス病, 19 牛免疫不全ウイルス感染症.)
    近代出版 2019年03月
  • 北大など、動物用の免疫チェックポイント阻害薬を開発 ー 目指すは病気の違いを越えた汎用免疫療法 ー
    今内 覚 (担当:その他)
    日経バイオテクONLINE 2017年10月
  • テレビ・ドクター4 よく分かる乳牛の病気100選
    今内 覚 (担当:分担執筆範囲:近年の重大疾病と予防策 地方病型(流行型)牛白血病、皮膚に異常を示す病気 散発性牛白血病の項)
    デーリィマン社 2017年10月
  • 動物衛生学
    今内 覚 (担当:分担執筆範囲:コラム 牛白血病)
    文永堂出版 2017年09月
  • 動物用ワクチンとバイオ医薬品 -新たな潮流-
    岡川朋弘, 今内 覚ほか (担当:分担執筆範囲:第1章 総論 4. 抗体医薬: 免疫チェックポイント阻害薬等)
    文永堂出版 2017年07月
  • 牛白血病防除の観点から
    今内 覚 (担当:その他範囲:検定検査乳S)
    検定検査乳S 34:7 2016年01月
  • 現代農業・牛白血病を出さない!尾鈴のBL対策
    宮崎県JA尾鈴 (担当:その他範囲:牛白血病を出さない!尾鈴のBL対策)
    農山漁村文化協会 2015年05月
  • 獣医免疫学
    今内 覚 ほか (担当:分担執筆範囲:第13章 ワクチン)
    緑書房 2015年
  • 新製品の登場で注目集める動物用医薬品
    今内 覚 (担当:その他)
    日経バイオテク 2014年09月
  • 牛病学〈第三版〉
    今内 覚 (担当:分担執筆範囲:V. 感染症の制御 3) 免疫疲弊化と免疫賦活化)
    近代出版 2013年

