Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Veterinary Medicine

Affiliation (Master)

• Faculty of Veterinary Medicine

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Profile and Settings

Affiliation

• Hokkaido University, Laboratory of Infectious Diseases, Faculty of Veterinary Medicine,, Professor

Profile and Settings

• Name (Japanese)

  Ohashi

• Name (Kana)

  Kazuhiko

• Name

  200901030602809832

Affiliation

• Hokkaido University, Laboratory of Infectious Diseases, Faculty of Veterinary Medicine,, Professor

Achievement

Research Interests

• 細胞生物学   感染免疫   

Research Areas

• Life sciences / Veterinary medicine

Research Experience

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

Education

•        - 1993  コーネル大学大学院
•        - 1993  Graduate School of Veterinary Medicine, Cornell University  Graduate School, Division of Veterinary Medicine
•        - 1985  Hokkaido University
•        - 1985  Hokkaido University  Graduate School, Division of Veterinary Medicine
•        - 1983  Hokkaido University  School of Veterinary Medicine
•        - 1983  Hokkaido University  Faculty of Veterinary Medicine

Awards

• 2008 日本獣医学会賞
  

Published Papers

• In Vivo Characterization of the Anti-Glutathione S-Transferase Antibody Using an In Vitro Mite Feeding Model

  Shwe Yee Win, Hikari Seo, Fumiya Horio, Sotaro Fujisawa, Jumpei Sato, Yoshinosuke Motai, Takumi Sato, Eiji Oishi, Akira Taneno, Lat Lat Htun, Saw Bawm, Tomohiro Okagawa, Naoya Maekawa, Satoru Konnai, Kazuhiko Ohashi, Shiro Murata

  Vaccines 12 (2) 148 - 148 2024/01/30 

  Poultry red mites (Dermanyssus gallinae, PRMs), tropical fowl mites (Ornithonyssus bursa, TFMs), and northern fowl mites (O. sylviarum, NFMs) are blood-feeding pests that debilitate poultry worldwide. Glutathione S-transferase (GST) plays an important role in the detoxification and drug metabolism of mites. However, research on avian mite GSTs as vaccine antigens is still lacking. Therefore, we aimed to evaluate the potential of avian mite GSTs for vaccine development. We identified GST genes from TFMs and NFMs. We prepared recombinant GST (rGST) from TFMs, NFMs, and PRMs, and assessed their protein functions. Moreover, we evaluated the cross-reactivity and acaricidal effect of immune plasma against each rGST on TFMs, NFMs, and PRMs. The deduced amino acid sequences of GSTs from TFMs and NFMs were 80% similar to those of the PRMs. The rGSTs exhibited catalytic activity in conjugating glutathione to the 1-chloro-2,4-dinitrobenzene substrate. Immune plasma against each rGST showed cross-reactivity with rGST from different mite species. Moreover, the survival rate of PRMs fed with immune plasma against the rGST of TFMs and NFMs was significantly lower than that of the control plasma. These results demonstrate the potential application of GST as an antigen for the development of a broad-spectrum vaccine against avian mites.
• Characterization of a Very Short Meq Protein Isoform in a Marek's Disease Virus Strain in Japan.

  Yoshinosuke Motai, Shiro Murata, Jumpei Sato, Akihito Nishi, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi

  Veterinary sciences 11 (1) 2024/01/20 

  Marek's disease virus (MDV) causes malignant lymphoma (Marek's disease; MD) in chickens. The Meq protein is essential for tumorigenesis since it regulates the expression of host and viral genes. Previously, we reported that the deletion of the short isoform of Meq (S-Meq) decreases the pathogenicity of MDV. Recently, we identified a further short isoform of Meq (very short isoform of Meq, VS-Meq) in chickens with MD in Japan. A 64-amino-acid deletion was confirmed at the C-terminus of VS-Meq. We measured the transcriptional regulation by VS-Meq in three gene promoters to investigate the effect of VS-Meq on protein function. Wild-type VS-Meq decreased the transrepression of the pp38 promoter but did not alter the transactivation activity of the Meq and Bcl-2 promoters. The deletion in VS-Meq did not affect the activity of the pp38 promoter but enhanced the transactivation activities of the Meq and Bcl-2 promoters. Collectively, the deletion of VS-Meq potentially enhanced the activity of the Meq promoter, while other amino acid sequences in wild-type VS-Meq seemed to affect the weak transrepression of the pp38 promoter. Further investigation is required to clarify the effects of these changes on pathogenicity.
• Development of a high-affinity anti-bovine PD-1 rabbit-bovine chimeric antibody using an efficient selection and large production system.

  Tomohiro Okagawa, Satoru Konnai, Shinya Goto, Yamato Sajiki, Otgontuya Ganbaatar, Kei Watari, Hayato Nakamura, Cai-Xia Wang, Taro Tachibana, Yukinari Kato, Yayoi Kameda, Junko Kohara, Nobuhiro Terasaki, Manabu Kubota, Akira Takeda, Hirofumi Takahashi, Yasuhiko Suzuki, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi

  Veterinary research 54 (1) 82 - 82 2023/09/27 

  Immune checkpoint molecules PD-1/PD-L1 cause T-cell exhaustion and contribute to disease progression in chronic infections of cattle. We established monoclonal antibodies (mAbs) that specifically inhibit the binding of bovine PD-1/PD-L1; however, conventional anti-PD-1 mAbs are not suitable as therapeutic agents because of their low binding affinity to antigen. In addition, their sensitivity for the detection of bovine PD-1 is low and their use for immunostaining PD-1 is limited. To address these issues, we established two anti-bovine PD-1 rabbit mAbs (1F10F1 and 4F5F2) and its chimeric form using bovine IgG1 (Boch1D10F1), which exhibit high binding affinity. One of the rabbit mAb 1D10F1 binds more strongly to bovine PD-1 compared with a conventional anti-PD-1 mAb (5D2) and exhibits marked inhibitory activity on the PD-1/PD-L1 interaction. In addition, PD-1 expression in bovine T cells could be detected with higher sensitivity by flow cytometry using 1D10F1. Furthermore, we established higher-producing cells of Boch1D10F1 and succeeded in the mass production of Boch1D10F1. Boch1D10F1 exhibited a similar binding affinity to bovine PD-1 and the inhibitory activity on PD-1/PD-L1 binding compared with 1D10F1. The immune activation by Boch1D10F1 was also confirmed by the enhancement of IFN-γ production. Finally, Boch1D10F1 was administered to bovine leukemia virus-infected cows to determine its antiviral effect. In conclusion, the high-affinity anti-PD-1 antibody developed in this study represents a powerful tool for detecting and inhibiting bovine PD-1 and is a candidate for PD-1-targeted immunotherapy in cattle.
• Enhanced Systemic Antitumour Immunity by Hypofractionated Radiotherapy and Anti-PD-L1 Therapy in Dogs with Pulmonary Metastatic Oral Malignant Melanoma.

  Tatsuya Deguchi, Naoya Maekawa, Satoru Konnai, Ryo Owaki, Kenji Hosoya, Keitaro Morishita, Motoji Nakamura, Tomohiro Okagawa, Hiroto Takeuchi, Sangho Kim, Ryohei Kinoshita, Yurika Tachibana, Madoka Yokokawa, Satoshi Takagi, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  Cancers 15 (11) 2023/06/01 

  Although immune checkpoint inhibitors (ICIs), such as the anti-programmed death-ligand 1 (PD-L1) antibody, have been developed for the treatment of canine malignant melanoma, desirable clinical efficacies have not been achieved. Recent studies in humans have suggested that radiation therapy (RT) combined with ICIs induces robust systemic antitumour immunity in patients with cancer. This study retrospectively examined the therapeutic efficacy of combination therapy (hypofractionated RT and anti-PD-L1 antibody [c4G12]) in dogs with pulmonary metastatic oral malignant melanoma. The intrathoracic clinical benefit rate (CBR)/median overall survival (OS) in the no RT (n = 20, free from the effect of RT), previous RT (n = 9, received RT ≤8 weeks prior to the first c4G12 dose), and concurrent RT (n = 10, c4G12 therapy within ±1 week of the first RT fraction) groups were 10%/185 days, 55.6%/283.5 days (p < 0.05 vs. no RT group), and 20%/129 days (p > 0.05 vs. no RT group), respectively. The adverse events were considered to be tolerable in the combination therapy. Thus, hypofractionated RT before the initiation of c4G12 therapy can be an effective approach for enhancing the therapeutic efficacy of immunotherapy, with acceptable safety profiles. Further prospective clinical studies are required to confirm the findings of this study.
• Molecular characterization of immunoinhibitory factors PD-1/PD-L1 in sheep.

  Wisa Tiyamanee, Satoru Konnai, Tomohiro Okagawa, Yutaro Nojima, Otgontuya Ganbaatar, Naoya Maekawa, Rie Hasebe, Yumiko Kagawa, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  Veterinary immunology and immunopathology 261 110609 - 110609 2023/05/11 

  Sheep have been used as a large animal experimental model for studying infectious diseases. However, due to a lack of staining antibodies and reagents, immunological studies on sheep have not progressed. The immunoinhibitory receptor programmed death-1 (PD-1) is expressed on T lymphocytes. The interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. We previously reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections using anti-bovine PD-L1 monoclonal antibodies (mAbs). Furthermore, we found that blocking antibodies against PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy of cattle. However, the immunological role of the PD-1/PD-L1 pathway in chronic diseases of sheep remains unknown. In this study, we identified cDNA sequences of ovine PD-1 and PD-L1 and examined the cross-activity of anti-bovine PD-L1 mAbs against ovine PD-L1 as well as the expression of PD-L1 in ovine listeriosis. The amino acid sequences of ovine PD-1 and PD-L1 share a high degree of identity and similarity with homologs from ruminants and other mammalian species. Anti-bovine PD-L1 mAb recognized ovine PD-L1 on lymphocytes in the flow cytometric assay. Furthermore, an immunohistochemical staining confirmed the PD-L1 expression on macrophages in the brain lesions of ovine listeriosis. These findings indicated that our anti-PD-L1 mAb would be useful for analyzing the ovine PD-1/PD-L1 pathway. Further research is needed to determine the immunological role of PD-1/PD-L1 in chronic diseases such as BLV infection through experimental infection of sheep.
• Investigation of peripheral blood responses in chickens infested with Dermanyssus gallinae.

  Sotaro Fujisawa, Shiro Murata, Takumi Sato, Eiji Oishi, Akira Taneno, Satoru Konnai, Kazuhiko Ohashi

  Parasitology international 95 102754 - 102754 2023/04/22 

  Among haematophagous ectoparasites that infest chickens, poultry red mite (Dermanyssus gallinae, PRM) is one of the most serious threats to poultry farms. Mass PRM infestation causes various health problems in chickens, resulting in significant productivity reduction in the poultry industry. Despite the efficiency of acaricides for controlling PRMs, the emergence of acaricide-resistant PRMs represents a challenging setback. Infestation with haematophagous ectoparasites, such as PRMs, induces inflammatory and haemostatic reactions in the host. Therefore, we aimed to explore the gene expression in chicken peripheral blood cells to elucidate host responses against PRM infestation in detail. RNA sequencing of blood-fed PRMs was performed, and the levels of the chicken-derived transcripts obtained from the ingested blood cells were analysed. Genes encoding haemoglobin subunits were found to be significantly more expressed, suggesting that PRM infestation causes anaemia in chickens. Additionally, the mRNA and plasma concentrations of CC chemokine ligand 4 and β2 microglobulin among the immune-related molecules were found to be significantly higher in PRM-infested chickens compared with non-infested animals. These results suggest that PRM infestation induce inflammation in chicken. Further studies are warranted to better understand the influence of PRM infestation on the host physiological states, including immunity.
• Analysis of gene expression in poultry red mite, Dermanyssus gallinae, by RNAscope in situ hybridization.

  Hikari Seo, Shiro Murata, Osamu Ichii, Takashi Namba, Shwe Yee Win, Takumi Sato, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi

  The Journal of veterinary medical science 85 (5) 532 - 535 2023/03/14 

  The poultry red mite (PRM; Dermanyssus gallinae) is a hematophagous ectoparasite that mainly infests chickens, and its infestation causes significant economic losses to the poultry industry. In this study, we examined the use of RNAscope-based in situ hybridization (ISH) to characterize gene expression in PRM. We analyzed the mRNA expression of Dermanyssus gallinaecathepsin D-1 (Dg-CatD-1) and Dermanyssus gallinae cystatin (Dg-Cys). RNAscope ISH analysis revealed that mRNA expression of Dg-CatD-1 was observed in the digestive tract, and Dg-Cystatin mRNA was expressed in the ovaries in addition to the digestive tract. RNAscope ISH could be applicable for the analysis of gene expression in each tissue of PRM and is an effective method to investigate the characteristics of target genes.
• Enhancement of Vaccine-Induced T-Cell Responses by PD-L1 Blockade in Calves.

  Tomohiro Okagawa, Satoru Konnai, Hayato Nakamura, Otgontuya Ganbaatar, Yamato Sajiki, Kei Watari, Haruka Noda, Mitsuru Honma, Yukinari Kato, Yasuhiko Suzuki, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi

  Vaccines 11 (3) 2023/03/01 

  Interactions between programmed death 1 (PD-1) and PD-ligand 1 (PD-L1) cause functional exhaustion of T cells by inducing inhibitory signals, thereby attenuating effector functions of T cells. We have developed an anti-bovine PD-L1 blocking antibody (Ab) and have demonstrated that blockade of the interaction between PD-1 and PD-L1 reactivates T-cell responses in cattle. In the present study, we examined the potential utility of PD-1/PD-L1-targeted immunotherapy in enhancing T-cell responses to vaccination. Calves were inoculated with a hexavalent live-attenuated viral vaccine against bovine respiratory infections in combination with treatment with an anti-PD-L1 Ab. The expression kinetics of PD-1 in T cells and T-cell responses to viral antigens were measured before and after vaccination to evaluate the adjuvant effect of anti-PD-L1 Ab. PD-1 expression was upregulated in vaccinated calves after the administration of a booster vaccination. The activation status of CD4+, CD8+, and γδTCR+ T cells was enhanced by the combination of vaccination and PD-L1 blockade. In addition, IFN-γ responses to viral antigens were increased following combinatorial vaccination with PD-L1 blockade. In conclusion, the blockade of the PD-1/PD-L1 interaction enhances T-cell responses induced by vaccination in cattle, indicating the potential utility of anti-PD-L1 Ab in improving the efficacy of current vaccination programs.
• Suppressive modulation of host immune responses by Dermanyssus gallinae infestation.

  Sotaro Fujisawa, Shiro Murata, Masayoshi Isezaki, Shwe Yee Win, Takumi Sato, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi

  Poultry science 102 (4) 102532 - 102532 2023/01/26 

  The poultry red mite (Dermanyssus gallinae, PRM) is a blood-sucking ectoparasite in chickens and is one of the most serious threats to poultry farms. Mass infestation with PRMs causes various health problems in chickens, resulting in significant productivity reduction in the poultry industry. Infestation with hematophagous ectoparasites, such as ticks, induces host inflammatory and hemostatic reactions. On the other hand, several studies have reported that hematophagous ectoparasites secrete various immunosuppressants from their saliva to suppress host immune responses to maintain blood sucking. Here, we examined the expression of cytokines in peripheral blood cells to investigate whether PRM infestation affects immunological states in chickens. In PRM-infested chickens, anti-inflammatory cytokines, IL-10 and TGF-β1, and immune checkpoint molecules, CTLA-4 and PD-1, were highly expressed compared to noninfested chickens. PRM-derived soluble mite extracts (SME) upregulated the gene expression of IL-10 in peripheral blood cells and HD-11 chicken macrophages. In addition, SME suppressed the expression of interferons and inflammatory cytokines in HD-11 chicken macrophages. Moreover, SME induces the polarization of macrophages into anti-inflammatory phenotypes. Collectively, PRM infestation could affect host immune responses, especially suppress the inflammatory responses. Further studies are warranted to fully understand the influence of PRM infestation on host immunity.
• Combined Immune Checkpoint Blockade Enhances Antiviral Immunity against Bovine Leukemia Virus.

  Hayato Nakamura, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Yamato Sajiki, Kei Watari, Kana Kamitani, Maya Saito, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  Journal of virology 97 (1) e0143022  2023/01/04 

  Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) in cattle and is widespread in many countries, including Japan. Recent studies have revealed that the expression of immunoinhibitory molecules, such as programmed death-1 (PD-1) and PD-ligand 1, plays a critical role in immunosuppression and disease progression during BLV infection. In addition, a preliminary study has suggested that another immunoinhibitory molecule, T-cell immunoglobulin domain and mucin domain-3 (TIM-3), is involved in immunosuppression during BLV infection. Therefore, this study was designed to further elucidate the immunoinhibitory role of immune checkpoint molecules in BLV infection. TIM-3 expression was upregulated on peripheral CD4+ and CD8+ T cells in BLV-infected cattle. Interestingly, in EBL cattle, CD4+ and CD8+ T cells infiltrating lymphomas expressed TIM-3. TIM-3 and PD-1 were upregulated and coexpressed in peripheral CD4+ and CD8+ T cells from BLV-infected cattle. Blockade by anti-bovine TIM-3 monoclonal antibody increased CD69 expression on T cells and gamma interferon (IFN-γ) production from peripheral blood mononuclear cells from BLV-infected cattle. A syncytium formation assay also demonstrated the antiviral effects of TIM-3 blockade against BLV infection. The combined inhibition of TIM-3 and PD-1 pathways significantly enhanced IFN-γ production and antiviral efficacy compared to inhibition alone. In conclusion, the combined blockade of TIM-3 and PD-1 pathways shows strong immune activation and antiviral effects and has potential as a novel therapeutic method for BLV infection. IMPORTANCE Enzootic bovine leukosis caused by bovine leukemia virus (BLV) is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BLV-host interactions are complex. Previously, it was found that immune checkpoint molecules, such as PD-1, suppress BLV-specific Th1 responses as the disease progresses. To date, most studies have focused only on how PD-1 facilitates escape from host immunity in BLV-infected cattle and the antiviral effects of the PD-1 blockade. In contrast, how T-cell immunoglobulin domain and mucin domain-3 (TIM-3), another immune checkpoint molecule, regulates anti-BLV immune responses is rarely reported. It is also unclear why PD-1 inhibition alone was insufficient to exert anti-BLV effects in previous clinical studies. In this study, the expression profile of TIM-3 in T cells derived from BLV-infected cattle suggested that TIM-3 upregulation is a cause of immunosuppression in infected cattle. Based on these results, anti-TIM-3 antibody was used to experimentally evaluate its function in influencing immunity against BLV. Results indicated that TIM-3 upregulation induced by BLV infection suppressed T-cell activation and antiviral cytokine production. Some T cells coexpressed PD-1 and TIM-3, indicating that simultaneous inhibition of PD-1 and TIM-3 with their respective antibodies synergistically restored antiviral immunity. This study could open new avenues for treating bovine chronic infections.
• Safety and clinical efficacy of an anti-PD-L1 antibody (c4G12) in dogs with advanced malignant tumours.

  Naoya Maekawa, Satoru Konnai, Kenji Hosoya, Sangho Kim, Ryohei Kinoshita, Tatsuya Deguchi, Ryo Owaki, Yurika Tachibana, Madoka Yokokawa, Hiroto Takeuchi, Yumiko Kagawa, Satoshi Takagi, Hiroshi Ohta, Yukinari Kato, Satoshi Yamamoto, Keiichi Yamamoto, Yasuhiko Suzuki, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi

  PloS one 18 (10) e0291727  2023 

  Immune checkpoint inhibitors (ICIs) have been developed for canine tumour treatment, and pilot clinical studies have demonstrated their antitumour efficacy in dogs with oral malignant melanoma (OMM). Although ICIs have been approved for various human malignancies, their clinical benefits in other tumour types remain to be elucidated in dogs. Here, we conducted a clinical study of c4G12, a canine chimeric anti-PD-L1 antibody, to assess its safety and efficacy in dogs with various advanced malignant tumours (n = 12) at the Veterinary Teaching Hospital of Hokkaido University from 2018 to 2023. Dogs with digit or foot pad malignant melanoma (n = 4), osteosarcoma (n = 2), hemangiosarcoma (n = 1), transitional cell carcinoma (n = 1), nasal adenocarcinoma (n = 1), B-cell lymphoma (n = 1), or undifferentiated sarcoma (n = 2) were treated with 2 or 5 mg/kg c4G12 every 2 weeks. Treatment-related adverse events of any grade were observed in eight dogs (66.7%), including elevated aspartate aminotransferase (grade 3) in one dog (8.3%) and thrombocytopenia (grade 4) in another dog (8.3%). Among dogs with target disease at baseline (n = 8), as defined by the response evaluation criteria for solid tumours in dogs (cRECIST), one dog with nasal adenocarcinoma and another with osteosarcoma experienced a partial response (PR), with an objective response rate of 25.0% (2 PR out of 8 dogs; 95% confidence interval: 3.2-65.1%). These results suggest that c4G12 is safe and tolerable and shows antitumor effects in dogs with malignant tumours other than OMM. Further clinical studies are warranted to identify the tumour types that are most likely to benefit from c4G12 treatment.
• Characterization of cysteine proteases from poultry red mite, tropical fowl mite, and northern fowl mite to assess the feasibility of developing a broadly efficacious vaccine against multiple mite species.

  Shwe Yee Win, Shiro Murata, Sotaro Fujisawa, Hikari Seo, Jumpei Sato, Yoshinosuke Motai, Takumi Sato, Eiji Oishi, Akira Taneno, Lat Lat Htun, Saw Bawm, Tomohiro Okagawa, Naoya Maekawa, Satoru Konnai, Kazuhiko Ohashi

  PloS one 18 (7) e0288565  2023 

  Infestation with poultry red mites (PRM, Dermanyssus gallinae) causes anemia, reduced egg production, and death in serious cases, resulting in significant economic losses to the poultry industry. As a novel strategy for controlling PRMs, vaccine approaches have been focused upon and several candidate vaccine antigens against PRMs have been reported. Tropical (TFM, Ornithonyssus bursa) and northern (NFM, Ornithonyssus sylviarum) fowl mites are also hematophagous and cause poultry industry problems similar to those caused by PRM. Therefore, ideal antigens for anti-PRM vaccines are molecules that cross-react with TFMs and NFMs, producing pesticidal effects similar to those against PRMs. In this study, to investigate the potential feasibility of developing vaccines with broad efficacy across mite species, we identified and characterized cysteine proteases (CPs) of TFMs and NFMs, which were previously reported to be effective vaccine antigens of PRMs. The open reading frames of CPs from TFMs and NFMs had the same sequences, which was 73.0% similar to that of PRMs. Phylogenetic analysis revealed that the CPs of TFMs and NFMs clustered in the same clade as CPs of PRMs. To assess protein functionality, we generated recombinant peptidase domains of CPs (rCP-PDs), revealing all rCP-PDs showed CP-like activities. Importantly, the plasma obtained from chickens immunized with each rCP-PD cross-reacted with rCP-PDs of different mites. Finally, all immune plasma of rCP-PDs reduced the survival rate of PRMs, even when the plasma was collected from chickens immunized with rCP-PDs derived from TFM and NFM. Therefore, CP antigen is a promising, broadly efficacious vaccine candidate against different avian mites.
• Potential of ferritin 2 as an antigen for the development of a universal vaccine for avian mites, poultry red mites, tropical fowl mites, and northern fowl mites.

  Shwe Yee Win, Shiro Murata, Sotaro Fujisawa, Hikari Seo, Jumpei Sato, Yoshinosuke Motai, Takumi Sato, Eiji Oishi, Akira Taneno, Lat Lat Htun, Saw Bawm, Tomohiro Okagawa, Naoya Maekawa, Satoru Konnai, Kazuhiko Ohashi

  Frontiers in veterinary science 10 1182930 - 1182930 2023 

  INTRODUCTION: Poultry red mites (PRMs, Dermanyssus gallinae), blood-sucking ectoparasites, are a threat to the poultry industry because of reduced production caused by infestation. In addition, tropical fowl mites (TFMs, Ornithonyssus bursa) and northern fowl mites (NFMs, Ornithonyssus sylviarum) are hematophagous, distributed in various regions, genetically and morphologically close to PRMs, and cause similar problems to the poultry industry. Vaccine approaches have been studied for PRM control, and several molecules have been identified in PRMs as candidates for effective vaccine antigens. The development of an anti-PRM vaccine as a universal vaccine with broad efficacy against avian mites could improve the productivity of poultry farms worldwide. Molecules that are highly conserved among avian mites and have critical functions in the physiology and growth of mites could be ideal antigen candidates for the development of universal vaccines. Ferritin 2 (FER2), an iron-binding protein, is critical for the reproduction and survival of PRMs and has been reported as a useful vaccine antigen for the control of PRMs and a candidate for the universal vaccine antigen in some tick species. METHOD AND RESULTS: Herein, we identified and characterized FER2 in TFMs and NFM. Compared with the sequence of PRM, the ferroxidase centers of the heavy chain subunits were conserved in FER2 of TFMs and NFMs. Phylogenetic analysis revealed that FER2 belongs to clusters of secretory ferritins of mites and other arthropods. Recombinant FER2 (rFER2) proteins from PRMs, TFMs, and NFMs exhibited iron-binding abilities. Immunization with each rFER2 induced strong antibody responses in chickens, and each immune plasma cross-reacted with rFER2 from different mites. Moreover, mortality rates of PRMs fed with immune plasma against rFER2 from TFMs or NFMs, in addition to PRMs, were higher than those of control plasma. DISCUSSION: rFER2 from each avian mite exhibited anti-PRM effects. This data suggests that it has the potential to be used as an antigen candidate for a universal vaccine against avian mites. Further studies are needed to access the usefulness of FER2 as a universal vaccine for the control of avian mites.
• In vitro evaluation of Lactiplantibacillus plantarum HOKKAIDO strain, effective lactic acid bacteria for calf diarrhea.

  Mari Ikehata, Satoru Konnai, Tomohiro Okagawa, Kentaro Abe, Mitsuru Honma, Toru Kitamura, Naoya Maekawa, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  Frontiers in veterinary science 10 1145445 - 1145445 2023 

  Calf diarrhea adversely affects growth and sometimes results in mortality, leading to severe economic losses to the cattle industry. Antibiotics are useful in the treatment against bacterial diarrhea, but not against viral, protozoan, and antibiotic-resistant bacterial diarrhea. Therefore, there are growing requirements for a novel control method for calf diarrhea. Probiotics have been considered promising candidates for preventive and supportive therapy for calf diarrhea for many years. A recent study has revealed that Lactiplantibacillus plantarum HOKKAIDO strain (Lp-HKD) reduces intestinal pathology and the severity of diarrhea in bovine rotavirus (BRV)-infected calves. Lp-HKD is known to enhance the function of human immune cells and expected to be used as probiotics for humans. Therefore, it is hypothesized that Lp-HKD modulates antiviral immune response in cattle and provide the clinical benefits in BRV-infected calves. However, the detailed mechanism of Lp-HKD-induced immunomodulation remains unknown. Thus, this study aimed to elucidate the immunomodulatory and antiviral effects of Lp-HKD in cattle. Cultivation assay of bovine peripheral blood mononuclear cells (PBMCs) showed that live and heat-killed Lp-HKD stimulates the production of interleukin-1β (IL-1β), IL-6, IL-10, and interferon-γ (IFN-γ) from PBMCs. Stimulation by heat-killed Lp-HKD yielded stronger cytokine production than stimulation by the live Lp-HKD. Additionally, CD14+ monocytes were identified as major producers of IL-1β, IL-6, and IL-10 under Lp-HKD stimulation; however, IFN-γ was mainly produced from immune cells other than CD14+ monocytes. Depletion of CD14+ monocytes from the PBMCs cultivation strongly decreased cytokine production induced by heat-killed Lp-HKD. The inhibition of toll-like receptor (TLR) 2/4 signaling decreased IL-1β and IL-6 production induced by live Lp-HKD and IL-1β, IL-6, and IFN-γ production induced by heat-killed Lp-HKD. Furthermore, live or heat-killed Lp-HKD also activated T cells and their production of IFN-γ and tumor necrosis factor-α. Then, culture supernatants of bovine PBMCs treated with heat-killed Lp-HKD demonstrated antiviral effects against BRV in vitro. In conclusion, this study demonstrated that Lp-HKD activates the functions of bovine immune cells via TLR2/4 signaling and exerts an antiviral effect against BRV through the induction of antiviral cytokines. Lp-HKD could be useful for the prevention and treatment of calf diarrhea through its immune activating effect.
• Molecular characterization of feline immune checkpoint molecules and establishment of PD-L1 immunohistochemistry for feline tumors.

  Naoya Maekawa, Satoru Konnai, Yumie Asano, Takumi Otsuka, Eri Aoki, Hiroto Takeuchi, Yukinari Kato, Mika K Kaneko, Shinji Yamada, Yumiko Kagawa, Maki Nishimura, Satoshi Takagi, Tatsuya Deguchi, Hiroshi Ohta, Takayuki Nakagawa, Yasuhiko Suzuki, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi

  PloS one 18 (1) e0281143  2023 

  Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has progressed dramatically in the past decade, partly due to the success of immunotherapies using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1 therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1 and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 share high sequence identities (66-83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor-ligand binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established an anti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PD-L1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in a feline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm, FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-γ stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1 expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5, 80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors.
• Characterization of SpsQ from Staphylococcus pseudintermedius as an affinity chromatography ligand for canine therapeutic antibodies.

  Hiroto Takeuchi, Chie Nakajima, Satoru Konnai, Naoya Maekawa, Tomohiro Okagawa, Masaru Usui, Yutaka Tamura, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  PloS one 18 (1) e0281171  2023 

  Coagulase-positive Staphylococci express protein A, which binds to host antibodies, to evade the immune system. Taking advantage of its specific binding to antibodies, protein A from Staphylococcus aureus, which is called SpA, is commonly used as an affinity chromatography ligand for human therapeutic antibodies. However, among four canine IgG subclasses (A, B, C, and D), only IgG-B binds to SpA strongly and establishing an efficient and robust purification scheme for canine therapeutic antibodies whose IgG subclass is A, C, or D remains difficult and depends on finding a suitable substitute to SpA. S. pseudintermedius, a major coagulase-positive Staphylococci found in dogs, expresses spsQ gene which is orthologous to S. aureus spa. We hypothesized that to serve S. pseudintermedius to better adapt to the dog immune system, SpsQ would bind to canine IgGs stronger than SpA, making it a better affinity chromatography ligand for canine therapeutic antibodies. To characterize SpsQ, we first determined the spsQ nucleotide sequence from S. pseudintermedius isolates. Based on the identified sequence, we prepared recombinant proteins containing the immunoglobulin-binding domains of SpA (r-SpA) and SpsQ (r-SpsQ) and determined their binding capacity for each canine IgG subclass. The binding capacity of r-SpsQ for IgG-B was almost as high as that of r-SpA. Interestingly, while both r-SpsQ and r-SpA showed no binding to IgG-C, the binding capacity of r-SpsQ for IgG-A and IgG-D was significantly higher than that of r-SpA. Finally, we performed affinity chromatography using r-SpsQ- or r-SpA-immobilized resin and revealed that the recovery rates of IgG-A and IgG-D using r-SpsQ were significantly higher than those using r-SpA. Our findings indicate that SpsQ has a strong potential to be used as an affinity chromatography ligand for canine therapeutic antibodies of subclass A, B, and D.
• Diagnosis and Early Prediction of Lymphoma Using High-Throughput Clonality Analysis of Bovine Leukemia Virus-Infected Cells

  Tomohiro Okagawa, Honami Shimakura, Satoru Konnai, Masumichi Saito, Takahiro Matsudaira, Naganori Nao, Shinji Yamada, Kenji Murakami, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi

  Microbiology Spectrum 10 (6) 2022/10/13 

  Bovine leukemia virus (BLV) infection causes aggressive B-cell lymphoma in cattle and sheep. The virus has spread to farms around the world, causing significant economic damage to the livestock industry.
• Prostaglandin E2-Induced Immune Suppression via Cytotoxic T-Lymphocyte Antigen 4 in Paratuberculosis.

  Yamato Sajiki, Satoru Konnai, Kei Watari, Tomohiro Okagawa, Akina Tanaka, Satoko Kawaji, Reiko Nagata, Naoya Maekawa, Yasuhiko Suzuki, Yukinari Kato, Shiro Murata, Yasuyuki Mori, Kazuhiko Ohashi

  Infection and immunity 90 (10) e0021022  2022/09/14 

  Paratuberculosis is a chronic enteritis of ruminants caused by the facultative intracellular pathogen Mycobacterium avium subsp. paratuberculosis. The Th1 response inhibits the proliferation of M. avium subsp. paratuberculosis during the early subclinical stage. However, we have previously shown that immune inhibitory molecules, such as prostaglandin E2 (PGE2), suppress M. avium subsp. paratuberculosis-specific Th1 responses as the disease progresses. To date, the mechanism underlying immunosuppression during M. avium subsp. paratuberculosis infection has not been elucidated. Therefore, in the present study, we investigated the function of cytotoxic T-lymphocyte antigen 4 (CTLA-4) expressed by peripheral blood mononuclear cells (PBMCs) from cattle with paratuberculosis because CTLA-4 expression is known to be elevated in T cells under an M. avium subsp. paratuberculosis experimental infection. M. avium subsp. paratuberculosis antigen induced CTLA-4 expression in T cells from cattle experimentally infected with M. avium subsp. paratuberculosis. Interestingly, both PGE2 and an E prostanoid 4 agonist also induced CTLA-4 expression in T cells. In addition, a functional assay with a bovine CTLA-4-immunogobulin fusion protein (CTLA-4-Ig) indicated that CTLA-4 inhibited gamma interferon (IFN-γ) production in M. avium subsp. paratuberculosis-stimulated PBMCs, while blockade by anti-bovine CTLA-4 monoclonal antibody increased the secretion of IFN-γ and tumor necrosis factor alpha production in these PBMCs. These preliminary findings show that PGE2 has immunosuppressive effects via CTLA-4 to M. avium subsp. paratuberculosis. Therefore, it is necessary to clarify in the future whether CTLA-4-mediated immunosuppression facilitates disease progression of paratuberculosis in cattle.
• 乳酸菌Lactobacillus plantarum HOKKAIDO株による免疫制御機序の解明

  池端 麻里, 今内 覚, 岡川 朋弘, 阿部 健太郎, 本間 満, 北村 亨, 前川 直也, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [DI1A - 01] 1347-8621 2022/09
• ネコ腫瘍組織におけるprogrammed death ligand 1(PD-L1)の発現解析

  青木 絵理, 今内 覚, 前川 直也, 岡川 朋弘, 竹内 寛人, 佐藤 純平, 浅野 裕美恵, 大塚 拓海, 賀川 由美子, 加藤 幸成, 高木 哲, 鈴木 定彦, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [DI1A - 04] 1347-8621 2022/09
• 鼻疽の診断抗原の探索

  市川 世識, 飯沼 由希帆, 岡川 朋弘, 前川 直也, 村田 史郎, 今内 覚, 木下 優太, 丹羽 秀和, 青島 圭佑, 小林 篤史, 大橋 和彦, 木村 享史

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [DI1A - 09] 1347-8621 2022/09
• 広範な有効性を示すワクチン開発のための鳥類寄生ダニ由来システインプロテアーゼの特性解析(Characterization of cysteine proteases from avian mites to develop a vaccine with broad efficacy)

  Win Shwe Yee, 村田 史郎, 藤澤 宗太郎, 瀬尾 光里, 佐藤 純平, 佐藤 匠, 大石 英司, 種子野 章, 岡川 朋弘, 前川 直也, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 165回 [E3P - 02] 1347-8621 2022/09
• ワクモ(Dermanyssus gallinae)由来シスタチン様分子の抗ワクモワクチン抗原としての評価

  藤澤 宗太郎, 村田 史郎, 伊勢崎 政美, 有泉 拓馬, 佐藤 匠, 大石 英司, 種子野 章, 前川 直也, 岡川 朋弘, 市居 修, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [E3P - 01] 1347-8621 2022/09
• ワクモ由来の2種類のアスパラギン酸ペプチダーゼの性状解析

  瀬尾 光里, 村田 史郎, Shwe Yee Win, 藤澤 宗太郎, 北條 巧, 伊勢崎 政美, 佐藤 匠, 大石 英司, 種子野 章, 市居 修, 前川 直也, 岡川 朋弘, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [E3P - 03] 1347-8621 2022/09
• 乳酸菌Lactobacillus plantarum HOKKAIDO株による免疫制御機序の解明

  池端 麻里, 今内 覚, 岡川 朋弘, 阿部 健太郎, 本間 満, 北村 亨, 前川 直也, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [DI1A - 01] 1347-8621 2022/09
• ネコ腫瘍組織におけるprogrammed death ligand 1(PD-L1)の発現解析

  青木 絵理, 今内 覚, 前川 直也, 岡川 朋弘, 竹内 寛人, 佐藤 純平, 浅野 裕美恵, 大塚 拓海, 賀川 由美子, 加藤 幸成, 高木 哲, 鈴木 定彦, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [DI1A - 04] 1347-8621 2022/09
• ワクモ(Dermanyssus gallinae)由来シスタチン様分子の抗ワクモワクチン抗原としての評価

  藤澤 宗太郎, 村田 史郎, 伊勢崎 政美, 有泉 拓馬, 佐藤 匠, 大石 英司, 種子野 章, 前川 直也, 岡川 朋弘, 市居 修, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [E3P - 01] 1347-8621 2022/09
• 広範な有効性を示すワクチン開発のための鳥類寄生ダニ由来システインプロテアーゼの特性解析(Characterization of cysteine proteases from avian mites to develop a vaccine with broad efficacy)

  Win Shwe Yee, 村田 史郎, 藤澤 宗太郎, 瀬尾 光里, 佐藤 純平, 佐藤 匠, 大石 英司, 種子野 章, 岡川 朋弘, 前川 直也, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 165回 [E3P - 02] 1347-8621 2022/09
• ワクモ由来の2種類のアスパラギン酸ペプチダーゼの性状解析

  瀬尾 光里, 村田 史郎, Shwe Yee Win, 藤澤 宗太郎, 北條 巧, 伊勢崎 政美, 佐藤 匠, 大石 英司, 種子野 章, 市居 修, 前川 直也, 岡川 朋弘, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [E3P - 03] 1347-8621 2022/09
• 鼻疽の診断抗原の探索

  市川 世識, 飯沼 由希帆, 岡川 朋弘, 前川 直也, 村田 史郎, 今内 覚, 木下 優太, 丹羽 秀和, 青島 圭佑, 小林 篤史, 大橋 和彦, 木村 享史

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [DI1A - 09] 1347-8621 2022/09
• Suppressive effects of Ixodes persulcatus sialostatin L2 against Borrelia miyamotoi-stimulated immunity.

  Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Masayoshi Isezaki, Shinji Yamada, Takuya Ito, Kozue Sato, Hiroki Kawabata, Carlos Logullo, Itabajara da Silva Vaz Jr, Shiro Murata, Kazuhiko Ohashi

  Ticks and tick-borne diseases 13 (4) 101963 - 101963 2022/07 

  Borrelia miyamotoi infection is an emerging tick-borne disease that causes hard tick-borne relapsing fever. B. miyamotoi is transmitted through the bite of ticks, including Ixodes persulcatus. Although accumulating evidence suggests that tick salivary proteins enhance the infectivity of other tick-borne pathogens, the association of B. miyamotoi with tick-derived proteins remains unknown. In this study, the effect of I. persulcatus sialostatin L2 (Ip-sL2), a tick-derived cystatin, on specific immunity to B. miyamotoi was preliminarily investigated in vitro. Mice were immunized with heat-killed B. miyamotoi and in vitro analyses of the splenocytes of the immunized mice indicated that the expression levels of the activation markers of CD11c+ and CD3+ cells were significantly upregulated by B. miyamotoi stimulation. Spleen cells from B. miyamotoi-immunized mice were used to determine whether Ip-sL2 regulates murine immune responses against B. miyamotoi. Treatment with Ip-sL2 in vitro inhibited the activation of CD11c+ and CD3+ cells as well as inflammatory cytokine production by cultured splenocytes. These findings show that Ip-sL2 has modulatory effects on murine immune responses to B. miyamotoi. Therefore, it is necessary to clarify in the future whether Ip-sL2 is involved in the enhanced infectivity of B. miyamotoi.
• Exploration of serum biomarkers in dogs with malignant melanoma receiving anti-PD-L1 therapy and potential of COX-2 inhibition for combination therapy.