講演・口頭発表等

  • 免疫療法 〜免疫チェックポイント阻害剤について〜
    今内 覚
    北海道大学One Healthフロンティア卓越大学院プログラムOne Health Relay Report https://onehealth.vetmed.hokudai.ac.jp/news/220 2020年03月 その他
  • 動物の難病(がん・感染症)に対する創薬研究
    今内 覚
    日本学術振興会 科研費 研究成果トピックス https://www.jsps.go.jp/j-grantsinaid/37_topics/data/10101-40396304.pdf 2020年03月 メディア報道等
  • 動物用バイオ医薬品の開発と応用研究  [招待講演]
    今内 覚
    農林水産省動物医薬品検査所 令和元年度第1回特別講演会 2020年01月 口頭発表(招待・特別)
  • 牛白血病の新たな制御方法、抗ウイルス効果の確認に成功 -牛の難治性疾病に対する応用に期待-  [通常講演]
    今内 覚ら
    農林水産2019年10大ニュース https://www.affrc.maff.go.jp/docs/press/191224.html 2019年12月 メディア報道等
  • 牛白血病の最新知見  [招待講演]
    今内 覚
    北海道獣医師会宗谷支部令和元年度新技術講習会 2019年11月 口頭発表(招待・特別)
  • 牛白血病の最新知見〜北海道大学臨床検査診断結果に基づく実践的対策の紹介〜  [招待講演]
    今内 覚
    みなみ北海道農業共済組合南部家畜診療センター講習会 2019年11月 口頭発表(招待・特別)
  • 抗生剤に頼らないプロバイオティクスを応用した新規家畜疾病予防法の開発 〜発酵哺乳飼料による子牛の下痢抑制効果〜  [招待講演]
    今内 覚
    みなみ北海道農業共済組合南部家畜診療センター講習会 2019年11月 口頭発表(招待・特別)
  • 動物の難病(がん・感染症)に対する 創薬研究(新薬の開発)  [招待講演]
    今内 覚
    国民との科学・技術対話 2019年10月 口頭発表(招待・特別)
  • 動物用抗体医薬品の開発および臨床研究  [招待講演]
    今内 覚
    公益社団法人日本動物用医薬品協会第51回学術講習会 2019年10月 口頭発表(招待・特別)
  • 動物難治性疾病に対する創薬研究  [招待講演]
    今内 覚
    WISE特別セミナー2019 2019年10月 口頭発表(招待・特別)
  • Effects of bovine tumor necrosis factor alpha decoy receptors on cell death and inflammatory cytokine kinetics: potential for bovine inflammation therapy  [通常講演]
    Fujisawa S, Konnai S, Okagawa T, Maekawa N, Goto S, Murata S, Ohashi K
    International Veterinary Immunology Symposium 2019 2019年08月 口頭発表(一般)
  • Functional analysis of bovine CTLA-4 in bovine leukemia virus infection  [通常講演]
    Watari K, Konnai S, Okagawa T, Maekawa N, Goto S, Murata S, Ohashi K
    International Veterinary Immunology Symposium 2019 2019年08月 口頭発表(一般)
  • Development of a sensitive PD-L1 immunohistochemistry of canine cancers and clinical efficacy of an anti-PD-L1 antibody in canine oral malignant melanoma with pulmonary metastasis  [通常講演]
    Maekawa N, Konnai S, Okagawa T, Murata S, Ohashi K
    International Veterinary Immunology Symposium 2019 2019年08月 口頭発表(招待・特別)
  • Immunomodulatory effects of sialostatin L and sialostatin L2 from Ixodes persulcatus Schulze, Taiga tick  [通常講演]
    Satoru Konnai, Yamato Sajiki, Tomohiro Okagawa, Naoya Maekawa, Masayoshi Isezaki, Shiro Murata, Kazuhiko Ohashi
    International Veterinary Immunology Symposium 2019 2019年08月 口頭発表(一般)
  • The induction of immunosuppression via prostaglandin E2 and the enhancement of anti-bacterial effects by anti-PD-L1 antibody combined with COX-2 inhibitor in Mycoplasma bovis infection  [通常講演]
    Goto S, Konnai S, Okagawa T, Maekawa N, Murata S, Ohashi K
    International Veterinary Immunology Symposium 2019 2019年08月 口頭発表(一般)
  • Contribution of prostaglandin E2 to disease progression and enhancement of antiviral effects by anti-PD-L1 antibody combined with COX-2 inhibitor in bovine leukemia virus infection  [通常講演]
    Sajiki Y, Konnai S, Okagawa T, Maekawa N, Murata S, Ohashi K
    International Veterinary Immunology Symposium 2019 2019年08月 口頭発表(招待・特別)
  • Establishment of anti-bovine PD-1 chimeric antibody and a pilot clinical study  [通常講演]
    Satoru Konnal, Tomohiro Okagawa, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi
    International Veterinary Immunology Symposium 2019 2019年08月 口頭発表(招待・特別)
  • ウシマイコプラズマ感染症における免疫疲弊化  [招待講演]
    今内 覚
    日本マイコプラズマ学会第46会学術集会 2019年05月 口頭発表(招待・特別)
  • 牛白血病臨床検査診断結果に基づく実践的対策  [招待講演]
    今内 覚
    上川青年獣医師会特別講演 2019年05月 口頭発表(招待・特別)
  • 獣医学領域における感染症の制御  [招待講演]
    今内 覚
    微生物化学研究所特別講演 2019年05月 口頭発表(招待・特別)
  • 免疫チェックポイントを標的としたイヌ免疫療法の試み  [招待講演]
    今内 覚
    麻布大学獣医学部臨床特別セミナー 2019年04月 口頭発表(招待・特別)
  • Developing immunotherapy to save animals  [招待講演]
    Satoru Konnai
    Research Highlight: https://www.global.hokudai.ac.jp/blog/developing-immunotherapy-to-save-animals/ 2019年04月 メディア報道等
  • 動物難治性疾病に対する創薬研究  [招待講演]
    今内 覚
    東北大学大学院医学系研究院抗体創薬研究分野 第6回抗体創薬研究セミナー 2019年03月 口頭発表(招待・特別)
  • Six scientists whose transformative work reaches across borders.  [招待講演]
    Satoru Konnai
    Nature index: https://www.nature.com/articles/d41586-019-00831-5 2019年03月 メディア報道等
  • 牛白血病の最新知見とウイルス量を基盤とした対策について  [招待講演]
    今内 覚
    大分県畜産協会家畜生産農場清浄化支援事業講習会 2019年03月 口頭発表(招待・特別)
  • 静かなる脅威・牛白血病 〜最新知見から〜  [招待講演]
    今内 覚
    日本中央競馬会(JRA)畜産振興事業シンポジウム 地方病性牛白血病・清浄化へのアプローチ ~牛白血病清浄化に向けて今しなければならないこと~ 2019年03月 口頭発表(基調)
  • 家畜の慢性感染症制御の難しさ 〜免疫から逃れる病原体たち〜  [招待講演]
    今内 覚
    第9回大動物臨床研究会東京シンポジウム 2019年03月 口頭発表(招待・特別)
  • 動物難治性疾病に対する創薬研究 〜免疫チェックポイントを標的としたイヌ免疫療法の試み〜  [招待講演]
    今内 覚
    第15回日本獣医内科学アカデミー学術大会 2019年02月 口頭発表(招待・特別)
  • Immune checkpoint inhibitors-related diseases in animals: Application of immunotherapy targeting the immune checkpoints for veterinary medicine  [招待講演]
    Satoru KONNAI
    Seminario Especial in Universidade Federal do Rio Grande do Sul 2019年02月 口頭発表(招待・特別)
  • 動物の難病(がん・感染症)に対する 創薬研究(新薬の開発)  [招待講演]
    今内 覚
    国民との科学・技術対話 2019年01月 口頭発表(招待・特別)
  • 牛白血病臨床検査診断結果に基づく実践的対策  [招待講演]
    今内 覚
    平成30年度小平町和牛生産改良組合牛白血病研修会 2019年01月 口頭発表(招待・特別)
  • 動物難治性疾病に対する創薬研究 〜免疫チェックポイントを標的とした新規免疫療法の試み〜  [招待講演]
    今内 覚
    大阪市立大学大学院工学研究科 医工・生命工学教育研究センター第1回セミナー 2018年12月 口頭発表(招待・特別)
  • イヌ難治性腫瘍疾患に対する免疫チェックポイントを 標的とした新規免疫療法の試み  [通常講演]
    今内 覚, 前川直也, 細谷謙次, 金 尚昊, 高木 哲, 賀川由美子, 岡川朋弘, 村田史郎, 大橋和彦
    北海道小動物獣医師会年次大会2018 2018年11月 口頭発表(一般)
  • 牛白血病の最新知見、現状と対策  [招待講演]
    今内 覚
    兵庫県畜産協会平成30年度食の安全・消費者の信頼確保対策事業平成30年度牛白血病講習会 2018年10月 口頭発表(招待・特別)
  • 動物難治性疾病に対する創薬研究 〜免疫学的解析を基盤とした新規制御法の開発〜  [招待講演]
    今内 覚
    第161回日本獣医学会学術集会微生物学分科会シンポジウム「感染症対策の新たな突破口を探る」 2018年09月 口頭発表(招待・特別)
  • 牛白血病の最新知見 〜臨床データーからの更新知見〜  [招待講演]
    今内 覚
    平成30年度みなみ北海道農業共済組合第1回診療等体験発表会 特別講演 2018年07月 口頭発表(招待・特別)
  • 免疫チェックポイントを標的とした動物用抗体医薬の開発  [招待講演]
    今内 覚
    第2回ヒトと伴侶動物の比較医学研究会 ヒト医療と獣医療の先端技術を駆使した次世代のがん治療〜がん撲滅への新たな挑戦〜 2018年06月 口頭発表(招待・特別)
  • 牛難治性疾病における免疫疲弊化機序の 解析と制御法への応用  [招待講演]
    今内 覚
    第74回九州・山口病性鑑定協議会 2018年06月 口頭発表(招待・特別)
  • 動物難治性疾病に対する創薬研究  [招待講演]
    今内 覚
    北里大学獣医学部特定講義 2018年06月 口頭発表(招待・特別)
  • 牛白血病の最新知見 現状と対策  [招待講演]
    今内 覚
    国立大学法人岐阜大学応用生物科学部附属家畜衛生地域連携教育研究センター家畜衛生講演会 2018年06月 口頭発表(招待・特別)
  • 牛白血病の最新知見  [招待講演]
    今内 覚
    鹿児島県獣医師会 産業動物部会曽於・肝属地区獣医師講習会 2018年03月 口頭発表(招待・特別)
  • 牛白血病の最新知見 帯広食肉衛生検査所との共同研究成果から  [招待講演]
    今内 覚
    北海道獣医師会十勝支部公衆衛生講習会 2018年03月 口頭発表(招待・特別)
  • 牛白血病の疫学と公衆衛生分野での留意点  [招待講演]
    今内 覚
    北海道獣医師会平成29年度獣医公衆衛生講習会 2018年03月 口頭発表(招待・特別)
  • 地方病性牛白血病対策 について  [招待講演]
    今内 覚
    平成29年度岩手県牛白血病防疫推進講習会 2018年01月 口頭発表(招待・特別)
  • 牛の免疫応答を利用した難治性疾病の新規制御法開発 〜免疫チェックポイントを標的とした動物用抗体創薬〜  [招待講演]
    今内 覚
    家畜衛生フォーラム2017 「抗菌剤に頼らない新しい家畜疾病の制御法 -モデルとしての難治性・慢性疾病克服のための研究-」 2017年12月 口頭発表(招待・特別)
  • Immune exhaustion during chronic diseases in animals  [招待講演]