  Naoya Maekawa, Satoru Konnai, Yumie Asano, Yamato Sajiki, Tatsuya Deguchi, Tomohiro Okagawa, Kei Watari, Hiroto Takeuchi, Satoshi Takagi, Kenji Hosoya, Sangho Kim, Hiroshi Ohta, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  Scientific reports 12 (1) 9265 - 9265 2022/06/03 

  Immune checkpoint inhibitors (ICIs) such as anti-PD-L1 antibodies are widely used to treat human cancers, and growing evidence suggests that ICIs are promising treatments for canine malignancies. However, only some canine oral malignant melanoma (OMM) cases respond to ICIs. To explore biomarkers predictive of survival in dogs with pulmonary metastatic OMM receiving the anti-PD-L1 antibody c4G12 (n = 27), serum concentrations of prostaglandin E2 (PGE2), cytokines, chemokines, and growth factors were measured prior to treatment initiation. Among 12 factors tested, PGE2, interleukin (IL)-12p40, IL-8, monocyte chemotactic protein-1 (MCP-1), and stem cell factor (SCF) were higher in OMM dogs compared to healthy dogs (n = 8). Further, lower baseline serum PGE2, MCP-1, and vascular endothelial growth factor (VEGF)-A concentrations as well as higher IL-2, IL-12, and SCF concentrations predicted prolonged overall survival. These observations suggest that PGE2 confers resistance against anti-PD-L1 therapy through immunosuppression and thus is a candidate target for combination therapy. Indeed, PGE2 suppressed IL-2 and interferon (IFN)-γ production by stimulated canine peripheral blood mononuclear cells (PBMCs), while inhibition of PGE2 biosynthesis using the COX-2 inhibitor meloxicam in combination with c4G12 enhanced Th1 cytokine production by PBMCs. Thus, serum PGE2 may be predictive of c4G12 treatment response, and concomitant use of COX-2 inhibitors may enhance ICI antitumor efficacy.
• Estradiol-induced immune suppression via prostaglandin E2 during parturition in bovine leukemia virus-infected cattle

  Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Junko Kohara, Atsushi Nitanai, Hirofumi Takahashi, Kentaro Kubota, Hiroshi Takeda, Shiro Murata, Kazuhiko Ohashi

  PLOS ONE 17 (3) e0263660 - e0263660 2022/03/09 

  Immune suppression during pregnancy and parturition is considered a risk factor that is related to the progression of bovine chronic diseases, such as bovine leukosis, which is caused by bovine leukemia virus (BLV). Our previous studies have demonstrated that prostaglandin E₂ (PGE₂) suppresses BLV-specific Th1 responses and contributes to the disease progression during BLV infection. Although PGE₂ reportedly plays important roles in the induction of parturition, PGE₂ involvement in immune suppression during parturition is unknown. To investigate its involvement, we analyzed PGE₂ kinetics and Th1 responses in BLV-infected pregnant cattle. PGE₂ concentrations in sera were increased, whereas IFN-γ responses were decreased before delivery. PGE₂ is known to suppress Th1 immune responses in cattle. Thus, these data suggest that PGE₂ upregulation inhibits Th1 responses during parturition. We also found that estradiol was important for PGE₂ induction in pregnant cattle. In vitro analyses indicated that estradiol suppressed IFN-γ production, at least in part, via PGE₂/EP4 signaling. In vivo analyses showed that estradiol administration significantly influenced the induction of PGE₂ production and impaired Th1 responses. Our data suggest that estradiol-induced PGE₂ is involved in the suppression of Th1 responses during pregnancy and parturition in cattle, which could contribute to the progression of BLV infection.
• In vitro evaluation of a cysteine protease from poultry red mites, Demanyssus gallinae, as a vaccine antigen for chickens

  Takuma Ariizumi, Shiro Murata, Sotaro Fujisawa, Masayoshi Isezaki, Takumi Sato, Eiji Oishi, Akira Taneno, Osamu Ichii, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi

  Poultry Science 101 (3) 101638 - 101638 0032-5791 2022/03
• Effect of Insertion and Deletion in the Meq Protein Encoded by Highly Oncogenic Marek’s Disease Virus on Transactivation Activity and Virulence

  Jumpei Sato, Shiro Murata, Zhiyuan Yang, Benedikt B. Kaufer, Sotaro Fujisawa, Hikari Seo, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Nikolaus Osterrieder, Mark S. Parcells, Kazuhiko Ohashi

  Viruses 14 (2) 382 - 382 2022/02/14 

  Marek’s disease virus (MDV) causes malignant lymphoma in chickens (Marek’s disease, MD). Although MD is currently controlled by vaccination, MDV strains have continuously increased in virulence over the recent decades. Polymorphisms in Meq, an MDV-encoded oncoprotein that serves as a transcription factor, have been associated with the enhanced virulence of the virus. In addition, insertions and deletions in Meq have been observed in MDV strains of higher virulence, but their contribution to said virulence remains elusive. In this study, we investigated the contribution of an insertion (L-Meq) and a deletion in the Meq gene (S-Meq) to its functions and MDV pathogenicity. Reporter assays revealed that both insertion and deletion enhanced the transactivation potential of Meq. Additionally, we generated RB-1B-based recombinant MDVs (rMDVs) encoding each Meq isoform and analyzed their pathogenic potential. rMDV encoding L-Meq indueced the highest mortality and tumor incidence in infected animals, whereas the rMDV encoding S-Meq exhibited the lowest pathogenicity. Thus, insertion enhanced the transactivation activity of Meq and MDV pathogenicity, whereas deletion reduced pathogenicity despite having increased transactivation activity. These data suggest that other functions of Meq affect MDV virulence. These data improve our understanding of the mechanisms underlying the evolution of MDV virulence.
• Enhancement of interleukin-2 production by bovine peripheral blood mononuclear cells treated with the combination of anti-programmed death-ligand 1 and cytotoxic T lymphocyte antigen 4 chimeric monoclonal antibodies.

  Kei Watari, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Yamato Sajiki, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  The Journal of veterinary medical science 84 (1) 6 - 15 2022/01/07 

  Our previous studies demonstrate the therapeutic efficacy against bovine diseases of an anti-bovine programmed death-ligand 1 (PD-L1) chimeric antibody. In humans, PD-1 and PD-L1 antibodies are more effective when combined with an antibody targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and these combination therapies are therefore clinically used. Here we generated an anti-bovine CTLA-4 chimeric antibody (chAb) to enhance the therapeutic efficacy of the PD-L1 antibody. We further analyzed the effects of dual blockade of CTLA-4 and PD-1 pathways on T-cell responses. The established anti-bovine CTLA-4 chAb showed comparable blocking activity on the binding of bovine CTLA-4 to CD80 and CD86 as the anti-bovine CTLA-4 mouse monoclonal antibody. Anti-bovine CTLA-4 chAb also significantly increased IL-2 production from bovine peripheral blood mononuclear cells (PBMCs). Further, the combination of anti-CTLA-4 chAb with anti-PD-L1 chAb significantly upregulated IL-2 production by PBMCs. These results suggest that the combination of antibodies have higher potential to enhance immune responses against pathogens compared with single administration.
• Characterization of a Novel Cysteine Protease Inhibitor from Poultry Red Mites: Potential Vaccine for Chickens

  Sotaro Fujisawa, Shiro Murata, Masayoshi Isezaki, Takuma Ariizumi, Takumi Sato, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Osamu Ichii, Satoru Konnai, Kazuhiko Ohashi

  Vaccines 9 (12) 1472 - 1472 2021/12/13 

  Poultry red mite (PRM; Dermanyssus gallinae) is a hazardous, blood-sucking ectoparasite of birds that constitutes a threat to poultry farming worldwide. Acaricides, commonly used in poultry farms to prevent PRMs, are not effective because of the rapid emergence of acaricide-resistant PRMs. However, vaccination may be a promising strategy to control PRM. We identified a novel cystatin-like molecule in PRMs: Dg-Cys. Dg-Cys mRNA expression was detected in the midgut and ovaries, in all stages of life. The PRM nymphs that were artificially fed with the plasma from chickens that were immunized with Dg-Cys in vitro had a significantly reduced reproductive capacity and survival rate. Moreover, combination of Dg-Cys with other antigen candidates, like copper transporter 1 or adipocyte plasma membrane-associated protein, enhanced vaccine efficacies. vaccination and its application as an antigen for cocktail vaccines could be an effective strategy to reduce the damage caused by PRMs in poultry farming.
• Tick saliva-induced programmed death-1 and PD-ligand 1 and its related host immunosuppression

  Yamato Sajiki, Satoru Konnai, Yoshinori Ikenaka, Kevin Christian Montecillo Gulay, Atsushi Kobayashi, Luís Fernando Parizi, Benvindo Capela João, Kei Watari, Sotaro Fujisawa, Tomohiro Okagawa, Naoya Maekawa, Carlos Logullo, Itabajara da Silva Vaz, Shiro Murata, Kazuhiko Ohashi

  Scientific Reports 11 (1) 1063 - 1063 2021/12 

  The tick Rhipicephalus microplus is a harmful parasite of cattle that causes considerable economic losses to the cattle breeding industry. Although R. microplus saliva (Rm-saliva) contains several immunosuppressants, any association between Rm-saliva and the expression of immunoinhibitory molecules, such as programmed death (PD)-1 and PD-ligand 1 (PD-L1), has not been described. In this study, flow cytometric analyses revealed that Rm-saliva upregulated PD-1 expression in T cells and PD-L1 expression in CD14⁺ and CD11c⁺ cells in cattle. Additionally, Rm-saliva decreased CD69 expression in T cells and Th1 cytokine production from peripheral blood mononuclear cells. Furthermore, PD-L1 blockade increased IFN-γ production in the presence of Rm-saliva, suggesting that Rm-saliva suppresses Th1 responses via the PD-1/PD-L1 pathway. To reveal the upregulation mechanism of PD-1/PD-L1 by Rm-saliva, we analyzed the function of prostaglandin E₂ (PGE₂), which is known as an inducer of PD-L1 expression, in Rm-saliva. We found that Rm-saliva contained a high concentration of PGE₂, and PGE₂ treatment induced PD-L1 expression in CD14⁺ cells in vitro. Immunohistochemical analyses revealed that PGE₂ and PD-L1 expression was upregulated in tick-attached skin in cattle. These data suggest that PGE₂ in Rm-saliva has the potential to induce the expression of immunoinhibitory molecules in host immune cells.
• Differential expression of PEPCK isoforms is correlated to Aedes aegypti oogenesis and embryogenesis

  Renato Martins da Silva, Wagner Oliveira Vital, Ronald Sodre Martins, Jorge Moraes, Helga Gomes, Christiano Calixto, Satoru Konnai, Kazuhiko Ohashi, Itabajara da Silva Vaz, Carlos Logullo

  Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology 256 1879-1107 2021/10/01 

  The mosquito Aedes aegypti undertakes a shift in carbohydrate metabolism during embryogenesis, including an increase in the activity of phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, at critical steps of embryo development. All eukaryotes studied to date present two PEPCK isoforms, namely PEPCK-M (mitochondrial) and PEPCK-C (cytosolic). In A. aegypti, however, these proteins are so far uncharacterized. In the present work we describe two A. aegypti PEPCK isoforms by sequence alignment, protein modeling, and transcription analysis in different tissues, as well as PEPCK enzymatic activity assays in mitochondrial and cytoplasmic compartments during oogenesis and embryogenesis. First, we characterized the protein sequences compared to other organisms, and identified conserved sites and key amino acids. We also performed structure modeling for AePEPCK(M) and AePEPCK(C), identifying highly conserved structural sites, as well as a signal peptide in AePEPCK(M) localized in a very hydrophobic region. Moreover, after blood meal and during mosquito oogenesis and embryogenesis, both PEPCKs isoforms showed different transcriptional profiles, suggesting that mRNA for the cytosolic form is transmitted maternally, whereas the mitochondrial form is synthesized by the zygote. Collectively, these results improve our understanding of mosquito physiology and may yield putative targets for developing new methods for A. aegypti control.
• In vitro characterization of adipocyte plasma membrane-associated protein from poultry red mites, Dermanyssus gallinae, as a vaccine antigen for chickens

  Sotaro Fujisawa, Shiro Murata, Masaki Takehara, Julia Aoyama, Ayu Morita, Masayoshi Isezaki, Shwe Yee Win, Takuma Ariizumi, Takumi Sato, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Osamu Ichii, Satoru Konnai, Kazuhiko Ohashi

  Vaccine 39 (41) 6057 - 6066 0264-410X 2021/10 

  The poultry red mite (Dermanyssus gallinae; PRM) is a blood-sucking ectoparasite of chickens that is a threat to poultry farming worldwide and significantly reduces productivity in the egg-laying industry. Chemical acaricides that are widely used in poultry farms for the prevention of PRMs are frequently ineffective due to the emergence of acaricide-resistant PRMs. Therefore, alternative control methods are needed, and vaccination is a promising strategy for controlling PRMs. A novel adipocyte-plasma membrane-associated protein-like molecule (Dg-APMAP) is highly expressed in blood-fed PRMs according to a previous RNA sequencing analysis. Here, we attempted to identify the full sequence of Dg-APMAP, study its expression in different life stages of PRMs, and evaluate its potential as a vaccine antigen. Dg-APMAP mRNA was expressed in the midgut and ovaries, and in all life stages regardless of feeding states. Importantly, in vitro feeding of PRMs with plasma derived from chickens immunized with the recombinant protein of the extracellular region of Dg-APMAP significantly reduced their survival rate in nymphs and adults, which require blood meals. Our data suggest that the host immune responses induced by vaccination with Dg-APMAP could be an effective strategy to reduce the suffering caused by PRMs in the poultry industry.
• Research Note: Characterization of S-Meq containing the deletion in Meq protein's transactivation domain in a Marek's disease virus strain in Japan.

  Shiro Murata, Eiji Yamamoto, Natsumi Sakashita, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi

  Poultry science 100 (11) 101461 - 101461 2021/09/02 

  Marek's disease virus (MDV) causes malignant lymphomas in chickens (Marek's disease; MD). Although MD has caused significant economic losses to the poultry industry, currently, its occurrence in the field is effectively controlled by vaccination. However, the genetic characteristics of MDV strains have changed, and the poultry industry has experienced MD outbreaks in vaccinated chickens because of enhanced virulence. Meq, an oncoprotein of MDV, is a key transcription factor correlated with the tumorigenesis in MD. Animal experiments using recombinant MDV revealed that distinct polymorphisms in Meq affect the virulence of MDV strains. Meq containing an insertion or deletion is present in some MDV strains. In the 2010s, field strains that encode Meq containing the deletion (S-Meq) were reported. In this study, we characterized the genetic features of S-Meq detected in a Japanese MDV strain and analyzed its transactivation activity to investigate S-Meq's protein function. S-Meq lacked 41 amino acids, and the deletion was at the same position as those observed in other countries. In addition, S-Meq exhibited higher transactivation activity than Meq from other MDV strains circulating in Japan. These results suggest that the deletion in the transactivation domain may enhance the Meq protein's function. Further investigation is needed to clarify whether the deletion in the transactivation domain of Meq affects MDV's virulence.
• ウシCTLA-4ならびにPD-L1を標的とした抗体併用法による免疫増強効果の検討

  渡 慧, 今内 覚, 岡川 朋弘, 前川 直也, 村田 史郎, 鈴木 定彦, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [DIO - 4] 1347-8621 2021/09
• 牛伝染性リンパ腫発症母牛における子宮内感染の解析

  嶋倉 穂南, 今内 覚, 岡川 朋弘, 中村 隼人, 神谷 可菜, 齋藤 麻矢, 前川 直也, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [DVO - 5] 1347-8621 2021/09
• マレック病ウイルスMeqタンパク質における挿入および欠損配列の病原性への影響

  佐藤 純平, 村田 史郎, 藤澤 宗太郎, 瀬尾 光里, 前川 直也, 岡川 朋弘, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [DVO - 25] 1347-8621 2021/09
• ワクモ(Dermanyssus gallinae)由来システインプロテアーゼの抗ワクモワクチン抗原としての評価

  有泉 拓馬, 村田 史郎, 藤澤 宗太郎, 伊勢崎 政美, 佐藤 匠, 大石 英司, 種子野 章, 市居 修, 前川 直也, 岡川 朋弘, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [EO - 6] 1347-8621 2021/09
• 鼻疽に対する診断用抗原の探索および血清診断法の確立

  飯沼 由希帆, 岡川 朋弘, 市川 世識, 陸 拾七, 前川 直也, 村田 史郎, 今内 覚, 木村 亨史, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [DIO - 2] 1347-8621 2021/09
• Expression and functional analysis of swine PD-1/PD-L1 pathway(和訳中)

  Otgontuya Ganbaatar, 今内 覚, 岡川 朋弘, 野島 裕太郎, 前川 直也, 市川 世識, 小林 篤史, 芝原 友幸, 柳川 洋二郎, 鈴木 定彦, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 164回 [DIO - 3] 1347-8621 2021/09
• ウシCTLA-4ならびにPD-L1を標的とした抗体併用法による免疫増強効果の検討

  渡 慧, 今内 覚, 岡川 朋弘, 前川 直也, 村田 史郎, 鈴木 定彦, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [DIO - 4] 1347-8621 2021/09
• 牛伝染性リンパ腫発症母牛における子宮内感染の解析

  嶋倉 穂南, 今内 覚, 岡川 朋弘, 中村 隼人, 神谷 可菜, 齋藤 麻矢, 前川 直也, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [DVO - 5] 1347-8621 2021/09
• マレック病ウイルスMeqタンパク質における挿入および欠損配列の病原性への影響

  佐藤 純平, 村田 史郎, 藤澤 宗太郎, 瀬尾 光里, 前川 直也, 岡川 朋弘, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [DVO - 25] 1347-8621 2021/09
• ワクモ(Dermanyssus gallinae)由来システインプロテアーゼの抗ワクモワクチン抗原としての評価

  有泉 拓馬, 村田 史郎, 藤澤 宗太郎, 伊勢崎 政美, 佐藤 匠, 大石 英司, 種子野 章, 市居 修, 前川 直也, 岡川 朋弘, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [EO - 6] 1347-8621 2021/09
• Programmed death‐ligand 1 expression in swine chronic infections and enhancement of interleukin‐2 production via programmed death‐1/programmed death‐ligand 1 blockade

  Otgontuya Ganbaatar, Satoru Konnai, Tomohiro Okagawa, Yutaro Nojima, Naoya Maekawa, Yoshiki Ichikawa, Atsushi Kobayashi, Tomoyuki Shibahara, Yojiro Yanagawa, Hidetoshi Higuchi, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  Immunity, Inflammation and Disease 2050-4527 2021/08/20
• Prostaglandin-related immune suppression in cattle

  Y. Sajiki, S. Konnai, Y. Ikenaka, T. Okagawa, N. Maekawa, C. Logullo, I. da Silva Vaz, S. Murata, K. Ohashi

  Veterinary Immunology and Immunopathology 236 110238 - 110238 0165-2427 2021/06 

  Prostaglandins (PGs) are lipid mediators derived from arachidonic acid by several enzymes including cyclooxygenase (COX)-1 and COX-2. We have previously shown that PGE2 regulates immune responses, such as Th1 cytokine production and T-cell proliferation, in cattle. However, it is still unclear whether other PGs are involved in the regulation of immune responses in cattle. Here, immunosuppressive profiles of PGs (PGA1, PGB2, PGD2, PGE2, PGF1α and PGF2α) were firstly examined using bovine peripheral blood mononuclear cells (PBMCs). In addition to PGE2, PGA1 significantly inhibited Th1 cytokine production from PBMCs in cattle. Further analyses focusing on PGA1 revealed that treatment with PGA1 in the presence of concanavalin A (con A) downregulated CD69, an activation marker, and IFN-γ expression in both CD4+ and CD8+ T cells. Sorted CD3+ T cells stimulated with con A were cultivated with PGA1, and IFN-γ and TNF-α concentrations decreased upon PGA1 treatment. Taken together, these results suggest that the treatment with PGA1 in vitro inhibits T-cell activation, especially Th1 cytokine production, in cattle.
• Selection of reference genes for quantitative PCR analysis in poultry red mite (Dermanyssus gallinae).

  Takuma Ariizumi, Shiro Murata, Sotaro Fujisawa, Masayoshi Isezaki, Naoya Maekawa, Tomohiro Okagawa, Takumi Sato, Eiji Oishi, Akira Taneno, Satoru Konnai, Kazuhiko Ohashi

  The Journal of veterinary medical science 83 (4) 558 - 565 2021/04/09 

  Poultry red mites (PRMs, Dermanyssus gallinae) are harmful ectoparasites that affect farmed chickens and cause serious economic losses in the poultry industry worldwide. Acaricides are used for PRM control; however, some PRMs have developed acaricide-resistant properties, which have indicated the need for different approaches for PRM control. Therefore, it is necessary to elucidate the biological status of PRMs to develop alternative PRM control strategies. Quantitative polymerase chain reaction (qPCR) allows analysis of the biological status at the transcript level. However, reference genes are preferable for accurate comparison of expression level changes given the large variation in the quality of the PRM samples collected in each farm. This study aimed to identify candidate reference genes with stable expression levels in the different blood feeding states and life stages of PRMs. First, we selected candidates based on the following criteria: sufficient expression intensity and no significant expression difference between fed and starved states. We selected and characterized seven candidate reference genes. Among them, we evaluated the gene expression stability between the starved and fed states using RefFinder; moreover, we compared their expression levels in each life-stage and identified two reference genes, Elongation factor 1-alpha (ELF1A)-like and apolipophorins-like. Finally, we evaluated the utility of the candidates as reference genes, and the use of ELF1A-like and apolipophorins-like successfully normalized ATP synthase subunit g -like gene expression. Thus, ELF1A-like and apolipophorins-like could be suitable reference genes in PRMs.
• Direct evidence of the preventive effect of milk replacer–based probiotic feeding in calves against severe diarrhea

  Fumi Kayasaki, Tomohiro Okagawa, Satoru Konnai, Junko Kohara, Yamato Sajiki, Kei Watari, Otgontuya Ganbaatar, Shinya Goto, Hayato Nakamura, Honami Shimakura, Erina Minato, Atsushi Kobayashi, Manabu Kubota, Nobuhiro Terasaki, Akira Takeda, Haruka Noda, Mitsuru Honma, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi

  Veterinary Microbiology 254 108976 - 108976 0378-1135 2021/03 

  Diarrhea is a major cause of death in calves and this is linked directly to economic loss in the cattle industry. Fermented milk replacer (FMR) has been used widely in clinical settings for calf feeding to improve its health and growth. However, the protective efficacy of FMR on calf diarrhea remains unclear. In this study, we verified the preventive effects of FMR feeding on calf diarrhea using an experimental infection model of bovine rotavirus (BRV) in newborn calves and a field study in dairy farms with calf diarrhea. In addition, we evaluated the protective efficacy of lactic acid bacteria-supplemented milk replacer (LAB-MR) in an experimental infection model. In the experimental infection, calves fed FMR or high-concentrated LAB-MR had diarrhea, but the water content of feces was lower and more stable than that of calves fed normal milk replacer. The amount of milk intake also decreased temporarily, but recovered immediately in the FMR- and LAB-MR-fed calves. As compared with the control calves, FMR- or LAB-MR-fed calves showed less severe or reduced histopathological lesions of enteritis in the intestinal mucosa. In a field study using dairy calves, FMR feeding significantly reduced the incidence of enteritis, mortality from enteritis, duration of a series of treatment for enteritis, number of consultations, and cost of medical care for the disease. These results suggest that feeding milk replacer-based probiotics to calves reduces the severity of diarrhea and tissue damage to the intestinal tract caused by BRV infection and provides significant clinical benefits to the prevention and treatment of calf diarrhea.
• PD-L1 immunohistochemistry for canine cancers and clinical benefit of anti-PD-L1 antibody in dogs with pulmonary metastatic oral malignant melanoma.

  Naoya Maekawa, Satoru Konnai, Maki Nishimura, Yumiko Kagawa, Satoshi Takagi, Kenji Hosoya, Hiroshi Ohta, Sangho Kim, Tomohiro Okagawa, Yusuke Izumi, Tatsuya Deguchi, Yukinari Kato, Satoshi Yamamoto, Keiichi Yamamoto, Mikihiro Toda, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  NPJ precision oncology 5 (1) 10 - 10 2021/02/12 

  Immunotherapy targeting programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) represents promising treatments for human cancers. Our previous studies demonstrated PD-L1 overexpression in some canine cancers, and suggested the therapeutic potential of a canine chimeric anti-PD-L1 monoclonal antibody (c4G12). However, such evidence is scarce, limiting the clinical application in dogs. In the present report, canine PD-L1 expression was assessed in various cancer types, using a new anti-PD-L1 mAb, 6C11-3A11, and the safety and efficacy of c4G12 were explored in 29 dogs with pulmonary metastatic oral malignant melanoma (OMM). PD-L1 expression was detected in most canine malignant cancers including OMM, and survival was significantly longer in the c4G12 treatment group (median 143 days) when compared to a historical control group (n = 15, median 54 days). In dogs with measurable disease (n = 13), one dog (7.7%) experienced a complete response. Treatment-related adverse events of any grade were observed in 15 dogs (51.7%). Here we show that PD-L1 is a promising target for cancer immunotherapy in dogs, and dogs could be a useful large animal model for human cancer research.
• Intrauterine infection with Mycobacterium avium subsp. paratuberculosis in pregnant cattle diagnosed with Johne's disease

  Yukiko Taniguchi, Satoru Konnai, Shinichi Sakakibara, Ayano Yamamoto, Tomohiro Okagawa, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi

  JAPANESE JOURNAL OF VETERINARY RESEARCH 69 (1) 51 - 55 0047-1917 2021/02 

  Johne's disease (JD) is caused by infection with Mycobacterium avium subsp. paratuberulosis (MAP). We confirmed the intrauterine infection of MAP in 22 pregnant cattle diagnosed with JD in Hokkaido, Japan. MAP was isolated from the umbilical cord (3/22: 13.6%) or caruncle (6/22: 27.3%) derived from the pregnant dams. Furthermore, dams with MAP from which MAP was isolated were also found to have a high mount of MAP or detected bacterial load in their feces. Fetuses of the tested dams indicated positive polymerase chain reaction (PCR) results for the MAP gene (17/22:77.3%) in several tissues. MAP was isolated from the PCR-positive sites of dams detected with high levels of bacteria (6/ 22: 27.3%). These results indicate that MAP infection in pregnant cattle must be prevented as it is important for JD control.
• Cross-species reactivity of antibodies against Ixodes persulcatus ferritin 2 to Rhipicephalus microplus

  Marina Amaral Xavier, Satoru Konnai, Luis Fernando Parizi, Naftaly Wangombe Githaka, Masayoshi Isezaki, Shinya Goto, Sotaro Fujisawa, Shinji Yamada, Tomohiro Okagawa, Naoya Maekawa, Carlos Logullo, Itabajara da Silva Vaz Jr, Shiro Murata, Kazuhiko Ohashi

  JAPANESE JOURNAL OF VETERINARY RESEARCH 69 (1) 57 - 65 0047-1917 2021/02 

  There are several studies that confirm the possibility of developing a vaccine against tick infestations. An immuno-bioinformatics approach was used to identify conserved antigenic regions between Ixodes persulcatus and Rhipicephalus microplus ferritin 2 ( FER2) in order to compose a novel putative vaccine. In addition, R. microplus were fed on blood containing antibodies anti-recombinant FER2 from I. persulcatus (rIp-FER2). The results revealed that anti-ferritin antibodies led to a decrease in the engorgement weight of the R. microplus females. Conservation of the predicted antigenic regions in different tick species suggests that this protein could be useful to develop a vaccine for cross-species protection.
• Characterization of a copper transporter 1 from Dermanyssus gallinae as a vaccine antigen

  Sotaro Fujisawa, Shiro Murata, Masayoshi Isezaki, Takuma Ariizumi, Takumi Sato, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Osamu Ichii, Satoru Konnai, Kazuhiko Ohashi

  Parasitology 0031-1820 2021 

  Poultry red mites (Dermanyssus gallinae, PRM) are dangerous ectoparasites that infest chickens and threaten the poultry industry worldwide. PRMs usually develop resistance to chemical acaricides, necessitating the development of more effective preventive agents, and vaccination could be an alternative strategy for controlling PRMs. The suitability of plasma membrane proteins expressed in the midguts as vaccine antigens was evaluated because these molecules are exposed to antibodies in the ingested blood and the binding of antibodies could potentially induce direct damage to midgut tissue and indirect damage via inhibition of the functions of target molecules. Therefore, in the present study, a copper transporter 1-like molecule (Dg-Ctr1) was identified and its efficacy as a vaccine antigen was assessed in vitro. Dg-Ctr1 mRNA was expressed in the midguts and ovaries and in all the life stages, and flow cytometric analysis indicated that Dg-Ctr1 was expressed on the plasma membrane. Importantly, nymphs fed on plasma derived from chickens immunized with the recombinant protein of the extracellular region of Dg-Ctr1 showed a significant reduction in the survival rate. These data indicate that the application of Dg-Ctr1 as a vaccine antigen could reduce the number of nymphs in the farms, contributing to reduction in the economic losses caused by PRMs in the poultry industry. To establish an effective vaccination strategy, the acaricidal effects of the combined use of Dg-Ctr1 with chemical acaricides or other vaccine antigens must be examined.
• Canine Transforming Growth Factor-β Receptor 2-Ig: A Potential Candidate Biologic for Melanoma Treatment That Reverses Transforming Growth Factor-β1 Immunosuppression.

  Hiroto Takeuchi, Satoru Konnai, Naoya Maekawa, Satoshi Takagi, Hiroshi Ohta, Noboru Sasaki, Sangho Kim, Tomohiro Okagawa, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  Frontiers in veterinary science 8 656715 - 656715 2021 

  Cancer cells can evade host immune systems via multiple mechanisms. Transforming growth factor beta 1 (TGF-β1) is an immunosuppressive cytokine that induces regulatory T cell (Tregs) differentiation and is involved in immune evasion mechanisms in cancer. The inhibition of the TGF-β1 signaling pathway can suppress cancer progression and metastasis through the modulation of anticancer immune responses. However, to best of our knowledge, no implementation of treatments targeting TGF-β1 has been reported in dog cancers. This study aimed to examine whether TGF-β1 is upregulated in canine cancers. We measured TGF-β1 concentrations in culture supernatants of canine melanoma cell lines and in serum samples from dogs with oral malignant melanoma. TGF-β1 production was observed in several cell lines, and serum TGF-β1 levels were elevated in dogs with oral malignant melanoma. Interestingly, the addition of recombinant TGF-β1 to canine peripheral blood mononuclear cell cultures decreased Th1 cytokine production and increased differentiation of CD4+CD25+Foxp3+ lymphocytes, suggesting that TGF-β1 is immunosuppressive in canine immune systems. We developed a decoy receptor for TGF-β, namely TGF-βRII-Ig, by identifying an open reading frame of the canine TGFBR2 gene. TGF-βRII-Ig was prepared as a recombinant fusion protein of the extracellular region of canine TGF-βRII and the Fc region of canine IgG-B. As expected, TGF-βRII-Ig bound to TGF-β1. In the presence of TGF-β1, the treatment with TGF-βRII-Ig increased Th1 cytokine production and decreased the differentiation of CD4+CD25+Foxp3+ lymphocytes. Our results suggest that TGF-βRII-Ig competitively inhibits the immunosuppressive effects of TGF-β1 and thereby activates immune responses. This study demonstrated the potential of TGF-βRII-Ig as a novel biologic for canine melanoma.
• Characterisation of a cysteine protease from poultry red mites and its potential use as a vaccine for chickens.

  Shiro Murata, Ayaka Taniguchi, Masayoshi Isezaki, Sotaro Fujisawa, Eishi Sakai, Akira Taneno, Osamu Ichii, Takuya Ito, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi

  Parasite (Paris, France) 28 9 - 9 2021 

  Poultry red mites (PRMs, Dermanyssus gallinae) are ectoparasites that negatively affect farmed chickens, leading to serious economic losses worldwide. Acaricides have been used to control PRMs in poultry houses. However, some PRMs have developed resistance to acaricides, and therefore different approaches are required to manage the problems caused by PRMs. Vaccination of chickens is one of the methods being considered to reduce the number of PRMs in poultry houses. In a previous study, a cysteine protease, Deg-CPR-1, was identified as a candidate vaccine against PRMs distributed in Europe. In this study, we investigated the characteristics of Deg-CPR-1. A phylogenetic analysis revealed that Deg-CPR-1 is closely related to the digestive cysteine proteases of other mite species, and it was classified into a cluster different from that of chicken cathepsins. Deg-CPR-1 of PRMs in Japan has an amino acid substitution compared with that of PRMs in Europe, but it showed efficacy as a vaccine, consistent with previous findings. Deg-CPR-1 exhibited cathepsin L-like enzyme activity. In addition, the Deg-CPR-1 mRNA was expressed in the midgut and in all stages of PRMs that feed on blood. These results imply that Deg-CPR-1 in the midgut may have important functions in physiological processes, and the inhibition of its expression may contribute to the efficacy of a Deg-CPR-1-based vaccine. Further research is required to fully understand the mechanisms of vaccine efficacy.
• The enhancement of Th1 immune response by anti-PD-L1 antibody in cattle infected with Mycobacterium avium subsp. paratuberculosis

  Yamato SAJIKI, Satoru KONNAI, Reiko NAGATA, Satoko KAWAJI, Hayato NAKAMURA, Sotaro FUJISAWA, Tomohiro OKAGAWA, Naoya MAEKAWA, Yukinari KATO, Yasuhiko SUZUKI, Shiro MURATA, Yasuyuki MORI, Kazuhiko OHASHI

  Journal of Veterinary Medical Science 83 (2) 162 - 166 0916-7250 2021 

  Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteritis of ruminants. Previous studies have shown that programmed death-ligand 1 (PD-L1) is associated with the disease progression, and PD-L1 blockade activates MAP-specific Th1 responses in vitro. Here, we performed anti-PD-L1 antibody administration using 2 MAP-infected cattle at the late subclinical stage of infection. After administration, bacterial shedding was reduced or maintained at a low level. Additionally, MAP-specific Th1 cytokine production was upregulated, and CD69 expression was increased in T cells. Collectively, the treatment has a potential as a novel control method against Johne's disease.
• A TLR7 agonist activates bovine Th1 response and exerts antiviral activity against bovine leukemia virus

  Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Hayato Nakamura, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  Developmental & Comparative Immunology 114 103847 - 103847 0145-305X 2021/01 

  Bovine leukemia virus (BLV) infection is a bovine chronic infection caused by BLV, a member of the genus Deltaretrovirus. In this study, we examined the immunomodulatory effects of GS-9620, a toll-like receptor (TLR) 7 agonist, in cattle (Bos taurus) and its therapeutic potential for treating BLV infection. GS-9620 induced cytokine production in peripheral blood mononuclear cells (PBMCs) as well as CD80 expression in CD11c+ cells and increased CD69 and interferon (IFN)-γ expressions in T cells. Removing CD11c+ cells from PBMCs decreased CD69 expression in T cells in the presence of GS-9620. These results suggest that TLR7 agonism promotes T-cell activation via CD11c+ cells. Analyses using PBMCs from BLV-infected cattle revealed that TLR7 expression in CD11c+ cells was upregulated during late-stage BLV infection. Furthermore, GS-9620 increased IFN-γ and TNF-α production and inhibited syncytium formation in vitro, suggesting that GS-9620 may be used to treat BLV infection.
• The Suppression of Th1 Response by Inducing TGF-β1 From Regulatory T Cells in Bovine Mycoplasmosis

  Yamato Sajiki, Satoru Konnai, Shinya Goto, Tomohiro Okagawa, Kosuke Ohira, Honami Shimakura, Naoya Maekawa, Satoshi Gondaira, Hidetoshi Higuchi, Motoshi Tajima, Yuki Hirano, Junko Kohara, Shiro Murata, Kazuhiko Ohashi

  Frontiers in Veterinary Science 7 609443 - 609443 2020/12/02 

  Regulatory T cells (Tregs) regulate immune responses and maintain host immune homeostasis. Tregs contribute to the disease progression of several chronic infections by oversuppressing immune responses via the secretion of immunosuppressive cytokines, such as transforming growth factor (TGF)-β and interleukin-10. In the present study, we examined the association of Tregs with Mycoplasma bovis infection, in which immunosuppression is frequently observed. Compared with uninfected cattle, the percentage of Tregs, CD4⁺CD25^highFoxp3⁺ T cells, was increased in M. bovis-infected cattle. Additionally, the plasma of M. bovis-infected cattle contained the high concentrations of TGF-β1, and M. bovis infection induced TGF-β1 production from bovine immune cells in in vitro cultures. Finally, we analyzed the immunosuppressive effects of TGF-β1 on bovine immune cells. Treatment with TGF-β1 significantly decreased the expression of CD69, an activation marker, in T cells, and Th1 cytokine production in vitro. These results suggest that the increase in Tregs and TGF-β1 secretion could be one of the immunosuppressive mechanisms and that lead to increased susceptibility to other infections in terms of exacerbation of disease during M. bovis infection.
• Molecular detection and genetic characterization of infectious laryngotracheitis virus in poultry in Myanmar

  Zhiyuan Yang, Shiro Murata, Sotaro Fujisawa, Masaki Takehara, Ken Katakura, Myint Myint Hmoon, Shwe Yee Win, Saw Bawm, Satoru Konnai, Kazuhiko Ohashi

  BMC Veterinary Research 16 (1) 453 - 453 2020/12 

  Avian infectious laryngotracheitis (ILT) is a highly contagious viral disease that causes severe economic losses to the poultry industry worldwide. In Southeast Asian countries, including Myanmar, poultry farming is a major industry. Although it is known that infectious respiratory pathogens, including infectious laryngotracheitis virus (ILTV), are a major threat to poultry farms, there are no data currently available on the epidemiology of ILTV in Myanmar. Therefore, in this study, we conducted a molecular detection of ILTV in 20 poultry farms in Myanmar. Of the 57 tested oropharyngeal swabs, 10 were positive for ILTV by polymerase chain reaction of a 647 bp region of the thymidine kinase (TK) gene, giving a prevalence of ILTV of 17.5% (10/57). Further sequencing analysis of infected cell protein 4 (ICP4) gene and glycoprotein B, G, and J (gB, gG, and gJ) genes indicated that these isolates were field strains. Phylogenetic analysis revealed that the Myanmar strains clustered together in a single branch and were closely related to other reference strains isolated from Asian countries. This study demonstrated the presence of ILTV in poultry farms in Myanmar. The genetic characterization analysis performed provides the fundamental data for epidemiological studies that monitor circulating strains of ILTV in Myanmar.
• Enhanced Immunotherapeutic Efficacy of Anti–PD-L1 Antibody in Combination with an EP4 Antagonist

  Yamato Sajiki, Satoru Konnai, Zimeng Cai, Kensuke Takada, Tomohiro Okagawa, Naoya Maekawa, Sotaro Fujisawa, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  ImmunoHorizons 4 (12) 837 - 850 2020/12/01 

  Combination treatment approaches are increasingly considered to overcome resistance to immunotherapy targeting immunoinhibitory molecules such as programmed death (PD)-1 and PD-ligand 1 (PD-L1). Previous studies have demonstrated that the therapeutic efficacy of anti-PD-L1 Abs is enhanced by combination treatment with cyclooxygenase-2 inhibitors, through downregulation of the immunosuppressive eicosanoid PGE2, although the underlying mechanism remains unclear. In this study, we show that serum PGE2 levels are upregulated after anti-PD-L1 Ab administration in a bovine model of immunotherapy and that PGE2 directly inhibits T cell activation via its receptor E prostanoid (EP) 4. Additionally, anti-PD-L1 Ab induces TNF-α production and TNF-α blockade reduces PGE2 production in the presence of anti-PD-L1 Ab, suggesting that anti-PD-L1 Ab-induced TNF-α impairs T cell activation by PGE2 upregulation. Our studies examining the therapeutic potential of the dual blockade of PD-L1 and EP4 in bovine and murine immune cells reveal that the dual blockade of PD-L1 and EP4 significantly enhances Th1 cytokine production in vitro. Finally, we show that the dual blockade decreases tumor volume and prolongs survival in mice inoculated with the murine lymphoma cell line EG7. Altogether, these results suggest that TNF-α induced by anti-PD-L1 Ab treatment is associated with T cell dysfunction via PGE2/EP4 pathway and that the dual blockade of PD-L1 and EP4 should be considered as a novel immunotherapy for cancer.
• Genetic characterization of a Marek’s disease virus strain isolated in Japan

  Shiro Murata, Yuka Machida, Masayoshi Isezaki, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi

  Virology Journal 17 (1) 186 - 186 2020/12 

  Marek’s disease virus (MDV) causes malignant lymphomas in chickens (Marek’s disease, MD). MD is currently controlled by vaccination; however, MDV strains have a tendency to develop increased virulence. Distinct diversity and point mutations are present in the Meq proteins, the oncoproteins of MDV, suggesting that changes in protein function induced by amino acid substitutions might affect MDV virulence. We previously reported that recent MDV isolates in Japan display distinct mutations in Meq proteins from those observed in traditional MDV isolates in Japan, but similar to those in MDV strains isolated from other countries. To further investigate the genetic characteristics in Japanese field strains, we sequenced the whole genome of an MDV strain that was successfully isolated from a chicken with MD in Japan. A phylogenetic analysis of the meq gene was also performed. Phylogenetic analysis revealed that the Meq proteins in most of the Japanese isolates were similar to those of Chinese and European strains, and the genomic sequence of the Japanese strain was classified into the Eurasian cluster. Comparison of coding region sequences among the Japanese strain and MDV strains from other countries revealed that the genetic characteristics of the Japanese strain were similar to those of Chinese and European strains. The MDV strains distributed in Asian and European countries including Japan seem to be genetically closer to each other than to MDV strains from North America. These findings indicate that the genetic diversities of MDV strains that emerged may have been dependent on the different vaccination-based control approaches.
• PD-L1 expression in equine malignant melanoma and functional effects of PD-L1 blockade

  Otgontuya Ganbaatar, Satoru Konnai, Tomohiro Okagawa, Yutaro Nojima, Naoya Maekawa, Erina Minato, Atsushi Kobayashi, Ryo Ando, Nobuya Sasaki, Daisuke Miyakoshi, Osamu Ichii, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  PLOS ONE 15 (11) e0234218 - e0234218 2020/11/20 

  Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on lymphocytes. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. In our previous studies, we have developed anti-bovine PD-L1 monoclonal antibodies (mAbs) and reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Furthermore, we found that blocking antibodies that target PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway for chronic equine diseases, including tumors, remains unclear. In this study, we identified cDNA sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 mAbs against EqPD-L1 using in vitro assays. In addition, we evaluated the expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). The amino acid sequences of EqPD-1 and EqPD-L1 share a considerable identity and similarity with homologs from non-primate species. Two clones of the anti-bovine PD-L1 mAbs recognized EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine PD-1/PD-L1. Of note, immunohistochemistry confirmed the PD-L1 expression in EMM tumor tissues. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These findings showed that our anti-PD-L1 mAbs would be useful for analyzing the equine PD-1/PD-L1 pathway. Further research is warranted to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horses.
• Identification and functional analysis of ferritin 2 from the Taiga tick Ixodes persulcatus Schulze

  Naftaly Wang'ombe Githaka, Satoru Konnai, Masayoshi Isezaki, Shinya Goto, Marina Amaral Xavier, Sotaro Fujisawa, Shinji Yamada, Tomohiro Okagawa, Naoya Maekawa, Carlos Logullo, Itabajara da Silva Vaz, Shiro Murata, Kazuhiko Ohashi