    Satoru KONNAI
    The 20th Joint Symposium Between Seoul National University and Hokkaido University 2017年11月 口頭発表(招待・特別)
  • 牛難治性疾病に対する多機能型バイオ医薬(抗体医薬)の創出と発展的応用  [招待講演]
    今内 覚
    農林水産業・食品産業科学技術研究推進事業 「知」の集積と活用の場ポスターセッション 2017年11月 口頭発表(招待・特別)
  • 動物用バイオ医薬品の開発  [招待講演]
    今内 覚
    東北大学大学院医学系研究科抗体創薬研究分野 第二回抗体創薬研究セミナー 2017年11月 口頭発表(招待・特別)
  • 増加している牛白血病 - 北海道での現状と対策について -  [招待講演]
    今内 覚
    第22回北海道肉牛研究会大会 2017年10月 口頭発表(招待・特別)
  • Epidemiological and immunological study for intractable infectious diseases in Mongolian livestock  [招待講演]
    Satoru KONNAI
    JICA Project: Infectious Diseases Seminar 2017 in Mongolia 2017年10月 口頭発表(招待・特別)
  • 動物の難病に対する新規治療法の開発  [招待講演]
    今内 覚
    国民との科学・技術対話 2017年10月 公開講演,セミナー,チュートリアル,講習,講義等
  • Theileria parva感染症における免疫チェックポイント因子の動態解析  [通常講演]
    今内 覚, 岡川朋弘, 山田慎二, Martin Simuunza, Timothy Connelley, Ivan Morrison, 村田史郎, 大橋和彦
    第160回日本獣医学会学術集会 2017年09月 口頭発表(一般)
  • Role of Inhibitory Molecules in Bovine Chronic Infectious Diseases and as Target for Therapy  [招待講演]
    Satoru KONNAI
    宮崎大学産業動物防疫リサーチセンター(CADIC)特別セミナー 2017年09月 口頭発表(招待・特別)
  • Therapeutic intervention in cancer and chronic infections in animals: Antibody mediated manipulation of PD-1/PD-L1 interaction  [招待講演]
    Satoru KONNAI
    Hokkaido University & Colorado State University Kick-off Symposium 2017 2017年07月 口頭発表(招待・特別)
  • 牛白血病の最新知見  [招待講演]
    今内 覚
    平成29年度第108回上川家畜衛生研究会 2017年07月 口頭発表(招待・特別)
  • 動物における免疫チェックポイント標的抗体による治療戦略  [招待講演]
    今内 覚
    化学及血清療法研究所 三風会 2017年06月 口頭発表(招待・特別)
  • マダニ唾液由来因子の病原体伝播における役割  [招待講演]
    今内 覚
    第90回日本細菌学会総会シンポジウム わが国におけるダニ媒介性細菌感染症研究の現状 2017年03月 口頭発表(招待・特別)
  • 動物難治性疾病における免疫回避機序の解明 〜免疫チェックポイントを標的とした動物用医薬品開発〜  [通常講演]
    今内 覚
    第2回北海道大学・部局横断シンポジウム 『免疫・癌・感染』 2017年03月 口頭発表(招待・特別)
  • Immune exhaustion during chronic infections in cattle  [通常講演]
    Satoru KONNAI
    Seminar in the Loslin institute, University of Edinburgh, UK 2017年02月 公開講演,セミナー,チュートリアル,講習,講義等
  • 牛白血病撲滅対策  [招待講演]
    今内 覚
    公益社団法人千葉県畜産協会牛白血病感染拡大防止研修会 ー牛白血病の感染拡大防止を図るためー 2017年01月 口頭発表(招待・特別)
  • 獣医療における抗体医薬の開発と現状  [招待講演]
    今内 覚
    徳島大学先端酵素学研究所免疫制御学セミナー 2016年12月 口頭発表(招待・特別)
  • 牛の感染免疫に関する最新の知見  [招待講演]
    今内 覚
    平成28年度岩手県獣医師会職域部会合同研修会 2016年12月 口頭発表(招待・特別)
  • 家畜法定伝染病ウシアナプラズマ病の免疫回避機構の解析  [通常講演]
    今内 覚
    第23回 リケッチア研究会 2016年12月 口頭発表(一般)
  • 牛白血病の最新の知見について  [招待講演]
    今内 覚
    神奈川県畜産技術協会生物科学安全研究所支部地方競馬益金補助事業主催講演会 2016年11月 口頭発表(招待・特別)
  • Immunotherapy for canine malignant cancers  [招待講演]
    Satoru KONNAI
    The 19th Joint Symposium Between Seoul National University and Hokkaido University 〜Advanced Veterinary Sciences: From Bench to Clinic〜 2016年11月 口頭発表(招待・特別)
  • 牛白血病の現状と対策  [招待講演]
    今内 覚
    日高町和牛生産改良組合防疫技術講習会 2016年11月 口頭発表(招待・特別)
  • 免疫チェックポイントを標的とする免疫療法とは  [招待講演]
    今内 覚
    平成28年宮崎大学テニュアトラック推進機構主催セミナー 2016年11月 口頭発表(招待・特別)
  • 牛白血病対策  [招待講演]
    今内 覚
    八紘牧場衛生対策会議 2016年10月 公開講演,セミナー,チュートリアル,講習,講義等
  • 牛白血病について  [招待講演]
    今内 覚
    平成28年度第3回標茶町技連学習会 2016年10月 口頭発表(招待・特別)
  • Identification of immunomodulatory factors in Ixodes persulcatus Schulze ‘Taiga tick’, the vector for Lyme disease and relapsing fever in Japan  [招待講演]
    Satoru KONNAI
    XXV International Congress of Entomology (ICE-2016) 2016年09月 口頭発表(招待・特別)
  • Development of novel strategy for disease control in animal-engineering animal antibodies for development of novel therapeutics  [招待講演]