  Ticks and Tick-borne Diseases 11 (6) 101547 - 101547 1877-959X 2020/11 

  Ferritin 2 (FER2) is an iron storage protein, which has been shown to be critical for iron homeostasis during blood feeding and reproduction in ticks and is therefore suitable as a component for anti-tick vaccines. In this study, we identified the FER2 of Ixodes persulcatus, a major vector for zoonotic diseases such as Lyme borreliosis and tick-borne relapsing fever in Japan, and investigated its functions. Ixodes persulcatus-derived ferritin 2 (Ip-FER2) showed concentration-dependent iron-binding ability and high amino acid conservation, consistent with FER2s of other tick species. Vaccines containing the recombinant Ip-FER2 elicited a significant reduction of the engorgement weight of adult I. persulcatus. Interestingly, the reduction of engorgement weight was also observed in Ixodes ovatus, a sympatric species of I. persulcatus. In silico analyses of FER2 sequences of I. persulcatus and other ticks showed a greater similarity with I. scapularis and I. ricinus and lesser similarity with Hyalomma anatolicum, Haemaphysalis longicornis, Rhipicephalus microplus, and R. appendiculatus. Moreover, it was observed that the tick FER2 sequences possess conserved regions within the primary structures, and in silico epitope mapping analysis revealed that antigenic regions were also conserved, particularly among Ixodes spp ticks. In conclusion, the data support further protective tick vaccination applications using the Ip-FER2 antigens identified herein.
• 牛白血病ウイルス感染症における免疫抑制受容体TIM-3の発現解析および機能解析

  中村 隼人, 今内 覚, 岡川 朋弘, 佐治木 大和, 渡 慧, 神谷 可菜, 齋藤 麻矢, 前川 直也, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 163回 221 - 221 1347-8621 2020/10
• ネコCytotoxic T-lymphocyte antigen-4(CTLA-4)を標的とした新規免疫抑制剤の開発における基礎的検討

  大塚 拓海, 今内 覚, 前川 直也, 渡 慧, 岡川 朋弘, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 163回 221 - 221 1347-8621 2020/10
• ワクモにおける定量的PCR法確立のための内在性コントロール遺伝子の探索

  有泉 拓馬, 村田 史郎, 藤澤 宗太郎, 伊勢崎 政美, 前川 直也, 岡川 朋弘, 種子野 章, 大石 英司, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 163回 248 - 248 1347-8621 2020/10
• 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 2020/06 [Refereed][Not invited]
  
• 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.  2020/03 [Refereed][Not invited]
  
• Expression Analysis of Canine CMTM6 and CMTM4 as Potential Regulators of the PD-L1 Protein in Canine Cancers

  Hiroto Takeuchi, Satoru Konnai, Naoya Maekawa, Erina Minato, Atsushi Kobayashi, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi

  Front Vet Sci 7 330 - 330 2020 [Refereed][Not invited]
   

  Cancer is one of the most significant causes of death in dogs. Antibody drugs targeting the PD-1/PD-L1 axis represent a promising immunotherapy for both human and canine cancers. However, the regulation mechanisms of PD-L1 expression in canine cancers require further investigation to better understand the resistance mechanisms to anti-PD-L1 therapy. Recent reports have shown that CMTM6 and CMTM4 are critical regulators of PD-L1 protein expression in human cancer cells. By preventing PD-L1 from lysosome-mediated degradation, CMTM6 maintains PD-L1 expression on the cell surface. However, the literature has not reported on CMTM6 and CMTM4 in dogs, and their functions are completely unknown. To reveal a regulation mechanism of PD-L1 in canine cancers, this study firstly identified the gene sequences of CMTM6 and CMTM4. Then, the expression analysis of these proteins was performed by immunohistochemistry. Furthermore, the functions of CMTM6 and CMTM4 in regulating PD-L1 expression were examined by gene knockdown of CMTM6 and CMTM4. Canine CMTM6 and CMTM4 displayed high amino acid sequence identities compared with those of humans and mice. An immunohistochemical analysis using cross-reactive antibodies revealed that canine malignant melanoma and osteosarcoma express CMTM6, CMTM4, and PD-L1 simultaneously. Gene knockdown of CMTM6 and CMTM4 with RNA interference significantly reduced the cell surface expression of PD-L1 in a canine cell line. These results suggest that CMTM6 and CMTM4 are regulators of PD-L1 expression in canine cancers and could serve as potential therapeutic targets to enhance antitumor immunity.
• 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 in press  2020/01 [Refereed][Not invited]
  
• Upregulation of PD-L1 expression by prostaglandin E2 and the enhancement of IFN-γ by anti-PD-L1 antibody combined with a COX-2 inhibitor in Mycoplasma bovis infection

  Goto S, Konnai S, Hirano Y, Kohara J, Okagawa T, Maekawa N, Sajiki Y, Watari K, Minato E, Kobayashi A, Gondaira S, Higuchi H, Koiwa M, Tajima M, Taguchi E, Uemura R, Yamada S, Kaneko M, Kato Y, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K

  Front Vet Sci 7 12 - 12 2020 [Refereed][Not invited]
   

  Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E2 (PGE2) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE2 in immune dysfunction and the relationship between PGE2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE2 and the PD-1/PD-L1 pathway.
• Clinical efficacy of the combined treatment of anti-PD-L1 rat-bovine chimeric antibody with a COX-2 inhibitor in calves infected with Mycoplasma bovis

  Goto S, Konnai S, Hirano Y, Kohara J, Okagawa T, Maekawa N, Sajiki Y, Watari K, Minato E, Kobayashi A, Gondaira S, Higuchi H, Koiwa M, Tajima M, Taguchi E, Ishida M, Uemura R, Yamada S, Kaneko M, Kato Y, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K

  Jpn J Vet Res 2020 [Refereed][Not invited]
  
• Molecular detection and genetic characterization of Mycoplasma gallisepticum, Mycoplama synoviae, and infectious bronchitis virus in poultry in Myanmar

  Sotaro Fujisawa, Shiro Murata, Masaki Takehara, Ken Katakura, Myint Myint Hmoon, Shwe Yee Win, Kazuhiko Ohashi

  BMC Veterinary Research 15 (1) 261 - 261 2019/12 

  BACKGROUND: In Southeast Asian countries, including Myanmar, poultry farming is a major industry. In order to manage and maintain stable productivity, it is important to establish policies for biosecurity. Infectious respiratory diseases are a major threat to poultry farming. Avian influenza and Newcastle disease have been reported in Myanmar, but no scientific information is available for other respiratory pathogens, such as mycoplasmas and infectious bronchitis virus (IBV). Identifying the genotypes and serotypes of IBVs is especially important to inform vaccination programs. In this study, we detected Mycoplasma gallisepticum (MG), M. synoviae (MS), and IBV in several poultry farms in Myanmar. RESULTS: Samples were collected from 20 farms in three major poultry farming areas in Myanmar, and MG, MS, and IBV were detected on two, four, and eight farms, respectively, by polymerase chain reaction. Phylogenetic analysis revealed that the observed MG and MS isolates were not identical to vaccine strains. Three different genotypes of IBV were detected, but none was an unknown variant. CONCLUSIONS: Mycoplasmas and IBV were detected on poultry farms in Myanmar. Periodic surveillance is required to establish the distribution of each pathogen, and to institute better vaccine protocols.
• 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  2019/09 [Refereed][Not invited]
  
• 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, Kato Y, Takahashi H, Terasaki N, Takeda A, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K

  J Immunol 203 (5) 1313 - 1324 2019/09 [Refereed][Not invited]
   

  Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.
• ミャンマーの養鶏場におけるマイコプラズマ、鶏伝染性気管支炎ウイルス、鶏伝染性喉頭気管炎ウイルスの分子学的検出と遺伝学的性状解析(Molecular detection and genetic characterization of mycoplasmas, infectious bronchitis virus and infectious laryngotracheitis virus in poultry farms in Myanmar)

  Yang Zhiyuan, 藤澤 宗太郎, 村田 史郎, 竹原 昌生, 片倉 賢, Myint Myint Hmoon, Shwe Yee Win, Saw Bawm, Lat Lat Htun, Ye Htut Aung, Mar Mar Win, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 162回 411 - 411 1347-8621 2019/08
• イヌ腫瘍組織およびイヌ腫瘍由来細胞株におけるイヌHER2の発現解析

  吉武 志江奈, 今内 覚, 前川 直也, 賀川 由美子, 西村 麻紀, 岡川 朋弘, 鈴木 定彦, 高木 哲, 中川 貴之, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 162回 380 - 380 1347-8621 2019/08
• イヌ腫瘍由来細胞株におけるCKLF-like MARVEL transmembrane domain containing protein 6(CMTM6)および4(CMTM4)の発現解析

  竹内 寛人, 今内 覚, 岡川 朋弘, 前川 直也, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 162回 380 - 380 1347-8621 2019/08
• BLV感染症に対するCOX-2阻害剤と抗PD-L1抗体併用法の抗ウイルス効果の検討

  佐治木 大和, 今内 覚, 岡川 朋弘, 前川 直也, 後藤 伸也, 小原 潤子, 山田 慎二, 加藤 幸成, 鈴木 定彦, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 162回 381 - 381 1347-8621 2019/08
• Mycoplasma bovis感染症に対する抗PD-L1キメラ抗体を用いた臨床試験

  後藤 伸也, 今内 覚, 平野 佑気, 小原 潤子, 岡川 朋弘, 前川 直也, 鈴木 定彦, 加藤 幸成, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 162回 381 - 381 1347-8621 2019/08
• RNA-Seqを用いたワクモ(Dermanyssus gallinae)の吸血状態別における遺伝子発現解析

  藤澤 宗太郎, 村田 史郎, 竹原 昌生, 伊勢崎 政美, 小川 遼, 宇野 有紀子, 種子野 章, 前川 直也, 岡川 朋弘, 今内 覚, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 162回 412 - 412 1347-8621 2019/08
• COX-2阻害剤の併用による抗PD-L1抗体を用いたイヌ腫瘍免疫療法の効果増強に向けた基礎的検討

  前川 直也, 今内 覚, 浅野 裕美恵, 岡川 朋弘, 高木 哲, 村田 史郎, 大橋 和彦

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 162回 460 - 460 1347-8621 2019/08
• 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 0047-1917 2019/08 [Refereed][Not invited]
   

  One of the known transmission pathways of bovine leukemia virus (BLV) is bloodsucking insects. Against this background, this study investigated changes in BLV seroconversion in cattle by season on three private farms in Northern Hokkaido. Study results showed that no BLV seroconversion was observed during winter, a season without horse flies, and that all seroconversions occurred during summer. Thus, we collected horse flies which were observed sucking blood on cattle in public grazed fields and performed BLV detection tests in the insects. The tests showed that 75% of the total collected horse flies were BLV-positive. These results suggested the existence of vectors such as horse flies in grazing filed (in summer) was a risk factor of the spread of BLV infection in Northern Hokkaido.
• 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  2019/04 [Refereed][Not invited]
  
• 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

  Scientific Rep. 9 4753  2019/03 [Refereed][Not invited]
  
• 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  2019/02 [Refereed][Not invited]
  
• 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 0047-1917 2018/08 [Refereed][Not invited]
   

  The immuno-inhibitory molecules PD-1, PD-L1, TIM-3, GAL-9, LAG-3, and CTLA-4 from blood samples of Mongolian native cattle and yak were characterized through cloning and sequencing. As these molecules are involved in cell-mediated immune responses, identifying the differences in their reactions against the pathogens found in bovine species may be beneficial. The amino acid sequences of these molecules were predicted for the purpose of characterizing their functional domains, such as the signal peptide, extracellular domain, transmembrane region, and intracellular domain. Amino acid alignment showed that the sequences of these immuno-inhibitory molecules from Mongolian native cattle and yak were highly homologous to those from other bovine species. As a preliminary application of the genetic information, we conducted expression analysis of PD-L1 in bovine viral diarrhea virus (BVDV)-infected yak by using real-time polymerase chain reaction, and PD-L1 mRNA expression in peripheral blood mononuclear cells derived from BVDV-infected yak was significantly upregulated compared to that of uninfected-yak. Further studies are necessary to assess whether these molecules play roles in disease progression during chronic infection of Mongolian native cattle and yak.
• 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  2018/06 [Refereed][Not invited]
  
• 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  2018/04 [Refereed][Not invited]
  
• 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  1556-6811 2018/04 [Refereed][Not invited]
   

  Bovine leukemia is classified into two types: enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). EBL is caused by infection with bovine leukemia virus (BLV), which induces persistent lymphocytosis and B-cell lymphoma in cattle after a long latent period. Although it has been demonstrated that BLV-associated lymphoma occurs predominantly in adult cattle of >3 to 5 years, suspicious cases of EBL onset in juvenile cattle were recently reported in Japan. To investigate the current status of bovine leukemia in Japan, we performed immunophenotypic analysis of samples from 50 cattle that were clinically diagnosed as having bovine leukemia. We classified the samples into five groups on the basis of the analysis and found two different types of EBL: classic EBL (cEBL), which has the familiar phenotype commonly known as EBL, and polyclonal EBL (pEBL), which exhibited neoplastic proliferation of polyclonal B cells. Moreover, there were several atypical EBL cases even in cEBL, including an early onset of EBL in juvenile cattle. A comparison of the cell marker expressions among cEBL, pEBL, and B-cell-type SBL (B-SBL) revealed characteristic patterns in B-cell leukemia, and these patterns could be clearly differentiated from those of healthy phenotypes, whereas it was difficult to discriminate between cEBL, pEBL, and B-SBL only by the expression patterns of cell markers. This study identified novel characteristics of bovine leukemia that should contribute to a better understanding of the mechanism underlying tumor development in BLV infection.
• Molecular detection and phylogenetic analysis of Ehrlichia canis in a Philippine dog

  Maekawa N, Konnai S, Balbin MM, Mingala CN, Gicana KRB, Bernando FAEM, Murata S, Ohashi K

  Ticks Tick Borne Dis. 9 (2) 266 - 269 2018/02 [Refereed][Not invited]
   

  Canine monocytic ehrlichiosis (CME), caused by a rickettsial bacterium, Ehrlichia canis, is distributed worldwide, particularly in tropical and subtropical regions. Transmission of E. canis is primarily mediated by the vector tick, Rhipicephalus sanguineus sensu lato and the bacteria then infect and replicate in monocytes and macrophages. Many cases are seen in veterinary hospitals and treated routinely; however, the genetic variation of E. canis strains found in the Philippines has been poorly investigated to date. In this study, the 16S rRNA gene and the gp200 gene of E. canis were detected by polymerase chain reaction from an infected dog in the Philippines, and the deduced amino acid sequence of the gp200 gene was subjected to a phylogenetic analysis. The Philippine genotype formed a cluster with the Taiwan genotype, and was somewhat divergent from the USA and Brazil strains. This suggested that E. canis underwent evolution in East and Southeast Asia, confirming the utility of the gp200 gene for the assessment of genetic relationships among strains.
• Expression profile of Rhipicephalus microplus vitellogenin receptor during oogenesis

  Seixas A, Alzugaray MF, Tirloni L, Parizi LF, Pinto AFM, Githaka NW, Konnai S, Ohashi K, Yates Iii JR, Termignoni C, da Silva Vaz I J

  Ticks Tick Borne Dis. 9 (1) 72 - 81 2018/01 [Refereed][Not invited]
  
• Correction for Nishimori et al., "Identification of an atypical enzootic bovine leukosis in Japan by using a novel classification of bovine leukemia based on immunophenotypic analysis"(Clinical and Vaccine Immunology (2017) 24: 9 (e00067-17) DOI: 10.1128/CVI.00067-17)

  Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Ayako Nakahara, Yuzumi Chiba, Masaho Ikeda, Shiro Murata, Kazuhiko Ohashi

  Clinical and Vaccine Immunology 24 (12) 1556-6811 2017/12 

  Volume 24, no. 9, e00067-17, 2017, https://doi.org/10.1128/CVI.00067-17. Page 12, lines 45 and 46: “JH primer, 5=-AGA CTA GTG AAG ACT CTC GGG TGT G-3=” should read “JH primer, 5=-AGA CTA GTG AGG AGA CGG TGA CC-3=.”
• Molecular epidemiological survey and genetic characterization of ovine gammaherpesvirus-2 in Mongolian livestock

  Ochirkhuu N, Konnai S, Odbileg R, Murata S, Ohashi K

  J Vet Med Sci. 公益社団法人 日本獣医学会 79 (12) 2040 - 2042 2017/12 [Refereed][Not invited]
   

  Sheep-associated malignant catarrhal fever (SA-MCF), caused by ovine gammaherpesvirus-2 (OvHV-2), is a fatal disease in all ruminants. The epidemiological survey and molecular characterization of OvHV-2 in Mongolian livestock were performed. Of 928 blood samples, 14 were positive for OvHV-2 in sheep and native cattle from Tsenkher County and in sheep from Lun County. Phylogenetic analyses revealed that the tegument gene of OvHV-2 sequences from Mongolian animals is identical to that in animals from Egypt, India, and Turkey, and is 98.0% similar to that in animals from Germany and Brazil. To our knowledge, this is the first confirmed report of OvHV-2 in Mongolian livestock, and could provide useful information for controlling SA-MCF.

• Intrauterine infection with bovine leukemia virus in pregnant dam with high viral load

  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 2017/12 [Refereed][Not invited]
   

  Enzootic bovine leukemia is caused by the bovine leukemia virus (BLV). BLV is transmitted vertically or horizontally through the transfer of infected cells via direct contact, through milk, insect bites and contaminated iatrogenic procedures. However, we lacked direct evidence of intrauterine infection. The purpose of this study was to confirm intrauterine BLV infection in two pregnant dams with high viral load by cesarean delivery. BLV was detected in cord and placental blood, and the BLV in the newborns showed 100% nucleotide identity with the BLV-env sequence from the dams. Notably, a newborn was seropositive for BLV but had no colostral antibodies. In this study, we presented a direct evidence of intrauterine BLV transmission in pregnant dam with a high proviral load. These results could aid the development of BLV control measures targeting viral load.
• Detection and molecular characterization of equine infectious anemia virus in Mongolian horses

  Sharav T, Konnai S, Ochirkhuu N, Ts EO, Mekata H, Sakoda Y, Umemura T, Murata S, Chultemdorj T, Ohashi K

  J Vet Med Sci. 公益社団法人 日本獣医学会 79 (11) 1884 - 1888 2017/11 [Refereed][Not invited]
   

  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.

• 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 2017/09 [Refereed][Not invited]
  
• Molecular Epidemiological Survey and Genetic Characterization of Anaplasma Species in Mongolian Livestock

  Ochirkhuu N, Konnai S, Odbileg R, Murata S, Ohashi K

  Vector Borne Zoonotic Dis. 17 (8) 539 - 549 2017/08 [Refereed][Not invited]
   

  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  2017/08 [Refereed][Not invited]
  
• Anti-Bovine Programmed Death-1 Rat-Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle

  Okagawa T, Konnai S, Nishimori A, Maekawa N, Ikebuchi R, Goto S, Nakajima C, Kohara J, Ogasawara S, Kato Y, Suzuki Y, Murata S, Ohashi K

  Front Immunol. 8 650 - 650 2017/06 [Refereed][Not invited]
   

  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.
• In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) rat-bovine chimeric antibody against bovine leukemia virus infection

  Nishimori A, Konnai S, Okagawa T, Maekawa N, Ikebuchi R, Goto S, Sajiki Y, Suzuki Y, Kohara J, Ogasawara S, Kato Y, Murata S, Ohashi K

  PLoS One. 12 (4) e0174916  2017/04 [Refereed][Not invited]
   

  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.
• Molecular and structural characterization of novel cystatins from the taiga tick Ixodes persulcatus

  Rangel CK, Parizi LF, Sabadin GA, Costa EP, Romeiro NC, Isezaki M, Githaka NW, Seixas A, Logullo C, Konnai S, Ohashi K, da Silva Vaz I J

  Ticks Tick Borne Dis. 8 (3) 432 - 441 2017/03 [Refereed][Not invited]
  
• Increase of cells expressing PD-1 and PD-L1 and enhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine mycoplasmosis.

  Goto, S, Konnai, S, Okagawa, T, Nishimori, A, Maekawa, N, Gondaira, S, Higuchi, H, Koiwa, M, Tajima, M, Kohara, J, Ogasawara, S, Kato, Y, Suzuki, Y, Murata, S, Ohashi, K

  Immunity, Inflammation and Disease 10 (3) 1 - 9 2017 [Refereed][Not invited]
   

  INTRODUCTION: Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway in immunosuppression in bovine mycoplasmosis. METHODS: In the initial experiments, we used enzyme-linked immunosorbent assay to measure interferon-γ (IFN-γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis. RESULTS: Expectedly, IFN-γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD-1+ CD4+ and PD-L1+ CD14+ cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD-1+ CD4+ and PD-1+ CD8+ T cells were negatively correlated with IFN-γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD-1/PD-L1 pathway in vitro by anti-bovine PD-1- and anti-bovine PD-L1 antibodies significantly upregulated the production of IFN-γ from anti-mycoplasma-specific cells. CONCLUSIONS: These results suggest that the PD-1/PD-L1 pathway could be involved in immune exhaustion of bovine mycoplasma-specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways.
• Isolation and purification of Gallid herpesvirus 2 strains currently distributed in Japan

  Machida Y, Murata S, Matsuyama-Kato A, Isezaki M, Taneno A, Sakai E, Konnai S, Ohashi K

  J Vet Med Sci. 公益社団法人 日本獣医学会 79 (1) 115 - 122 2017/01 [Refereed][Not invited]
   

  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.

• Immune exhaustion during chronic infections in cattle

  Konnai S, Murata S, Ohashi K

  J Vet Med Sci. 79 (1) 1 - 5 0916-7250 2017/01 [Refereed][Invited]
  
• A novel mechanism of functional cooperativity regulation by thiol redox status in a dimeric inorganic pyrophosphatase

  Costa EP, Façanha AR, Cruz CS, Silva JN, Machado JA, Carvalho GM, Fernandes MR, Martins R, Campos E, Romeiro NC, Githaka NW, Konnai S, Ohashi K, Vaz IS Jr, Logullo C

  Biochim Biophys Acta Gen Subj. 1861 (1 Pt A) 2922 - 2933 2017/01 [Refereed][Not invited]
  
• Cooperation of PD-1 and LAG-3 Contributes to T-Cell Exhaustion in Anaplasma marginale-Infected Cattle

  Okagawa T, Konnai S, Deringer JR, Ueti MW, Scoles GA, Murata S, Ohashi K, Brown WC

  Infect Immun. 84 (10) 2779 - 90 2016/09 [Refereed][Not invited]
   

  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-γ) 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 γδ T-cell populations gradually increased and also peaked at 5 wpi. A large increase in the percentage of LAG-3(+) γδ 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-γ 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.
• Molecular detection and characterization of bovine viral diarrhea virus in Mongolian cattle and yaks

  Ochirkhuu N, Konnai S, Odbileg R, Odzaya B, Gansukh S, Murata S, Ohashi K

  Arch Virol. 161 (8) 2279 - 83 2016/08 [Refereed][Not invited]
   

  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.
• Immunohistochemical Analysis of PD-L1 Expression in Canine Malignant Cancers and PD-1 Expression on Lymphocytes in Canine Oral Melanoma

  Maekawa N, Konnai S, Okagawa T, Nishimori A, Ikebuchi R, Izumi Y, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Kato Y, Murata S, Ohashi K

  PLoS One. 11 (6) e0157176  2016/06 [Refereed][Not invited]
   

  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.
• Detection of bovine leukemia virus and identification of its genotype in Mongolian cattle

  Ochirkhuu N, Konnai S, Odbileg R, Nishimori A, Okagawa T, Murata S, Ohashi K

  Arch Virol. 161 (4) 985 - 91 2016/04 [Refereed][Not invited]
   

  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.
• Direct polymerase chain reaction from blood and tissue samples for rapid diagnosis of bovine leukemia virus infection

  Nishimori A, Konnai S, Ikebuchi R, Okagawa T, Nakahara A, Murata S, Ohashi K

  J Vet Med Sci. 公益社団法人 日本獣医学会 78 (5) 791 - 6 0916-7250 2016/01 [Refereed][Not invited]
   

  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.
• 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 2016/01 [Refereed][Not invited]
   

  CD4(+)CD25(high)Foxp3(+) T cells suppress excess immune responses that lead to autoimmune and/or inflammatory diseases, and maintain host immune homeostasis. However, CD4(+)CD25(high)Foxp3(+) T cells reportedly contribute to disease progression by over suppressing immune responses in some chronic infections. In this study, kinetic and functional analyses of CD4(+)CD25(high)Foxp3(+) T cells were performed in cattle with bovine leukemia virus (BLV) infections, which have reported immunosuppressive characteristics. In initial experiments, production of the Th1 cytokines IFN-γ and TNF-α was reduced in BLV-infected cattle compared with uninfected cattle, and numbers of IFN-γ or TNF-α producing CD4(+) T cells decreased with disease progression. In contrast, IFN-γ production by NK cells was inversely correlated with BLV proviral loads in infected cattle. Additionally, during persistent lymphocytosis disease stages, NK cytotoxicity was depressed as indicated by low expression of the cytolytic protein perforin. Concomitantly, total CD4(+)CD25(high)Foxp3(+) T cell numbers and percentages of TGF-β(+) cells were increased, suggesting that TGF-β plays a role in the functional declines of CD4(+) T cells and NK cells. In further experiments, recombinant bovine TGF-β suppressed IFN-γ and TNF-α production by CD4(+) T cells and NK cytotoxicity in cultured cells. These data suggest that TGF-β from CD4(+)CD25(high)Foxp3(+) T cells is immunosuppressive and contributes to disease progression and the development of opportunistic infections during BLV infection.
• Identification and the preliminary in vitro characterization of IRIS homologue from salivary glands of Ixodes persulcatus Schulze

  Toyomane K, Konnai S, Niwa A, Githaka WN, Isezaki M, Yamada S, Ito T, Takano A, Ando S, Kawabata H, Murata S, Ohashi K

  Ticks and Tick-borne Diseases 7 (1) 119 - 125 2016 [Refereed][Not invited]
   

  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-γ ELISA showed that Ipis-1-Ig inhibits the proliferation and IFN-γ production of bovine peripheral blood mononuclear cells (PBMCs). Notably, Ipis-1-Ig inhibited the cell proliferation and production of IFN-γ 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.
• A preliminary survey of the seroprevalence of Mycobacterium avium subspecies paratuberculosis in Mongolian cattle

  Ochirkhuu N, Konnai S, Odbileg R, Murata S, Ohashi K

  Jpn J Vet Res. Graduate School of Veterinary Medicine, Hokkaido University 63 (4) 191 - 4 0047-1917 2015/11 [Refereed][Not invited]
   

  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.
• 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 2015/10 [Refereed][Not invited]
  
• Molecular epidemiological survey and genetic analysis of vector-borne infections of cattle in Luzon Island, the Philippines

  Ochirkhuu N, Konnai S, Mingala CN, Okagawa T, Villanueva M, Pilapil FM, Murata S, Ohashi K

  Vet Parasitol. 212 (3-4) 161 - 7 2015/09 [Refereed][Not invited]
   

  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.
• Expression of regulatory dendritic cell-related cytokines in cattle experimentally infected with Trypanosoma evansi

  Mekata H, Murata S, Mingala CN, Ohashi K, Konnai S

  J Vet Med Sci. 77 (8) 1017 - 9 2015/08 [Refereed][Not invited]
  
• Vaccination with cyclin-dependent kinase tick antigen confers protection against Ixodes infestation

  Gomes H, Moraes J, Githaka N, Martins R, Isezaki M, Vaz Ida S Jr, Logullo C, Konnai S, Ohashi K

  Vet Parasitol. 211 (3-4) 266 - 73 2015/07 [Refereed][Not invited]
  
• Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection

  Suzuki S, Konnai S, Okagawa T, Ikebuchi R, Nishimori A, Kohara J, Mingala CN, Murata S, Ohashi K

  Vet Immunol Immunopathol. 163 (3-4) 115 - 24 2015/02 [Refereed][Not invited]
   

  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-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-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-β 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-γ 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.
• Rhipicephalus microplus and Ixodes ovatus cystatins in tick blood digestion and evasion of host immune response

  Parizi LF, Sabadin GA, Alzugaray MF, Seixas A, Logullo C, Konnai S, Ohashi K, Masuda A, da Silva Vaz I J

  Parasit Vectors. 8 122  2015/02 [Refereed][Not invited]
  
• Non-classical gluconeogenesis-dependent glucose metabolism in Rhipicephalus microplus embryonic cell line BME26

  da Silva RM, Noce BD, Waltero CF, Costa EP, de Abreu LA, Githaka NW, Moraes J, Gomes HF, Konnai S, Vaz Ida S Jr, Ohashi K, Logullo C

  Int J Mol Sci. 16 (1) 1821 - 39 2015/01 [Refereed][Not invited]
  
• An investigation of binding ability of Ixodes persulcatus Schulze Salp15 with Lyme disease spirochetes.

  Murase Y, Konnai S, Yamada S, Githaka N, Isezaki M, Ito T, Takano A, Ando S, Kawabata H, Murata S, Ohashi K

  Insect Biochemistry and Molecular Biology 60 59 - 67 2015 [Refereed][Not invited]
  
• Influence of PD-L1 cross-linking on cell death in PD-L1-expressing cell lines and bovine lymphocytes

  Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K

  Immunology 142 (4) 551 - 61 2014/08 [Refereed][Not invited]
   

  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-γ (IFN-γ) production, whereas PD-1-Ig treatment decreased this cytokine production and cell proliferation. The IFN-γ 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.
• Expression analysis of programmed death ligand 2 in tumors caused by the avian oncovirus Marek's disease virus

  Matsuyama-Kato A, Murata S, Isezaki M, Takasaki S, Kano R, Konnai S, Ohashi K

  Arch Virol. 159 (8) 2123 - 6 2014/08 [Refereed][Not invited]
   

  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-γ 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.
• Reprolysin metalloproteases from Ixodes persulcatus, Rhipicephalus sanguineus and Rhipicephalus microplus ticks

  Ali A, Tirloni L, Isezaki M, Seixas A, Konnai S, Ohashi K, da Silva Vaz, Junior I, Termignoni C

  Exp Appl Acarol. 63 (4) 559 - 78 2014/08 [Refereed][Not invited]
  
• Differences in cellular function and viral protein expression between IgMhigh and IgMlow B-cells in bovine leukemia virus-infected cattle

  Ikebuchi R, Konnai S, Okagawa T, Nishimori A, Nakahara A, Murata S, Ohashi K

  J Gen Virol. 95 (Pt 8) 1832 - 42 2014/08 [Refereed][Not invited]
   

  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.
• Tick Surveillance for Relapsing Fever Spirochete Borrelia miyamotoi in Hokkaido, 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  1932-6203 2014/08 [Refereed][Not invited]
   

  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.
• Identification and characterization of bovine programmed death-ligand 2

  Nishimori A, Konnai S, Ikebuchi R, Okagawa T, Nakajima C, Suzuki Y, Mingala CN, Murata S, Ohashi K

  Microbiol Immunol. 58 (7) :388 - 97 2014/07 [Refereed][Not invited]
   

  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-γ 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-γ production from virus envelope peptides-stimulated PBMCs derived from bovine leukemia virus-infected cattle. Interestingly, PD-L2-Ig-induced IFN-γ 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.
• Expression of PD-L1 on canine tumor cells and enhancement of IFN-γ production from tumor-infiltrating cells by PD-L1 blockade

  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  2014/06 [Refereed][Not invited]
   

  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-γ 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.
• Identification and sequence characterization of novel Theileria genotypes from the waterbuck (Kobus defassa) in a Theileria parva-endemic area in Kenya

  Githaka N, Konnai S, Bishop R, Odongo D, Lekolool I, Kariuki E, Gakuya F, Kamau L, Isezaki M, Murata S, Ohashi K

  Vet Parasitol. 202 (3-4) 180 - 93 2014/03 [Refereed][Not invited]
   

  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 caffer) 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.
• Suppressive effects of neutrophil by Salp16-like salivary gland proteins from Ixodes persulcatus Schulze tick

  Hidano A, Konnai S, Yamada S, Githaka N, Isezaki M, Higuchi H, Nagahata H, Ito T, Takano A, Ando S, Kawabata H, Murata S, Ohashi K

  Insect Molecular Biology 23 (4) 466 - 474 0962-1075 2014 [Refereed][Not invited]
   

  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. © 2014 The Royal Entomological Society.
• Sequence characterization and immunogenicity of cystatins from the cattle tick Rhipicephalus (Boophilus) microplus

  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 2013/12 [Refereed][Not invited]
  
• Horizontal transmission of bovine leukemia virus from lymphocytotic cattle, and beneficial effects of insect vector control

  Ooshiro M, Konnai S, Katagiri Y, Afuso M, Arakaki N, Tsuha O, Murata S, Ohashi K

  Vet Rec. 173 (21) 527  2013/11 [Refereed][Not invited]
  
• Genotypic variations in field isolates of Theileria species infecting giraffes (Giraffa camelopardalis tippelskirchi and Giraffa camelopardalis reticulata) in Kenya

  Githaka N, Konnai S, Skilton R, Kariuki E, Kanduma E, Murata S, Ohashi K

  Parasitol Int. 62 (5) 448 - 53 2013/10 [Refereed][Not invited]
   

  Recently, mortalities among giraffes, attributed to infection with unique species of piroplasms were reported in South Africa. Although haemoparasites are known to occur in giraffes of Kenya, the prevalence, genetic diversity and pathogenicity of these parasites have not been investigated. In this study, blood samples from 13 giraffes in Kenya were investigated microscopically and genomic DNA extracted. PCR amplicons of the hyper-variable region 4 (V4) of Theileria spp. small subunit ribosomal RNA (18S rRNA) gene were hybridized to a panel of genus- and species-specific oligonucleotide probes by reverse line blot (RLB). Two newly designed oligonucleotide probes specific for previously identified Theileria spp. of giraffes found single infections in eight of the specimens and mixed infections in the remaining five samples. Partial 18S rRNA genes were successfully amplified from 9 samples and the PCR amplicons were cloned. A total of 28 plasmid clones representing the Kenyan isolates were analyzed in the present study and compared with those of closely-related organisms retrieved from GenBank. In agreement with RLB results, the nucleotide sequence alignment indicated the presence of mixed infections in the giraffes. In addition, sequence alignment with the obtained 18S rRNA gene sequences revealed extensive microheterogeneities within and between isolates, characterized by indels in the V4 regions and point mutations outside this region. Phylogeny with 18S rRNA gene sequences from the detected parasites and those of related organisms places Theileria of giraffes into two major groups, within which are numerous clades that include the isolates reported in South Africa. Collectively, these data suggest the existence of at least two distinct Theileria species among giraffes, and extensive genetic diversity within the two parasite groups.
• Identification and structural-functional analysis of cyclin-dependent kinases of the cattle tick Rhipicephalus (Boophilus) microplus

  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.  2013/10 [Refereed][Not invited]
  
• Expression analysis of Foxp3 in T cells from bovine leukemia virus infected cattle

  Suzuki S, Konnai S, Okagawa T, Ikebuchi R, Shirai T, Sunden Y, Mingala CN, Murata S, Ohashi K

  Microbiol Immunol. 57 (8) 600 - 4 2013/08 [Refereed][Not invited]
   

  In the present study, we monitored Foxp3(+) T cells in bovine leukemia virus (BLV)-infected cattle. By flow cytometric analysis, the proportion of Foxp3(+) CD4(+) cells from persistent lymphocytotic cattle was significantly increased compared to control and AL cattle. Interestingly, the proportion of Foxp3(+) CD4(+) cells correlated positively with the increased number of lymphocytes, virus titer and virus load, whereas it inversely correlated with IFN-γ mRNA expression, suggesting that Foxp3(+) CD4(+) T cells in cattle have a potentially immunosuppressive function. Further studies are necessary to elucidate the detailed mechanism behind the increased Treg during BLV infection.
• Blockade of bovine PD-1 increases T cell function and inhibits bovine leukemia virus expression in B cells in vitro.

  Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K

  Vet Res. 44 (1) 59 - 59 2013/07 [Refereed][Not invited]
   

  Programmed death-1 (PD-1) is a known immunoinhibitory receptor that contributes to immune evasion of various tumor cells and pathogens causing chronic infection, such as bovine leukemia virus (BLV) infection. First, in this study, to establish a method for the expression and functional analysis of bovine PD-1, hybridomas producing monoclonal antibodies (mAb) specific for bovine PD-1 were established. Treatment with these anti-PD-1 mAb enhanced interferon-gamma (IFN-γ) production of bovine peripheral blood mononuclear cells (PBMC). Next, to examine whether PD-1 blockade by anti-PD-1 mAb could upregulate the immune reaction during chronic infection, the expression and functional analysis of PD-1 in PBMC isolated from BLV-infected cattle with or without lymphoma were performed using anti-PD-1 mAb. The frequencies of both PD-1+ CD4+ T cells in blood and lymph node and PD-1+ CD8+ T cells in lymph node were higher in BLV-infected cattle with lymphoma than those without lymphoma or control uninfected cattle. PD-1 blockade enhanced IFN-γ production and proliferation and reduced BLV-gp51 expression and B-cell activation in PBMC from BLV-infected cattle in response to BLV-gp51 peptide mixture. These data show that anti-bovine PD-1 mAb could provide a new therapy to control BLV infection via upregulation of immune response.
• Expression analysis of Foxp3 in T-cells from bovine leukemia virus infected cattle.

  Suzuki S, Konnai S, Okagawa T, Ikebuchi R, Shirai T, Sunden Y, Mingala CN, Murata S, Ohashi K

  Microbiology and immunology 0385-5600 2013/06 [Refereed][Not invited]
  
• Characterization of Meq proteins from field isolates of Marek's disease virus in Japan

  Murata S, Hashiguchi T, Hayashi Y, Yamamoto Y, Matsuyama-Kato A, Takasaki S, Isezaki M, Onuma M, Konnai S, Ohashi K

  Infection Genetics And Evolution Elsevier Science Bv 16 137 - 43 1567-1348 2013/06 [Refereed][Not invited]
   

  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.
• 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 2013/06 [Not refereed][Invited]
  
• Bovine whole-blood culture as a tool for the measurement of endotoxin activities in Gram-negative bacterial vaccines

  Imamura S, Nakamizo M, Kawanishi M, Nakajima N, Yamamoto K, Uchiyama M, Hirano F, Nagai H, Kijima M, Ikebuchi R, Mekata H, Murata S, Konnai S, Ohashi K

  Vet Immunol Immunopathol. 153 (1-2) 153 - 8 2013/05 [Refereed][Not invited]
  
• Isolation, cloning, and pathologic analysis of Trypanosoma evansi field isolates

  Mekata H, Konnai S, Mingala CN, Abes NS, Gutierrez CA, Dargantes AP, Witola WH, Inoue N, Onuma M, Murata S, Ohashi K

  Parasitol Res. 112 (4) 1513 - 21 2013/04 [Refereed][Not invited]
  
• The conserved role of the AKT/GSK3 axis in cell survival and glycogen metabolism in Rhipicephalus (Boophilus) microplus embryo tick cell line BME26

  de Abreu LA, Calixto C, Waltero CF, Della Noce BP, Githaka NW, Seixas A, Parizi LF, Konnai S, Vaz Ida S, Ohashi K, Logullo C

  Biochim Biophys Acta. 1830 (3) 2574 - 82 2013/03 [Refereed][Not invited]
  
• Identification and partial characterization of a gut Rhipicephalus appendiculatus cystatin

  Imamura S, Konnai S, Yamada S, Parizi LF, Githaka N, Vaz Ida S Jr, Murata S, Ohashi K

  Ticks Tick Borne Dis. 4 (1-2) 138 - 44 2013/02 [Refereed][Not invited]
   

  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 12.8kDa. 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.
• Enhanced expression of LAG-3 on lymphocyte subpopulations from persistently lymphocytotic cattle infected with bovine leukemia virus

  Konnai S, Suzuki S, Shirai T, Ikebuchi R, Okagawa T, Sunden Y, Mingala CN, Onuma M, Murata S, Ohashi K

  Comp Immunol Microbiol Infect Dis. 36 (1) 63 - 9 2013/01 [Refereed][Not invited]
   

  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-γ 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.
• Prevalence of Lyme Borrelia in Ixodes persulcatus Ticks from an Area with a Confirmed Case of Lyme Disease

  MURASE Yusuke, KONNAI Satoru, GITHAKA Naftaly, HIDANO Arata, TAYLOR Kyle, ITO Takuya, TAKANO Ai, ANDO Shuji, KAWABATA Hiroki, TSUBOTA Toshio, MURATA Shiro, OHASHI Kazuhiko

  The Japanese Journal of Veterinary Science JAPANESE SOCIETY OF VETERINARY SCIENCE 75 (2) 215 - 218 0916-7250 2013 [Refereed][Not invited]
   

  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. persulcatus 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 rrf(5S)-rrl(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. persulcatus ticks were coinfected with Anaplasma phagocytophilum and B. garinii. The difference between the number of obtained and expected co-infections was significant (χ²=4.32, P=0.038).
• The quest for a universal vaccine against ticks: cross-immunity insights

  Parizi LF, Githaka NW, Logullo C, Konnai S, Masuda A, Ohashi K, da Silva Vaz I J

  Vet J. 194 (2) 158 - 65 2012/11 [Refereed][Not invited]
  
• Molecular detection and characterization of potentially new Babesia and Theileria species/variants in wild felids from Kenya

  Githaka N, Konnai S, Kariuki E, Kanduma E, Murata S, Ohashi K

  Acta Trop. 124 (1) 71 - 8 2012/10 [Refereed][Not invited]
   

  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 18S ribosomal RNA (18S 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.
• Transcriptional profiling of inflammatory cytokine genes in African buffaloes (Syncerus caffer) infected with Theileria parva

  Okagawa T, Konnai S, Mekata H, Githaka N, Suzuki S, Kariuki E, Gakuya F, Kanduma E, Shirai T, Ikebuchi R, Ikenaka Y, Ishizuka M, Murata S, Ohashi K

  Vet Immunol Immunopathol. 148 (3-4) 373 - 9 2012/08 [Refereed][Not invited]
  
• Surveillance of Marek's disease virus in migratory and sedentary birds in Hokkaido, Japan

  Murata S, Hayashi Y, Kato A, Isezaki M, Takasaki S, Onuma M, Osa Y, Asakawa M, Konnai S, Ohashi K

  Vet J. Elsevier 192 (3) 538 - 40 1090-0233 2012/06 [Refereed][Not invited]
   

  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.
• Kinetics of regulatory dendritic cells in inflammatory responses during Trypanosoma evansi infection

  Mekata H, Konnai S, Mingala CN, Abes NS, Gutierrez CA, Dargantes AP, Witola WH, Inoue N, Onuma M, Murata S, Ohashi K

  Parasite Immunol. 34 (6) 318 - 29 2012/06 [Refereed][Not invited]
  
• Molecular characterization of immunoinhibitory factors PD-1/PD-L1 in chickens infected with Marek's disease virus

  Matsuyama-Kato A, Murata S, Isezaki M, Kano R, Takasaki S, Ichii O, Konnai S, Ohashi K

  Virol J. 9 94  2012/05 [Refereed][Not invited]
  
• Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection

  Okagawa T, Konnai S, Ikebuchi R, Suzuki S, Shirai T, Sunden Y, Onuma M, Murata S, Ohashi K

  Vet Res. 43 (1) 45 - 45 2012/05 [Refereed][Not invited]
   

  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-γ 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-γ 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.
• Molecular cloning and characterization of Th1 and Th2 cytokines of African buffalo (Syncerus caffer)

  Suzuki S, Konnai S, Okagawa T, Githaka NW, Kariuki E, Gakuya F, Kanduma E, Shirai T, Ikebuchi R, Ikenaka Y, Ishizuka M, Murata S, Ohashi K

  Int J Immunogenet. 39 (2) 170 - 82 1744-3121 2012/04 [Refereed][Not invited]
   

  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-γ, and Th2 cytokines IL-4 and IL-10. The cloned cDNA of IL-2, IL-4, IL-10 and IFN-γ 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-γ 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. © 2011 Blackwell Publishing Ltd.
• Identification of TROSPA homologue in Ixodes persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan.

  Konnai S, Yamada S, Imamura S, Nishikado H, Githaka N, Ito T, Takano A, Kawabata H, Murata S, Ohashi K

  Ticks and Tick-borne Diseases 2 75 - 77 2012 [Refereed][Not invited]
  
• Molecular cloning of bovine lymphocyte activation gene-3 and its expression characteristics in bovine leukemia virus-infected cattle

  Shirai T, Konnai S, Ikebuchi R, Okagawa T, Suzuki S, Sunden Y, Onuma M, Murata S, Ohashi K

  Vet Immunol Immunopathol. 144 (3-4) 462 - 7 2011/12 [Refereed][Not invited]
   

  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.
• Increase of cells expressing PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via PD-L1 blockade

  Ikebuchi R, Konnai S, Shirai T, Sunden Y, Murata S, Onuma M, Ohashi K

  Vet Res. 42 103 - 103 2011/09 [Refereed][Not invited]
   

  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.
• Analysis of transcriptional activities of the Meq proteins present in highly virulent Marek's disease virus strains, RB1B and Md5

  Murata S, Okada T, Kano R, Hayashi Y, Hashiguchi T, Onuma M, Konnai S, Ohashi K

  Virus Genes. 43 (1) 66 - 71 2011/08 [Refereed][Not invited]
   

  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.
• Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks

  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 0378-1135 2011/05
• Molecular identification and expression analysis of lipocalins from blood feeding taiga tick, Ixodes persulcatus Schulze

  Satoru Konnai, Hideto Nishikado, Shinji Yamada, Saiki Imamura, Takuya Ito, Misao Onuma, Shiro Murata, Kazuhiko Ohashi

  Experimental Parasitology 127 (2) 467 - 474 0014-4894 2011/02 

  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.
• Cross immunity with Haemaphysalis longicornis glutathione S-transferase reduces an experimental Rhipicephalus (Boophilus) microplus infestation

  Luís Fernando Parizi, Kiyoko Uemura Utiumi, Saiki Imamura, Misao Onuma, Kazuhiko Ohashi, Aoi Masuda, Itabajara da Silva Vaz

  Experimental Parasitology 127 (1) 113 - 118 0014-4894 2011/01
• Characterization of CTLA-4, PD-1 and PDL-1 of swamp and riverine type water buffaloes

  Claro N. Mingala, Satoru Konnai, Ryoyo Ikebuchi, Kazuhiko Ohashi

  Comparative Immunology, Microbiology and Infectious Diseases 34 (1) 55 - 63 0147-9571 2011/01
• Virological and Serological Studies of Porcine Respiratory Coronavirus Infection on a Japanese Farm

  MEKATA Hirohisa, KONNAI Satoru, SIMUUNZA Martin, CHEMBENSOFU Mwelwa, KANO Rika, WITOLA William H., TEMBO Mwase E., CHITAMBO Harrison, INOUE Noboru, ONUMA Misao, OHASHI Kazuhiko

  The Japanese Journal of Veterinary Science JAPANESE SOCIETY OF VETERINARY SCIENCE 70 (9) 923 - 928 0916-7250 2008 

  The prevalence of trypanosome infections in tsetse flies, Glossina pallidipes, collected from 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 in 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.
  
• A Preliminary Examination of Data-base System Collecting, Stocking, Processing and Publishing Information about Potential Pathogens for Causing Outbreak of Wild Birds.

  Osa Yuichi, Akamatsu Rika, Takada Masayuki, Ohashi Kazuhiko, Okazaki Katsunori, Kaneko Masami, Endoh Daiji, Asakawa Mitsuhiko, Tsubota Toshio, Asano Makoto

  Abstracts of the Annual Meeting of the Ecological Society of Japan 日本生態学会 52 432 - 432 2005 

  生態学会２００４年大会において「野生鳥類の大量死リスク評価につながる病原体データベースの基本コンセプトについて」を発表し、病原体情報の収集分析のために、広域サンプリング_-_＞病原体タイプの同定_-_＞情報管理・蓄積_-_＞情報解析_-_＞情報公開及び活用といった基本的なコンセプト（設計図）を提示した。
  本発表においては、これらのコンセプトに基づいたシステムの実際的な運用例等を示すとともに、２００３年度に実施したサンプリングにより把握できた野生鳥類における病原体保有動態について中間的な報告を行う。また、実際に罹病あるいは死亡した個体に関する病原体（死因）情報のフォーマットや、「情報の共通化・共有化」等の具体的な運用面の問題についても述べる。さらに野生鳥類の移動_-_分散様式・病原体の伝播_-_発病様式・湖沼等の生息環境の空間構造・人間活動との相互作用を視野においた野生鳥類大量死発生予測モデリングについて手法検討を行う。
• Cloning and sequence analysis of llama cytokines related to cell-mediated immunity

  Raadan Odbileg, Sung-Il Lee, Reiko Yoshida, Kyung-Soo Chang, Kazuhiko Ohashi, Chihiro Sugimoto, Misao Onuma

  Veterinary Immunology and Immunopathology 99 (1-2) 1 - 10 0165-2427 2004 

  In order to characterize the T helper 1 (Th1) cytokines of llama, we have cloned several llama cytokine genes and compared them to those of other mammalian species. The cDNAs encoding for interleukin (IL)-2, interferon (IFN)γ, IL-12p35 and IL-12p40 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-2, IFN-γ, IL-12 p35 and IL-12p40 were found to be 465, 501, 669 or 993 bp in length, with open reading frames encoding 154, 166, 222 or 330 amino acids, respectively. Homology analyses of nucleotide and deduced amino sequences of llama IL-2, IFN-γ, IL-12p35 and IL-12p40 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla, which includes pig and cattle. © 2004 Elsevier B.V. All rights reserved.
• 日本におけるニューカッスル病ウイルス株のヌクレオカプシド蛋白遺伝子の分子的特徴と制限酵素-based rapid pathotyping法の開発(Molecular characterization of the nucleocapsid protein gene of Newcastle disease virus strains in Japan and development of a restriction enzyme-based rapid pathotyping method)

  ファン・ハンミン, 張 景洙, 真瀬 昌司, 大橋 和彦, 小沼 操

  日本獣医学会学術集会講演要旨集 135回 127 - 127 1347-8621 2003/03
• Protective effect of orally administered human interferon (HuIFN)-alpha against systemic Listeria monocytogenes infection and a practical advantage of HUIFN-alpha derived from transgenic potato plant

  K Ohya, T Matsumura, N Itchoda, K Ohashi, M Onuma, C Sugimoto

  PLANT BIOTECHNOLOGY 2002 AND BEYOND 389 - 391 2003 [Refereed][Not invited]
   

  Type I interferon (IFN-alpha/beta) is the first cytokine used for clinical applications against viral and neoplasmic diseases. Usually it is administrated by subcutaneous and intramuscular injection, but several studies have reported that orally administered Type I IFN is also effective against viral and autoimmune diseases. Therefore, we examined whether orally administered human IFN (HuIFN)-alpha can augment protection against systemic bacterial infection using Listeria monocytogenes infection in mice as an experimental model. Daily oral administrations for 6 days of 1000 international units (IU) of purified natural HuIFN-a reduced bacterial burden in spleen and liver from L. monocytogenes-infected mice. This effect was observed in the middle phase of L. monocytogenes infection, but not in the early phase of the infection. Effects of oral administration of HuIFN-alpha expressed in potato plant were also examined in this infection model. Daily oral administrations of extracts of the transgenic potato tuber for 6 days decreased bacterial burden in the spleen. Lower doses of HuIFN-alpha in the extracts (20 IU/mouse/day) exerted a protective effect at almost the same level as the results achieved by the administration of 1000 IU of HuIFN-alpha. This result may be due to the 'bioencapsulation' effect for HuIFN-alpha by plant compartmentalization, which is one of the advantages of the plant expression system over other expression systems of recombinant proteins. Our present observation indicates the transgenic plants expressing cytokines can be used as feed/food and their additives in order to enhance natural immune responses in humans and animals.
• Erratum: Seroprevalence and molecular evidence for the presence of bovine immunodeficiency virus in Brazilian cattle (Japanese Journal of Veterinary Research (2002) vol. 50 (1) (9-16))

  Sothy Meas, Farias Jeronimo Ruas, Tatsufumi Usui, Yoshiyuki Teraoka, Albert Mulenga, Kyung-Soo Chang, Aoi Masuda, Claudo Roberto Madruga, Kazuhiko Ohashi, Misao Onuma

  Japanese Journal of Veterinary Research 50 (2-3) 145  0047-1917 2002/11
• BLV-Tax発現DNAワクチンのウイルス増殖抑制効果

  笛吹 達史, 今内 覚, 田島 茂, 渡来 仁, 大橋 和彦, 間 陽子, 小沼 操

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 133回 95 - 95 1347-8621 2002/03
• Seroprevalence and molecular evidence for the presence of bovine immunodeficiency virus in Brazilian cattle

  Sothy Meas, Farias Jeronimo Ruas, Tatsufumi Usui, Yoshiyuki Teraoka, Albert Mulenga, Kyung-Soo Chang, Aoi Masuda, Claudo Roberto Madruga, Kazuhiko Ohashi, Misao Onuma

  The Japanese journal of veterinary research 50 (1) 9 - 16 0047-1917 2002 

  Data on the worldwide distribution of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) is limited. A prevalence study of antibodies to BIV and BLV was conducted in six different cattle herds in Brazil. Out of a total of 238 sera analyzed, 11.7% were found positive for anti-BIV p26 antibodies as determined by Western blot analysis, 2.1% were positive for anti-BLV gp51 antibodies as detected by immunodiffusion test. Peripheral blood mononuclear cells from BIV seropositive cattle were found to have BIV-provirus DNA, as detected by nested polymerase chain reaction. A nucleotide sequence corresponding to a 298 bp fragment of the BIV pol gene was also analyzed. Amino acid sequences of these Brazilian pol gene products showed 98.0 to 100% homology to the American strain BIV R29, 97.0 to 99.0% to Japanese BIV isolates, and divergence ranged from 0 to 4.0% among Brazilian BIV isolates. This evidence of the presence of BIV and BLV infections in Brazil should be considered a health risk to Brazilian cattle populations and a potential causative agent of chronic disease in cattle.
• Host Immune Responses in the Course of Bovine Leukemia Virus Infection (Immunology)

  KABEYA Hidenori, OHASHI Kazuhiko, ONUMA Misao

  The journal of veterinary medical science 社団法人日本獣医学会 63 (7) 703 - 708, s・iii 0916-7250 2001/07 

  Bovine leukemia virus (BLV) is a type C retrovirus infecting bovine B cells and causing enzootic bovine leukosis. Since it takes long periods to develop the disease, it is believed that BLV and host immune responses are closely related. In this review, the accumulated data showing close relationship between BLV and host immune responses are summarized in 4 sections. First, we discuss the role of cell-mediated immunity in protecting hosts from BLV infection. Second, several reports showing the relationship between the disease progression and the change of cytokine profiles are summarized. In the third section, we have focused on tumor necrosis factor α (TNFα) and its two types of receptors, and the possible involvement of TNF α in the BLV-induced leukemogenesis is discussed. The expression of TNFα has been shown to be regulated by major histocompatibility complex (MHC) haplotype. The resistance to BLV infection is supposed to be established by some innate factors, which are closely related to MHC haplotype. Finally, we propose that a breeding strategy based on the MHC haplotype could be a good approach to control BLV infection. This review includes some recent data from us and other groups.
• Seroprevalence of Bovine Immunodeficiency Virus in Dairy and Beef Cattle Herds in Korea

  Kyoung-Oh Cho, Sothy Meas, Nam-Yong Park, Yong-Hwan Kim, Yoon-Kyu Lim, Daiji Endoh, Sung-Il Lee, Kazuhiko Ohashi, Chihiro Sugimoto, Misao Onuma

  Journal of Veterinary Medical Science 61 (5) 549 - 551 1782-155X 1999/05 [Refereed][Not invited]
   

  Infection of bovine immunodeficiency virus (BIV), a lentivirus, is thought to sporadically occur throughout the world, but seroepidemiological surveys concerning the incidence of BIV are limited and have not been undertaken in Korea. A total of 266 sera from different twenty dairy (Holstein) and twenty-six Korean native beef (Hanwoo) farms of the south-western part of Korea was analyzed for the presence of anti-BIV antibodies by Western blotting. Thirty five percent and 33% of dairy and beef cattle, respectively, were BIV-seropositive. By nested polymerase chain reaction, it was confirmed that these seropositive cows had provirus in the peripheral blood mononuclear cells. To demonstrate the correlation with BIV and bovine leukemia virus (BLV) infection, these sera were also analyzed for anti-BLV antibodies by immunodiffusion test, resulting in high prevalence of BLV infection but relatively a few dual infections. We report herein the first serological detection of antibodies to BIV in Korea.
• Pathogenesis of Marek's Disease(MD) and Possible Mechanisms of Immunity Induced by MD Vaccine.

  MORIMURA Toshifumi, OHASHI Kazuhiko, SUGIMOTO Chihiro, ONUMA Misao

  The Japanese Journal of Veterinary Science JAPANESE SOCIETY OF VETERINARY SCIENCE 60 (1) 1 - 8 0916-7250 1998 

  Marek's disease (MD) is a lymphoproliferative disease of chicken, which is characterized by malignant T cell-lymphoma formation. This disease can be effectively prevented by vaccination with attenuated MD virus (MDV), apathogenic MDV or herpesvirus of turkey. MD vaccines are ones of a few vaccines which can prevent virus-induced tumor among mammalian and avian species. To determine the roles of T cell subsets in the protection mechanism, chickens vaccinated with an attenuated MDV (CVI988) were depleted of either CD4⁺ or CD8⁺T cells by neonatal thymectomy and injections of monoclonal antibodies against chicken CD4 or CD8 molecules and then challenged with an oncogenic MDV. These birds were effectively protected from MDV-induced tumors. However, virus titers in CD4⁺T cells, which are the main target cells for MDV-latent infection and subsequent transformation, were much higher in CD8-deficient vaccinated chickens than in untreated vaccinated chickens at the early stage of the latent phase. These results suggested that CD8⁺T cell responses induced by the MD vaccine are essential for anti-virus but not anti-tumor effects. Here, we will discuss how the attenuated vaccine prevents chickens from lymphoma-formation by an oncogenic MDV.

MISC

• 牛伝染性リンパ腫の診断と早期発症予測法の開発

  富永みその, 今内覚, 岡川朋弘, 神谷可菜, 齋藤麻矢, 安富一郎, 目堅博久, 前川直也, 村田史郎, 大橋和彦  北海道獣医師会雑誌  67-  (8)  2023
• Chimeric provirus of bovine leukemia virus/ SMAD family member 3 in cattle with enzootic bovine leukosis

  直亨則, 直亨則, 岡川朋弘, 野尻直未, 今内覚, 今内覚, 嶋倉穂南, 富永みその, 吉田初佳, 西山依里, 松平崇弘, 前川直也, 村田史郎, 村田史郎, 村松正道, 村松正道, 大橋和彦, 大橋和彦, 斎藤益満  日本ウイルス学会学術集会プログラム・予稿集(Web)  70th-  2023
• 免疫チェックポイント分子Programmed death-ligand1(PD-L1)を標的とした抗体薬による犬の鼻腔内腺癌に対する免疫療法の検討

  多田佳史, 前川直也, 今内覚, 細谷謙次, 大脇稜, 竹内寛人, 賀川由美子, 高木哲, 高木哲, 鈴木定彦, 鈴木定彦, 岡川朋弘, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  166th-  2023
• 牛伝染性リンパ腫ウイルス感染症における免疫チェックポイント因子TIM-3およびPD-L1を標的とした抗ウイルス効果の検討

  中村隼人, 今内覚, 岡川朋弘, 前川直也, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  166th-  2023
• クローナリティ解析による地方病性牛伝染性リンパ腫に対する発症予測法の開発

  富永みその, 岡川朋弘, 嶋倉穂南, 斎藤益満, 松平崇弘, 直亨則, 山田慎二, 村上賢二, 前川直也, 村田史郎, 大橋和彦, 今内覚  日本獣医学会学術集会講演要旨集  166th-  2023
• マレック病ウイルスMeqタンパク質における挿入配列による病原性増強機構の解明

  佐藤純平, 村田史郎, YEE Win Shwe, 瀬尾光里, もたい吉之介, 前川直也, 岡川朋弘, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  166th-  2023
• 新たに認められた欠損配列を持つマレック病ウイルスmeq遺伝子の性状解析

  もたい吉之介, 村田史郎, 佐藤純平, 西明仁, 前川直也, 岡川朋弘, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  166th-  2023
• 牛伝染性リンパ腫ウイルス感染牛における分娩が及ぼす免疫抑制メカニズムの解析

  富永みその, 今内覚, 佐治木大和, 岡川朋弘, 小原潤子, 似内厚之, 高橋博文, 窪田健太郎, 武田休史, 前川直也, 村田史郎, 大橋和彦  北海道獣医師会雑誌  66-  (8)  2022
• 牛伝染性リンパ腫の病態発生機序解析を基盤とする新制御法の試み

  今内覚, 岡川朋弘, 前川直也, 村田史郎, 大橋和彦  家畜衛生学雑誌  47-  (3)  2021
• Expression and functional analysis of swine PD-1 /PD-L1 pathway

  OTGONTUYA Ganbaatar, 今内覚, 岡川朋弘, 野島裕太郎, 前川直也, 市川世識, 小林篤史, 芝原友幸, 柳川洋二郎, 鈴木定彦, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• ワクモ(Dermanyssus gallinae)の吸血に対する宿主免疫応答の解明

  藤澤宗太郎, 村田史郎, 伊勢崎政美, 佐藤匠, 大石英司, 種子野章, 前川直也, 岡川朋弘, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• 動物用抗体医薬品の開発および臨床研究

  今内 覚, 岡川 朋弘, 前川 直也, 中島 千絵, 鈴木 定彦, 山本 啓一, 戸田 幹洋, 村田 史郎, 大橋 和彦  JVPA digest : 日本動物用医薬品協会会報  (67)  1  -10  2020/02
• 牛白血病の病態発生機序解析を基盤とする新制御法の試み

  今内覚, 岡川朋弘, 前川直也, 村田史郎, 大橋和彦  畜産技術  (784)  2020
• 分子標的薬-将来の展望 免疫チェックポイント阻害薬を用いた腫瘍免疫療法

  前川直也, 今内覚, 岡川朋弘, 村田史郎, 大橋和彦  Veterinary Oncology  7-  (2)  2020
• ネコCytotoxic T-lymphocyte antigen-4(CTLA-4)を標的とした新規免疫抑制剤の開発における基礎的検討

  大塚拓海, 今内覚, 前川直也, 渡慧, 岡川朋弘, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  163rd-  2020
• ワクモにおける定量的PCR法確立のための内在性コントロール遺伝子の探索

  有泉拓馬, 村田史郎, 藤澤宗太郎, 伊勢崎政美, 前川直也, 岡川朋弘, 種子野章, 大石英司, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  163rd-  2020
• 牛白血病ウイルス感染症における免疫抑制受容体TIM-3の発現解析および機能解析

  中村隼人, 今内覚, 岡川朋弘, 佐治木大和, 渡慧, 神谷可菜, 齋藤麻矢, 前川直也, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  163rd-  2020
• ウシマイコプラズマ感染症における免疫疲弊化

  今内 覚, 後藤伸也, 岡川朋弘, 前川直也, 村田 史郎, 大橋和彦  日本マイコプラズマ学会雑誌  46-  31  -33  2019/12  [Not refereed][Invited]
  
• Tick factors associated pathogen transmission ~Tick immunosuppressants in saliva~

  Satoru Konnal  Med. Entomol. Zool.  70-  (4)  189  -197  2019/12  [Refereed][Invited]
  
• 免疫チェックポイント分子Programmed death‐ligand 1(PD‐L1)を標的とする抗体薬による免疫療法が奏功した肺転移のある口腔内悪性黒色腫の犬の1例

  竹内寛人, 前川直也, 今内覚, 高木哲, 細谷謙次, 賀川由美子, 岡川朋弘, 鈴木定彦, 鈴木定彦, 山本啓一, 山本啓一, 村田史郎, 大橋和彦  北海道獣医師会雑誌  63-  (8)  343  2019/08/09  [Not refereed][Not invited]
  
• ロタウイルス実験感染子牛モデルを用いた3.5倍発酵代用乳の腸炎抑制効果の検証

  茅先史, 今内覚, 久保田学, 岡川朋弘, 佐治木大和, 渡慧, 小原潤子, 前川直也, 村田史郎, 大橋和彦  北海道獣医師会雑誌  63-  (8)  302  2019/08/09  [Not refereed][Not invited]
  
• 免疫学的解析を基盤とした動物用創薬研究

  Satoru Konnal  臨床獣医  37-  (3)  37  -41  2019/03  [Not refereed][Invited]
  
• ワクモ由来Adipocyte plasma membrane-associated proteinの抗ワクモワクチン抗原としての評価

  森田鮎, 竹原昌生, 村田史郎, 伊勢崎政美, 藤澤宗太郎, 種子野章, 酒井英史, 宇野有紀子, 小川遼, 市居修, 前川直也, 岡川朋弘, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  162nd-  2019
• 動物難治性疾病に対する創薬研究

  今内 覚, 岡川朋弘, 前川直也, 中島千絵, 鈴木定彦, 山本啓一, 戸田幹洋, 村田史郎, 大橋和彦  MPアグロジャーナル  35-  (10)  22  -25  2018/10  [Not refereed][Invited]
  
• 免疫チェックポイント分子を標的とした腫瘍免疫療法 抗PD-1/PD-L1抗体療法

  前川直也, 今内 覚, 村田史郎, 大橋和彦, 高木 哲  獣医畜産新報  71-  (9)  659  -663  2018/09  [Not refereed][Invited]
  
• 牛白血病における新規病態進行評価マーカーの検索

  渡慧, 今内覚, 佐治木大和, 岡川朋弘, 前川直也, 後藤伸也, 村田史郎, 大橋和彦  北海道獣医師会雑誌  62-  (8)  282  2018/08/24  [Not refereed][Not invited]
  
• ヨーネ病におけるプロスタグランジンE₂(PGE₂)動態解析及びCOX‐2阻害剤の免疫活性化効果の検討

  佐治木大和, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 後藤伸也, 永田礼子, 川治聡子, 森康行, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  341  2018/08/21  [Not refereed][Not invited]
  
• 肺転移のある悪性黒色腫罹患犬に対するPD‐L1を標的とした抗体薬の治療効果

  前川直也, 今内覚, 高木哲, 細谷謙次, 賀川由美子, 岡川朋弘, 和泉雄介, 出口辰弥, 鈴木定彦, 山本啓一, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  429  2018/08/21  [Not refereed][Not invited]
  
• マレック病ウイルスの病原性進化機構の解明―病原性試験や全ゲノム解析の試み

  大橋和彦, 村田史郎, 町田柚香, 伊勢崎政美, 今内覚  日本獣医学会学術集会講演要旨集  161st-  216  2018/08/21  [Not refereed][Not invited]
  
• 免疫抑制因子PD‐1およびPD‐L1を標的としたイヌ腫瘍に対する新規免疫療法の検討

  前川直也, 今内覚, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  233  2018/08/21  [Not refereed][Not invited]
  
• 動物難治性疾病の免疫学的解析を基盤とした新規制御法の開発

  今内覚, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  202  2018/08/21  [Not refereed][Not invited]
  
• 地方病性牛白血病の簡易鑑別診断基準の確立と若齢発症牛の実態調査

  田中晶菜, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 戸塚知恵, 千葉由純, 池田昌穂, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  353  2018/08/21  [Not refereed][Not invited]
  
• ウシCytotoxic T‐lymphocyte antigen‐4(CTLA‐4)の機能解析および慢性感染症における発現解析

  渡慧, 今内覚, 岡川朋弘, 前川直也, 後藤伸也, 佐治木大和, 村田史郎, 鈴木定彦, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  340  2018/08/21  [Not refereed][Not invited]
  
• イヌCytotoxic T‐lymphocyte antigen‐4(CTLA‐4)を標的としたバイオ医薬品の開発に向けた基礎的検討

  石原悠太郎, 今内覚, 岡川朋弘, 前川直也, 鈴木定彦, 大田寛, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  430  2018/08/21  [Not refereed][Not invited]
  
• Mycoplasma bovis感染症における免疫抑制機序の解析

  後藤伸也, 今内覚, 岡川朋弘, 前川直也, 佐治木大和, 渡慧, 樋口豪紀, 小岩政照, 田島誉士, 鈴木定彦, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  342  2018/08/21  [Not refereed][Not invited]
  
• 地方病性牛白血病若齢発症牛におけるプロウイルス挿入部位の網羅的解析

  岡川朋弘, 今内覚, 西森朝美, 田中晶菜, 前川直也, 戸塚知恵, 千葉由純, 池田昌穂, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  351  2018/08/21  [Not refereed][Not invited]
  
• ミャンマー連邦共和国における鶏の吸血性外部寄生虫の分布状況調査

  竹原昌生, 村田史郎, 片倉賢, MYINT MYINT Hmoon, SHWE YEE Win, SAW Bawm, LAT LAT Htun, YE HTUT Aung, MAR MAR Win, 伊勢崎政美, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  375  2018/08/21  [Not refereed][Not invited]
  
• ワクモ(Dermanyssus gallinae)由来カテプシンL用タンパク質の性状解析とワクチン抗原としての評価

  村田史郎, 伊勢崎政美, 谷口綾香, 北條巧, 種子野章, 酒井英史, 宇野有紀子, 矢吹卓也, 市居修, 伊東拓也, 今内覚, 大橋和彦  衛生動物  69-  (2)  103  2018/06/25  [Not refereed][Not invited]
  
• 獣医療における抗体医薬の現状

  今内 覚, 村田史郎, 大橋和彦  家畜診療  65-  (4)  233  -240  2018/04  [Not refereed][Invited]
  
• COX‐2阻害薬と抗PD‐L1抗体の併用によるイヌ腫瘍疾患の制御法の検討

  浅野裕美恵, 今内覚, 岡川朋弘, 前川直也, 西森朝美, 後藤伸也, 高木哲, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  371  2017/08/30  [Not refereed][Not invited]
  
• PD‐L1を標的としたイヌ用キメラ抗体医薬の作製と悪性黒色腫および未分化肉腫罹患犬における抗腫瘍効果の検討

  前川直也, 今内覚, 高木哲, 賀川由美子, 岡川朋弘, 西森朝美, 池渕良洋, 和泉雄介, 出口辰弥, 加藤幸成, 鈴木定彦, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  460  2017/08/30  [Not refereed][Not invited]
  
• 日本に分布するマレック病ウイルス野外株の病原性の検討

  村田史郎, 種子野章, 酒井英史, 町田柚香, 松山あゆ美, 伊勢崎政美, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  405  2017/08/30  [Not refereed][Not invited]
  
• マレック病ウイルス弱毒株を用いた新規抗ワクモワクチン開発に向けた基礎研究

  青山珠里愛, 村田史郎, 伊勢崎政美, 種子野章, 酒井英史, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  405  2017/08/30  [Not refereed][Not invited]
  
• 牛マイコプラズマ感染症における免疫抑制機序の解明

  後藤伸也, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 樋口豪紀, 小岩政照, 田島誉士, 小原潤子, 加藤幸成, 鈴木定彦, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  370  2017/08/30  [Not refereed][Not invited]
  
• 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

  今内覚, 岡山朋弘, 山田慎二, SIMUUNZA Martin, CONNELLEY Timothy, MORRISON Ivan, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  338  2017/08/30  [Not refereed][Not invited]
  
• 牛白血病ウイルス(BLV)感染症においてプロスタグランジンE₂(PGE₂)が及ぼす免疫抑制機序の解明

  佐治木大和, 今内覚, 西森朝美, 岡川朋弘, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  370  2017/08/30  [Not refereed][Not invited]
  
• BLV感染症をモデルとした抗ウシPD‐L1キメラ抗体の臨床応用試験

  西森朝美, 今内覚, 岡川朋弘, 前川直也, 池渕良洋, 後藤伸也, 佐治木大和, 鈴木定彦, 小原潤子, 小笠原諭, 加藤幸成, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  370  2017/08/30  [Not refereed][Not invited]
  
• 牛白血病ウイルス(BLV)感染ハイリスク牛における初めての子宮内感染直接証明

  佐治木 大和, 今内 覚, 西森 朝美, 岡川 朋弘, 永野 昌志, 小原 潤子, 村田 史郎, 大橋 和彦  北海道獣医師会雑誌  61-  (8)  280  -280  2017/08  [Not refereed][Not invited]
  
• マダニ唾液由来因子の病原体伝播における役割

  今内覚, 伊東拓也, 高野愛, 川端寛樹, 安藤秀二, 村田史郎, 大橋和彦  日本細菌学雑誌(Web)  72-  (1)  35(J‐STAGE)  2017  [Not refereed][Not invited]
  
• ウシCytotoxic T‐lymphocyte antigen‐4(CTLA‐4)の機能解析と特異抗体の樹立

  渡慧, 今内覚, 前川直也, 岡川朋弘, 西森朝美, 鈴木定彦, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  159th-  365  -365  2016/08/30  [Not refereed][Not invited]
  
• 家畜における免疫チェックポイント関連因子の遺伝子比較解析とウシ用抗体を用いた動物種横断的研究

  野島裕太郎, 今内覚, 西森朝美, 前川直也, 岡川朋弘, 森康行, 賀川由美子, 鈴木定彦, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  159th-  364  2016/08/30  [Not refereed][Not invited]
  
• シュルツェマダニ(Ixodes persulcatus)由来sialostatinL2の機能解析

  越智晶絵, 今内覚, 伊東拓也, 川端寛樹, 高野愛, 安藤秀二, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  159th-  339  2016/08/30  [Not refereed][Not invited]
  
• 抗ワクモ(Dermanyssus gallinae)ワクチン開発に向けたワクモ由来フェリチン2の性状解析

  伊勢崎政美, 村田史郎, 酒井英史, 矢吹卓也, 種子野章, 市居修, 伊東拓也, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  159th-  399  2016/08/30  [Not refereed][Not invited]
  
• ウシ腫瘍壊死因子(TNF‐α)デコイレセプターによる新規抗炎症剤の開発

  藤澤宗太郎, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 村田史郎, 鈴木定彦, 大橋和彦  日本獣医学会学術集会講演要旨集  159th-  364  2016/08/30  [Not refereed][Not invited]
  
• マレック病ウイルス日本分離株の全ゲノム解析

  村田史郎, 町田柚香, 伊勢崎政美, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  159th-  399  2016/08/30  [Not refereed][Not invited]
  
• 牛白血病への対策を練る〜牛白血病について〜

  今内 覚, 村田 史郎, 大橋 和彦  養牛の友  7-  (484)  32  -36  2016/07  [Not refereed][Invited]
  
• 増加する牛白血病とその対策

  今内 覚, 村田 史郎, 大橋 和彦  農家の友  68-  (2)  108  -110  2016/02  [Not refereed][Invited]
  
• イヌ腫瘍浸潤リンパ球におけるPD‐1発現解析と免疫チェックポイント分子を標的とした新規腫瘍免疫療法の検討

  前川直也, 今内覚, 池渕良洋, 岡川朋弘, 足立真実, 高木哲, 賀川由美子, 賀川由美子, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  405  2015/08/30  [Not refereed][Not invited]
  
• シュルツェマダニ(Ixodes persulcatus)唾液腺由来免疫抑制因子sL2の同定および機能解析

  越智晶絵, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  301  2015/08/30  [Not refereed][Not invited]
  
• 牛白血病ウイルス感染症における疲弊化T細胞の動態とその再活性化

  岡川朋弘, 今内覚, 西森朝美, 前川直也, 池渕良洋, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  324  2015/08/30  [Not refereed][Not invited]
  
• 日本に分布するマレック病ウイルス野外株の分離

  町田柚香, 村田史郎, 伊勢崎政美, 酒井英史, 種子野章, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  354  2015/08/30  [Not refereed][Not invited]
  
• 日本国内における牛白血病若齢発症牛の実態調査と表現型解析

  西森朝美, 今内覚, 池渕良洋, 岡川朋弘, 中原綾子, 千葉由純, 戸塚知恵, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  336  2015/08/30  [Not refereed][Not invited]
  
• ワクモ(Dermanyssus gallinae)由来カテプシンL様タンパク質の性状解析とワクチン抗原としての評価

  北條巧, 谷口綾香, 村田史郎, 伊勢崎政美, 酒井英史, 矢吹卓也, 寺田晴菜, 種子野章, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  353  2015/08/30  [Not refereed][Not invited]
  
• マレック病ウイルスmeq遺伝子に認められた欠損配列

  村田史郎, 山本英次, 坂下奈津美, 松山あゆ美, 伊勢崎政美, 町田柚香, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  354  2015/08/30  [Not refereed][Not invited]
  
• シュルツェマダニ(Ixodes persulcatus)由来Salp15因子によるライム病ボレリア伝播促進機能の解析

  村瀬優介, 今内覚, 伊東拓也, 高野愛, 川端寛樹, 安藤秀二, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  301  2015/08/30  [Not refereed][Not invited]
  
• マレック病ウイルス癌遺伝子(Meq)と病原性との関連

  高木道浩, 村田史郎, 今内覚, 大橋和彦  鶏病研究会報  50-  (4)  199  -205  2015/02/25  [Not refereed][Not invited]
  
• 牛白血病ウイルス感染牛におけるナチュラル・キラー細胞の動態および機能解析

  中原綾子, 今内覚, 池渕良洋, 岡川朋弘, 西森朝美, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  157th-  388  2014/08/11  [Not refereed][Not invited]
  
• ウシ制御性T細胞の機能解析および慢性感染症における動態解析

  大平浩輔, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 中原綾子, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  157th-  388  2014/08/11  [Not refereed][Not invited]
  
• イヌ免疫抑制因子PD‐1およびPD‐L1を標的とした新規腫瘍免疫療法の検討

  前川直也, 今内覚, 池渕良洋, 岡川朋弘, 足立真実, 高木哲, 賀川由美子, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  157th-  483  2014/08/11  [Not refereed][Not invited]
  
• Direct PCR法を用いた全血からの牛白血病ウイルス迅速簡易診断法の検討

  西森朝美, 今内覚, 池渕良洋, 岡川朋弘, 大平浩輔, 中原綾子, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  157th-  424  2014/08/11  [Not refereed][Not invited]
  
• 野生鳥類―畜産防疫の手引き~牛舎に侵入する野生鳥類~

  長雄一, 大越安吾, 平井綱雄, 藤井啓, 大橋和彦, 村田史郎, 遠藤大二, 金子正美, 田中克佳, 浅川満彦  酪農ジャーナル  67-  (8)  29  -31  2014/08/01  [Not refereed][Not invited]
  
• シュルツェマダニ(lxodes persulcatus)由来因子の宿主免疫抑制機能の解析

  今内覚, 伊東拓也, 川端寛樹, 高野愛, 安藤秀二, 村田史郎, 大橋和彦  大原綜合病院年報  53-  65  2013/12/15  [Not refereed][Not invited]
  
• 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  動物ワクチン-バイオ医薬品研究会ニュースレター  8-  18  -19  2013/12  [Not refereed][Invited]
  
• 牛白血病ウイルスの感染予防対策

  今内 覚, 村田 史郎, 大橋 和彦  DAIRYMAN  63-  (9)  42  -43  2013/09  [Not refereed][Invited]
  
• イヌ免疫抑制因子PD‐1およびPD‐L1の同定と新規免疫療法樹立への基礎研究

  前川直也, 今内覚, 池渕良洋, 岡川朋弘, 高木哲, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  156th-  257  2013/08/30  [Not refereed][Not invited]
  
• 牛白血病ウイルス感染牛由来IgM^high B細胞とIgM^low B細胞のウイルス抗原発現解析及び性状解析

  池渕良洋, 今内覚, 岡川朋弘, 西森朝美, 中原綾子, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  156th-  271  2013/08/30  [Not refereed][Not invited]
  
• マレック病腫瘍病変形成における免疫抑制因子発現機序の検討

  松山あゆ美, 村田史郎, 伊勢崎政美, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  156th-  257  2013/08/30  [Not refereed][Not invited]
  
• シュルツェマダニ由来免疫抑制因子の機能解析

  豊間根耕地, 今内覚, 伊東拓也, 川端寛樹, 高野愛, 安藤秀二, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  156th-  235  2013/08/30  [Not refereed][Not invited]
  
• 全血培養法によるエンドトキシン活性測定の有用性の検討

  今村彩貴, 中溝万里, 川西路子, 池渕良洋, 目堅博久, 村田史郎, 今内覚, 大橋和彦, 中島奈緒, 山本欣也, 内山万利小, 平野文哉, 永井英貴, 木島まゆみ  日本獣医学会学術集会講演要旨集  156th-  260  2013/08/30  [Not refereed][Not invited]
  
• 国内に潜在すると考えられる新興回帰熱に関する疫学調査研究

  高野愛, 川端寛樹, 大久保(佐藤)梢, 中尾稔, 伊東拓也, TAYLOR Kyle, 李景利, 坪田敏男, 今内覚, 吉村英紘, 豊間根耕地, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  156th-  304  2013/08/30  [Not refereed][Not invited]
  
• ウシ免疫抑制因子PD‐L1およびPD‐L2の組換えタンパク質作製と免疫機能解析

  西森朝美, 今内覚, 池渕良洋, 岡川朋弘, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  156th-  257  2013/08/30  [Not refereed][Not invited]
  
• 牛白血病発生農場におけるサシバエ種の同定とウイルスの検出

  今内覚, 豊間根耕地, 小池菜々子, 伊東拓也, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  156th-  231  2013/08/30  [Not refereed][Not invited]
  
• シュルツェマダニにおける病原体伝播関連因子の同定.

  今内覚, 川端寛樹, 伊東拓也, 高野愛, 安藤秀二, 村田史郎, 大橋和彦  衛生動物  64-  (2)  112  2013/06/15  [Not refereed][Not invited]
  
• スーラ病の分子疫学調査および野外分離株を用いた比較病原性解析と病態発生機序の解明

  目堅博久, 今内覚, MINGALA Claro N, 井上昇, 村田史郎, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  155th-  81  2013/03/04  [Not refereed][Not invited]
  
• RNA用試料抽出・保存用濾紙のRNAウイルスPCR検出への利用

  松山智慧麿, 遠藤大二, 大橋和彦, 今内覚, 村田史郎, 有川二郎, 吉松組子, 水谷哲也, 林正信  日本獣医学会学術集会講演要旨集  155th-  229  2013/03/04  [Not refereed][Not invited]
  
• シュルツェマダニ由来因子の病原体伝播における機能解析

  今内覚, 川端寛樹, 伊東拓也, 高野愛, 安藤秀二, 村田史郎, 大橋和彦  大原綜合病院年報  52-  105  -106  2012/12/15  [Not refereed][Not invited]
  
• 感染症と免疫の基礎 臨床現場で役立つ免疫の知識

  大橋 和彦, 村田 史郎, 今内 覚  臨床獣医  11-  50  -52  2012/11  [Not refereed][Invited]
  
• 感染症と免疫の基礎 細胞性免疫

  今内 覚, 村田 史郎, 大橋 和彦  臨床獣医  10-  42  -44  2012/10  [Not refereed][Invited]
  
• 感染症と免疫の基礎 液性免疫

  村田 史郎, 今内 覚, 大橋 和彦  臨床獣医  9-  60  -62  2012/09  [Not refereed][Invited]
  
• ワクモDermanyssus gallinae由来発現遺伝子の網羅的解析

  伊勢崎政美, 村田史郎, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  154th-  203  2012/08/31  [Not refereed][Not invited]
  
• フィリピン共和国におけるTrypanosoma evansi野外流行株の分離および比較病原性解析

  今内覚, 目堅博久, CLARO Mingala, NANCY Abes, ALAN Dargantes, 井上昇, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  154th-  213  2012/08/31  [Not refereed][Not invited]
  
• マレック病ウイルスワクチン接種鶏における免疫抑制因子PD‐1およびPD‐L1の発現解析

  高崎紗蘭, 村田史郎, 松山あゆ美, 伊勢崎政美, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  154th-  234  2012/08/31  [Not refereed][Not invited]
  
• 牛白血病ウイルス感染牛における免疫抑制因子Tim‐3/Gal‐9の発現解析および機能解析

  岡川朋弘, 今内覚, 池渕良洋, 鈴木紗織, 寸田祐嗣, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  154th-  233  2012/08/31  [Not refereed][Not invited]
  
• マレック病感染鶏由来腫瘍病変部における免疫抑制因子PD‐1/PD‐L1発現の形態学的解析

  村田史郎, 松山あゆ美, 伊勢崎政美, 高崎紗蘭, 市居修, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  154th-  234  2012/08/31  [Not refereed][Not invited]
  
• シュルツェマダニ(Ixodes persulcatus)唾液腺由来免疫抑制因子sL2の同定および発現解析

  高田春奈, 今内覚, NAFTALY Githaka, 伊勢崎政美, 伊東拓也, 安藤秀二, 川端寛樹, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  154th-  204  2012/08/31  [Not refereed][Not invited]
  
• ウシの免疫抑制受容体PD‐1に対するモノクローナル抗体の作製および免疫活性化能の検討

  池渕良洋, 今内覚, 岡川朋弘, 横山和正, 中島千絵, 鈴木定彦, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  154th-  233  2012/08/31  [Not refereed][Not invited]
  
• 増加している牛白血病 〜北海道での現状と対策について〜

  今内 覚, 村田 史郎, 大橋 和彦  北海道獣医師会雑誌.  56-  1  -7  2012/07  [Not refereed][Invited]
  
• ウシ難治性疾病に対する多機能型新規治療法の開発

  岡川朋弘, 今内覚, 池渕良洋, 鈴木紗織, 村田史郎, 大橋和彦  動物ワクチン-バイオ医薬品研究会ニュースレター  5-  21  -23  2012/06  [Not refereed][Invited]
  
• 地方病性牛白血病腫瘍組織における免疫抑制因子PD‐L1の発現とその役割

  寸田祐嗣, 竹林賢作, 今内覚, 池渕良洋, 大橋和彦, 落合謙爾, 梅村孝司  日本獣医学会学術集会講演要旨集  153rd-  203  2012/03/01  [Not refereed][Not invited]
  
• A comparative analysis of cytokine profile of Theileria parva infection in Bovidae

  Okagawa T, Konnai S, Githaka N, Mekata H, Suzuki S, Kariuki E, Skilton R, Ishizuka M, Murata S, Ohashi K  Japanese Journal of Veterinary Parasitology  10-  (1-2)  37  2012/03  [Not refereed][Not invited]
  
• Babesia ovata感染宿主有核細胞の培養系産物を抗原としたELISAによる抗体検出系

  児玉道, 荻原喜久美, 納谷裕子, 村田史郎, 今内覚, 大橋和彦, 岸川正剛  日本獣医学会学術集会講演要旨集  153rd-  223  2012/03/01  [Not refereed][Not invited]
  
• 牛難治性疾病に対する免疫抑制因子を標的とした治療型ワクチンの開発

  今内覚, 池渕良洋, 岡川朋弘, 鈴木紗織, 白井達哉, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  153rd-  117  2012/03/01  [Not refereed][Not invited]
  
• 牛白血病ウイルス感染牛における免疫抑制因子PD‐1/PL‐L1の発現解析および機能解析

  池渕良洋, 今内覚, 村田史郎, 大橋和彦  日本獣医師会獣医学術学会年次大会講演要旨集  2011-  45  2012/01/20  [Not refereed][Not invited]
  
• ウシTim-3の発現・機能解析と新規疾病制御法への応用展開

  岡川朋弘, 今内覚, 池渕良洋, 鈴木紗織, 村田史郎, 大橋和彦  動物ワクチン-バイオ医薬品研究会ニュースレター  4-  27  -29  2011/12  [Not refereed][Invited]
  
• 牛白血病の感染予防対策 最終回 4 ある農場における清浄化の取り組み事例

  今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  Dairy Jpn  56-  (15)  63  -65  2011/12/01  [Not refereed][Not invited]
  
• 牛白血病の感染予防対策 —診療・生産現場からの声に対して—

  今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  Dairy Japan  12-  62  -64  2011/12  [Not refereed][Invited]
  
• 牛白血病の感染予防対策 3 牛白血病に対する風評被害と間違った知識とは?