    Satoru KONNAI
    Texas A&M University, College of Veterinary Medicine, Veterinary Pathology Seminar Series 2016年09月 口頭発表(招待・特別)
  • Engineering antibodies for development of novel therapeutics and vaccine for livestock  [招待講演]
    Satoru KONNAI
    1st International Symposium on Livestock Biotechnology 2016年08月 口頭発表(招待・特別)
  • Increases of cells expressing PD-1 and PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via its blockade  [通常講演]
    Satoru KONNAI
    International Veterinary Immunology Symposium 2016 2016年08月 ポスター発表
  • Research on immune checkpoint in veterinary medicine  [招待講演]
    Satoru KONNAI
    Joint Workshop on Livestock Disease Research in University of Sydney 2016年08月 公開講演,セミナー,チュートリアル,講習,講義等
  • むしからうつる感染症  [招待講演]
    今内 覚
    札幌幌南ロータリークラブ市民講話 2016年08月 公開講演,セミナー,チュートリアル,講習,講義等
  • 牛白血病における免疫応答 〜免疫学的解析に基づく新規動物用バイオ医薬品の開発〜  [招待講演]
    今内 覚
    第20回大動物臨床教育セミナー 2016年05月 口頭発表(招待・特別)
  • 牛白血病について  [招待講演]
    今内 覚
    平成28年度JA新はこだて酪農生産部会長万部支部講習会 2016年04月 口頭発表(招待・特別)
  • 牧野における牛白血病対策  [招待講演]
    今内 覚
    後志家畜自衛防疫推進協議会産業動物飼養管理講習会 2016年04月 口頭発表(招待・特別)
  • 牛白血病の免疫学的解析  [招待講演]
    今内 覚
    理化学研究所シンポジウム 2016年03月 口頭発表(招待・特別)
  • 牛白血病の制御法について  [招待講演]
    今内 覚
    道東NOSAI牛白血病拡大防止事業教育講座 2016年03月 公開講演,セミナー,チュートリアル,講習,講義等
  • 牛白血病の感染防御対策  [招待講演]
    今内 覚
    道東NOSAI牛白血病拡大防止対策事業教育講座 2016年03月 公開講演,セミナー,チュートリアル,講習,講義等
  • 牛白血病における免疫性状と制御方法の開発  [招待講演]
    今内 覚
    平成27年度日本獣医師会獣医学術学会年次大会特別企画シンポジウム「牛白血病の現状と課題 -清浄化に向けて-」 2016年02月 口頭発表(招待・特別)
  • 牛白血病の現状と対策について  [招待講演]
    今内 覚
    静岡県東部家畜保健衛生推進協議会畜産講演会 2016年02月 口頭発表(招待・特別)
  • 牛白血病における免疫応答 〜免疫学的解析に基づく新規疾病予防法の開発〜  [招待講演]
    今内 覚
    大動物臨床研究会第6回東京シンポジウム 2016年02月 口頭発表(招待・特別)
  • 獣医医療における抗体医薬の開発と現状  [招待講演]
    今内 覚
    東北大学大学院医学系研究科特別講義 2016年01月 口頭発表(招待・特別)
  • Development of therapeutic antibodies targeting immunoinhibitory molecules in veterinary medicine  [招待講演]
    Satoru KONNAI
    IPR/CRED/PDIS Joint International Seminar -From protein structural science to development of therapeutics- 2016年01月 口頭発表(招待・特別)
  • 動物難治性疾病に対するバイオ医薬品の開発  [招待講演]
    今内 覚
    JA全農家畜衛生研究所特別講演 2015年11月 口頭発表(招待・特別)
  • 牛白血病の現状と対策  [招待講演]
    今内 覚
    青森県上十三地区家畜衛生推進協議会H27年度畜産講習会 2015年11月 口頭発表(招待・特別)
  • 初乳の衛生管理について 〜牛白血病防除の観点から〜  [招待講演]
    今内 覚
    第23回乳房炎防疫対策研究会 2015年11月 口頭発表(招待・特別)
  • 牛白血病の最新知見  [招待講演]
    今内 覚
    第2回ミニ宮崎しゃくなげ会研修会 2015年11月 口頭発表(招待・特別)
  • 牛白血病における免疫応答  [招待講演]
    今内 覚
    第39回大動物臨床研究会シンポジウム 2015年11月 口頭発表(招待・特別)
  • 牛白血病 最近の知見と対策について  [招待講演]
    今内 覚
    大分県畜産協会特別講演 2015年09月 口頭発表(招待・特別)
  • 牛白血病の病態発生機序および制御法に関する研究  [招待講演]
    今内 覚
    第158回日本獣医学会学術集会特別企画 2015年09月 口頭発表(招待・特別)
  • 牛白血病に認められる細胞性免疫の抑制 - PL牛を優先淘汰する意義 -  [招待講演]
    今内 覚
    2015年度 牛臨床寄生虫研究会・九州沖縄産業動物臨床研究会合同研究集会 2015年07月 口頭発表(招待・特別)
  • 地方病型牛白血病の対策と新規制御法研究の現状  [招待講演]
    今内 覚
    長野県獣医師会産業動物臨床部会講習会 2015年04月 口頭発表(招待・特別)
  • 牛白血病の対策  [招待講演]
    今内 覚
    北海道公共牧場会職員春期研修会 2015年04月 口頭発表(招待・特別)
  • 慢性感染症における免疫応答と免疫回避機構  [招待講演]
    今内 覚
    新潟県農業共済組合連合会平成26年度家畜診療等技術講習会 2015年03月 口頭発表(招待・特別)
  • 牛白血病への対策について  [招待講演]
    今内 覚
    新潟県農業共済組合連合会平成26年度家畜診療等技術講習会 2015年03月 口頭発表(招待・特別)
  • 牛白血病の現状について  [招待講演]
    今内 覚
    平成26年度放牧衛生技術検討会ならびに釧路地区家畜自衛防疫研修会 2015年02月 口頭発表(招待・特別)
  • 牛白血病ウイルス(BLV)感染症の現状と対策  [招待講演]
    今内 覚
    北海道獣医師会十勝支部産業動物講習会 2015年02月 口頭発表(招待・特別)