  今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  Dairy Jpn  56-  (14)  38  -40  2011/11/01  [Not refereed][Not invited]
  
• 牛白血病の感染予防対策 牛白血病に対する風評被害と間違った知識とは？

  今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  Dairy Japan  11-  57  -58  2011/11  [Not refereed][Invited]
  
• 牛白血病の感染予防対策 —今からできる予防策とは？—

  今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  Dairy Japan  10-  34  -36  2011/10  [Not refereed][Invited]
  
• 現場で活かす酪農技術 牛白血病の感染予防対策 1 牛白血病はなぜ増えたのか?

  今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  Dairy Jpn  56-  (11)  53  -55  2011/09/01  [Not refereed][Not invited]
  
• 牛白血病の感染予防対策 —牛白血病はなぜ増えたのか？—

  今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  Dairy Japan  9-  37  -39  2011/09  [Not refereed][Invited]
  
• シュルツェマダニ(Ixodes persulcatus)唾液腺由来免疫抑制因子の同定および発現解析

  丹羽彩乃, 今内覚, GITHAKA Naftary, 伊東拓也, 川端寛樹, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  152nd-  207  2011/08/31  [Not refereed][Not invited]
  
• マレック病ウイルス強毒株由来Meqおよび弱毒株由来L‐Meqによるbcl‐2遺伝子発現調節の比較

  村田史郎, 伊勢崎政美, 松山あゆ美, 高崎紗蘭, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  152nd-  241  2011/08/31  [Not refereed][Not invited]
  
• ウシ免疫抑制受容体Tim‐3の同定と牛白血病ウイルス感染牛における発現解析

  岡川朋弘, 今内覚, 池渕良洋, 鈴木紗織, 寸田祐嗣, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  152nd-  224  2011/08/31  [Not refereed][Not invited]
  
• 牛白血病ウイルス感染牛における制御性T細胞の動態解析

  鈴木紗織, 今内覚, 池渕良洋, 岡川朋弘, 寸田祐嗣, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  152nd-  224  2011/08/31  [Not refereed][Not invited]
  
• トリパノソーマ原虫感染において制御性樹状細胞は炎症反応を抑制して病態の改善に関与する

  目堅博久, 今内覚, 井上昇, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  152nd-  205  2011/08/31  [Not refereed][Not invited]
  
• Theileria parva原虫感染牛属のサイトカイン発現比較解析

  今内覚, 岡川朋弘, NAFTALY Githaka, 目堅博久, 鈴木紗織, EDWARD Kariuki, ROBERT Skilton, 石塚真由美, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  152nd-  204  2011/08/31  [Not refereed][Not invited]
  
• シュルツェマダニ(Ixodes persulcatus)におけるライム病ボレリア伝播関連因子の同定.

  今内覚, 村瀬優介, 伊東拓也, 高野愛, 川端寛樹, 村田史郎, 大橋和彦  衛生動物  62-  (2)  133  2011/06/15  [Not refereed][Not invited]
  
• バイオセキュリティを見直す 4)感染牛を把握し,伝播リスクを抑える ウイルスを原因とする牛白血病の防疫

  今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  Dairyman  61-  (6)  30  -31  2011/06/01  [Not refereed][Not invited]
  
• ウイルスを原因とする牛白血病の防疫（感染牛を把握し、伝播リスクを抑える）

  今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  DAIRYMAN  61-  30  -31  2011/06  [Not refereed][Invited]
  
• Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks

  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  2011/05  [Not refereed][Not invited]
   

  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.
• 牛の難治性疾病に対する多機能型ワクチン戦略

  今内覚, 池渕良洋, 寸田祐嗣, 小沼操, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  151st-  120  2011/03/01  [Not refereed][Not invited]
  
• 牛末梢血よりTheileria orientalisとBabesia ovataとの混合感染有核細胞培養系の樹立

  児玉道, 荻原喜久美, 納屋裕子, 久保正法, 榊原伸一, 村田史郎, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  151st-  217  2011/03/01  [Not refereed][Not invited]
  
• ベクターコントロールによる東海岸熱新規防除法の探索

  山田慎二, 今内覚, 今村彩貴, SIMMUNZA Martin, NANBOTA Andrew, 小沼操, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  151st-  79  2011/03/01  [Not refereed][Not invited]
  
• 牛白血病の感染防御対策

  今内 覚, 田島誉士, 小沼 操, 村田史郎, 大橋和彦  北海道しゃくなげ会会報  50-  10  -12  2011/02  [Not refereed][Invited]
  
• 国内の牛白血病の現状と今後の対策について

  今内 覚, 田島 誉士, 村田 史郎, 大橋 和彦  牛臨床寄生虫研究会誌  2-  2  -3  2011/02  [Not refereed][Invited]
  
• Molecular identification and expression analysis of lipocalins from blood feeding taiga tick, Ixodes persulcatus Schulze

  Satoru Konnai, Hideto Nishikado, Shinji Yamada, Saiki Imamura, Takuya Ito, Misao Onuma, Shiro Murata, Kazuhiko Ohashi  EXPERIMENTAL PARASITOLOGY  127-  (2)  467  -474  2011/02  [Not refereed][Not invited]
   

  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.
• 獣医学における保全医学の展開―生物多様性と野生動物感染症―マガンなどの野生水禽の疾病抵抗性とマレック病ウイルスの分布

  大橋和彦, 松原綾子, 村田史郎, 今内覚  獣医畜産新報  (1074)  21  -26  2011/01/01  [Not refereed][Not invited]
  
• 牛白血病の正体と予防を考察する

  今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  HOLSTEIN MAGAZINE  505-  4  -7  2011/01  [Not refereed][Invited]
  
• Cross immunity with Haemaphysalis longicornis glutathione S-transferase reduces an experimental Rhipicephalus (Boophilus) microplus infestation

  Luis Fernando Parizi, Kiyoko Uemura Utiumi, Saiki Imamura, Misao Onuma, Kazuhiko Ohashi, Aoi Masuda, Itabajara da Silva Vaz  EXPERIMENTAL PARASITOLOGY  127-  (1)  113  -118  2011/01  [Not refereed][Not invited]
   

  Recombinant Glutathione S-transferase of Haemaphysalis longicornis (rGST-Hl) was expressed in Escherichia coli, purified by affinity chromatography and used in the immunization of cattle. Western blot analysis showed positive antibody response in cattle immunized with rGST-Hl. The tests also showed that immunized bovine sera recognize native Rhipicephalus micro plus proteins in different tissue extracts. Furthermore, the vaccine potential of rGST-Hl was investigated against infestation of Hereford cattle by R. microplus. Vaccination of cattle with rGST-Hl conferred partial cross-protection immunity against R. microplus. Considering the effect on number of engorged ticks, egg laying capacity and egg fertility, the overall efficacy of vaccination was of 57%, as compared with control group. (C) 2010 Elsevier Inc. All rights reserved.
• Characterization of CTLA-4, PD-1 and PDL-1 of swamp and riverine type water buffaloes

  Claro N. Mingala, Satoru Konnai, Ryoyo Ikebuchi, Kazuhiko Ohashi  COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES  34-  (1)  55  -63  2011/01  [Not refereed][Not invited]
   

  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.
• 難治性慢性疾病における免疫疲弊化機序の解明と新規治療法への応用―獣医医療への応用にむけて―

  池渕良洋, 今内覚, 寸田祐輔, 小沼操, 村田史郎, 大橋和彦  日生研たより  56-  (6)  77  -81  2010/11/01  [Not refereed][Not invited]
  
• フィリピン共和国で分離したTrypanosoma evansiの病原性比較解析

  目堅博久, 今内覚, MINGALA Claro, DARGANTES Alan, 井上昇, 小沼操, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  150th-  191  2010/09/01  [Not refereed][Not invited]
  
• シュルツェマダニ(Ixodes persulcatus)由来Salp15様因子の性状解析

  村瀬優介, 今内覚, 伊藤拓也, 高野愛, 川端寛樹, 小沼操, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  150th-  193  2010/09/01  [Not refereed][Not invited]
  
• アフリカスイギュウ(Syncerus caffer)のTh1,Th2および炎症性サイトカイン遺伝子の同定

  岡川朋弘, 鈴木紗織, 今内覚, GITHAKA Naftali, KARIUKI Edward, GAKUYA Francis, ESTHER Kanduma, 石塚真由美, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  150th-  212  2010/09/01  [Not refereed][Not invited]
  
• マレック病ウイルス感染鶏における免疫抑制受容体PD‐1とそのリガンドPD‐L1の発現解析

  河東あゆ美, 村田史郎, 加納里佳, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  150th-  214  2010/09/01  [Not refereed][Not invited]
  
• 牛白血病ウイルス感染牛における免疫抑制因子PD‐1/PD‐L1の発現解析と機能解析

  池渕良洋, 今内覚, 白井達哉, 寸田祐嗣, 小沼操, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  150th-  214  2010/09/01  [Not refereed][Not invited]
  
• シュルツェマダニ(Ixodes persulcatus)由来Salp16の宿主免疫抑機構解析

  今内覚, 肥田野新, 村瀬優介, 伊東拓也, 川端寛樹, 樋口豪起, 小沼操, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  150th-  193  2010/09/01  [Not refereed][Not invited]
  
• ウシ免疫抑制因子LAG‐3の同定と牛白血病ウイルス感染牛における発現解析

  白井達哉, 今内覚, 池渕良洋, 寸田祐嗣, 小沼操, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  150th-  214  2010/09/01  [Not refereed][Not invited]
  
• ワクモDermanyssus gallinaeに対する新規防除法開発のためのトランスクリプトーム解析

  伊勢崎政美, 村田史郎, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  150th-  193  2010/09/01  [Not refereed][Not invited]
  
• マガンおよび鶏におけるinterferon‐γ遺伝子プロモーター活性の比較解析

  松原綾子, 村田史郎, 加納里佳, 橋口知幸, 伊勢崎政美, 河東あゆ美, 小沼操, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  150th-  215  2010/09/01  [Not refereed][Not invited]
  
• 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―

  今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  北海道獣医師会雑誌  54-  (8)  378  2010/08/10  [Not refereed][Not invited]
  
• 難治性慢性疾病における免疫疲弊化機序の解明と新規治療法への応用〜獣医医療への応用に向けて〜

  池渕良洋, 今内 覚, 寸田祐嗣, 小沼 操, 村田史郎, 大橋和彦  NIBS Letter  56-  (6)  7  -11  2010/06  [Not refereed][Invited]
  
• 増加傾向にある牛白血病の現状と対策 〜診療現場からの声に対して〜

  今内 覚, 田島 誉士, 小沼 操, 村田 史郎, 大橋 和彦  産業動物臨床医学雑誌  1-  (2)  110  -114  2010/06  [Not refereed][Invited]
  
• Two novel Salp15-like immunosuppressant genes from salivary glands of Ixodes persulcatus Schulze tick

  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  2010/06  [Not refereed][Not invited]
   

  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.
• Molecular cloning and expression analysis of bovine programmed death-1

  Ryoyo Ikebuchi, Satoru Konnai, Yuji Sunden, Misao Onuma, Kazuhiko Ohashi  MICROBIOLOGY AND IMMUNOLOGY  54-  (5)  291  -298  2010/05  [Not refereed][Not invited]
   

  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.
• 増加傾向にある牛白血病の現状と対策 〜診療・生産現場からの声に対して〜

  今内 覚, 田島 誉士, 小沼 操, 大橋 和彦  家畜診療  57-  (4)  201  -207  2010/04  [Not refereed][Invited]
  
• マガンなどの野生水禽の疾病抵抗性とマレック病ウイルスの分布

  大橋和彦, 松原綾子, 村田史郎, 今内覚  日本獣医学会学術集会講演要旨集  149th-  198  2010  [Not refereed][Not invited]
  
• The detection and characterization of the meq variants of Marek's diseases virus

  Ohashi K, Murata S, Okada T, Konnai S  Animal Viruses  16-  117  -132  2010/01  [Refereed][Invited]
  
• Molecular detection of trypanosomes in cattle in South America and genetic diversity of Trypanosoma evansi based on expression-site-associated gene 6

  Hirohisa Mekata, Satoru Konnai, William H. Witola, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi  INFECTION GENETICS AND EVOLUTION  9-  (6)  1301  -1305  2009/12  [Not refereed][Not invited]
   

  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.
• PCR-based detection of blood parasites in cattle and adult Rhipicephalus appendiculatus ticks

  Shinji Yamada, Satoru Konnai, Saiki Imamura, Martin Simuunza, Mwelwa Chembensofu, Amos Chota, Andrew Nambota, Misao Onuma, Kazuhiko Ohashi  VETERINARY JOURNAL  182-  (2)  352  -355  2009/11  [Not refereed][Not invited]
   

  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.
• Comparative immunogenicity of Haemaphysalis longicornis and Rhipicephalus (Boophilus) microplus calreticulins

  Luis Fernando Parizi, Herbert Rech, Carlos Alexandre Sanchez Ferreira, Saiki Imamura, Kazuhiko Ohashi, Misao Onuma, Aoi Masuda, Itabajara da Silva Vaz  VETERINARY PARASITOLOGY  164-  (2-4)  282  -290  2009/10  [Not refereed][Not invited]
   

  The ticks Rhipicephalus (Boophilus) microplus and Haemaphysalis longicornis are blood-sucking ectoparasites of bovines, causing serious damages to the livestock production. The main control method for these ticks is based on acaricides. However, the use of vaccines has been studied as a promising control strategy. Calreticulin (CRT) is a multifunctional, predominantly intracellular protein present in almost all cells of animals. The secretion of CRT during feeding might be linked to the modulation of the parasite-host interaction. In the present study, recombinant CRTs of R. microplus (rBmCRT) and H. longicornis (rHlCRT) were expressed in Escherichia coli and purified by ion exchange chromatography and used for the immunization of bovines and mouse. ELISA demonstrated that both rCRTs are recognized by the sera of immunized bovines. In silica, despite the difference in amino acid sequences, antigenic index analysis of HlCRT and BmCRT using the Jameson-Wolf algorithm indicated that both proteins were very similar in antigenicity index, although six different epitopes between the tick CRTs have been inferred. These data were corroborated by competitive ELISA analyses, which suggest the presence of different epitopes within the proteins. Western blot analyses showed that anti-rBmCRT and anti-rHlCRT bovine sera also recognized the native proteins in larvae extracts and, moreover, sera of bovines immunized with saliva and extract of salivary glands recognized both recombinant CRTs. Thus, mouse and bovine immune system recognized rCRTs, resulting in the production of antibodies with similar specificity for both recombinant proteins, although different epitopes could be distinguished between rBmCRT and rHlCRT. (C) 2009 Elsevier B.V. All rights reserved.
• Cloning and characterization of Rhipicephalus appendiculatus voraxin alpha and its effect as anti-tick vaccine

  Shinji Yamada, Satoru Konnai, Saiki Imamura, Takuya Ito, Misao Onuma, Kazuhiko Ohashi  VACCINE  27-  (43)  5989  -5997  2009/10  [Not refereed][Not invited]
   

  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.
• Classification of new BVDV isolates from Philippine water buffalo using the viral E2 region

  Claro N. Mingala, Satoru Konnai, Motoshi Tajima, Misao Onuma, Kazuhiko Ohashi  JOURNAL OF BASIC MICROBIOLOGY  49-  (5)  495  -500  2009/10  [Not refereed][Not invited]
   

  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.
• Quantification of water buffalo (Bubalus bubalis) cytokine expression in response to inactivated foot-and-mouth disease (FMD) vaccine

  Claro N. Mingala, Satoru Konnai, Fe A. Venturina, Misao Onuma, Kazubliko Ohashi  RESEARCH IN VETERINARY SCIENCE  87-  (2)  213  -217  2009/10  [Not refereed][Not invited]
   

  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.
• Identification and characterization of antimicrobial peptide, defensin, in the taiga tick, Ixodes persulcatus

  Y. Saito, S. Konnai, S. Yamada, S. Imamura, H. Nishikado, T. Ito, M. Onuma, K. Ohashi  INSECT MOLECULAR BIOLOGY  18-  (4)  531  -539  2009/08  [Not refereed][Not invited]
   

  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.
• Effect of vaccination with a recombinant metalloprotease from Haemaphysalis longicornis

  Saiki Imamura, Itabajara da Silva Vaz, Satoru Konnai, Shinji Yamada, Chie Nakajima, Misao Onuma, Kazuhiko Ohashi  EXPERIMENTAL AND APPLIED ACAROLOGY  48-  (4)  345  -358  2009/08  [Not refereed][Not invited]
   

  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.
• Development and application of a quantitative real-time PCR for the diagnosis of Surra in water buffaloes

  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  2009/07  [Not refereed][Not invited]
   

  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.
• Development of an anti-tick vaccine to protect cattle against tick-borne transmission of Theileria parva

  Saiki Imamura, Satoru Konnai, Chie Nakajima, Shinji Yamada, Yuko Ito, Kazuhiko Ohashi, Misao Onuma  Veterinary Immunology and Immunopathology  128-  278  -279  2009/05  [Not refereed][Not invited]
  
• Comparative moleculo-immunological analysis of swamp- and riverine-type water buffaloes responses

  Claro N. Mingala, Satoru Konnai, Libertado C. Cruz, Misao Onuma, Kazuhiko Ohashi  CYTOKINE  46-  (2)  273  -282  2009/05  [Not refereed][Not invited]
   

  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.
• Microarray Analysis of Host Immune Responses to Marek's Disease Virus Infection in Vaccinated Chickens

  Rika Kano, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi  JOURNAL OF VETERINARY MEDICAL SCIENCE  71-  (5)  603  -610  2009/05  [Not refereed][Not invited]
   

  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.
• Expression and activity of glycogen synthase kinase during vitellogenesis and embryogenesis of Rhipicephalus (Boophilus) microplus

  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  2009/05  [Not refereed][Not invited]
   

  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.
• Cytokine profiles in chickens infected with virulent and avirulent Marek's disease viruses: Interferon-gamma is a key factor in the protection of Marek's disease by vaccination

  Rika Kano, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi  MICROBIOLOGY AND IMMUNOLOGY  53-  (4)  224  -232  2009/04  [Not refereed][Not invited]
   

  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.
• Suppression of cell proliferation and cytokine expression by HL-p36, a tick salivary gland-derived protein of Haemaphysalis longicornis

  Satoru Konnai, Chie Nakajima, Saiki Imamura, Shinji Yamada, Hideto Nishikado, Michi Kodama, Misao Onuma, Kazuhiko Ohashi  IMMUNOLOGY  126-  (2)  209  -219  2009/02  [Not refereed][Not invited]
   

  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.
• シュルツェマダニにおけるライム病ボレリア伝播関連遺伝子の同定

  今内覚, 村瀬優介, 山田慎二, 肥田野新, 伊東拓也, 川端寛樹, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  148th-  2009
• 牛白血病ウイルス感染症の現状と対策—清浄化への道、まず新たな感染を防ぐことからー

  今内 覚, 田島誉士, 小沼 操, 大橋和彦  北獣会誌  53-  (9)  529  -534  2009  [Not refereed][Invited]
  
• Quantitative Analysis of Cytokine mRNA Expression and Protozoan DNA Load in Theileria parva-Infected Cattle

  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  2009/01  [Not refereed][Not invited]
   

  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.
• Rhipicephalus appendiculatus: Characterization of a testis-associated protein

  Shinji Yamada, Yuko Ito, Saiki Imamura, Satoru Konnai, Takuya Ito, Misao Onuma, Kazuhiko Ohashi  EXPERIMENTAL PARASITOLOGY  120-  (4)  337  -342  2008/12  [Not refereed][Not invited]
   

  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.
• 採卵鶏の神経膠腫から分離されたトリ白血病ウイルスの病原性

  唯野剛史, 落合謙爾, 畑井仁, 越智章仁, 小桜利恵, 大橋和彦, 寸田祐嗣, 梅村孝司  日本獣医学会学術集会講演要旨集  146th-  160  2008/09/05  [Not refereed][Not invited]
  
• Theileria parva感染牛に認められた炎症性サイトカインストーム

  山田慎二, 今内覚, 今村彩貴, SIMUUNZA Martin, CHEMBENSOFU Mwelwa, AMOS Chota, NAMBOTA Andrew, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  146th-  170  2008/09/05  [Not refereed][Not invited]
  
• マレック病ウイルスmeq遺伝子の多型に伴うMeqタンパクの転写活性化能の変化

  橋口知幸, 村田史郎, 岡田宰, 加納里佳, 今内覚, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  146th-  189  2008/09/05  [Not refereed][Not invited]
  
• 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  2008/09  [Not refereed][Not invited]
   

  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.
• Serological survey of Toxoplasma gondii in wild waterfowl in Chukotka, Kamchatka, Russia and Hokkaido, Japan

  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  2008/08  [Not refereed][Not invited]
   

  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.
• Detection of Trypanosoma brucei in field-captured tsetse flies and identification of host species fed on by the infected flies

  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  2008/08  [Not refereed][Not invited]
   

  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.
• Effects of anti-tick cocktail vaccine against Rhipicephalus appendiculatus

  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  2008/08  [Not refereed][Not invited]
   

  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.
• Prevalence of fowl glioma-inducing virus in chickens of zoological gardens in Japan and nucleotide variation in the env gene

  Hitoshi Hatai, Kenji Ochiai, Mariko Murakami, Syunsuke Imanishi, Yukiko Tomioka, Takeshi Toyoda, Kazuhiko Ohashi, Takashi Umemura  JOURNAL OF VETERINARY MEDICAL SCIENCE  70-  (5)  469  -474  2008/05  [Not refereed][Not invited]
   

  Fowl glioma-inducing virus (FGV), which belongs to subgroup A of avian leukosis virus (ALV), is tumorigenic in the nervous system. In a zoological garden in Japan, approximately 40% of chickens, including Japanese fowls, were infected with FGV. Because this zoological garden plays a role as a major supplier of Japanese fowl for other zoological gardens, FGV infection is suspected to have spread among ornamental chickens. In this study, the prevalence of the disease was examined in a total of 129 chickens in three other zoological gardens by nested polymerase chain reaction (PCR), reverse transcription nested PCR and enzyme-linked immunosorbent assay. Twenty-six to 56 percent of the fowls in each of the examined gardens were positive by nested PCR. The phylogenetic analysis revealed that the 3' untranslated region, including the specific sequence of FGV, of the 14 isolated ALVs showed high sequence identity and a close relationship with FGV. In addition, the env gene of the isolates frequently showed mutations and deletions of nucleotides. These results suggest that FGV is prevalent among ornamental chickens kept in zoological gardens in Japan.
• 神経系に腫瘍原性を持つトリレトロウイルスの疫学的検索

  越智章仁, 落合謙爾, 畑井仁, 唯野剛史, 大橋和彦, 梅村孝司  日本獣医学会学術集会講演要旨集  145th-  174  2008/03/07  [Not refereed][Not invited]
  
• A survey of abortifacient infectious agents in livestock in Luzon, the Philippines, with emphasis on the situation in a cattle herd with abortion problems

  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  2008/03  [Not refereed][Not invited]
   

  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.
• Cytokine responses in camels (Camelus bactrianus) vaccinated with Brucella abortus strain 19 vaccine

  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  2008/02  [Not refereed][Not invited]
   

  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.
• 弱毒および強毒株マレック病ウイルス感染鶏における免疫応答の比較

  加納里佳, 橋口知幸, 岡田宰, 村田史郎, 今内覚, 小沼操, 大橋和彦  日本ウイルス学会北海道支部夏季シンポジウム講演抄録  42nd-  14  2008  [Not refereed][Not invited]
  
• 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  2008/01  [Not refereed][Not invited]
   

  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.
• A recombinant avian leukosis virus associated with fowl glioma in layer chickens in Japan

  Hitoshi Hatai, Kenji Ochiai, Katsue Nagakura, Syunsuke Imanishi, Akihiro Ochi, Rie Kozakura, Masaaki Ono, Masanobu Goryo, Kazuhiko Ohashi, Takashi Umemura  AVIAN PATHOLOGY  37-  (2)  127  -137  2008  [Not refereed][Not invited]
   

  Fowl glioma is characterized by multiple nodular growth of astrocytes, and fowl glioma-inducing virus belonging to avian leukosis virus has been isolated from Japanese bantam as a causal agent. Subcutaneous neoplasms of the head and neck have been reported in layer chickens since 2003 in Japan, and fowl glioma concurred in these affected layers. In the present study, the histopathology of 240 layers, including 18 layers with subcutaneous neoplasms and 222 layers kept with the affected layers, was performed to clarify the characteristics of fowl glioma in layers. Microscopically, 103 layers showed non-suppurative encephalitis, and four layers had locally extensive proliferation or multiple nodules of astrocytes. Gliomas concurred in 11 layers with subcutaneous neoplasms and occurred independently in three layers. In addition, two layers had locally extensive proliferation of small, round cells in the cerebrum. The fowl glioma-inducing virus genome was not detected in the affected brains by nested polymerase chain reaction. Ten isolates were obtained from the affected brains. By nucleotide sequencing of the env gene, SU coding regions of these isolates were most closely related to myeloblastosis-associated virus-like viruses, but TM regions showed the highest similarity to endogenous viral (ev) loci. The genome of one isolate mainly consisted of ev loci and contained several parts of other avian leukosis/sarcoma viruses. These results show that the causal avian leukosis virus of fowl glioma is not just fowl glioma-inducing virus and that different avian leukosis virus strains having oncogenicity in the central nervous system by recombination are spread in layers in Japan.
• 帯広近郊,ロシアならびにカムチャッカにおける野生水禽類のToxoplasma gondii抗体保有状況

  村尾岳洋, 加納里佳, 村田史郎, 今内覚, 大橋和彦, 小沼操, 前田龍一郎, 小俣吉孝  日本野生動物医学会大会・講演要旨集  13th-  88  2007/09/06  [Not refereed][Not invited]
  
• Development of a nested polymerase chain reaction method to detect oncogenic Marek's disease virus from feather tips

  Shiro Murata, Kyung-Soo Chang, Sung-Il Lee, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi  JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION  19-  (5)  471  -478  2007/09  [Not refereed][Not invited]
   

  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.
• コイタマダニにおけるオスダニ由来因子の同定と発現解析

  山田慎二, 今村彩貴, 今内覚, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  144th-  69  2007/08/27  [Not refereed][Not invited]
  
• コイタマダニ由来新規シスタチン遺伝子のクローニングと性状解析

  今村彩貴, 山田慎二, 今内覚, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  144th-  69  2007/08/27  [Not refereed][Not invited]
  
• シュルツェマダニ由来defensin遺伝子のクローニングおよび発現解析

  斉藤洋一, 今内覚, 西門秀人, 山田慎二, 今村彩貴, 森亜紀奈, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  144th-  67  2007/08/27  [Not refereed][Not invited]
  
• マレック病ウイルス弱毒株由来L‐Meqと強毒株由来Meqの機能における比較解析

  村田史郎, 岡田宰, 加納里佳, 小沼操, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  144th-  83  2007/08/27  [Not refereed][Not invited]
  
• 弱毒および強毒マレック病ウイルス感染鶏における免疫応答の比較

  加納里佳, 村田史郎, 岡田宰, 今内覚, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  144th-  77  2007/08/27  [Not refereed][Not invited]
  
• 帯広近郊,ロシアならびにカムチャッカにおける野生カモ類のトキソプラズマ抗体保有状況

  村尾岳洋, 加納里佳, 村田史郎, 今内覚, 大橋和彦, 小沼操, 前田龍一郎, 小俣吉孝  日本獣医学会学術集会講演要旨集  144th-  65  2007/08/27  [Not refereed][Not invited]
  
• マレック病ウイルス発癌遺伝子meqのスプライス産物Δmeqの発現・性状解析

  岡田宰, 村田史郎, 加納里佳, 高木道浩, 今内覚, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  144th-  83  2007/08/27  [Not refereed][Not invited]
  
• Detection of the virulent Marek's disease virus genome from feather tips of wild geese in Japan and the Far East region of Russia

  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  2007/08  [Not refereed][Not invited]
   

  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.
• Identification and characterization of a novel spliced form of the meq transcript in lymphoblastoid cell lines derived from Marek's disease tumours

  Tsukasa Okada, Michihiro Takagi, Shiro Murata, Misao Onuma, Kazuhiko Ohashi  JOURNAL OF GENERAL VIROLOGY  88-  (8)  2111  -2120  2007/08  [Not refereed][Not invited]
   

  In tumour cell lines established from Marek's disease (MID) lymphomas L-meq is consistently expressed. It contains a 180 bp insertion encoding additional copies of the proline-rich repeat in the meq open reading frame and its product may contribute to the maintenance of MID virus (MDV) latency. In this study, we identified a novel spliced form of the meq transcript in MD-derived lymphoblastoid cell lines and in MDV-infected cells. This transcript, termed Delta meq, encodes an N-terminal 98 aa of the Meq protein and lacks part of the basic leucine zipper (bZIP) and transactivation domains. In MID cell lines, transcription of L-meq was significantly downreguiated, while that of the Delta meq transcript was upregulated during apoptosis. These observations were also confirmed at the protein expression level. Reporter assays using meq- and interleukin-2 (IL-2)-promoter-driven luciferase vectors revealed that AMeq suppressed transactivation by L-Meq or Meq in a dose-dependent manner. Immunoprecipitation confirmed that AMeq was associated with L-Meq or Meq physically. These results suggest that AMeq could be involved in 2006 apoptosis in MID cell lines as it works as a negative regulator of L-Meq and Meq by direct interaction.
• Attachment duration required for Rhipicephalus appendiculatus to transmit Theileria parva to the host

  Satoru Konnai, Shinji Yamada, Saiki Imamura, Martin Simuunza, Mwelwa Chembensof, Amos Chota, Andrew Nambota, Kazuhiko Ohashi, Misao Onuma  Vector-Borne and Zoonotic Diseases  7-  (2)  241  -248  2007/06  [Not refereed][Not invited]
   

  Theileria parva, the agent of East Coast fever (ECF), is transmitted to the host during the blood meal feeding of Rhipicephalus appendiculatus ticks. In order to investigate the relationship between the attachment duration of R. appendiculatus and the transmission of T. parva, infected adult ticks were allowed to attach to naive mice for variable lengths of time. Attached ticks and host animal's back skin biopsies from the tick attachment site were collected daily, starting from 24 hours post-tick attachment, and used for seminested polymerase chain reaction (PCR) detection of T. parva. T. parva-infected ticks started to transmit the parasites from 72 hours post-tick attachment. As expected, the transmission of T. parva from ticks to mouse skin increased with duration of tick attachment. Transmission of the parasites was 77.7%, 100%, 85.5%, and 100% on day 4, 5, 6, and 7 post-tick attachment, respectively, as could be detected from mice skin biopsies taken from T. parva-infected ticks' attachment sites. These results have important implications for our understanding of early events in the transmission of T. parva and would help in the development of effective pharmacologic substances and/or vaccines against ticks. © Mary Ann Liebert, Inc.
• Interferon-gamma expression associated with suppression of bovine leukemia virus at the early phase of infection in sheep

  Tatsufumi Usui, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma  AIDS RESEARCH AND HUMAN RETROVIRUSES  23-  (4)  609  -609  2007/04  [Not refereed][Not invited]
  
• Tumor necrosis factor-alpha genetic polymorphism may contribute to progression of bovine leukemia virus infection

  Satoru Konnai, Tatsufumi Usui, Manabu Ikeda, Junko Kohara, Kosuke Okada, Kazuhiko Ohashi, Misao Onuma  AIDS RESEARCH AND HUMAN RETROVIRUSES  23-  (4)  608  -609  2007/04  [Not refereed][Not invited]
  
• Molecular cloning, sequencing and phylogenetic analysis of inflammatory cytokines of swamp type buffalo contrasting with other bubaline breeds

  Claro N. Mingala, Raadan Odbileg, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma  COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES  30-  (2)  119  -131  2007/03  [Not refereed][Not invited]
   

  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.
• Interferon-gamma expression associated with suppression of bovine leukemia virus at the early phase of infection in sheep

  Tatsufumi Usui, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY  115-  (1-2)  17  -23  2007/01  [Not refereed][Not invited]
   

  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.
• Comparative assessment of Th1 and Th2 cytokines of swamp type buffalo and other bubaline breeds by molecular cloning, sequencing and phylogenetics

  Claro N. Mingala, Raadan Odbileg, Satoru Konnal, Kazuhiko Ohashi, Misao Onuma  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY  113-  (3-4)  348  -356  2006/10  [Not refereed][Not invited]
   

  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.
• Complete cDNA sequences and phylogenetic analyses of the Th1 and Th2 cytokines of the bactrian camel (Camelus bactrianus)

  Raadan Odbileg, Byambaa Purevtseren, Zayat Batsukh, Satoru Konnaii, Kazuhiko Ohashi, Misao Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  68-  (9)  941  -946  2006/09  [Not refereed][Not invited]
   

  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.
• Theileria parva感染Rhipicephalus appendiculatusによる宿主への実験的伝播

  今内覚, 山田慎二, 今村彩貴, MARTIN Simuunza, MWELWA Chembensofu, ANDREW Nambota, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  142nd-  67  2006/08/31  [Not refereed][Not invited]
  
• マレック病由来腫瘍細胞株におけるMeqバリアント(変異体)の機能解析

  岡田宰, 高木道浩, 村田史郎, 加納里佳, 林裕子, 今内覚, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  142nd-  80  2006/08/31  [Not refereed][Not invited]
  
• マレック病ウイルスの強毒化とmeq遺伝子の多型との関連

  村田史郎, 岡田宰, 加納里佳, 林裕子, 今内覚, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  142nd-  80  2006/08/31  [Not refereed][Not invited]
  
• The presence of the p53 transcripts with truncated open reading frames in Marek's disease tumor-derived cell lines

  Michihiro Takagi, Kazuhiko Ohashi, Toshifumi Morimura, Chihiro Sugimoto, Misao Onuma  LEUKEMIA RESEARCH  30-  (8)  987  -992  2006/08  [Not refereed][Not invited]
   

  Several kinds of the p53 transcripts in which their open reading frames (ORFs) were truncated (ranging from 101 to 765 bp) were identified in Marek's disease (MD)-derived tumor cell lines as well as avian leukosis- and reticuloendotheliosis-derived ones, detected by nested RT-PCR and subsequent nucleotide sequence analysis. In these ORFs, regions encoding the proline-rich and DNA-binding domains of the p53 protein were frequently deleted, and many of these deletions were found to cause frame shift. Western blot analysis using anti-p53 monoclonal antibodies revealed that multiple p53 isoform proteins with various molecular weights including 45-46, 35 and 28 kDa were expressed in these tumor cell lines, though the p53 protein with a molecular weight of 49 kDa was detected in chicken embryo fibroblasts transformed by the SV40 T antigen as a control. Since no deletions were found in the p53 gene of these MD tumor cell lines, truncations in the p53 ORFs observed in this study might result from alternative splicing of the p53 gene. (c) 2005 Elsevier Ltd. All rights reserved.
• Expression of tumor necrosis factor-alpha in IgM(+) B-cells from bovine leukemia virus-infected lymphocytotic sheep

  Tatsufumi Usui, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY  112-  (3-4)  296  -301  2006/08  [Not refereed][Not invited]
   

  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.
• Acquisition and transmission of Theileria parva by vector tick, Rhipicephalus appendiculatus

  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  2006/08  [Not refereed][Not invited]
   

  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.
• Tumor necrosis factor-alpha genetic polymorphism may contribute to progression of bovine leukemia virus-infection

  Satoru Konnai, Tatsufumi Usui, Manabu Ikeda, Junko Kohara, Toh-ichi Hirata, Kosuke Okada, Kazuhiko Ohashi, Misao Onuma  MICROBES AND INFECTION  8-  (8)  2163  -2171  2006/07  [Not refereed][Not invited]
   

  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.
• The effect of TAO expression on PCD-like phenomenon development and drug resistance in Trypanosoma brucei

  A Tsuda, WH Witola, S Konnai, K Ohashi, M Onuma  PARASITOLOGY INTERNATIONAL  55-  (2)  135  -142  2006/06  [Not refereed][Not invited]
   

  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.
• The presence of a short form of p53 in chicken lymphoblastoid cell lines during apoptosis

  Michihiro Takagi, Tamami Takeda, Yuki Asada, Chihiro Sugimoto, Misao Onuma, Kazuhiko Ohashi  JOURNAL OF VETERINARY MEDICAL SCIENCE  68-  (6)  561  -566  2006/06  [Not refereed][Not invited]
   

  To examine the roles of a short form of p53 in the regulation of apoptosis in chicken lymphoblastoid tumor cell lines derived from Marek's disease (MD) and avian leukosis (AL), the expressions of the p53 proteins were analyzed in these cell lines in which apoptosis was chemically induced. In MSB 1-0 derived from MD, the expression of a 40 kDa protein of p53 was decreased and that of a 32 kDa protein, a short form of p53, was increased during apoptosis induced by actinomycin D. In 1104B1 derived from AL, the expressions of 42 and 32 kDa of p53 were increased during the apoptosis. The short form of p53 was undetectable in these cell lines when apoptosis was blocked by the pretreatment with endonuclease inhibitor, Zn2+, protease inhibitors, TPCK and TLCK, or caspase inhibitor, Z-VAD-FMK. In the transcriptional level, the expressions of bcl-2 and 1AP were decreased in these cell lines during actinomycin D-induced apoptosis, but no change was detected in the expression level of p53. These results suggest that, in these chicken tumors, the short form of p53 could play a role in the initiation of apoptosis induced by the chemotherapeutic compound, and that the regulation of its expression may be important for the maintenance of transformation status.
• A novel gene encoding a thrombin inhibitory protein in a cDNA library from Haemaphysalis longicornis salivary gland

  C Nakajima, S Imamura, S Konnai, S Yamada, H Nishikado, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  68-  (5)  447  -452  2006/05  [Not refereed][Not invited]
   

  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.
• Two serine protease inhibitors (serpins) that induce a bovine protective immune response against Rhipicephalus appendiculatus ticks

  S Imamura, B Namangala, T Tajima, ME Tembo, J Yasuda, K Ohashi, M Onuma  VACCINE  24-  (13)  2230  -2237  2006/03  [Not refereed][Not invited]
   

  We have previously undertaken preliminary characterization of two Rhipicephalus appendiculutus serine protease inhibitors (RAS-1 and -2) as anti-tick vaccine candidates. In this study, to clarify this hypothesis, we generated and further characterized recombinant RAS-1 and -2 (rRAS-1 and -2) and tested their potency as a cocktail anti-tick vaccine in cattle. RT-PCR analysis showed that RAS-1 and -2mRNA transcripts are expressed during all life cycle stages of ticks, independent of sex. As judged by SDS-PAGE rRAS-1 and -2 migrated as a molecular weight of around 64 and 60 kDa protein, respectively, considering that the expression vector produced a recombinant protein fused with 18-22 kDa TRX protein. RAS-1 and -2 were found not to be secreted into the bite site as determined by the reactivity of anti-tick saliva sera to rRAS-1 and -2, suggesting that both proteins are concealed antigens. Vaccination of cattle with a combination of rRAS-1 and -2 conferred significant protective immunity against ticks, resulting in 61.4% reduction in nymph engorgement rate, and in 28 and 43% increased mortality rate in adult female and male ticks, respectively. This is the first report on an anti-tick vaccine trial using a combination of two different serpins derived from R. appendiculatus, and using cattle as a natural host. (c) 2005 Elsevier Ltd. All rights reserved.
• Tumor necrosis factor-alpha up-regulation in spontaneously proliferating cells derived from bovine leukemia virus-infected cattle