  • 慢性感染症に認められる免疫疲弊  [招待講演]
    今内 覚
    平成26年度日本獣医師会獣医学術集会年次大会特別企画 シンポジウム 2015年02月 口頭発表(招待・特別)
  • Increases of cells expressing PD-1 and PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via its blockade  [招待講演]
    Satoru Konnal
    Keystone Symposia on Immunity to Veterinary Pathogens: Informing Vaccine Development 2015年01月 口頭発表(招待・特別)
  • 牛白血病 ー最新知見・現状と対策ー  [招待講演]
    今内 覚
    平成26年度留萌和牛振興協議会研修会 2015年01月 口頭発表(招待・特別)
  • 感染症と免疫細胞の役割  [招待講演]
    今内 覚
    中央畜産会平成26年度海外家畜伝染病等危機管理対策強化講習会 第一次診療臨床診断技術強化講習会 2014年12月 口頭発表(招待・特別)
  • 牛白血病の現状と対策  [招待講演]
    今内 覚
    北海道獣医師会道南支部産業動物講習会 2014年11月 口頭発表(招待・特別)
  • 牛白血病の現状と感染免疫  [招待講演]
    今内 覚
    平成26年度千葉県農業共済組合連合会紫葉会技術懇談会 2014年11月 口頭発表(招待・特別)
  • 牛白血病の現状と北海道大学の取り組み  [招待講演]
    今内 覚
    日本ウイルス学会北海道支部 第 48 回 夏季シンポジウム 2014年07月 口頭発表(招待・特別)
  • 牛の難治性疾病に対する新規ワクチン戦略  [通常講演]
    今内 覚
    農業・食品産業技術総合研究機構 生物系特定産業技術研究支援センター 2013年度研究成果発表会 2014年03月 口頭発表(招待・特別)
  • 牛白血病の新規制御法の開発と清浄化モデルの構築  [招待講演]
    今内 覚
    北海道の元気創出フォーラム~知的財産を活かした地域潜在力の発掘に向けて~ 2014年02月 口頭発表(招待・特別)
  • 獣医医療における免疫抑制機序を標的とした新規疾病制御法の開発  [招待講演]
    今内 覚
    大阪大学微生物病研究所セミナー 2014年01月 口頭発表(招待・特別)
  • 牛白血病の病態発生機序の解明  [招待講演]
    今内 覚
    平成25年度動物衛生研究所北海道支所集談会 2013年12月 口頭発表(招待・特別)
  • 牛白血病 ー最新知見・現状と対策ー  [招待講演]
    今内 覚
    北海道しりべし獣医師会産業動物飼養管理講習会 2013年11月 口頭発表(招待・特別)
  • 牛白血病ウイルスの感染防御対策  [招待講演]
    今内 覚
    平成25年宮崎県畜産技術研修会特別講演 2013年11月 口頭発表(招待・特別)
  • 牛白血病ウイルス(BLV)感染症の現状と対策  [招待講演]
    今内 覚
    埼玉県獣医師会北支部・埼玉県八日会・しゃくなげ会埼玉県支部・埼玉県畜産技術振興会合同定例研修会 2013年11月 口頭発表(招待・特別)
  • 家畜臨床免疫学研究: 牛白血病における病態発生機序の解明と新規制御法の確立  [招待講演]
    今内 覚
    岐阜大学大学院連合獣医学研究科 平成25年度若手研究者育成プログラム 2013年11月 口頭発表(招待・特別)
  • 牛白血病における病態発生機序の解明と新規制御法の確立に関する研究  [招待講演]
    今内 覚
    微生物化学研究所特別講演 2013年10月 口頭発表(招待・特別)
  • 牛白血病の感染防御対策  [招待講演]
    今内 覚
    北海道獣医師会留萌支部平成25年度新技術講習会 2013年09月 口頭発表(招待・特別)
  • ウシ難治性感染症の現状と対策および新規制御法の開発  [招待講演]
    今内 覚
    ゼノアック 家畜感染症研修会 2013年09月 口頭発表(招待・特別)
  • 気をつけよう 虫さされによる感染症  [招待講演]
    今内 覚
    札幌幌南ロータリークラブ市民講話 2013年08月 公開講演,セミナー,チュートリアル,講習,講義等
  • 牛白血病の現状と対策  [招待講演]
    今内 覚
    化学及血清療法研究所第30回家畜衛生講習会並びに研究協議会 2013年08月 口頭発表(招待・特別)
  • 牛白血病の現状と対策について  [招待講演]
    今内 覚
    NOSAI山形平成25年度家畜診療所技術研究会 2013年07月 口頭発表(招待・特別)
  • 農場の感染症リスクコントロール  [招待講演]
    今内 覚
    NOSAI山形平成25年度家畜診療所職員研修会 2013年07月 口頭発表(招待・特別)
  • 牛白血病 ー最新知見、現状と対策ー  [招待講演]
    今内 覚
    第104回上川家畜衛生研究会 2013年07月 口頭発表(招待・特別)
  • 感染症(牛白血病)について 現状と対策  [招待講演]
    今内 覚
    第35回鹿児島・宮崎しゃくなげ会合同技術研修会 2013年07月 口頭発表(基調)
  • 牛白血病清浄化対策について  [招待講演]
    今内 覚
    宮崎県尾鈴農業協同組合BL対策研修会 2013年06月 口頭発表(招待・特別)
  • 牛白血病の対策について  [招待講演]
    今内 覚
    北海道獣医師会牛白血病対策検討委員会 2013年03月 口頭発表(招待・特別)
  • 牛白血病ウイルス(BLV)感染症の 現状と対策  [招待講演]
    今内 覚
    第51回愛知県獣医師会学術研究発表会特別講演 2013年03月 口頭発表(招待・特別)
  • Role of Inhibitory Molecules in Bovine Chronic Infectious Diseases and as Target for Therapy  [招待講演]
    Satoru Konnal
    Philippines Carabao Center Special Seminar 2013年03月 口頭発表(招待・特別)
  • 牛難治性疾病に対する免疫抑制因子を標的とした新規制御法の開発  [招待講演]
    今内 覚
    日本大学生物資源科学部 動物医科学研究センター特別セミナー 2013年01月 口頭発表(招待・特別)
  • 牛白血病ウイルス感染症の伝播阻止トライアルについて  [招待講演]
    今内 覚
    本川牧場疾病対策講習会 2012年12月 口頭発表(招待・特別)
  • 牛白血病ウイルス感染症の現状と対策  [招待講演]
    今内 覚
    沖縄県獣医師会家畜衛生畜産部会沖縄畜産技術者協会特別講演 2012年12月 口頭発表(招待・特別)
  • 牛の白血病について  [招待講演]
    今内 覚
    北海道獣医師会胆振支部特別講演 2012年11月 口頭発表(招待・特別)
  • 牛白血病ウイルス感染症の現状と対策  [招待講演]
    今内 覚
    宮崎県獣医師会学術委員会産業動物部門研修会 2012年11月 口頭発表(招待・特別)
  • 牛白血病の現状と対策  [招待講演]
    今内 覚
    埼玉県獣医師会特別講演 2012年10月 口頭発表(招待・特別)
  • 牛慢性疾患の新たなる治療法の可能性 -免疫抑制因子を標的とした新規治療薬の開発-  [招待講演]
    今内 覚
    酪農学園大学大動物研究セミナー 2012年09月 口頭発表(招待・特別)
  • 牛の白血病について  [招待講演]
    今内 覚
    北海道獣医師会根室支部特別講演 2012年05月 口頭発表(招待・特別)
  • 牛難治性疾病に対する新規戦略(多機能型ワクチンによる牛疾病制御の試み)  [招待講演]
    今内 覚
    第153回日本獣医学会学術集会微生物分科会シンポジウム 2012年03月 口頭発表(招待・特別)
  • 牛白血病のコントロールについて〜ある農場における清浄化の取り組み事例から〜  [招待講演]
    今内 覚
    平成23年度 北海道家畜保健衛生所病性鑑定技術検討会 2012年01月 口頭発表(招待・特別)
  • 牛白血病の現状と感染予防対策について  [招待講演]
    今内 覚
    道央農業振興公社 畜産研修会 2011年12月 口頭発表(招待・特別)
  • 牛の難治性疾病に対する多機能型ワクチン戦略  [招待講演]
    今内 覚
    第51回家畜衛生学会シンポジウム 2011年11月 口頭発表(招待・特別)
  • 国内の牛白血病の現状と今後の対策について  [招待講演]
    今内 覚
    牛臨床寄生虫研究会・家畜臨床学会ジョイントシンポジウム 2011年10月 口頭発表(招待・特別)
  • 牛白血病の現状とその対策  [招待講演]
    今内 覚
    栃木県獣医師会特別講演 2011年10月 口頭発表(招待・特別)
  • 牛の難治性疾病に対する多機能型ワクチン戦略  [招待講演]
    今内 覚
    宮崎大学農学部特別シンポジウム(集談会) 2011年09月 口頭発表(招待・特別)
  • 牛白血病の現場でのコントロールについて  [招待講演]
    今内 覚
    宮崎大学農学部特別シンポジウム(教育プログラム) 2011年09月 口頭発表(招待・特別)
  • Research on host-pathogen interactions for establishment of novel strategies for disease control  [招待講演]
    今内 覚
    Special seminar in Federal University of Rio Grande do Sul 2011年08月 口頭発表(招待・特別)
  • Research on host-pathogen interactions for establishment of novel strategies for disease control  [招待講演]
    今内 覚
    Special seminar in Northern Fluminense State University Darcy Ribeiro (Campos campas) 2011年07月 口頭発表(招待・特別)
  • Research on host-pathogen interactions for establishment of novel strategies for disease control  [招待講演]
    今内 覚
    Special seminar in Northern Fluminense State University Darcy Ribeiro (Makae campus) 2011年07月 口頭発表(招待・特別)
  • 牛白血病ウイルス感染症の現状と対策  [招待講演]
    今内 覚
    福岡県家畜保健衛生所講習会 2011年02月 口頭発表(招待・特別)
  • 牛白血病の感染防御対策  [招待講演]
    今内 覚
    第43回北海道しゃくなげ会 2011年02月 口頭発表(一般)
  • 生産現場における白血病対策 —現状と課題—  [招待講演]
    今内 覚
    平成22年度日本獣医師会 獣医学術学会年次大会 2011年02月 口頭発表(招待・特別)
  • 増加傾向にある牛白血病の現状と対策  [招待講演]
    今内 覚
    日本家畜臨床学会第41回学術集会 2010年11月 口頭発表(招待・特別)
  • 原虫病疾病制御のための新戦略 -ダニワクチン開発-  [招待講演]
    今内 覚
    日本家畜衛生学会 2010年11月 口頭発表(招待・特別)
  • 増加傾向にある牛白血病の現状と対策  [招待講演]
    今内 覚
    平成22年度北海道地区家畜診療技術研修会 2010年09月 口頭発表(招待・特別)
  • メガファームにおける取り組み紹介 —牛白血病ウイルス感染伝播阻止トライアルの実例—  [招待講演]
    今内 覚
    平成22年度獣医学術北海道地区学会シンポジウム 2010年09月 口頭発表(招待・特別)
  • Pro-inflammatory cytokine storm in Theileria parva-infected cattle  [招待講演]
    今内 覚
    The 9th International Veterinary Immunology Symposium (Concurrent Session) 2010年08月 シンポジウム・ワークショップパネル(指名)
  • 牛白血病ウイルス感染症の現状と対策  [招待講演]
    今内 覚
    北海道獣医師会オホーツク支部大動物講習会2 2010年07月 口頭発表(招待・特別)
  • 牛白血病ウイルス感染症の現状と対策  [招待講演]
    今内 覚
    北海道獣医師会オホーツク支部大動物講習会1 2010年07月 口頭発表(一般)
  • 新規動物用ワクチンならびに新規治療法の開発  [招待講演]
    今内 覚
    日本生物科学研究所シンポジウム 2010年03月 口頭発表(招待・特別)
  • 新規動物用ワクチンならびに新規治療法の開発  [招待講演]
    今内 覚
    動物用ワクチンーバイオ医薬研究会改組設立記念シンポジウム 2010年03月 口頭発表(招待・特別)
  • PD-1/PD-L1システム制御による牛白血病ワクチン戦略  [招待講演]
    今内 覚
    平成21年度日本獣医師会学会年次大会・特別企画 2010年01月 口頭発表(招待・特別)
  • 牛難治性慢性感染症における免疫疲弊化機序の解明と免疫賦活化の試み  [招待講演]
    今内 覚
    第4回日本家畜臨床感染症研究会総会・学術集会 2009年12月 口頭発表(招待・特別)
  • 牛白血病ウイルス感染症の現状と対策  [招待講演]
    今内 覚
    北海道獣医師会根室支部大動物臨床部会講演会 2009年11月 口頭発表(招待・特別)
  • 牛白血病ウイルス(BLV)感染症対策  [招待講演]
    今内 覚
    第60回北海道獣医師大会産業動物獣医学会シンポジウム 2009年09月 口頭発表(招待・特別)
  • ダニのEST解析とダニワクチンの現状  [招待講演]
    今内 覚
    第15回 ダニと疾患のインタフェースに関するセミナー(SADI) 教育講演 2007年05月 口頭発表(招待・特別)