  S Konnai, T Usui, M Ikeda, J Kohara, T Hirata, K Okada, K Ohashi, M Onuma  ARCHIVES OF VIROLOGY  151-  (2)  347  -360  2006/02  [Not refereed][Not invited]
   

  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.
• Expression of alternative oxidase inhibits programmed cell death-like phenomenon in bloodstream form of Trypanosoma brucei rhodesiense

  A Tsuda, WH Witola, K Ohashi, M Onuma  PARASITOLOGY INTERNATIONAL  54-  (4)  243  -251  2005/12  [Not refereed][Not invited]
   

  Trypanosoma brucei rhodesiense is one of the causative agents of African Trypanosomiasis. Programmed cell death (PCD) is fundamental in the development, homeostasis and immune mechanisms of multicellular organisms. It has been shown that, other than occurring in multicellular organisms, the PCD phenomenon also takes place in unicellular organisms. In the present study, we have found that under high-density axenic culture conditions, bloodstream form of T h. rhodesiense depicts a PCD-like phenomenon. We investigated the association of the PCD-like phenomenon with expression of trypanosome alternative oxidase (TAO) under low-temperature stress conditions. We observed that bloodstream form of T b. rhodesiense did not show any PCD but had Lip-regulated expression of TAO. Inhibition of TAO by the addition of ascofranone caused the development of PCD in bloodstream T h. rhodesiense under low-temperature stress, implying that expression of TAO may contribute to the inhibition of PCD. (c) 2005 Elsevier Ireland Ltd. All rights reserved.
• Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis

  HM Pham, S Konnai, T Usui, KS Chang, S Murata, M Mase, K Ohashi, M Onuma  ARCHIVES OF VIROLOGY  150-  (12)  2429  -2438  2005/12  [Not refereed][Not invited]
   

  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.
• Nested polymerase chain reaction for detection of the avian leukosis virus causing so-called fowl glioma

  H Hatai, K Ochiai, Y Tomioka, T Toyoda, K Hayashi, M Anada, M Kato, A Toda, K Ohashi, E Ono, T Kimura, T Umemura  AVIAN PATHOLOGY  34-  (6)  473  -479  2005/12  [Not refereed][Not invited]
   

  The complete nucleotide sequence of the avian leukosis virus causing so-called fowl glioma has been previously determined. Primers were designed for detection of the fowl glioma-causal virus (FGV) based on the 3' untranslated region of the viral genome. The provirus and viral RNA of FGV were specifically detected in various organs and tissues, including feather pulp, from experimentally infected birds using nested polymerase chain reaction (PCR) and reverse transcription nested PCR. The prevalence of FGV was evaluated in 131 Japanese fowls of a zoological garden in Japan based on the detection of the FGV genome in feather pulp using PCR and the detection of viral antigen in faeces by enzyme-linked immunosorbent assay. FGV proviral DNA was detected in feather pulp of 52 birds (39.7%) by nested PCR. Later, nine dead birds from among the 52 were histologically diagnosed as having fowl glioma and found to have the proviral DNA in the affected brain. These results demonstrated that the PCR-based detection of FGV in feather pulp is useful for epidemiological studies on fowl glioma.
• Identification and characterization of peptides binding to Newcastle disease virus by phage display

  M Ozawa, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  67-  (12)  1237  -1241  2005/12  [Not refereed][Not invited]
   

  Three individual peptide sequences, EVSHPKVG, WVTTSNQNV, and SGGSNRSP, which have potentials to bind to Newcastle disease virus (NDV), were identified by the biopanning method using phage display technology. The binding specificities of these peptides presented on phages were confirmed by ELISA competition assay using chicken anti-NDV antiserum. The synthetic peptides designed based on these results partially neutralized the infection of NDV in vitro. The peptide-motives identified here have the potential to lead to the identification of novel molecules that inhibit the NDV infection independent of the immune system.
• Theileria orientalis: Cloning a cDNA encoding a protein similar to thiol protease with haemoglobin-binding activity

  WY He, K Ohashi, C Sugimoto, M Onuma  EXPERIMENTAL PARASITOLOGY  111-  (3)  143  -153  2005/11  [Not refereed][Not invited]
   

  A gene encoding a protein (Tocp1) from Theileria orientalis was isolated from a cDNA library and the deduced amino acid sequence of Tocp1 has 476 amino acids. The primary structure of Tocp1 is similar to eukaryotic thiol proteases (EC 3.4.22.-), but no enzymatic activity was observed with the substitution of essential cysteine at the cysteine active site for glycine. Southern blot analysis showed that multiple genes similar to Tocp1 were present in the parasite genome. Sequence analysis of the genome of the parasite showed that there are at least five different genes similar to Tocp1. Tocpl transcripts were detected in the T. orientalis piroplasma by Northern blot analysis. Western blot analysis showed that Tocp1 was expressed in the piroplasm of T. orientalis. To address the role of Tocpl in the life cycle of T. orientalis, Tocp1 was expressed using pET32 expression system. Binding affinity to haemoglobin was demonstrated by enzyme-linked immunosorbent assay. (c) 2005 Elsevier Inc. All rights reserved.
• Acquired resistance to berenil in a cloned isolate of Trypanosoma evansi is associated with upregulation of a novel gene, TeDR40

  WH Witola, A Tsuda, N Inoue, K Ohashi, M Onuma  PARASITOLOGY  131-  635  -646  2005/11  [Not refereed][Not invited]
   

  Drug resistance is now a severe and increasing problem in trypanosomes, but molecular details of mechanisms of resistance are only beginning to unveil. There is urgent need to clearly elucidate the different mechanisms of drug resistance in trypanosomes in order to circumvent existing resistance problems and avoid emergence of resistance to the next generation drugs. In this study, we cloned and characterized I novel gene, TeDR40, whose expression is associated with resistance to berenil in Trypanosoma evansi. Expression analysis showed that the gene was at least 1000-fold upregulated in resistant parasites and the encoded protein appeared to have a ubiquitous cellular localization. To investigate the association of TeDR40 with berenil-resistance, we genetically modified wild-type berenil-sensitive T. evansi for inducible overexpression of the TeDR40 gene. Induction of over-expression of TeDR40 in T. evansi led to decreased (P<0(.)01) sensitivity to berenil. Our findings indicate a possible correlation between over-expression of a novel gene, TeDR40, and reduced sensitivity to berenil in an in vitro-cultured clonal line of T. evansi.
• Random sequencing of cDNA library derived from partially-fed adult female Haemaphysalis longicornis salivary gland

  C Nakajima, ID Vaz, S Imamura, S Konnai, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  67-  (11)  1127  -1131  2005/11  [Not refereed][Not invited]
   

  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.
• Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

  S Konnai, T Usui, M Ikeda, J Kohara, T Hirata, K Okada, K Ohashi, M Onuma  VIROLOGY  339-  (2)  239  -248  2005/09  [Not refereed][Not invited]
   

  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.
• Molecular cloning and phylogenetic analysis of inflammatory cytokines of Camelidae (llama and camel)

  R Odbileg, S Konnai, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  67-  (9)  921  -925  2005/09  [Not refereed][Not invited]
   

  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.
• コイタマダニ由来セルピンの抗ダニカクテルワクチン効果

  今村彩貴, BONIFACE Mamangala, 今内覚, 中島千絵, MWASE Tembo, 田島朋子, 安田準, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  140th-  84  2005/08/31  [Not refereed][Not invited]
  
• コイタマダニにおける精巣由来オス因子の探索

  伊藤裕子, 今内覚, 今村彩貴, 中島千絵, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  140th-  79  2005/08/31  [Not refereed][Not invited]
  
• フタトゲチマダニ由来新規メタロプロテアーゼ遺伝子のクローニングと性状解析

  今村彩貴, 今内覚, 中島千絵, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  140th-  78  2005/08/31  [Not refereed][Not invited]
  
• ザンビア共和国における牛およびRhipicephalus appendiculatusからのTheileria parvaの検出

  今内覚, 今村彩貴, 中島千絵, SIMUNZA Martin, AMOS Chota, CHEMBENSOFU Mwelwa, ANDOREU Nambota, 安田準, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  140th-  84  2005/08/31  [Not refereed][Not invited]
  
• 鶏およびマガン由来マレック病ウイルスの遺伝子解析とその比較

  村田史郎, 山本裕己, 岡田宰, 浅川満彦, 長雄一, 池内俊雄, 今内覚, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  140th-  108  2005/08/31  [Not refereed][Not invited]
  
• Detection of the meq gene in the T cell subsets from chickens infected with Marek's disease virus serotype 1

  KS Chang, K Ohashi, SI Lee, M Takagi, M Onuma  JAPANESE JOURNAL OF VETERINARY RESEARCH  53-  (1-2)  3  -11  2005/08  [Not refereed][Not invited]
   

  The meq gene was thought to be only detected in Marek's disease virus serotype 1 (MDV 1) including a very virulent strain, Md5, while L-meq, in which a 180-bp sequence is inserted into the meq open reading frame, is found in other strains of MDV I, such as CVI 988/R6. However, both meq and L-meq were previously detected by PCR in chickens infected with MDV I, suggesting that MDV 1 may consists of at least two subpopulations, one with meq, the other with L-meq. To further analyze these subpopulations, we analyzed the time course changes in distribution of these subpopulations among T cell subsets from chickens infected with MDV1. Both meq and L-meq were detected in CD4(+) and CD8(+) T cells infected with strain ME or CVI 988/R6. The shift in MDV subpopulations from one displaying meq to the other displaying L-meq and/or the conversion from meq to L-meq occurred mainly in the CD8(+) T cell subset from Md5-infected chickens. PCR products corresponding to L-meq rather than meq were frequently amplified from the CD8(+) T cell subset from CVI 988/R 6 -infected chickens. These results suggest that a dominant sub-population of MDV 1 changes depending on the T cell subsets, and that L-meq is dominantly present in the CD8(+) T cells which play a role in the clearance of pathogenic agents.
• Ability of orally administered IFN-alpha-containing transgenic potato extracts to inhibit Listeria monocytogenes infection

  K Ohya, T Matsumura, N Itchoda, K Ohashi, M Onuma, C Sugimoto  JOURNAL OF INTERFERON AND CYTOKINE RESEARCH  25-  (8)  459  -466  2005/08  [Not refereed][Not invited]
   

  Type I interferons (IFN-alpha/beta) were originally thought to be antiviral cytokines, but it has recently been reported that they also play an important role in potentiating innate and adaptive immune responses. Moreover, several studies have shown that the oral administration of type I IFN ameliorates various biologic activities. Here, we studied the ability of orally administered IFN-alpha to protect mice from systemic Listeria monocytogenes infection. Daily oral administration of purified natural IFN-alpha at a concentration of 1000 international units (IU)/20 mu l reduced the bacterial burden in infected organs. We also examined the protective effect of IFN-alpha expressed in transgenic potato plants. A much lower concentration of IFN-alpha (20 IU/20 mu l) in the plant extracts was almost as protective as much higher concentrations of purified natural IFN-alpha. Our observations indicate that transgenic cytokine-expressing plants can be used prophylactically as edible pharmaceuticals to enhance systemic defense responses in humans and animals.
• Cloning and characterization of cDNA encoding a prohibitin-like protein from Theileria orientalis

  WY He, K Ohashi, C Sugimoto, M Tsuji, M Onuma  JAPANESE JOURNAL OF VETERINARY RESEARCH  53-  (1-2)  27  -35  2005/08  [Not refereed][Not invited]
   

  A cDNA clone encoding a prohibitin-like protein (Toprh) was isolated from a piroplasm cDNA library of Theileria orientalis and its nucleotide sequence was determined. An open reading frame, encoding a polypeptide of 278 amino acid residues, was found in Toprh cDNA sequence. An intron of 89 bp was identified when this cDNA clone was compared with the Toprh gene in the genome of T orientalis. The deduced amino acid sequence of Toprh shares 93.8, 93.1 and 69.1% identities with the prohibitins of T parva (from chromosome 1), T annulata (from chromosome 1), and Plasmodium falciparum, (from chromosome 10), respectively. By Western blot analysis, Toprh was found to be expressed in the piroplasm stage of the parasites.
• Cloning and sequence analysis of llama (lama glama) Th2 (IL-4, IL-10 and IL-13) cytokines

  R Odbileg, SI Lee, K Ohashi, M Onuma  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY  104-  (3-4)  145  -153  2005/04  [Not refereed][Not invited]
   

  This paper describes the cloning and sequence analysis of the cDNAs encoding the T helper (Th) 2 cytokines of llama including interleukin-4 (IL-4), IL-10 and IL-13. The cDNAs encoding for IL-4, IL-10 and IL-13 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-4, IL-10 and IL-13 were found to be 402, 537 and 411 bp in length, with open reading frames encoding 133, 178 or 136 amino acids, respectively. Homology analyses of nucleotide and deduced amino acid sequences of llama IL-4, IL-10 and IL-13 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla (pig, cattle) and Perissodactyla (horse). (c) 2004 Elsevier B.V. All rights reserved.
• Loop-mediated isothermal amplification for rapid detection of Newcastle disease virus

  HM Pham, C Nakajima, K Ohashi, M Onuma  JOURNAL OF CLINICAL MICROBIOLOGY  43-  (4)  1646  -1650  2005/04  [Not refereed][Not invited]
   

  We have evaluated a diagnostic system based on the loop-mediated isothermal amplification (LAMP) assay for the rapid, simple, and sensitive detection of Newcastle disease virus (NDV) directly from culture isolates as well as clinical samples. By using one set of specific primers targeting the fusion protein gene, the LAMP assay rapidly amplified the target gene within 2 h, requiring only a regular laboratory water bath or heat block for reaction. The results obtained from testing the genomes of 38 NDV strains, other different viruses, and clinical samples of experimentally infected chickens showed that LAMP was as sensitive and specific as nested PCR. All LAMP-positive samples were positive by nested PCR. The LAMP assay is faster than nested PCR, cost-effective, and easy to perform. Our results clearly demonstrate that the LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV infection.
• Multiple perineuriomas in chicken (Gallus gallus domesticus)

  T Toyoda, K Ochiai, K Ohashi, Y Tomioka, T Kimura, T Umemura  VETERINARY PATHOLOGY  42-  (2)  176  -183  2005/03  [Not refereed][Not invited]
   

  Intraneural perineurioma is an extremely rare condition characterized by perineurial cell proliferation within peripheral nerve (PN) sheaths. In the veterinary field, this entity has been reported only in a dog. We examined multiple enlargements of PNs in 11 chickens (Gallus gallus domesticus) (9 Japanese bantams and 2 specific pathogen-free White Leghorn), which were inoculated with an avian leukosis virus (ALV) causing so-called fowl glioma. All chickens clinically exhibited progressive leg paralysis. Lumbosacral plexus, brachial plexus, and/or spinal ganglion were commonly affected, and these nerves contained a diffuse proliferation of spindle cells arranged concentrically in characteristic onion bulb-like structures surrounded by residual axons and myelin sheaths. The spindle cells were immunohistochemically negative for S-100alpha/beta protein. Electron microscopy revealed that these cells were characterized by short bipolar cytoplasmic processes, occasional cytoplasmic pinocytotic vesicles, and discontinuous basal laminae. These features are consistent with those of intraneural perineurioma. Furthermore, the specific sequence of the ALV was detected in the PN lesions of 8/11 (73%) birds by polymerase chain reaction. These results indicate that the multiple intraneural perineuriomas of chicken may be associated with the ALV-A causing fowl glioma.
• Relationship of bovine viral diarrhea virus persistent infection to incidence,of diseases on dairy farms based on bulk tank milk test by RT-PCR

  T Kozasa, M Tajima, Yasutomi, I, K Sano, K Ohashi, M Onuma  VETERINARY MICROBIOLOGY  106-  (1-2)  41  -47  2005/03  [Not refereed][Not invited]
   

  The prevalence of bovine viral diarrhea virus (BVDV) in dairy herds in Hokkaido, Japan, was estimated by reverse transcription polymerase chain reaction (RT-PCR) using bulk tank milk samples. Sixteen out of 265 dairy herds were identified as BVDV positive, and at least one persistently infected (PI) cattle was recognized in each of the positive herds except for two herds of which, owners did not agree to examine individual cows. The proportion of positive herds with a history of BVDV PI was significantly higher than that with no history of BVDV PI (odds ratio (OR) 4.25, 95% confidence interval (0) 1.471-12.278, p = 0.004). The herds examined for BVDV were divided into two groups, high and low disease incidence groups based on the occurrence of diseases such as diarrhea, pneumonia or abortion in the past I year. The BVDV positive herds in the high disease incidence group were significantly more than that in the low disease incidence group (OR 2.92, CI 1.110-7.683, p = 0.024). It was observed that there were significantly (p = 0.008) more PI calves or heifers in farms of high disease incidence group than in farms of low disease incidence group. These results suggested that bulk tank milk test was available method for the detection of PI animals in dairy herds, and the existence of PI non-lactating cows in herd correlated with the incidence of diseases of the diarrhea or respiratory disorders. (c) 2004 Elsevier B.V. All rights reserved.
• Quantification of llama inflammatory cytokine mRNAs by real-time RT-PCR

  R Odbileg, S Konnai, T Usui, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  67-  (2)  195  -198  2005/02  [Not refereed][Not invited]
   

  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.
• Molecular cloning and sequence analysis of cDNAs encoding for Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus actins

  ID Vaz, S Imamura, C Nakajima, FC de Cardoso, CAS Ferreira, G Renard, A Masuda, K Ohashi, N Onuma  VETERINARY PARASITOLOGY  127-  (2)  147  -155  2005/01  [Not refereed][Not invited]
   

  The nucleotide and deduced amino acid sequences of the actins from ticks, Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus, have been determined. Nucleotide sequence analysis showed open reading frames of 1128-nucleotide-long encoding proteins of 376 amino acids with a predicted molecular weight of 41.82 kDa each. Comparison between the nucleic acid and deduced amino acid sequences as well as structural and phylogenetic analyses of thew genes confirmed the high similarity among actins from ticks in comparison to other species. (C) 2004 Elsevier B.V. All rights reserved.
• A serine protease inhibitor (serpin) from Haemaphysalis longicornis as an anti-tick vaccine

  S Imamura, ID Vaz, M Sugino, K Ohashi, M Onuma  VACCINE  23-  (10)  1301  -1311  2005/01  [Not refereed][Not invited]
   

  The application of anti-tick vaccine has been shown to be the most promising alternative tick control strategy compared to the current use of acaricides that suffer from a number of limitations. The success of this strategy is dependent on the cloning, and characterization of tick molecules involved in the mediation of tick central physiological roles. Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a cDNA with high similarity in the reactive center loop (RCL) to representative serpin, heparin cofactor If. We have named this novel gene as Haemaphysalis longicornis serpin-2 (HLS2). RT-PCR analysis showed that HLS2 mRNA transcripts are not expressed in salivary glands but in hemolymph by feeding ticks. HLS2 was not introduced into the bite site as measured by Western blot analysis. The activated partial thromboplastin time (APTT) and the thrombin inhibitory assay using recombinant HLS2 (rHLS2) demonstrated prolonged coagulation time and inhibition of thrombin activity. These results suggested that HLS2 is present only in hemolymph of the feeding ticks and the function of HLS2 is homeostasis in tick physiological compartment. Vaccination of rabbits with rHLS2 conferred protective immunity against ticks, resulting in 44.6 and 43.0% mortality in nymphal and adult ticks, respectively. These results show that rHLS2 could be an important candidate as a component of a cocktail anti-tick vaccine. (C) 2004 Elsevier Ltd. All rights reserved.
• Genetic variability in ESAG6 genes among Trypanosoma evansi isolates and in comparison to other Trypanozoon members

  WH Witola, N Sarataphan, N Inoue, K Ohashi, M Onuma  ACTA TROPICA  93-  (1)  63  -73  2005/01  [Not refereed][Not invited]
   

  Bloodstream trypanosomes take up iron needed for their propagation through the transferrin receptor that, in Trypanosoma brucei, is encoded by expression-site-associated genes (ESAGs), ESAG6 and 7 genes located in variant surface glycoprotein expression sites. ESAG6 and 7 genes in different expression sites have been shown to encode transferrin receptors with varying affinities for polymorphic transferrins. T brucei could cope with the different host transferrins by switching between expression sites. ESAG6- and 7-encoded transferrin receptor appear to be present in Trypanosoma evansi but the genes have not yet been characterized. In this study, we cloned and sequenced different members of ESAG6 genes in seven isolates of T evansi from geographically distinct localities in Thailand. We assessed the intra- and inter-species genetic variability in the transferrin receptor gene regions involved in transferrin binding and established that T evansi, like T brucei, has widely diverse ESAG6 genes. In addition, T evansi possess a clade of ESAG6 variants not observed in T brucei and different T evansi strains share at least two conserved variants. We further noted that T evansi possesses all the reported T equiperdum ESAG6 variants as a subset. Our findings depict a correlation between the genetic diversity in the transferrin-binding regions of ESAG6 genes with the broad host range of T evansi and T brucei compared to the narrow host range of Trypanosoma equiperdum. (C) 2004 Elsevier B.V. All rights reserved.
• Cloning and sequence analysis of llama cytokines related to cell-mediated immunity (vol 99, pg 1, 2004)

  R Odbileg, SI Lee, R Yoshida, KS Chang, K Ohashi, C Sugimoto, M Onuma  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY  102-  (1-2)  91  -+  2004/11  [Not refereed][Not invited]
   

  In order to characterize the T helper 1 (Th1) cytokines of llama, we have cloned several llama cytokine genes and compared them to those of other mammalian species. The cDNAs encoding for interleukin (IL)-2, interferon (IFN)gamma, IL-12p35 and IL-12p40 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-2 IFN-gamma, IL-12p35 and IL-12p40 were found to be 465, 501, 669 or 993 bp in length, with open reading frames encoding 154 166, 222 or 330 amino acids, respectively. Homology analyses of nucleotide and deduced amino sequences of llama IL-2, IFN-gamma, IL-12p35 and IL-12p40 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla, which includes pig and cattle. (C) 2004 Elsevier B.V. All rights reserved.
• 発現遺伝子解析によるダニ由来有用物質の探索

  中島千絵, 今村彩貴, 今内覚, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  138th-  65  2004/08/01  [Not refereed][Not invited]
  
• Molecular characterization of the nucleocapsid protein gene of Newcastle disease virus strains in Japan and development of a restriction enzyme-based rapid identifying method

  HM Pham, KS Chang, M Mase, K Ohashi, M Onuma  ARCHIVES OF VIROLOGY  149-  (8)  1559  -1569  2004/08  [Not refereed][Not invited]
   

  Nucleocapsid protein (NP) gene of Newcastle disease virus (NDV) strains mainly isolated in Japan from 1930 to 2001 was genetically characterized. By deduced amino acid sequence comparison, the N-terminal region (from 1 to 401 residues) of the NP protein was found to be highly conserved, while the C-terminal region was highly variable among the NDV isolates. A phylogenetic tree construct based on the nucleotide sequence of the complete NP gene revealed that the old (prior to 1970s) and the new (after 1980s) isolates could be classified into two major different groups, i.e., a group comprising virulent strains, and another group composed of avirulent strains. By restriction enzyme analysis using Pst I, none of the virulent strains were cleaved, while avirulent strains were cleaved. The results may be useful for simple primary screening test for differentiating NDV isolates.
• 見えてきたウイルス発癌 マレック病ウイルスによる潜伏感染・腫よう化におけるL‐meq遺伝子の役割

  大橋和彦, CHANG K‐S, LEE S‐I, 村田史郎, 高木道浩, 小沼操  獣医畜産新報  (996)  594  -595  2004/07/01  [Not refereed][Not invited]
  
• Cloning, expression and partial characterization of a Haemaphysalis longicornis and a Rhipicephalus appendiculatus glutathione S-transferase

  ID Vaz, S Imamura, K Ohashi, M Onuma  INSECT MOLECULAR BIOLOGY  13-  (3)  329  -335  2004/06  [Not refereed][Not invited]
   

  The ticks Haemaphysalis longicornis and Rhipicephalus appendiculatus are important parasites worldwide. The current method for control of cattle ticks involves the use of chemicals. Nevertheless, parasite resistance is an ever increasing global problem. Glutathione S-transferases (GSTs) play a central role in detoxication of xenobiotic and endogenous compounds. Several authors have noted that an increase in GST activity is associated with resistance to insecticides and acaricides. In the present study, we report the cloning and expression of GST cDNAs from H. longicornis and R. appendiculatus. In addition, we determine the effect of three acaricides (ethion, deltamethrin and diazinon) on the enzymatic activity of rGSTs.
• RNA-interference silencing of the adenosine transporter-1 gene in Trypanosoma evansi confers resistance to diminazene aceturate

  WH Witola, N Inoue, K Ohashi, M Onuma  EXPERIMENTAL PARASITOLOGY  107-  (1-2)  47  -57  2004/05  [Not refereed][Not invited]
   

  Drug resistance of trypanosomes is now a problem, but its underlying mechanisms are not fully understood. Cellular uptake of the major trypanocidal drugs is thought to occur through an adenosine transporter. The adenosine transporter-1 gene, TbAT1, encoding a P2-like nucleoside transporter has previously been cloned from Trypanosoma brucei brucei, and when expressed in yeast, it showed very similar substrate specificity to the P2-nucleoside transporter, but could not transport diamidines (pentamidine and diminazene). We have cloned and sequenced a similar gene (TevAT1) from Trypanosoma evansi and found it to have 99.7% identity to the TbAT1 gene. To elucidate the role of the TevAT1 gene on diamidine trypanocidal effect, we genetically engineered T evansi for conditional knock-out of the TevAT1 gene by RNA interference (RNAi). Induction of the RNAi resulted in 10-fold depletion of TevAT1 mRNA, with concomitantly significant resistance to diminazene aceturate (berenil). The induced parasites propagated normally and attained peak cell density at an in vitro concentration of berenil, 5.5-fold higher than the IC100 of the wild-type. TevAT1 knock-out had no effect on the trypanocidal activity of suramin and antrycide, but conferred some resistance to samorin. Our findings validate the significance of the TevAT1 adenosine transporter-1 gene in mediating the trypanocidal effect of diamidines in T evansi. Further, we show for the first time that RNAi gene silencing in T evansi can be induced using plasmids designed for T brucei. We also demonstrate the usefulness of real-time PCR in rapidly quantifying mRNA levels in trypanosomes. (C) 2004 Elsevier Inc. All rights reserved.
• Evidence for bovine immunodeficiency virus infection in cattle in Zambia

  S Meas, M Nakayama, T Usui, Y Nakazato, J Yasuda, K Ohashi, M Onuma  JAPANESE JOURNAL OF VETERINARY RESEARCH  52-  (1)  3  -8  2004/05  [Not refereed][Not invited]
   

  We report herein on the first evidence for the presence of bovine immunodeficiency virus (BIV) in Zambia. Serological surveillance of BIV and bovine leukemia virus (BLV) was conducted in traditional cattle herds in Zambia. Out of a total of 262 sera analyzed, 11.4% were found positive for anti-BIV p26 antibodies as determined by Western blot analysis, while 5.0% were positive for anti-BLV gp51 antibodies as detected by immunodiffusion test. Peripheral blood mononuclear cells from BIV seropositive cattle were found to have BIV-provirus DNA, as detected by nested polymerase chain reaction. A nucleotide sequence corresponding to a 298 bp fragment of the BIV pol gene was also analyzed. Amino acid sequences of these Zambian pol gene products showed 98.0 to 100% homology to the American strain BIV R29,97.0 to 99.0% to Japanese BIV isolates, and divergence ranged from 0.0 to 2.0% among Zambian BIV isolates.
• マレック病ウイルスによる潜伏感染・腫よう化におけるL‐meq遺伝子の役割

  大橋和彦, CHANG K‐S, LEE S‐I, 村田史郎, 高木道浩, 小沼操  日本獣医学会学術集会講演要旨集  137th-  86  2004/03/01  [Not refereed][Not invited]
  
• Genome sequence analysis of the avian retrovirus causing so-called fowl glioma and the promoter activity of the long terminal repeat

  Y Tomioka, K Ochiai, K Ohashi, E Ono, T Toyoda, T Kimura, T Umemura  JOURNAL OF GENERAL VIROLOGY  85-  (3)  647  -652  2004/03  [Not refereed][Not invited]
   

  So-called fowl glioma is a retroviral infectious disease caused by avian leukosis virus subgroup A (ALV-A). We determined the complete nucleotide sequence of the virus genome. The full-length sequence was consistent with a genetic organization typical of a replication-competent type C retrovirus lacking viral oncogenes. The coding sequences were well conserved with those of replication-competent viruses, but the 3' noncoding regions including LTR were most related to those of replication-defective sarcoma viruses. The U3 region of the LTR had a few deletions and several point mutations compared to that of other ALVs. The promoter activities of the LTRs of glioma-inducing ALV and ALV-A standard strain, RAV-1, were equivalent in chick embryo fibroblasts (CEF), while that of glioma-inducing ALV was significantly lower than that of RAV-1 in human astrocytic cells. These subtle differences of the promoter activity of the LTR may be related to the induction of glial neoplasm.
• Detecting the Neutralizing Antibody in Bulk-milk Whey To Investigate Prevalence of Infection with the Bovine Viral-diarrhea Virus

  KOZASA Takashi, TAJIMA Motoshi, SANO Kimihiro, YASUTOMI Ichiro, OHASHI Kazuhiko, ONUMA Misao  Journal of the Japan Veterinary Medical Association  57-  (11)  705  -709  2004  [Not refereed][Not invited]
   

  北海道の2地区196軒の酪農家からのバルク乳の乳清を用いたウィルス中和試験により、牛ウィルス性下痢ウィルス（BVDV）の浸潤状況を調査した。検査対象とした2地区は、バルク乳からBVDV遺伝子が検出されたA地区151軒と検出されなかったB地区45軒である。検査の結果、BVDV遺伝子検出の有無にかかわらず166軒（166/196＝84.7％）の酪農家の検体が16倍以上の中和抗体価を示した。また、そのうち疫学情報の入手が可能であった132軒中、64軒（48.5％）では過去にBVDV感染が摘発されたことがなく、ワクチン接種歴もなく、かつ自家生産牛のみの牛群であった。以上からこれらの地区には、感染経路は不明であるかBVDVが広く浸潤していることが明らかとなり、本感染症を牛群から清浄化するためには長期的な監視が必要であると考えられた。
• 臨床症状を指標とした牛ウイルス性下痢ウイルスの汚染状況調査

  日本獣医師会雑誌  57-  511  -514  2004  [Not refereed][Not invited]
  
• In ovo infection with an avian leukosis virus causing fowl glioma: viral distribution and pathogenesis

  Y Tomioka, K Ochiai, K Ohashi, T Kimura, T Umemura  AVIAN PATHOLOGY  32-  (6)  617  -624  2003/12  [Not refereed][Not invited]
   

  We have previously isolated an avian leukosis virus (ALV) from a chicken affected with so-called fowl glioma. A resistance-inducing factor test indicated that the isolate was classified into a subgroup A. The distribution and pathogenicity were investigated in C/O specific pathogen free chickens infected in ovo with this virus. Histologically, 11 of 12 (92%) infected birds had non-suppurative encephalitis and three birds (25%) showed the characteristic nodules of fowl glioma at 50 or 100 days of age. Non-suppurative myocarditis with matrix inclusions and atypical myocytes were also noted in nine (75%) of the birds and the ALV antigens were immunohistochemically detected in various general organs as well as the central nervous system and heart. The semi-quantitative determination of the proviral DNA and viral RNA supported the immunohistochemical results and indicated that the virus was likely to replicate especially in myocardial fibres. The isolated ALV failed to induce other neoplastic lesions in this line of chickens within the experimental period of 100 days, despite the broad tissue tropism throughout the body. These results confirmed that this virus was able to induce glioma in embryo-inoculated chickens.
• Protective effects of vaccination with bovine leukemia virus (BLV) tax DNA against BLV infection in sheep

  T Usui, S Konnai, S Tajima, S Watarai, Y Aida, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  65-  (11)  1201  -1205  2003/11  [Not refereed][Not invited]
   

  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.
• マレック病ウイルス感染鶏の羽材料からのmeq遺伝子の検出

  村田史郎, CHANG K‐S, LEE S‐I, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  136th-  135  2003/09/10  [Not refereed][Not invited]
  
• Production and characterization of monoclonal antibodies against midgut of ixodid tick, Haemaphysalis longicornis

  M Nakajima, M Kodama, H Yanase, T Iwanaga, A Mulenga, K Ohashi, M Onuma  VETERINARY PARASITOLOGY  115-  (4)  355  -363  2003/08  [Not refereed][Not invited]
   

  There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development. (C) 2003 Elsevier B.V. All rights reserved.
• The rapid quantitative analysis of bovine cytokine genes by real-time RT-PCR

  S Konnai, T Usui, K Ohashi, M Onuma  VETERINARY MICROBIOLOGY  94-  (4)  283  -294  2003/07  [Not refereed][Not invited]
   

  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.
• A serine proteinase inhibitor (serpin) from ixodid tick Haemaphysalis longicornis; cloning and preliminary assessment of its suitability as a candidate for a tick vaccine

  M Sugino, S Imamura, A Mulenga, M Nakajima, A Tsuda, K Ohashi, M Onuma  VACCINE  21-  (21-22)  2844  -2851  2003/06  [Not refereed][Not invited]
   

  Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a 1350 bp cDNA which encodes a 378 polypeptide with high sequence similarity to several known serpins. We have named this gene as Haemaphysalis longicornis serpin-1 (HLS1). Northern blotting and reverse transcription (RT)-PCR analysis of total RNA from unfed or partially fed whole ticks as well as dissected tick organs revealed that transcription of HLS1 mRNA was induced by blood meal feeding during the slow feeding phase (24-48 h post-attachment) only in the tick midguts. Vaccination of rabbits with recombinant HLS1 (rHLS1) expressed in Escherichia coli resulted. in 43.9 and 11.2% mortality of nymph and adult ticks which were fed on immunized rabbits. Polyclonal rabbit antibodies to tick saliva did not react with rHLS1, suggesting that native HLS1 was not secreted into the host during tick feeding. rHLS1 could be a potential candidate for a cocktail anti-tick vaccine. (C) 2003 Elsevier Science Ltd. All rights reserved.
• Evidence of bovine immunodeficiency virus in cattle in Turkey

  S Meas, Z Yilmaz, T Usui, S Torun, K Yesilbag, K Ohashi, M Onuma  JAPANESE JOURNAL OF VETERINARY RESEARCH  51-  (1)  3  -8  2003/05  [Not refereed][Not invited]
   

  A seroepidemiological study of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) infections was conducted in four different cattle herds in Turkey. A total of 300 blood samples were analyzed and 12.3% were found to be positive for anti-BIV p26 antibodies by Western blot analysis and 1.6% positive for anti-BLV gp51 antibodies by an immunodiffusion test. BIV infection was confirmed with the detection of BIV-provirus DNA using the nested polymerase chain reaction. This is the first evidence for the presence of BIV in cattle in Turkey.
• cDNA cloning of a Rab5 homologue in Babesia gibsoni

  JL Zhou, K Ohashi, M Yamasaki, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  65-  (4)  519  -521  2003/04  [Not refereed][Not invited]
   

  For studying protein trafficking in Babesia-infected erythrocyte, we describe the cloning of a Rab5, one of molecular marker for vesicle trafficking in eukaryotic cells, gene homologue in Babesia gibsoni (BgRab5). The full-length cDNA of BgRab5 is 1,020 bp long with an open reading frame encoding a protein of 220 amino acids. The deduced amino acid sequence of BgRab5 contained the highly conserved GTP-binding consensus sequence and shares about 40% homology with that of Rab5 from Plasmodium falciparum, Toxoplasma gondii, Dog, Lotus japonicusor, Oryza sativa. Northern blot analysis showed that the BgRab5 probe hybridized with a 1 kb band in total RNA from parasitized erythrocytes, that was consistent with the size of the BgRab5 full-length cDNA.
• Seroprevalence of bovine immunodeficiency virus and bovine leukemia virus in dairy and beef cattle in Hokkaido

  T Usui, S Meas, S Konnai, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  65-  (2)  287  -289  2003/02  [Not refereed][Not invited]
   

  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.
• Passive immunization with monoclonal antibodies: effects on Haemaphysalis longicornis tick infestation of BALB/c mice

  M Nakajima, H Yanase, T Iwanaga, M Kodama, K Ohashi, M Onuma  JAPANESE JOURNAL OF VETERINARY RESEARCH  50-  (4)  157  -163  2003/02  [Not refereed][Not invited]
   

  Tick vaccine development plays an important role in current tick control strategies. Previously, we have produced three different isotypes of monoclonal antibodies (mAbs) which recognized a midgut protein of adult Haemaphysalis longicornis. These mAbs, typed as IgG1, 2a, and 2b, reacted with a 76 kDa surface protein of midgut cells. We speculated that the 76 kDa protein may be an unknown antigen for a tick vaccine and the three mAbs may work as probes to clone the protein. In this study, to test whether these three isotypes have anti-tick effects and if so which works more effectively, we conducted passive immunization in BALB/c mice with each of the mAbs, and infested the mice with adult ticks. All isotypes significantly reduced the number of hatched larvae, compared to controls, however, no differences in the magnitude of the reduction were observed among the three.
• Diversity (polymorphism) of the meq gene in the attenuated Marek's disease virus (MDV) serotype 1 and MDV-transformed cell lines

  KS Chang, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  64-  (12)  1097  -1101  2002/12  [Not refereed][Not invited]
   

  The meq gene encoding a 339-amino-acid bZIP transactivator protein has been identified as a candidate oncogene of Marek's disease virus serotype I (MDV1). which induces malignant lymphomas in chickens. We have previously reported that, in addition to meq, L-meq, in which a 180-bp sequence is inserted into the region encoding the transactivation domain of meq, is also detected in chickens experimentally infected with MDV. To further analyze the diversity in meq, PCR was performed using a primer set which specifically amplify the proline-rich repeat (PRR) region in the transactivation domain of meq. In CV1988/R6, a vaccine strain of MDV1, and JM, an MDV I strain attenuated by prolonged passage in vitro, a major band of a 0.8 kb corresponding to L-meq as well as a minor band of 0.6 kb corresponding to meq was detected by PCR. Furthermore, extra 0.5- and 0.3-kb bands, corresponding to genes termed as short meq (S-meq). and very short meq (VS-meq), respectively, were also detected. These genes were also detected in MDV-transformed cell lines, MSB1 and MTB1. In Md5, an oncogenic MDV1, attenuated by prolonged passage in vitro, the 0.6-kb meq was consistently detected, and 0.5-kb S-meq was occasionally detected. This diversity in meq was due to the difference in the copy number of the PRR region: L-meq and meq contained 9 and 6 copies of PRR while 4 and 2 copies of PRR were present in S-meq and VS-meq, respectively. Thus, the meq gene is polymorphic in the attenuated MDV1 and the MDV-transformed cell lines, and gene products from different meq genes may have different functions from each other.
• Suppression of transcription activity of the MEQ protein of oncogenic Marek's disease virus serotype 1 (MDV1) by L-MEQ of non-oncogenic MDV1

  KS Chang, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  64-  (12)  1091  -1095  2002/12  [Not refereed][Not invited]
   

  Meq is one of the candidate oncogenes in the MDV1 genome. We previously reported a difference in the meq open reading frame (ORF) between oncogenic and non-oncogenic MDV1: L-meq. in which a 180-bp sequence is inserted into the meq ORF, is detected in non-oncogenic MDV1. To study the functions of a gene product of L-meq (L-MEQ), transactivation by L-MEQ was analyzed by dual luciferase assay using a reporter gene under the control of long (-1--873 bp) and short (-1--355 bp) meq promoter (LMP and SMP, respectively). LMP showed higher promoter function than SMP. L-MEQ transactivated the expression of the reporter gene, but less than MEQ did. In the presence of SNIP or the cytomegalovirus immediate-early promoter, the same or slightly higher transactivation was observed in cells cotransfected with both meq and L-meq than cells transfected only with meq. However, in the presence of LMP, lower transactivation was observed in cells cotransfected with both meq and L-meq than cells transfected only with meq, suggesting that L-MEQ can be a transrepressor. Replication of a vvMDV1 was enhanced in the cells with meq. Interestingly, however, replication of vvMDV1 was suppressed in the cells with L-meq or with both L-meq and meq, compared to untransfected cells. Thus, L-MEQ could suppress replication of vvMDV1 displaying the meq gene in coinfetced cells.
• Seroprevalence and molecular evidence for the presence of bovine immunodeficiency virus in Brazilian cattle (vol 50, pg 9, 2002)

  S Meas, J Ruas, NA Farias, T Usui, Y Teraoka, A Mulenga, KS Chang, A Masuda, CR Madruga, K Ohashi, M Onuma  JAPANESE JOURNAL OF VETERINARY RESEARCH  50-  (2-3)  145  -145  2002/11  [Not refereed][Not invited]
  
• Babesia gibsoni: molecular cloning and characterization of Rab6 and Rab11 homologues

  JL Zhou, A Mulenga, M Yamasaki, K Ohashi, Y Maede, M Onuma  EXPERIMENTAL PARASITOLOGY  101-  (4)  210  -214  2002/08  [Not refereed][Not invited]
   

  Members of the Rab subfamily of GTPases have been implicated as important components in vesicle trafficking in the eukaryotes, individual Rab proteins have a remarkable degree of specific subcellular localization. As a first step towards developing a set of compartment specific probes for studying protein trafficking in Babesia-infected erythrocyte, here we describe the cloning and characterization of Rab6 and Rab11 gene homologues in Babesia gibsoni (BgRab6 and BgRab11). The deduced amino acid sequence of both BgRab6 and BgRab11 contained the highly conserved GTP-binding consensus sequence and C-terminal cysteines. Northern blotting analysis of total RNA hybridized a 1.3 kb band on both BgRab6 and BgRab11 probed blots consistent with the expected size. Using a GTP-binding assay we demonstrated that Escherichia coli expressed recombinant BgRab6 and BgRab11 were able to bind GTP. BgRab6 and BgRab11 represent the first two molecular markers of B. gibsoni.
• The detection of the meq gene in chicken infected with Marek's disease virus serotype 1

  KS Chang, SI Lee, K Ohashi, A Ibrahim, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  64-  (5)  413  -417  2002/05  [Not refereed][Not invited]
   

  In the genome of strains of very virulent Marek's disease virus serotype 1 (vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF. termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CVI988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.
• Nonsuppurative myocarditis associated with so-called fowl glioma

  N Iwata, K Ochiai, K Hayashi, K Ohashi, T Umemura  JOURNAL OF VETERINARY MEDICAL SCIENCE  64-  (5)  395  -399  2002/05  [Not refereed][Not invited]
   

  C/O specific pathogen-free White Leghorn chickens were intracerebrally inoculated at one day of age with a brain homogenate of Japanese bantams (Gallus gallus domesticus) affected with fowl glioma. Histologically, six of eight inoculated chickens developed nonsuppurative meningoencephalitis in cerebrum and two of them had the characteristic lesions of fowl glioma. Hyperplastic lymphoid foci concomitantly developed in many organs of these birds, especially in the heart. Apart from these lymphoid foci, lymphocytic myocarditis was observed in all inoculated birds. Matrix inclusions were also noted in myocardial cells. Immunohistochemically, avian leukosis virus antigens were detected in reticular cells in the lymphoid foci, mesangial cells of the kidney, smooth muscle cells of the blood vessels, and myocardial cells, Of these tissues, the myocardium of all inoculated birds consistently showed strong reactivity for this antigens. The matrix inclusions were also positive for the antigens. These results suggest that the causal virus of fowl glioma has a high propensity to replicate, especially in myocardium and nonsuppurative myocarditis occurs associated with so-called fowl glioma.
• Avian retrovirus infection causes naturally occurring glioma: Isolation and transmission of a virus from so-called fowl glioma

  N Iwata, K Ochiai, K Hayashi, K Ohashi, T Umemura  AVIAN PATHOLOGY  31-  (2)  193  -199  2002/04  [Not refereed][Not invited]
   

  So-called fowl glioma is characterized by multiple nodular gliomatous growths associated with disseminated non-suppurative encephalitis. To investigate the possibility of the induction of the gliomatous lesions, chicks of Japanese bantams (Gallus gallus domesticus) and specific pathogen free chickens (C/O strain White Leghorn) were intracerebrally inoculated with a brain homogenate or culture supernatant from a bantam affected with fowl glioma. All bantams and 16 chickens (89%) in the inoculated groups showed non-suppurative encephalitis, and the 18 bantams (82%) and five chickens (28%) developed multiple nodules consisting of aggregations of astrocytes in the cerebrum. These astrocytes had avian leukosis virus (ALV) antigen. By Southern blot analysis, the ALV sequence was detected both in DNA prepared from the brains of the inoculated birds and in DNA from the inoculum. Ultrastructurally, tadpole-shaped particles, approximately 100 nm in diameter, were detected in the concentrated supernatant of the chicken embryo fibroblasts, and budding of the particles was noted. These results substantiated that fowl glioma of the bantams could be transmitted by intracerebral inoculation of the affected tissue and that the causal agent was an unidentified strain of ALV.
• Expression of biologically active human tumor necrosis factor-alpha in transgenic potato plant

  K Ohya, N Itchoda, K Ohashi, M Onuma, C Sugimoto, T Matsumura  JOURNAL OF INTERFERON AND CYTOKINE RESEARCH  22-  (3)  371  -378  2002/03  [Not refereed][Not invited]
   

  We report the successful insertion of the cDNA of human tumor necrosis factor-alpha (HuTNF-alpha) into the genome of potato plant species, Solanum tuberosum, using Agrobacterium tumefacience-mediated transformation. HuTNF-alpha is a known and essential cytokine mediating host defense against tumors and infectious diseases and an immunomodulating agent. To enhance the accumulation of foreign gene product expression in plant cells, the molecular design of the constructed HuTNF-alpha is presented. Transcription and translation of TNF-alpha in transformants were confirmed by Northern blot, RT-PCR, ELISA, and Western blot, respectively. Expression of the bioactive HuTNF-alpha in plant cells was confirmed by way of the cytotoxic effect of the extract obtained from the transformants against marine L929 cells. We think that the expression level of HuTNFalpha (15 mug/g potato plant tissue) obtained in the present study may be sufficient to induce responses/effects similar to those generated by TNF-alpha in human milk administered orally. We believe that the TNF-alpha expressed in edible potato plants has tremendous potential for clinical use in the areas of medicine and veterinary science. Its usefulness and applicability, therefore, need to be fully explored.
• Biological and pathological studies on Marek’s disease virus (strains Md5 and CVI988C/R6) using polymerase chain reaction (PCR).

  1st International Science Conference of Faculty of Veterinary Medicine  1  -13  2002  [Not refereed][Not invited]
  
• The L-meq gene detected in chicken cells infected with Marek's disease virus serotype 1.

  1st International Science Conference of Faculty of Veterinary Medicine  21  -26  2002  [Not refereed][Not invited]
  
• Vertical transmission of bovine leukemia virus and bovine immunodeficiency virus in dairy cattle herds

  S Meas, T Usui, K Ohashi, C Sugimoto, M Onuma  VETERINARY MICROBIOLOGY  84-  (3)  275  -282  2002/01  [Not refereed][Not invited]
   

  Vertical transmission of bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) was investigated in five dairy cattle herds in Hokkaido, where 36.1 and 17.0% of cattle were BLV and BIN seropositive, respectively, and 9.9% of dams were co-infected with both BIV and BLV. Twenty six cases of offspring born from dams infected with only BLV (17 cases) or with both BIN and BLV (9 cases) were examined for the presence of BLV and BIV before and after colostrum feeding by polymerase chain reaction (PCR) and syncytium assay. After birth, all calves were separated immediately from their dams. The offspring born from BLV-positive dams were BLV-negative before colostrum feeding, suggesting that no transplacental transmission had occurred. Thereafter, these offspring were fed colostrum or milk from their dams, but still remained BLV-negative. The other offspring born from BLV-positive dams were fed with BLV-negative colostrum, or with pasteurized BLV-positive colostrum. All these calves remained negative for BLV infection, suggesting that in utero transmission of BLV is negligible. In the case of offspring born from darns co-infected with BLV and BIV, calves were BIV-positive before colostrum feeding at I day after the birth, indicating in utero transmission of BIV. After colostrum feeding from their dams, newborn calves became BLV-positive. In addition, one calf was BLV-positive even before colostrum feeding. These results suggest that BIV can be transmitted to offspring in utero. and that BLV can be transmitted through colostrum or milk if dams are infected with both BIV and BLV. (C) 2002 Elsevier Science B.V. All rights reserved.
• 北海道宮島沼におけるマレック病感染マガン

  酪農大野生動物医学グループ, 富川 徹, 大橋 和彦, 小沼 操  Zoo and wildlife news = ズー・アンド・ワイルドライフニュース  (13)  28  -29  2001/12/01
• Survey of Theileria parasite infection in cattle in Cambodia and Vietnam using piroplasm surface protein gene-specific polymerase chain reaction

  M Inoue, D Van Nguyen, S Meas, K Ohashi, S Sen, C Sugimoto, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  63-  (10)  1155  -1157  2001/10  [Not refereed][Not invited]
   

  A survey of Theileria parasite infection in cattle in Cambodia and Vietnam was carried out by using allele-specific polymerase chain reaction. A total of 137 blood samples from draught animals in Cambodia and 40 blood samples from dairy cattle in Vietnam were analyzed. In Cambodia, 69 out of 137(50.4%) samples were PCR-positive containing mainly the Thai and the C type parasites. In Vietnam, 11(27.5%) samples were positive and all were of the Thai type parasite.
• Expression of two subtypes of human IFN-alpha in transgenic potato plants

  K Ohya, T Matsumura, K Ohashi, M Onuma, C Sugimoto  JOURNAL OF INTERFERON AND CYTOKINE RESEARCH  21-  (8)  595  -602  2001/08  [Not refereed][Not invited]
   

  Plant expression systems have advantages over other in vitro expression systems in terms of low production costs and low risk of contamination by animal viruses or bacterial endotoxins. In this study, cDNA encoding two subtypes of human interferon-alpha 2b and 8 (HuIFN-alpha 2b and HuIFN-alpha8) were introduced into potato plants (Solanum tuberosum) using Agrobacterium-mediated transformation. Transcription and translation of the inserted HuIFN-alpha cDNA were confirmed by Northern blot analysis and ELISA, respectively. Bioactivity of the products was assayed by inhibition of vesicular stomatitis virus (VSV) replication on a human amniotic cell line. However, because of the presence of substances in potato tissue extracts that were toxic to animal cells, successful demonstration of IFN bioactivity in the transformants; was achieved only after removal of such substances by dialysis. The maximum level of IFN activity in plant extracts was 560 IU/g of tissue. These results indicated that the HuIFN-alpha gene introduced into the potato plant was correctly translated and transcribed in plant cells. This report for the first time shows that biologically active animal cytokines with potential pharmaceutical applications could be expressed in transgenic potato plants.
• Tumor necrosis factor alpha and its receptors in experimentally bovine leukemia virus-infected sheep

  H Kabeya, A Fukuda, K Ohashi, C Sugimoto, M Onuma  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY  81-  (1-2)  129  -139  2001/08  [Not refereed][Not invited]
   

  To examine whether tumor necrosis factor alpha (TNF alpha) contributes to the pathogenesis of bovine leukemia virus (BLV) infection, the mRNA expression patterns of TNF alpha and its receptors, type I (TNF RI) and type 2 (TNF R2) were investigated. Sheep inoculated with BLV were divided into two groups: one was BLV-positive and the other BLV-negative based on the detection in peripheral blood mononuclear cells (PBMC). Expression of TNF R1 mRNA was down-regulated in PBMC from the BLV-positive compared to BLV-negative sheep. No difference was shown in the expression levels of TNF R2 mRNA between the two groups, Furthermore, proliferative responses of PBMC in the presence of TNF alpha were observed from the BLV-positive, but not BLV-negative sheep. Membrane-bound TNF alpha (mTNF alpha) is thought to be one of the ligands, inducing B-cell activation. Flow cytometric analysis demonstrated that the number of PBMC that were positive for mTNF alpha expression, was increased in the BLV-positive sheep. Thus, the expression of TNF alpha and its receptors may be closely associated with lymphocytosis induced by BLV. (C) 2001 Elsevier Science B.V, All rights reserved.
• cDNA cloning, characterization and vaccine effect analysis of Haemaphysalis longicornis tick saliva proteins

  A Tsuda, A Mulenga, C Sugimoto, M Nakajima, K Ohashi, M Onuma  VACCINE  19-  (30)  4287  -4296  2001/07  [Not refereed][Not invited]
   

  Immunological control of ticks is currently the only sustainable and practical alternative method to the current use of acaricides which has serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. We used a combination of immuno-screening of an adult tick cDNA library as well as the 3 and 5 rapid amplification of cDNA ends to clone two cDNAs, encoding tick saliva proteins from Haemaphysicalis longicornis. The two cDNAs herein named HL 34 and 35 are 1000 by each and encode polypeptides with 292 and 321 amino acid residues respectively. Northern blotting analysis of total RNA from ticks at different feeding stages revealed that expression of both HL 34 and HL35 mRNAs is induced during the slow feeding phase. We speculate that the functions of both genes are closely associated with blood feeding. Expression analysis by RT-PCR showed that both genes are expressed in other tick organs in addition to salivary glands. Recombinant HL 34 was successfully expressed in Escherichia coli and its suitability as a tick vaccine antigen was analyzed in rabbits. We propose that rHL34 could be a useful component of a cocktail tick vaccine. (C) 2001 Elsevier Science Ltd. All rights reserved.
• Characterization of two cDNAs encoding serine proteinases from the hard tick Haemaphysalis longicornis

  A Mulenga, C Sugimoto, G Ingram, K Ohashi, O Misao  INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY  31-  (8)  817  -825  2001/06  [Not refereed][Not invited]
   

  Host vaccination against tick infestation is at present the most practical and sustainable alternative tick control method to the current acaricide use which has serious limitations. However the success of this approach to control ticks depends upon the identification of target vaccine antigens. Members of the serine proteinase gene family may represent an interesting group of proteins to target as candidate antigens because of their involvement in regulation of many physiological functions and development processes in a wide range of organisms. We used RT-PCR with the 3' and 5' RACE to clone two cDNAs encoding full-length serine proteinases from the hard tick, Haemaphysalis longicornis. RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His(57) and Ser(195) conserved among most known serine proteinase. Gene specific primers designed from nucleotide sequences of the RT-PCR products were used to prime the 3' and 5' RACE. Southern blotting analysis showed that both HLSG-1 and -2 are single copy. The 2 cDNAs, HLSG-1 and -2 are 1.2 and 1.0 kb long in size with open reading frames encoding polypeptides with 37.7 and 31.2 kDa predicted molecular mass respectively. Northern blotting analysis of total RNA from unfed and partially fed whole ticks showed that the expression of mRNAs for both HLSG-1 and -2 was induced by blood feeding. Expression analysis by RT-PCR showed that both HLSG-1 and -2 are expressed in other tick organs in addition to salivary glands and midguts. The 6 serine proteinase consensus cyteine residues are well conseverd in both HLSG-1 and -2. We have discussed our findings with respect to tick vaccine development research. (C) 2001 Elsevier Science Ltd. All rights reserved.
• Heparin inhibits plaque formation by cell-free Marek's disease viruses in vitro

  SI Lee, K Ohashi, C Sugimoto, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  63-  (4)  427  -432  2001/04  [Not refereed][Not invited]
   

  The mechanisms of Marek's disease virus (MDV) entry to host cells have not yet been analyzed. Heparan sulfate (HS) on the cell surface serves as a receptor for several herpesviruses in mammalian species. in this study, we demonstrated that plaque formation by cell-free MDV is inhibited by the addition of soluble heparin to the cell culture. Moreover, pretreatment of susceptible cells, chicken embryo fibroblasts, with heparinase, partially reduced infectivity of the cell-free MDV. From these results, it was suggested that the MDV entry, at least in the case of cell-free MDV, is dependent on the presence of cell surface glycosaminoglycans, principally HS.
• Identification of new deleted forms of the p53 transcripts and their products in Marek's disease lymphoblastoid cell lines

  M Takagi, K Ohashia, T Takeda, Y Asada, Y Wakita, C Sugimoto, M Onuma, J Kawano, R Osawa, A Shimizu  CURRENT PROGRESS ON MAREK'S DISEASE RESEARCH  305  -312  2001  [Not refereed][Not invited]
   

  A 122-bp deletion form of the p53 transcript was found in Marek's disease (MD)-derived cell lines, MSB1 and MTB1. To determine whether this deletion is dependent on the cell cycle, the p53 transcripts expressed in three MSB1 subclones, MSB1-O, -clone 18 and -41C, and their gene products were analyzed by RT-PCR, nested-PCR and Western blotting. Throughout the cell cycle and even during apoptosis induced by actinomycin D, the deleted form of the p53 transcript was continuously expressed, and no difference was found in the expression pattern of the deleted form, suggesting that deletion in the p53 transcripts does not depend on the cell cycle. When apoptosis was induced, however, the p53 protein with a smaller size detected by anti-p53 monoclonal antibody was expressed in MSB1-O. Therefore, the translation of the smaller p53 protein from the deleted transcript may play a role in the initiation of apoptosis. In addition, two short forms of the p53 transcript, produced by the deletions in exon 8 and 10, and from exon 8 to 10, were further identified in MSB I subclones by nested-PCR. Thus, several kinds of deleted forms of the p53 transcripts are present in MD tumor cell lines though their roles in the tumorigenesis by MDV remain to be established.
• The kinetic changes of lymphocyte populations and cytokine profiles in chickens genetically susceptible and resistant to Marek's disease

  K Ohashi, Y Maeda, SI Lee, M Mizutani, C Sugimoto, M Onuma  CURRENT PROGRESS ON MAREK'S DISEASE RESEARCH  295  -301  2001  [Not refereed][Not invited]
   

  The differences in the kinetic changes of lymphocyte populations and cytokine profiles were studied between chickens genetically susceptible and resistant to Marek's disease (MD) during the early phase of MD virus (MDV) infection. A transient increase in the number of CD4(+) T cells was observed in both susceptible and resistant lines of chickens at 7 days post infection (pi), but no significant difference was shown in the degree of the increase between the two lines of chickens. Instead, higher titers of MDV were re-isolated from genetically susceptible than resistant chickens. Since the numbers of CD4(+) and CD8(+) T cells were constantly lower in the peripheral blood of the susceptible line of chickens after 10 days pi, the severity of this continuous depression may also be one of the factors determining the susceptibility to MD. The expression of cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, were also analyzed in both resistant and susceptible chickens by reverse-transcriptase polymerase chain reaction. Although the level of IFN-gamma expression was higher in susceptible than resistant chickens during the MDV infection, the relationship between the level of IFN-gamma expression and susceptibility to MD was not clarified in this study. In addition, there appeared to be little correlation between the time course of the change of lymphocyte populations and the expression levels of IL-2 after MDV infection in both resistant and susceptible chickens.
• Phylogenetic relationships of bovine immunodeficiency virus in cattle and buffaloes based on surface envelope gene sequences

  S Meas, K Ohashi, C Sugimoto, M Onuma  ARCHIVES OF VIROLOGY  146-  (5)  1037  -1045  2001  [Not refereed][Not invited]
   

  Isolates of bovine immunodeficiency virus (BIV) exhibit a striking genomic diversity, most of which are located in the viral envelope gene. Since this property of the BIV group of viruses may play an important role in the pathobiology of the virus, the surface envelope gene, particularly the conserved (C) 2, hypervariable (V) 1, V2 and C3 regions, of eleven different isolates from different environments with different bovine breeds naturally infected with BIV, including dairy cows in Japan, buffaloes in Pakistan and draught animals in Cambodia, were sequenced. When compared to the nucleotide sequence of American BIV isolates, all Asian BIV field isolates seem to be smaller, several base substitutions were observed in the V1 region, and deletions were also found in the V2 region of env gene in these samples. However, deduced amino acid sequences were not so different among isolates from different bovine breeds, suggesting that bovine susceptibility to BIV infection may not depend upon bovine breed or buffaloes. Moreover, phylogenetic analysis revealed that genotypes were distinct between Asian and American BIV isolates and these results also provide an information on the molecular epidemiology of naturally occurring BIV infection in cattle and buffaloes
• Insertion of a 178-BP sequence into the meq gene of Marek's disease virus serotype 1

  SI Lee, K Ohashi, M Takagi, KS Chang, C Sugimoto, M Onuma  CURRENT PROGRESS ON MAREK'S DISEASE RESEARCH  289  -294  2001  [Not refereed][Not invited]
   

  Serotype I strains of Marek's disease virus (MDV1), except attenuated vaccine strains, are known to cause lymphomas in visceral organs of infected chickens. We have previously found that a 178-bp sequence was inserted to the meq open reading frame (ORF) of strains CVI988/C and CVI988/R6, attenuated non-oncogenic MDV1 strains (9). To further determine if this insertion could be detected in other NMV1 strains, southern blot and nucleotide sequence analyses were done with total cellular DNA from MDV1-infected chicken embryo fibroblasts. The insertion of 178-bp sequence was detected in the meq ORF of strain JM, a mild MDV1 strain, and occurred at the exactly same position as observed in CVI988. This insertion was not detected in strain Md5 which had been passaged 17 times in vitro. The biological significance and function of this long meq gene is not yet elucidated, but this insertion is not CVI988-specific and can occur in oncogenic MDV1 strains.
• Seroprevalence of bovine immunodeficiency virus and bovine leukemia virus in draught animals in Cambodia

  S Meas, K Ohashi, S Tum, M Chhin, K Te, K Miura, C Sugimoto, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  62-  (7)  779  -781  2000/07  [Not refereed][Not invited]
   

  Since bovine immunodeficiency virus (BIV), known as bovine lentivirus, has been detected in dairy and beef cattle in various countries around the world, a prevalence study of antibodies to BIV and bovine leukemia virus (BLV) was conducted in draught animals in five provinces in Cambodia, where protozoan parasite infections were suspected in some animals. To clarify the status of draught animals including Haryana, Brahman, mixed-breed, local breed cattle and muscle water buffaloes, a total of 544 cattle and 42 buffaloes were tested, and 26.3 and 16.7%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting. There were 5.3% positive for anti-BLV antibodies detected by immunodiffusion test among the cattle, but no reactors among buffaloes and no dual infection for both BIV and BLV was determined in this study. Peripheral blood mononuclear cells from BIV-seropositive cattle were found to have BIV-provirus DNA, as detected by polymerase chain reaction and subsequent Southern blot hybridization. This is the First evidence for the presence of BIV and BLV infections in draught animals in tropical countries such as Cambodia. This wide distribution of BIV suggests its association with problems in animal health as reported worldwide, and that a primary BIV infection can predispose death of affected animals by other aggressive pathogens or stresses.
• Characterization of an 84 kDa protein inducing an immediate hypersensitivity reaction in rabbits sensitized to Haemaphysalis longicornis ticks

  A Mulenga, C Sugimoto, K Ohashi, M Onuma  BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE  1501-  (2-3)  219  -226  2000/06  [Not refereed][Not invited]
   

  An immunogenic 84 kDa protein was isolated and purified from whole tick extracts of Haemaphysalis longicornis larvae by a combination of ion exchange, reverse phase and hydrophobic interaction chromatographies. The protein, when injected intradermally into rabbits exposed to repeated tick feeding, induces an immediate cutaneous hypersensitivity reaction. It has been purified to homogeneity as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis and silver staining. Amino acid sequences for two peptides derived from proteolytic cleavage of p84 were scanned against known proteins on the SWISS-PROT database. A 7 residue motif, ISGWGNT present in one of the two peptides appeared conserved in both vertebrate and invertebrate trypsin-like serine proteinases, while another 7 amino acid motif, HVPAGQI present in the second peptide showed homology to an Escherichia coli ATP-binding protein. We have discussed our findings in relation to isolation and characterization of target antigens for tick vaccine candidates. (C) 2000 Elsevier Science B.V. All rights reserved.
• Infection of bovine immunodeficiency virus and bovine leukemia virus in water buffalo and cattle populations in Pakistan

  S Meas, J Seto, C Sugimoto, M Bakhsh, M Riaz, T Sato, K Naeem, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  62-  (3)  329  -331  2000/03  [Not refereed][Not invited]
   

  A survey of antibodies to bovine immunodeficiency virus (BIV) known as bovine lentivirus and bovine leukemia virus (BLV) was conducted with samples from water buffalo and cattle populations in Pakistan. A total of 370 water buffaloes and 76 cattle were tested, and 10.3% and 15.8%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting, while 0.8% of water buffaloes and no cattle were positive for anti-BLV antibodies determined by immunodiffusion test. BIV-seropositive water buffaloes and cattle were found to have BIV proviral DNA in the peripheral blood mononuclear cells determined by nested polymerase chain reaction. This is the first report of BIV infections in water buffaloes.
• Difference in the meq gene between oncogenic and attenuated strains of Marek's disease virus serotype 1

  SI Lee, M Takagi, K Ohashi, C Sugimoto, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  62-  (3)  287  -292  2000/03  [Not refereed][Not invited]
   

  Serotype 1 strains of Marek's disease virus (MDV1), except attenuated vaccine strains, are known to cause lymphomas in visceral organs of infected chickens. To know additional genetic differences between oncogenic and nononcogenic MDV1, polymerase chain reaction (PCR) was performed to amplify the meq gene of the viral genome. In addition to the 1,062-bp band including the native meq open reading frame (ORF), a 1.2-kb band was amplified from the DNA sample prepared from chick embryo fibroblast infected with an attenuated strain, CVI988, but not with oncogenic strains. Sequence analysis of the 1.2-kb band showed that a 178-bp sequence was inserted to the meg ORF of CVI988. This ORF could encode for the Meq protein with a different transactivator domain. Southern blot analysis also confirmed the insertion of the 178-bp sequence in the meg ORF of CVI988. This insertion of 178-bp sequence may explain the reason why CVI988 is not oncogenic.
• Differences in the kinetic changes of lymphocyte populations and cytokine profiles between chickens genetically susceptible and resistant to Marek's disease.

  Proceedings of the International Veterinary Cytokine and Vaccine Conference  318  -321  2000  [Not refereed][Not invited]
  
• Involovement of tumor necrosis factor a in the pathogenesis of bovine leukemia virus infection.

  Proceedings of the International Veterinary Cytokine and Vaccine Conference  155  -158  2000  [Not refereed][Not invited]
  
• Expression of biologically active molecules in transgenic potatoes.

  Proceedings of the International Veterinary Cytokine and Vaccine Conference  116  -119  2000  [Not refereed][Not invited]
  
• Tick saliva proteins interacting with the host immune response factors.

  Proceedings of the International Veterinary Cytokine and Vaccine Conference  93  -96  2000  [Not refereed][Not invited]
  
• Electron microscopic and immunohistochemical localization of Marek's disease (MD) herpesvirus particles in MD skin lymphomas

  KO Cho, K Ohashi, M Onuma  VETERINARY PATHOLOGY  36-  (4)  314  -320  1999/07  [Not refereed][Not invited]
   

  Skin lymphomas induced in 11 specific-pathogen-free chickens by inoculation at 1 day of age with Marek's disease virus (MDV) were biopsied weekly and examined by electron microscopy and immunohistochemistry. In the sequentially biopsied lymphomas, immature MDV particles (abortive replication) were found only in the nuclei of necrotic lymphoblasts within necrotizing neoplasms. The necrotizing lymphomas were observed in two of the 11 experimental birds and were associated with prominent vascular endothelial cell injury, including fibrinoid necrosis of blood vessels. Nonnecrotizing lymphomas biopsied sequentially from the 11 experimental birds did not contain virus particles of any kind in the lymphoblasts and had no distinct vascular lesions. Immunohistochemically, MDV early antigen (pp38), but not late antigens (glycoproteins B and C), was detected only in the necrotizing lymphomas. These findings indicate that abortive MDV replication mainly occurred in necrotic lymphoblasts, which might have been induced by ischemia.
• Evidence of neoplastic nature and viral aetiology of so-called fowl glioma

  K Ochiai, K Ohashi, T Mukai, T Kimura, T Umemura, C Itakura  VETERINARY RECORD  145-  (3)  79  -81  1999/07  [Not refereed][Not invited]
  
• Up-regulation of tumor necrosis factor alpha mRNA is associated with bovine-leukemia virus (BLV) elimination in the early phase of infection

  H Kabeya, K Ohashi, N Oyunbileg, Y Nagaoka, Y Aida, C Sugimoto, Y Yokomizo, M Onuma  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY  68-  (2-4)  255  -265  1999/05  [Not refereed][Not invited]
   

  Protective immune responses were analyzed in eight sheep vaccinated with BLV envelope peptides and experimentally infected with bovine-leukemia virus (BLV). Five of eight peptide-immunized sheep showed a high T-cell proliferative response to the BLV peptides and all of these were protected from the infection. The other three peptide-immunized sheep showed no T-cell proliferative responses to any BLV antigens similar to control sheep, though they also exhibited resistance to BLV challenge. To investigate other mechanisms which suppress BLV expansion in these non-responding sheep, we measured the levels of the cytokine expressions before, and after, BLV challenge using competitive reverse-transcriptase polymerase chain-reaction systems. It was revealed that the expression of tumor necrosis factor alpha (TNF alpha) was higher in BLV-resistant sheep than in BLV-susceptible sheep. Thus, TNF alpha expression rather than specific T-cell activity may play an important role in the protective mechanism against BLV infection, at least during the primary viremia phase. (C) 1999 Elsevier Science B.V. All rights reserved.
• Characterization of immune responses caused by bovine leukemia virus envelope peptides in sheep

  H Kabeya, K Ohashi, C Sugimoto, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  61-  (5)  475  -480  1999/05  [Not refereed][Not invited]
   

  To study the immunomodulative activity caused by bovine leukemia virus envelope (BLV Env) peptide, sheep were immunized with two kinds of Th-epitope peptides, peptide 98 (BLV Env 98-117), and 61 (BLV Env 61-78). Four of eight immunized sheep showed specific proliferative responses against both of the peptide stimulations. To characterize the cells responding to the peptides, peptide-specific cells were established From the responding sheep by the continuous stimulation of peripheral blood mononuclear cells (PBMCs) with either peptide 98 or 61 in vitro. The peptide 98-specific cells consisted of CD4-positive cells, whereas the peptide 61-specific cells consisted of CD8-positive cells and MHC class II-positive cells. In addition, cytokine profile analysis indicated that the peptide 98-stimulated cells expressed IFN-gamma but not IL-10, although the peptide 61-stimulated cells expressed IL-10 but not IFN-gamma. These results show that BLV envelope peptides 98 and 61 can modulate immune responses of sheep lymphocytes in different ways and may contribute to the pathogenesis of BLV infection.
• Molecular cloning of two Haemaphysalis longicornis cathepsin L-like cysteine proteinase genes

  A Mulenga, C Sugimoto, G Ingram, K Ohashi, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  61-  (5)  497  -502  1999/05  [Not refereed][Not invited]
   

  Immunological protection of mammalian hosts against tick infestation has been proposed as the most sustainable alternative tick control method to the current use of acaricides which has several limitations. The success of this method is dependent on the identification of key molecules for use as tick vaccine antigens. Proteolytic enzymes are involved in a wide range of cellular processes in eukaryotes such as development regulation and nutrition, thus they can be considered as good target antigens for a tick vaccine. In the present study we used primers designed based on the consensus amino acid motifs flanking the conserved active sites C-25 and N-175 present in all papain-like cysteine proteinases to amplify by polymerase chain reaction, sequence and characterize two Haemaphysalis longicornis tick cysteine proteinase genes. Based on the nucleotide and deduced amino acid sequences, both genes were identified as members of the cysteine proteinase gene family by presence in their sequences of consensus motifs flanking the conserved active sites C-25, H-150 and N-175 that are present in all papain-like cysteine proteinases. Both genes are about 1.2 kb in size and show high sequence homology predominantly to invertebrate cathepsin L-like cysteine proteinases.
• Seroprevalence of bovine immunodeficiency virus in dairy and beef cattle herds in Korea

  KO Cho, S Meas, NY Park, YH Kim, YK Lim, D Endoh, SI Lee, K Ohashi, C Sugimoto, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  61-  (5)  549  -551  1999/05  [Not refereed][Not invited]
   

  Infection of bovine immunodeficiency virus (BIV), a lentivirus, is thought to sporadically occur throughout the world, but seroepidemiological surveys concerning the incidence of BIV are limited and have not been undertaken in Korea. A total of 266 sera from different twenty dairy (Holstein) and twenty-six Korean native beef (Hanwoo) farms of the south-western part of Korea was analyzed for the presence of anti-BIV antibodies by Western blotting. Thirty five percent and 33% of dairy and beef cattle, respectively, were BIV-seropositive. By nested polymerase chain reaction, it was confirmed that these seropositive cows had provirus in the peripheral blood mononuclear cells. To demonstrate the correlation with BIV and bovine leukemia virus (BLV) infection, these sera were also analyzed for anti-BLV antibodies by immunodiffusion test, resulting in high prevalence of BLV infection but relatively a few dual infections. We report herein the first serological detection of antibodies to BIV in Korea.
• Molecular characterization of a Haemaphysalis longicornis tick salivary gland-associated 29-kilodalton protein and its effect as a vaccine against tick infestation in rabbits

  A Mulenga, C Sugimoto, Y Sako, K Ohashi, A Musoke, M Shubash, M Onuma  INFECTION AND IMMUNITY  67-  (4)  1652  -1658  1999/04  [Not refereed][Not invited]
   

  The use of tick vaccines in mammalian hosts has been shown to be the most promising alternative tick control method to current use of acaricides, which suffers from a number of limitations. However, the success of this method is dependent on the identification, cloning, and in vitro expression of tick molecules involved in the mediation of key physiological roles with respect to the biological success of a tick as a vector and pest. We have sequenced and characterized a Haemaphysalis longicornis tick salivary gland-associated cDNA coding for a 29-kDa extracellular matrix-like protein. This protein is expressed in both unfed and fed immature and mature H. longicornis ticks. The predicted amino acid sequence of p29 shows high homology to sequences of some known extracellular matrix like-proteins with the structural conservation similar to all known collagen proteins. Immunization with the recombinant p29 conferred a significant protective immunity in rabbits, resulting in reduced engorgement weight for adult ticks and up to 40 and 56% mortality in larvae and nymphs that fed on the immunized rabbits. We speculate that this protein is associated with formation of tick cement, a chemical compound that enables the tick to remain attached to the host, and suggest a role for p29 as a candidate tick vaccine molecule for the control of ticks. We have discussed our findings with respect to the search of tick molecules for vaccine candidates.
• Expression of bcl-2 and bcl-x genes in lymphocytes and tumor cell lines derived from MDV-infected chickens

  K Ohashi, T Morimura, M Takagi, SI Lee, KO Cho, H Takahashi, Y Maeda, C Sugimoto, M Onuma  ACTA VIROLOGICA  43-  (2-3)  128  -132  1999/04  [Not refereed][Not invited]
   

  To characterize the molecular events involved in both apoptosis and transformation process induced by Marek's disease virus (MDV), the expressions of the bcl-2 and bcl-x genes, ones of the dominant apoptosis-regulating genes, in Marek's disease (MD) tumor cell lines and cells prepared from MDV-infected chickens were analyzed. The expression of bcl-2 was down-regulated in both CD4(+) and CD8(+) T cells prepared from MDV-infected chickens at 3 weeks p.i. No bcl-2 transcript was detected in MD tumor-derived MSB1 and MTB1 cell lines, which had been established from primary MD tumors. On the other hand, the bcl-xL transcript whose product can also inhibit apoptosis was expressed in cell lines derived from MD. By the treatment with phorbol 12-myristate 13-acetate (PMA) and ionomycin, normal CD4(+) T cells were induced to express bcl-xS which can promote apoptosis, while bcl-xL was constitutively expressed in MD cell lines. Our results suggest that bcl-xL rather than bcl-2 might play an important role in the transformation process by MDV.
• Bovine leukaemia virus envelope peptides cause immunomodulation in BALB/c mice

  H Kabeya, K Ohashi, C Sugimoto, M Onuma  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY  68-  (1)  39  -48  1999/03  [Not refereed][Not invited]
   

  Immunomodulatory activity of two bovine leukaemia virus envelope (BLVEnv) derived peptides were examined in BALB/c mice. One is a peptide homologous to CKS-17 which is known as a 17-amino acid peptide derived from p15E of feline leukaemia virus (CKS-17/BLV), and the other is an 18-amino acid synthetic peptide of BLV Env 61-78 (pep61). Priming with CKS-17/BLV in vitro, as well as CKS-17, significantly suppressed the mitogen-induced proliferative responses of spleen cells in naive BALB/c mice. In addition, priming of spleen cells with pep61 in vitro and in vivo resulted in suppression of lipopolysaccaride-induced B-cell proliferative response. This suppression was partially due to the basic amino acid sequence in the peptide because if the pep61-derived peptide lacking Arg was used, this inhibitory activity was partially restored. In contrast, pep61 enhanced both concanavalin A-stimulated proliferative response and IL-2 production. These findings showed that pep61 may contribute to the modification of the host immune responses in the course of BLV infection. (C) 1999 Elsevier Science B.V. ALI rights reserved.
• Anti-viral and anti-tumor effects induced by an attenuated Marek's disease virus in CD4-or CD8-deficient chickens

  T Morimura, KO Cho, Y Kudo, Y Hiramoto, K Ohashi, M Hattori, C Sugimoto, M Onuma  ARCHIVES OF VIROLOGY  144-  (9)  1809  -1818  1999  [Not refereed][Not invited]
   

  By vaccination with an attenuated Marek's disease virus (MDV), strain CVI988, chickens ape protected from the development of T cell lymphoma caused by an oncogenic MDV. To clarify the role of T lymphocyte subsets in the protection mechanisms of this vaccine, vaccinated chickens were depleted of T cell subsets by neonatal thymectomy and injections of monoclonal antibodies specific to chicken CD4 and CD8 molecules, and then challenged with an oncogenic MDV, strain Md5. The MDV titers rescued from CD8(+) T cells, which are the main targets for latent infection and subsequent transformation by MDV, was much higher in the CD8-deficient vaccinated chickens than in untreated vaccinated chickens at the early stage of the latent phase. However, the neonatal vaccination prevented lymphoma formation by strain Md5 even in either CD4(+) or CD8(+) T cell-depleted chickens. These results suggest that specific CD8(+) T cell responses induced by the MD vaccine play a crucial role in the prevention of MDV infection during the latent phase, but may not be essential for the prevention of lymphoma formation by an oncogenic MDV.
• Re-isolation of Marek's disease virus from T cell subsets of vaccinated and non-vaccinated chickens

  SI Lee, K Ohashi, T Morimura, C Sugimoto, M Onuma  ARCHIVES OF VIROLOGY  144-  (1)  45  -54  1999  [Not refereed][Not invited]
   

  To know the effect of Marek's disease (MD) vaccines, we analyzed the distribution of MD virus (MDV) among T cell subsets from chickens vaccinated or non-vaccinated with MD vaccine and subsequently challenged with a virulent MDV. The challenged MDV was reisolated preferentially from CD4(+) T cells, and the average titers of challenged MDV rescued were significantly lower in vaccinated chickens compared to that of non-vaccinated chickens. In addition, it was also shown that different serotypes of MDV, CVI988 and SB-1, have remarkable difference in recovery rates of viruses from CD4(+) and CD8(+) T cells, though both CVI988 and SB-1 can reduce the infection rates of virulent MDV to splenocytes.
• Seroprevalence and field isolation of bovine immunodeficiency virus

  S Meas, H Kabeya, S Yoshihara, K Ohashi, S Matsuki, Y Mikami, C Sugimoto, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  60-  (11)  1195  -1202  1998/11  [Not refereed][Not invited]
   

  A seroprevalence study of bovine lentivirus, known as bovine immunodeficiency virus (BIV), was conducted in 12 different dairy herds in Hokkaido, where some herds were a high prevalence of bovine leukemia virus (BLV) infection. Amongst 611 cattle, 28.6% of cattle were BLV-seropositive, and 11.7% of cattle were seropositive for BIV, while 4.2% of cattle were seropositive for both BIV and BLV. For the isolation of BIV, 19 samples of peripheral blood mononuclear cells (PBMC) and one sample of milk-derived leukocytes were prepared from BIV-seropositive cows. These PBMC and leukocyte preparations were then co-cultivated with cc81 cells, a cat cell line transformed by mouse sarcoma virus. BIV was isolated from 17 PBMC and one milk-derived leukocyte samples. The isolated viruses showed slow replication and syncytia formation. Major core antigen, p26 from these isolates were reacted with anti-BIV (American isolate R-29) serum. In addition, proviral DNA was detected in blood and milk samples by nested polymerase chain reaction and subsequent Southern blot hybridization. Nucleotide sequence analysis of the amplified pol gene products showed its 99.0 to 99.7% homology to that of BIV R-29. These results indicate that the Japanese BIV isolates appear to be antigenically and genetically similar to the American R-29. Since BIV was isolated from milk samples, BIV could possibly be transmitted through milk. This is the first report of BIV isolation in Japan.
• Analysis of tumor suppressor gene p53 in chicken lymphoblastoid tumor cell lines and field tumors

  M Takagi, K Ohashi, T Morimura, C Sugimoto, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  60-  (8)  923  -929  1998/08  [Not refereed][Not invited]
   

  To determine whether there is any abnormalities of the p53 gene in chicken lymphoblastoid tumor cell lines derived from Mareks disease (MD), lymphoid leukosis, reticuloendotheliosis, and field tumors, some portions of p53 cDNA corresponding to core and C-terminal domains (nucleotide positions 277-1104 in the p53 open reading frame (ORF)) were sequenced. Several mutations were identified in both cell lines and field tumors. However, none of these mutations is localized at the "hot spot", which has been reported as the site for transformation-activating mutations. Moreover, partial cDNA clones with a 122-bp deletion in the p53 ORF were identified in two cell lines, MSB1 and MTB1 derived from MD tumors. Southern blot analysis showed that no deletion occurred in the genome of p53 in MSB1, indicating that deletion occurred at the transcriptional level. This deletion could cause a frame shift of the encoding p53 protein, possibly resulting in the generation of a functionally different p53 protein. However, we confirmed that p53 mRNA without deletion is also present in each of these cell lines. These mutations of the p53 gene and deletion in the p53 transcript may be ones of molecular changes specific to the transformation induced by MD virus.
• Cytology of feather pulp lesions from Marek's disease (MD) virus-infected chickens and its application for diagnosis and prediction of MD

  KO Cho, NY Park, D Endoh, K Ohashi, C Sugimoto, C Itakura, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  60-  (7)  843  -847  1998/07  [Not refereed][Not invited]
   

  Cytological changes of feather pulp lesions (FPL) sampled chronologically from. the same specific-pathogen free chickens inoculated with Marek's disease virus serotype 1 (MDV) were examined, comparing with their histological changes. The birds having Marek's disease (MD) lymphomas or nerve lesions exhibited the characteristic lesion changes on the cytological smears of FPL; the initial non-suppurative inflammatory to the late lymphomatous FPL. The birds having neither the MD visceral lymphomas nor the nerve lesions manifested only non-suppurative inflammatory FPL on the cytological smears throughout the experimental periods. Histological evaluation of FPL sampled from the same birds confirmed as above mentioned cytological results. From these results, the cytological evaluation of FPL proved to be an effective diagnostic and prognostic tool in foreseeing MD incidence.
• Epitope mapping of bovine leukemia virus transactivator protein tax

  N Sakakibara, H Kabeya, K Ohashi, C Sugimoto, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  60-  (5)  599  -605  1998/05  [Not refereed][Not invited]
   

  The immunogenicity of the bovine leukemia virus (BLV) transactivator protein (tax) was studied by mapping its B-cell and T-cell epitopes. Peptides (18 to 20-mer) overlapping by 10 amino acids, spanning whole amino acid sequence of BLVtax were synthesized. Reconbinant BLV tax protein was used to immunize two different strains of mice, C57BL/6 and BALB/c. B-cell and T-cell epitopes of recombinant BLVtax protein was determined by screening all the 30 synthetic peptides, against immune serum in ELISA for antibody reactivity, and against immune spleen cells in lymphocyte proliferation assay for T-cell stimulation. Peptides with amino acids at position 111-130 and 131-150 were T-cell epitopes for C57BL/6 and BALB/c mice immune cells, respectively. B-cell epitope was mapped to amino acid sequence at 261-280 in both strains of mice. These results imply that BLVtax protein contains some of BLV-immunodominant epitopes and this information may be applied for designing an effective peptide vaccine capable of inducing neutralizing antibodies as well as cellular immunity.
• Expression and characterization of dog CYP2D15 using Baculovirus expression system

  T Tasaki, A Nakamura, S Itoh, K Ohashi, Y Yamamoto, M Masuda, H Iwata, A Kazusaka, T Kamataki, S Fujita  JOURNAL OF BIOCHEMISTRY  123-  (1)  162  -168  1998/01  [Not refereed][Not invited]
   

  Dog CYP2D15 was expressed in Sf9 cells with a recombinant baculovirus. Infection of Sf9 insect cells with a recombinant dog CYP2D15-virus resulted in the expression of a protein which cross-reacted with a polyclonal antibody against a dog CYP2D15-specific peptide, The difference spectrum of CO-complex of reduced P450 of the infected cell microsomes had a maximal absorbance at 449 nm, The specific content of P450 was calculated to be 0.56 nmol/mg of Sf9 cell microsomal protein, Although the expressed dog CYP2D15 showed high catalytic activity for the hydroxylations of bunitrolol and imipramine at low substrate concentration (10 mu M), the catalytic activity for that of debrisoquine (50 mu M) was extremely low as compared with that of CYP2D from other species, Dog liver microsomes also showed bunitrolol and imipramine hydroxylase activities, but not debrisoquine hydroxylase activity at the same substrate concentrations, In addition, the expressed CYP2D showed high catalytic activity for imipramine N-demethylation, Thus, our study reveals that the expressed dog CYP2D15 engages in high catalytic activity and has a unique substrate specificity from other CYP2D subfamilies, Western blot analysis suggested that the dog CYP2D15 contents were less than 4% of the total liver P450 content, assuming that 100% of expressed CYP2D15 incorporated heme.
• Laboratory of Infectious Diseases (An Introduction of the four Departments composing the Graduate Scholl of Veterinary Medicine, Hokkaido University (2))

  ONUMA Misao, SUGIMOTO Chihiro, OHASHI Kazuhiko  Japanese journal of veterinary research  45-  (3)  172  -173  1997/11/28
• マレック病ウイルスによる腫瘍化とアポトーシス 相反する現象に発癌遺伝子 bcl-2 ファミリーが関与

  大橋 和彦, 守村 敏史, 高木 道浩, 杉本 千尋, 小沼 操  化学と生物  35-  (11)  748  -750  1997/11/25  [Not refereed][Not invited]
  
• Apoptosis in peripheral CD4＋ T cells and thymocytes by Marek's disease virus-infection.