その他活動・業績

受賞

  • 2019年06月 伊藤記念財団 第4回伊藤記念財団賞
     
    受賞者: 今内 覚
  • 2017年01月 北海道大学 平成28年度北海道大学研究総長賞
     
    受賞者: 今内 覚
  • 2015年09月 日本獣医学会 日本獣医学会賞
     
    受賞者: 今内 覚
  • 2014年01月 北海道 平成25年度北海道科学技術奨励賞
     
    受賞者: 今内 覚
  • 2005年06月 森永奉仕会 森永奉仕会賞
     
    受賞者: 今内覚
  • 2003年09月 日本獣医学会 獣医学奨励賞
     
    受賞者: 今内覚
  • 2003年09月 日本獣医学会 日本獣医学会会長賞
     
    受賞者: 今内覚

教育活動情報

主要な担当授業

  • 獣医科学・感染症学基礎科目 研究機器演習
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 伝染病学実習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 細菌、ウイルス、原虫、同定、診断
  • 獣医科学・感染症学基礎科目 免疫学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医疫学演習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 疫学研究手法、原因論、リスク分析、統計学
  • 獣医科学基礎科目B 研究機器演習
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 動物衛生学実習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 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
  • 獣医科学基礎科目B 免疫学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目 研究機器演習
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 獣医科学基礎科目 免疫学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • アドバンスト演習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部

大学運営

委員歴

  • 2019年 - 現在   農政推進協議会 家畜防疫対策緊急プロジェクト   委員
  • 2015年 - 現在   公益社団法人日本獣医学会   国際会議組織委員会 委員
  • 2014年 - 現在   プロダクションメディスン研究会   委員
  • 2014年 - 現在   大動物臨床研究会   特別理事
  • 2013年 - 現在   北海道獣医師会   牛白血病対策検討委員会 委員
  • 2010年 - 現在   動物用ワクチン-バイオ医薬品研究会   幹事
  • 2005年 - 2010年   動物サイトカイン研究会   事務局長


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