  Leukemia  11 Supplement 3-  206  -208  1997  [Not refereed][Not invited]
  
• A hypervariable region in VP1 of chicken infectious anemia virus mediates rate of spread and cell tropism in tissue culture

  RW Renshaw, C Soine, T Weinkle, PH OConnell, K Ohashi, S Watson, B Lucio, S Harrington, KA Schat  JOURNAL OF VIROLOGY  70-  (12)  8872  -8878  1996/12  [Not refereed][Not invited]
   

  Chicken infectious anemia virus (CIAV) is a unique infectious agent with an amino acid composition that has been found to be remarkably conserved even in isolates from different parts of the world, We have characterized field isolates of CIAV which vary significantly in terms of their abilities to replicate in culture, demonstrating a biological difference between isolates, Two sublines of MDCC-MSB1 cells that differ in their abilities to support CIAV mere identified. In the MSB1(S) subline the CIA-1 isolate of CIAV was found to be less cytopathogenic than the prototype Cux-1(C) isolate; the MSB1(L) subline, which supports Cux-1(C) replication, was found to be nonpermissive for CIA-1. Alignments of the VP1 sequences of previously examined isolates with those of the field isolates CIA-I and L-028 and the culture-adapted ConnB isolate revealed a previously unreported hypervariable region spanning amino acid positions 139 to 151. Chimeras of Cux-1 (C) and CIA-1 mere constructed to examine the potential for this region to affect cytopathogenicity, Transfer of a 316-bp region of Cus-1(C) open reading frame 1 into CIA-1 produced a virus with a cytopathogenic profile typical of Cux-1(C), indicating that one or both of the amino acid differences at positions 139 and 144 affect the rate of replication or the spread of infection. Transfection experiments with additional chimeras indicated that the inability of CIA-1 to replicate in MSB1(L) cells is mediated by a larger region of the genome which contains the hypervariable region in addition to upstream amino acid differences, Analysis of chimeras excluding the entire region of open reading frame 1 suggested the presence of a secondary mediator in the progression of infection in culture that R as localized to a region containing a single nucleotide difference which results in amino acid differences in both VP2 (V-153) and the nuclear localization signal of VP3 (C-118). Immunofluorescence assays indicated an increased cytoplasmic distribution of VP3 and a general lack of VP3-associated apoptatic bodies in infections of CIA-1 and chimeras containing V-153 or C-118, as opposed to a primarily nuclear distribution and association with well-formed apoptotic bodies in Cux-1(C)-infected cells.
• Immunomodulative effects of bovine immunodeficiency-like virus (BIV)-infection and mixed infection of BIV and bovine leukemia virus on sheep

  N Hirai, H Kabeya, K Ohashi, C Sugimoto, M Onuma  JAPANESE JOURNAL OF VETERINARY RESEARCH  44-  (3)  153  -163  1996/12  [Not refereed][Not invited]
   

  Experimental bovine immunodeficiency-like virus (BIV)-infection and mixed infection of BIV and bovine leukemia Virus (BLV) were performed on sheep. BIV proviral DNA and anti-BIV antibodies were persistently detected in all BIV-inoculated sheep. A slight increase in lymphocyte counts was observed in BIV-infected sheep, but the percentages of CD4(+) and CD8(+) cells in sheep peripheral blood mononuclear cells (PBMCs) were not significantly changed. A transient decrease in lymphocyte blastogenic response to concanavalin A was observed in two of three BIV-infected sheep at 3-6 months after inoculation. From 6 months after BLV-inoculation to sheep which were previously infected with BIV, the numbers of lymphocytes expressing a tumor-associated antigen (TAA) of bovine leukosis were increased compared to those of a sheep inoculated with BLV alone. The BLV titers in PBMCs and the antibody titers against BLV from sheep infected with both BIV and BLV were higher than those of a sheep inoculated with BLV alone.
• An effective peptide vaccine to eliminate bovine leukaemia virus (BLV) infested cells in carrier sheep

  H Kabeya, K Ohashi, K Ohishi, C Sugimoto, H Amanuma, M Onuma  VACCINE  14-  (12)  1118  -1122  1996/08  [Not refereed][Not invited]
   

  Protective effects of the gp51 of bovine leukaemia virus (BLV) expressed by a recombinant baculovirus (rgp51) and synthetic multiple antigenic peptides (MAP) of T-helper, T-cytotoxic, and B-cell epitopes of gp51 were investigated against BLV challenge. Two and three sheep were immunized with rgp51 and a mixture of peptides with Freund's complete adjuvant, respectively. BLV was detected from all the immunized sheep at 2 weeks and showed peak levels at 4 weeks after the challenge. However, in two sheep immunized with the mixed peptides, the titer of BLV gradually decreased and one sheep eliminated BLV completely at 28 weeks after the challenge. These two sheep showed higher lymphocyte proliferative responses against the immunized peptides than the other sheep, One of the sheep also showed the specific cytotoxic lymphocyte activity against the BLVgp51-expressing target in vitro. These results suggest the possibility of the peptide vaccine for elimination of BLV in carrier animals in vivo. Copyright (C) 1996 Elsevier Science Ltd.
• Detection of antibodies against bovine immunodeficiency-like virus in daily cattle in Hokkaido

  N Hirai, H Kabeya, K Ohashi, C Sugimoto, M Onuma  JOURNAL OF VETERINARY MEDICAL SCIENCE  58-  (5)  455  -457  1996/05  [Not refereed][Not invited]
   

  Serological survey of bovine immunodeficiency-like virus (BIV) infection was performed in cattle of 3 different farms in Hokkaido, where a relatively high seroprevalence was recorded for bovine leukemia virus (BLV). About a half of 120 cattle tested were seropositive for BLV, while 7.5% of the cattle were seropositive for BIV. Though increased numbers of leukocytes were frequently observed in BLV-seropositive cows, no such changes were observed in BIV-positive but BLV-negative cows. No correlation was demonstrated between BIV- and BLV-seroprevalence of the cattle.
• Re-isolation of Marek's disease virus from T cell subsets of vaccinated and nonvaccinated chickens

  Y Hiramoto, SI Lee, T Morimura, K Ohashi, C Sugimoto, M Onuma  CURRENT RESEARCH ON MAREK'S DISEASE  130  -135  1996  [Not refereed][Not invited]
   

  Marek's disease (Mn) lymphoma formation can be prevented by the vaccination of a live attenuated XID virus (MDV). We analyzed the effect of MD vaccination on the distribution of MDV among T cell subsets. Chickens vaccinated with CVI988 at one day of age were challenged with Md5 at 5 days of age. Non-vaccinated chickens were also challenged with Md5 at the same day. At 7, 14, 21 and 28 days after the Md5 challenge, MDV was re-isolated from CD4(+) and CD8(+) T cell subsets, and virus titers were compared each other. The fractionation of T cell subsets was achieved by positive immune-magnetic selection using monoclonal antibodies to either CD4 or CD8. The average titer of rescued MDV from vaccinated chickens was significantly power compared to that from nonvaccinated chickens. In addition, the titer of MDV isolated from CD4(+) T cells was consistently higher than that from CD8+ T cells, and these titers were decreased during the experimental period. Only very low titers of MDV were isolated from both CD4 and CD8(+) T cell subsets in vaccinated chickens at 21 and 28 days after challenge. These results suggest that CD4(+) T cell subset is preferentially infected with MDV, and that the infection to CD4(+) as well as CD8(+) T cells was suppressed by vaccination.
• Apoptosis is of CD4(+) T cell in vivo and nonresponsiveness of Cd4(+) and Cd8(+) T cells in vitro observed in chickens infected with Marek's disease virus

  T Morimura, K Ohashi, M Hattori, C Sugimoto, M Onuma  CURRENT RESEARCH ON MAREK'S DISEASE  335  -340  1996  [Not refereed][Not invited]
   

  We investigated whether T cell-immunosuppression induced by Marek's disease virus (MDV)-infection is due to apoptosis. DNA fragmentation analysis showed that CD4(+) but not CD8(+) T cells underwent apoptosis in infected chickens. Furthermore, splenic CD4(+) and CD8(+) T cells in infected chickens did not respond to the stimulation with phorbol 12-myristate 13-acetate plus ionomycin in vitro. This low response of T cells from MDV-infected chickens was at least partially due to apoptotic cell death. Thus, apoptosis observed in MDV-infected chickens may at least in part contribute to the immunosuppression induced by MDV.
• Apoptosis and CD8-down-regulation in the thymus of chickens infected with Marek's disease virus - Brief report

  T Morimura, K Ohashi, Y Kon, M Hattori, C Sugimoto, M Onuma  ARCHIVES OF VIROLOGY  141-  (11)  2243  -2249  1996  [Not refereed][Not invited]
   

  Marek's disease virus (MDV)-infected chickens show thymic atrophy during the acute phase of infection. We examined whether the thymic atrophy by MDV-infection was mediated by apoptosis. Apoptosis-specific DNA ladderings were clearly observed in thymocytes one week after MDV-infection. Histological and flow cytometry studies revealed that immature CD4(+)CD8(+)thymocytes underwent apototic cell death. In addition, the expression level of CD8 molecules on both CD4(-)CD8(+) and CD4(+)CD8(+) thymocyte populations was down-regulated in the infected chickens. These thymic changes might be involved in the pathogenesis of Marek's disease.
• Characterization of extrathymic T cells of chickens

  H Yamamoto, M Hattori, K Ohashi, C Sugimoto, M Onuma  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY  49-  (4)  375  -386  1996/01  [Not refereed][Not invited]
   

  The function of CD4(+) T cells in antibody production was examined by using T cell subset-depleted chickens. CD4- and CD8-depleted chickens, established by the combination of thymectomy and injection of T cell subset-specific monoclonal antibodies, were immunized with sheep red blood cells (SRBC). Titers of anti-SRBC antibody produced in CD4-depleted chickens were lower than those in control chickens, while no difference in the antibody production was observed between CD8-depleted and control chickens. In chickens depleted of both CD4(+) and CD8(+) T cells, the recovery of T cells in the periphery was demonstrated starting 3 weeks after T cell depletion. Those T cells recovered in the periphery predominantly expressed CD4 molecules. Although low titers of antibody against SRBC were detected in chickens depleted of both CD4(+) and CD8(+) T cells, an increase of anti-SRBC antibody production was coincidentally observed with the recovery of CD4(+) T cells in the periphery. These results suggest that CD4(+) T cells could differentiate in extrathymic environments in chickens, and have a helper function in antibody production similar to that of intrathymic T cells. These extrathymic T cells, however, showed a lower proliferative response to concanavalin A than intrathymic T cells, suggesting that these extrathymic T cells may have some properties distinct from intrathymic T cells.
• Analysis of chicken p53 gene in chicken lymphoblastoid tumor cell lines and field tumors

  M Takagi, K Ohashi, T Morimura, C Sugimoto, M Onuma  CURRENT RESEARCH ON MAREK'S DISEASE  251  -256  1996  [Not refereed][Not invited]
   

  To determine whether there is any abnormalities of the p53 gene in chicken lymphoblastoid tumor cell lines (derived from Marek's disease (MD), lymphoid leukosis and reticuloendotheliosis tumors) and field tumors, some portions of p53 cDNA corresponding to domains IT to V were sequenced. Several mutations were Identified in both cell lines and field tumors. However, none of these mutations is localized at the "hot spot", which has been regarded as the site for transformation-activating mutations. Moreover, the 122-bp deletion including exon eight of the p53 gene were shown in three cell lines; MSB1, MTB1 and HP1 derived from MD tumors. This deletion could cause a frame shift of the encoding p53 protein, possibly resulting in the generation of a functionally different p53 protein. However, we confirmed that p53 mRNA without deletion is also present in each of these cell lines. In addition, mutations of the: p53 gene were identified in these cell lines. Thus, mutations of the p53 gene and deletion in the p53 transcript may be induced by MD virus, Alternatively, this deletion may result from alternative splicing which may occur in the cell cycle-dependent manner.
• KINETIC-ANALYSIS OF T-CELLS AND ANTIBODY-PRODUCTION IN CHICKENS INFECTED WITH MAREKS-DISEASE VIRUS

  H YAMAMOTO, M HATTORI, K OHASHI, C SUGIMOTO, M ONUMA  JOURNAL OF VETERINARY MEDICAL SCIENCE  57-  (5)  945  -946  1995/10  [Not refereed][Not invited]
   

  In chickens inoculated with a Marek's disease (MD) vaccine and subsequently with virulent MD virus (MDV), CD4(+) T cell population was drastically decreased following a transient increase at 21 days after hatching (16 days after MDV infection). To elucidate the immune response after the decrease of CD4(+) T cell population, the antibody production against sheep red blood cells (SRBC) was examined in these chickens. Chickens challenged with a virulent MDV after MD vaccination produced lower titers of anti-SRBC antibody than untreated control chickens. Antibody production against SRBC was also lowered in vaccinated chickens or chickens challenged with a virulent MDV.
• Immunomodulation of peripheral T cells in chickens infected with Marek's disease virus: Involvement in immunosuppression

  T. Morimura, M. Hattori, K. Ohashi, C. Sugimoto, M. Onuma  Journal of General Virology  76-  (12)  2979  -2985  1995  [Not refereed][Not invited]
   

  Marek's disease virus (MDV) causes T cell immunosuppression in chickens during latent infection. Morphological changes specific to apoptosis were demonstrated in peripheral blood mononuclear cells (PBMC) of MDV-infected chickens at 2-3 weeks post-inoculation (p.i.). Analysis of DNA fragmentation in T cell subsets in the peripheral blood revealed that CD4+ T cells but not CD8+ T cells underwent apoptosis after MDV infection. The proportion of CD4+ T cells, but not that of CD8+ T cells, in the peripheral blood expanded transiently at 16 days p.i., and rapidly decreased 1 week later. The decrease in CD4+ T cells might be mediated by apoptosis, because a rapid reduction in CD4+ T cells was observed when these cells underwent apoptosis. Analysis of the T cell-receptor (TCR) repertoire of the peripheral blood showed that Vβ1 but not Vβ2-αβ TCR-bearing cells expanded at 16 days p.i., when the transient expansion of the CD4+ T cell population was observed in these chickens. Flow cytometric profiles also showed that the expression of CD8 was down-regulated after infection with MDV, but there was no difference in the expression level of CD4 molecules between normal and infected chickens. Northern blot analysis indicated that the down-regulation of CD8 occurred at the transcriptional level. These results suggest that both apoptosis of CD4+ T cells and down-regulation of CD8 molecules could contribute to the immunosuppression caused by MDV.
• Possible mechanisms of immuno-suppression in chickens induced by Marek’s disease virus.

  日仏獣医学雑誌  6-  89  -99  1995  [Not refereed][Not invited]
  
• CHARACTERIZATION OF MAREKS-DISEASE VIRUS BAMHI-A-SPECIFIC CDNA CLONES OBTAINED FROM A MAREKS-DISEASE LYMPHOBLASTOID CELL-LINE

  K OHASHI, PH OCONNELL, KA SCHAT  VIROLOGY  199-  (2)  275  -283  1994/03  [Not refereed][Not invited]
   

  A cDNA library was constructed from poly(A)(+) RNA fractions obtained from a Marek's disease (MD) lymphoblastoid cell line, MDCC-CU41, in which viral gene expression is very limited. Three independent groups (1, 2, and 3) of MD virus (MDV)-specific clones were obtained, which were mapped in the inverted repeat region of the BamHI-A fragment of the MDV genome. Northern blot analysis showed that probes prepared from these cDNA clones hybridized with several transcripts of different sizes in poly(A)(+) RNA of MDCC-CU41, although the amounts of these transcripts were relatively small compared to those in MDV lytically infected cells. Moreover, a small open reading frame, which can encode a 94-amino-acid protein, was identified in the A41 cDNA clone (Group 3). By RNase protection assays, the 1.2-kb Group 3 transcriptional unit has been defined. In indirect immunofluorescent antibody assays, antiserum against the bacterially expressed fusion protein, glutathione S-transferase-A41, reacted specifically with the cytoplasmic regions of MDV (strain RB1B)-infected chick kidney cells. However, MDCC-CU41 did not contain a detectable level of the protein determined by these methods. (C) 1994 Academic Press, Inc.
• CHARACTERIZATION OF A MAREKS-DISEASE VIRUS BAMHI-L-SPECIFIC CDNA CLONE OBTAINED FROM A MAREKS-DISEASE LYMPHOBLASTOID CELL-LINE

  K OHASHI, WP ZHOU, PH OCONNELL, KA SCHAT  JOURNAL OF VIROLOGY  68-  (2)  1191  -1195  1994/02  [Not refereed][Not invited]
   

  Two Marek's disease (MD) virus BamHI-L-specific cDNA clones were isolated from a cDNA library constructed from poly(A)(+) RNA fractions of an MD lymphoblastoid cell line, MDCC-CU41 (CU41). These clones were mapped to the region corresponding to the BamHI-Q(2), and -L, regions. These clones hybridized with 2.5-, 0.8-, and 0.6-kb transcripts prepared from CU41. The transcriptional unit of the 0.6-kb transcript was determined by RNase protection assays. An open reading frame encoding a 107-amino-acid polypeptide was identified in the 0.6-kb transcript. Reverse transcriptase-PCR demonstrated the presence of this transcript in both CU41 and a reticuloendotheliosis virus-transformed cell line latently infected with MD virus.
• cDNA clones derived from the Marek's disease tumor cell line MDCC-CU41.

  Proceedings of 19th World's Poultry Congress  54  -57  1992  [Not refereed][Not invited]
  
• ANALYSIS OF MAREKS-DISEASE TUMOR-ASSOCIATED SURFACE-ANTIGEN ON MDCC-MSB1-CLO18 CELLS

  M HORIE, K OHASHI, H KODAMA, T MIKAMI  INTERNATIONAL JOURNAL OF CANCER  47-  (2)  238  -243  1991/01  [Not refereed][Not invited]
   

  The physico-chemical nature of Marek's disease tumor-associated surface antigen (MATSA) on Marek's disease (MD) lymphoblastoid tumor cell line (MDCC-MSBI-clo.18) was examined by the cellular enzyme-linked immunosorbent assay (CELISA) and the sandwich enzyme-linked immunosorbent assay (ELISA) using an anti-MATSA immune serum or a monoclonal antibody (MAb) 2B9 developed against MATSA. Our results indicate that MATSA is a glycoprotein and 2B9 recognizes an antigenic site in the protein moiety of MATSA. MATSA was solubilized from MSBI-clo.18 cells by treatment with 0.5% Nonidet P-40, and purified by affinity chromatography coupling with 2B9 and further by ion exchange chromatography on diethylaminoethylcellulose (IECD). MATSA was eluted with 0.2 to 0.3 M KCl in IECD and the purity of MATSA was increased about 2,500-fold. The purified MATSA was shown to have a molecular weight (M(r)) of 70,000 by SDS-PAGE. The reactivity of purified MATSA with anti-thymus cell serum was examined. MATSA was detectable by anti-thymus cell serum, although 2B9, which was used to purify MATSA from MSBI-clo.18 cells, was not reactive to cells prepared from the thymus. However, MATSA was no longer detectable after the absorption of anti-thymus cell serum by chicken bursa cells. The absorption of anti-thymus cell serum by chicken red blood cells (RBC) had no effect on the reactivity against MATSA. These results suggest that MATSA may be a lymphocyte-specific antigen modified during leukemogenesis by Marek's disease virus (MDV).
• Cytotoxic activity of spleen cells from chickens with transplantable Marek's disease tumors.

  Proceedings of 3rd International Symposium on Marek's Disease  268  -275  1989  [Not refereed][Not invited]
  
• Suppression of NK activity of spleen cells by chicken fetal antigen present on Marek's disease lymphoblastoid cell line cells

  Kazuhiko Ohashi, Takeshi Mikami, Hiroshi Kodama, Hisao Izawa  International Journal of Cancer  40-  (3)  378  -382  1987  [Not refereed][Not invited]
   

  Chicken fetal antigen (CFA) expressed on the cell surface of Marek's disease (MD) lymphoblastoid cell line (MDCC‐MSBI) cells was purified by affinity chromatography using a monoclonal antibody (2H3). A 4‐hr 51Cr release assay was performed using spleen cells as effector cells to investigate the role of the CFA in the immune response of chickens, especially in relation to natural killer (NK) activity. Spleen cells of specific‐pathogen‐free (SPF) chickens showed reduced NK activity in the presence of CFA in vitro, as did those cells treated with MSBI soluble antigen. NK activity of spleen cells from chickens treated with CFA was also suppressed when compared to the cells from untreated or ovalbumin‐treated chickens. In addition, MSBI clo. 18 transplantable tumors grew progressively in some of the 14‐day‐old chickens treated with CFA, whereas the tumors regressed in age‐matched chickens with or without treatment by ovalbumin. Copyright © 1987 Wiley‐Liss, Inc., A Wiley Company
• MONOCLONAL-ANTIBODY TO CHICKEN FETAL ANTIGEN ON MAREKS-DISEASE LYMPHOBLASTOID CELL-LINE MDCC-MSB1

  K OHASHI, T MIKAMI, T HIGASHIHARA, H KODAMA, H IZAWA  CANCER RESEARCH  46-  (11)  5858  -5863  1986/11  [Not refereed][Not invited]
  
• Detection of cell-surface antigens on MDCC-MSB1 cells using monoclonal antibodies.

  2nd International Symposium on Marek's Disease  214  -235  1985  [Not refereed][Not invited]
  
• MONOCLONAL-ANTIBODIES AGAINST A MAREKS-DISEASE TUMOR-ASSOCIATED SURFACE-ANTIGEN

  T HIGASHIHARA, T MIKAMI, K OHASHI, H KODAMA, M ONUMA, H IZAWA  JAPANESE JOURNAL OF VETERINARY SCIENCE  46-  (5)  649  -658  1984  [Not refereed][Not invited]
  

Association Memberships

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

Research Projects

• Development of cocktail vaccines against poultry red mites using the recombinant turkey herpesvirus

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/04 -2027/03 

  Author : 村田 史郎, 今内 覚, 大橋 和彦
• The roles of polymorphism in Meq on the pathogenicity of Marek's disease virus and their application for the vaccine development

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/04 -2026/03 

  Author : 大橋 和彦, 村田 史郎, 今内 覚
• Development of evolving therapeutic technologies to address unmet medical needs for intractable diseases in animals

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/04 -2025/03 

  Author : 今内 覚, 村田 史郎, 大橋 和彦
• The search for the candidate molecule for the vaccine to control a wide range of ectoparasite species of chickens

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2020/04 -2023/03 

  Author : 大橋 和彦, 村田 史郎, 今内 覚

   

  ワクモやトリサシダニは、鳥類の代表的な外部寄生虫であり、鶏に貧血や削痩、産卵率低下など養鶏産業に大きな経済的被害をもたらしている。現在、その防除 には主に駆虫剤が使用されているが、薬剤耐性個体の出現や駆虫剤残存による衛生環境の悪化などが問題となっており、抗ダニワクチンなど新規防除法の開発が必要となっている。そこで本研究では、ワクモとトリサシダニ及び近縁なミナミトリサシダニを同時に防除できるダニ種横断的な新規“ユニバーサル”ワクチン の開発を目指して、RNA-SEQ解析や全ゲノム解析と相同性検索により、これらのダニ間で共通の非暴露型抗原の探索・同定を行い、 吸血したダニ個体に致死的に作用して鶏舎内の寄生虫個体数を持続的に減少できるワクチンへの応用を検討することを目的とした。 今年度は、昨年度実施したRNA-SEQによる発現する遺伝子群の網羅的解析の結果を元にFER2など数種類の候補遺伝子を選抜して、その組換え抗原の調製や機能解析を行った。in vitro feeding assayによる今後交差防御試験を行いワクチン候補抗原としての有用性を確認する。またいくつかのワクモ由来分子の抗ワクモワクチンとしての有用性を検討し、抗ワクモ効果（持続的な個体数の減少）を示す新規抗原を同定した。さらに複数種の抗原を用いたカクテル抗原が単独抗原よりも有効な抗ワクモ効果を示すことも明らかとなった。今後、これらの抗原についても“ユニバーサル”ワクチンへの応用を検討していく。
• Experimental study for practical application of cross-sectional cancer therapy in companion animals

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/04 -2022/03 

  Author : KONNAI SATORU

   

  Our previous studies demonstrated programmed cell death-ligand 1 (PD-L1) overexpression in some canine cancers, and suggested the therapeutic potential of a canine chimeric anti-PD-L1 monoclonal antibody (mAb). Thus, canine PD-L1 expression was assessed in various cancer types and the safety and efficacy of therapeutic anti-PD-L1 mAb were explored in dogs with pulmonary metastatic oral malignant melanoma (OMM). PD-L1 expression was detected in most canine malignant cancers including OMM, and survival was significantly longer in the treatment group when compared to a historical control group. We show that PD-L1 is a promising target for cancer immunotherapy in dogs.
• Development of a method to control poultry red mites using recombinant Marek's disease virus that secretes a vaccine antigen

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2021/03 

  Author : Murata Shiro

   

  Poultry red mites (PRMs, Dermanyssus gallinae) are haematophagous ectoparasites which causes serious economic losses to the poultry industry worldwide, and the development of novel methods to control PRMs is required. We have investigated the application of vaccination and have shown that immunization with vaccine antigens could contribute to the reduction in the number of PRMs. In addition, we investigate the application of virus vectors to develop easy-to-use vaccines for farmers. In this study, we designed the recombinant virus that secrets vaccine antigens to improve the efficiency of vaccines using the recombinant virus. To produce an antigen-secreting virus, we inserted the signal peptides of a virus-derived secretory protein to the virus genome. However, the secretion of antigens was not confirmed, although the expression was observed in infected cells. Further manipulation is required to generate effective vaccines using virus vectors for controlling PRMs.
• Analysis of the molecular mechanism for the increase in the virulence of Marek's disease virus

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2017/04 -2020/03 

  Author : Ohashi Kazuhiko

   

  Marek’s disease virus (MDV), a causative agents of Marek’s disease (MD), which is characterized by the formation of malignant lymphoma, appears to increase its virulence in the fields. To elucidate the molecular mechanisms for the increase in its virulence, we isolated MDVs from vaccinated chickens which still developed clinical MD, and analyzed their virological properties. MDV recently isolated in Japan caused clinical MD in inoculated chickens, although less virulent to chickens compared to virulent MDV strains isolated in United States. In addition, it is suggested that virus-derived factors other than polymorphisms in viral oncogene reported in the United States would be involved in the increase in MDV virulence.
• Analysis of genetic polymorphism of poultry red mites in the world and search for candidate molecules of anti-mite vaccines

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2019/03 

  Author : OHASHI Kazuhiko

   

  Poultry red mites (PRMs), Dermanyssus gallinae, are blood-sucking ectoparasites of chickens, which cause huge economic losses in the poultry industries worldwide. To establish a new effective control method for PRMs, we have searched for and identified candidate antigens which are not polymorphic among PRMs from different areas in the world to develop anti-PRM vaccines. Several antigens identified in this study showed anti-PRM effects determined by in vitro feeding assay using sera from immunized chickens.
• Comprehensive analysis of molecules expressed on the midgut of poultry red mites for the development of new anti-mite vaccines

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2018/03 

  Author : OHASHI Kazuhiko

   

  For the development of new vaccines against poultry red mite, Dermanyssus gallinae, a search was made for novel vaccine candidates expressed on the cell membrane of the midgut of the mite. Three kinds of molecules, whose expressions are predicted on the cell membrane, were identified, and their expressions on the midgut were confirmed by laser capture microdissection and RT-PCR. In addition, cathepsin D-like molecule, catD2, which was already identified in the course of the comprehensive analysis of genes expressed in the red mites done in our laboratory, was shown to be expressed in protonymph, deutonymph and adult and expressed regardless engorgement or starvation. The recombinant catD2 protein was not recognized by the sera from chickens fed by the red mites, suggesting that catD2 is a concealed antigen. Further analyses, including the detailed functions, the expression sites in red mites and the evaluation as vaccine antigens, are required to develop effective vaccines.
• Empirical research for bovine leukemia control strategy

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2018/03 

  Author : KONNAI SATORU

   

  Bovine leukemia virus (BLV) is highly endemic in many countries including Japan. In this study, we evaluated the risk for BLV transmission among the infected animals to establish the guideline for effective eradication of EBL. We found that BLV-infected cattle with persistent lymphocytosis (PL) or high proviral load or EBL are considered major sources of both horizontal and vertical BLV transmission. Furthermore, maternal proviral load is closely correlated with the frequency of vertical transmission to calves. However, no direct evidence of intrauterine BLV infection has been confirmed in infected cattle. We confirmed intrauterine BLV infection in two pregnant dams with high viral load by cesarean delivery, and BLV was detected in the newborns. In the study, we presented a direct evidence of intrauterine BLV transmission in pregnant dam with a high proviral load. These results could aid the development of BLV control measures targeting viral load.
• Studies on the molecular mechanisms for the increase in MDV virulence in the field

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : Kazuhiko Ohashi

   

  Marek’s disease virus (MDV), a causative agent of Marek’s disease (MD), which is characterized by the formation of malignant lymphomas, tends to increase its virulence in the fields. To clarify the molecular mechanism for the increase in MDV virulence, recent MDV strains isolated from vaccinated chickens which developed clinical MD were virologically and molecular biologically characterized. Several new diversities/mutations were detected in viral genes of these strains, and a plaque-purified MDV strain was shown to be pathogenic to chicks. However, phylogenetic analysis of the whole genome of the MDV strain and reference strains showed that domestic MDV may increase its virulence through unknown mechanisms which are different from those suggested previously in MDV strains isolated in the United States.
• Pathologic analysis of malignant infectious diseases in foreign livestock and its applications for establishment of novel control strategy.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2017/03 

  Author : Satoru KONNAI, Cruz L., Abes N., Mingala C., Skilton R., Bishop R., Nene V., Kanduma E., Githaka N., Gakuya F., Kariuki E., Simmunza M., Logullo C., Itabajara S., Brown W., Mulenga A., Morrison I., Connelley T., Whittington R., Silva K. de, Chultemdorj T., Sharav T., Odbileg R.

   

  Although there are many malignant infectious diseases in livestock involving immune abnormalities (impairments), the mechanisms underlying these diseases remain unknown. In this research, we analyzed the mechanism of immune abnormalities in the malignant infectious diseases associated with economic loss in livestock industry, such as bovine anaplasmosis, East Coast fever, Surra, bovine tuberculosis, Johne’s disease, bovine leukemia, Equine infectious anemia. Our analyses have shown that immunosuppressive factors, such as Programmed death 1 (PD-1) and Programmed death-ligand 1 (PD-L1), are involved in immune suppression seen in the malignant infectious diseases. Furthermore, in vitro blockade of the PD-1/PD-L1 pathway restored anti-pathogen-specific T-cell proliferation and cytokine production.
• Establishment of control measure for bovine leukemia based on risk analysis of virus transmission

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011/11 -2015/03 

  Author : KONNAI SATORU, OHASHI Kazuhiko, MURATA Shiro

   

  Bovine leukaemia virus (BLV) causes enzootic bovine leukosis (EBL) in cattle and represents a serious problem in Japanese meat and dairy industries. In this study, BLV from newly infected cattle was characterized to determine the virus source in a herd and a vector control measure was used to prevent the spread of the infection. The env sequences from positive conversion cattle showed 100% nucleotide identity with the sequence from the PL cattle with high provirus, suggesting that animals may serve as a reservoir of BLV infection. In the farms, where many blood-feeding stable flies were observed, vector control performed using a combination of insect repellents was very efficient as no new cases of BLV infection were observed after the treatment. This study indicates that BLV-infected cattle with high virus is one of high risk factors for the transmission, and vector-borne transmission might have been the major transmission route in the farm.
• 神経疾患を引き起こすレトロウイルスの海外拡散の実態とゲノム多様性

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2012/04 -2014/03 

  Author : 落合 謙爾, 大橋 和彦

   

  研究代表者は「トリのグリオーマ」がA型トリ白血病ウイルス(ALV-A)感染症であることを明らかにした。本課題の目的は神経系に病原性を示すALVの国内外の感染状況ならびに分離株のゲノム多様性と病原性の相関を明らかにすることである。初年度である今年度は国内各地で生産された日本鶏の疫学調査を実施するとともに,海外調査に適した材料採取方法を検討した。得られた成果は以下の通りである。1.国内16ヶ所で生産された日本鶏77羽中26内種を含む68羽(88.3%)がFGV特異的nestedPCRで陽性を示した。2.PCRで陽性となった48羽からALVの分離を試みたところ,8ヶ所,県別に見ると福井,静岡,島根,愛媛および高知の5県で生産された鶏からALV株が分離された。これらのうち,7株のnestedPCR検出領域はFGV特異配列と高い相同性(90～98%)を示した。3.一方,日本鶏にはFGV特異配列を持つ未知の内在性レトロウイルスが存在することが示唆された。このため,FGV特異的PCRの成績の判定だけでは正確な神経病原性ALVの感染率は得られないことがわかった。4.分離株のenvSU領域に基づく分子系統樹では福井,静岡,島根および愛媛で分離された5株がFGVまたはFGV変異株Sp53と同じクラスターに分類された。5.有精卵からのALV分離を試みたところ,ALVは分離されたが,分離率は孵化率に依存するため,海外調査ではこの採取方法を補助的に用いることにした。6.以上の成績から,海外調査を行うためにはFGV特異配列の有無にかかわらず日本鶏に感染するALVを分離し,これらのウイルスゲノムおよび病原性を解析する必要があることがわかった。
• Studies on the immunoinhibitory mechanisms of Marek's disease virus and its application for adjuvant

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011/04 -2014/03 

  Author : OHASHI KAZUHIKO, KONNAI Satoru, MURATA Shiro

   

  The programmed death 1 (PD-1) receptor and its ligands, programmed death ligand 1 (PD-L1) and 2 (PD-L2) are known to have an immunoinhibitory function which contribute to the pathogenesis of chronic infections and neoplastic diseases via the PD-1/PD-L pathway. In this study, it has been shown that chicken PD-1 and PD-L1/PD-L2 also have an immunoinhibitory function, such as inhibition of cytokine production. The expressions of PD-1 and PD-L2 were significantly higher in the spleens of infected chickens in the latent phase and in tumor lesions caused by Marek's disease virus (MDV). These results suggest that chicken PD-1/PD-L2 pathway is involved in the establishment of latency and tumor formation by MDV.
• レトロウイルスが引き起こす多様な神経障害の分子基盤

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2010/04 -2014/03 

  Author : 落合 謙爾, 大橋 和彦

   

  研究代表者は「トリのグリオーマ so-called fowl glioma」が A型トリ白血病ウイルス (ALV-A) 感染症であることを明らかにした。本課題の目的は代表者が分離した神経病原性 ALV の解析を通してレトロウイルス誘発性神経障害の分子基盤を解明することである。今年度得られた成績は以下の通りである。1.作製した FGV の感染性分子クローンおよび FGV の gag-pol をトリ白血病ウイルスベクター RCAS(A) のそれと入れ換えたキメラウイルスの神経系に対する病原性を感染実験により解析した。その結果，分子クローンおよびキメラウイルスは鶏に対しグリア結節および囲管性リンパ球浸潤といった非化膿性脳炎を引き起こすが，観察期間内にはグリオーマが誘発されないことがわかった。これは脳内で複製するウイルス量が低いためと推察された。2. 新たに FGV の env/3’LTR および ΔLTR をトリ白血病ウイルスベクター RCAS(A) のそれらと入れ換えたキメラウイルスを作製した。3.FGV 特異配列を欠き，内在性レトロウイルス ev-1 由来と考えられる ALV, Km_5666 株の中枢神経系に対する病原性を感染実験により解析した。感染実験鶏の脳には囲管性リンパ球浸潤，脳室周囲の未分化円形細胞の増殖，グリオーシス，およびグリオーマが誘発された。これら病変の程度は既報の Sp 株に比べて弱く，脳内ウイルス RNA 発現量は検索したいずれの日齢でも Sp-40 株のそれらと比較して低かった。4. 以上の成績から，FGV 特異配列はグリオーマ誘発能とは関連しないことが示唆された。また，グリオーマの形成には ALV 感染初期の脳内ウイルス量が関与することが推察された。
• Elucidation of the evasion mechanism of cytokine storm in disease resistant native livestock

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2010 -2012 

  Author : KONNAI Satoru, OHASHI Kazuhiko, CRUZ L., ABES N., MINGALA C., SKILTON R., BISHOP R., NENE V., KANDUMA E., GITHAKA N., GAKUYA F., KARIUKI E., SIMMUNZA M., LOGULLO C., ITABAJARA S.

   

  In African, Asian and South American countries, native livestock is relatively a disease-resistant animal, but they can be infected with infectious agents. Therefore, there is a need to investigate the immunological events during disease development. Our study has shown that, in T. evansi (causative agent for Sura disease) and T. parva (causative agent for East Cost fever)-infected native animals, there is an augmentation of a subset of regulatory DCs that act as potential regulators of the inflammatory responses including cytokine storm.
• Identification and characterization of factors involved in the resistance to Marek' s disease in chickens and wild waterfowls

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2011 

  Author : OHASHI Kazuhiko, KONNAI Satoru

   

  To study the molecular mechanism(s) for the natural resistance to Marek' s disease (MD) in wild waterfowls and genetically resistant lines of chickens, the regulatory mechanisms of the expression of interferon y (IFNy), which plays an important role in the cell-mediated immunity against MD virus (MDV), were analyzed by using the conventional luciferase reporter assay system. The promoter activity was more strongly repressed in white-fronted geese than chickens when Meq, an MDV oncoprotein, was co-expressed. This result suggests that, in white-fronted geese, reduced expression of IFNy by Meq at early after MDV infection could result in the lower degree of T cell activation (activated T cells are targets for MDV) to reduce the chances for the birds to develop MD.
• The origin and genome diversity of retroviruses causing neurological disorders

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2008 -2011 

  Author : OCHIAI Kenji, OHASHI Kazuhiko

   

  Fowl glioma is caused by fowl glioma-inducing virus (FGV), belonging to subgroup A of avian leucosis virus (ALV-A). To clarify how and where FGV emerged, the prevalence of FGV in European, Asian and Japanese native chickens were examined and the viral genome of the isolates were analyzed. The results suggest that FGV is likely to have appeared in Japanese fowls in Japan without a relationship to the past fowl glioma found in Western countries, and that FGV and FGV vatriants still spread in Japanese fowls, showing genome diversity mainly based on the recombination between these strains and avian endogenous/exogenous viruses.
• がん・病原微生物によるT細胞機能抑制の分子メカニズム解明とその制御

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2008 -2010 

  Author : 服部 雅一, 大橋 和彦, 杉本 千尋

   

  白血病の発症に伴いマウスの末梢において抗原不応答性に陥ったPD-1^+メモリー型ヘルパーT細胞(PD-1^+MP T細胞) が蓄積することから,他の癌においても同様の現象が見られるかどうかについて,マウス大腸癌細胞株Colon26を用いた移植癌モデルとメチルコラントレン (MCA) を用いた化学発がんモデルを用いて検討を行った。その結果,いずれのモデルにおいても癌組織中へのPD-1^+MP T細胞の蓄積が観察され,癌に普遍的な現象であることが強く示唆された。蓄積したPD-1^+MP T細胞は白血病で見られた細胞同様,抗原刺激に対し無反応であったが,前者とは異なりIL-2添加によりその反応性の回復が見られた。DNAマイクロアレイ解析を行ったところ,オステオポンチン (OPN) の発現上昇やゲノムオーガナイザーであるSATB1の発現低下が観察されたが,白血病のケースで観察されたC/EBPα転写因子の発現上昇は観察されなかった。このことはPD-1^+MP T細胞の抗原府応答性の原因としてSATB1の発現低下あるいはOPNの産生が関与していることを示唆している。PD-1^+MP T細胞は抗原レセプターを介したシグナルに対し増殖を示さなかったが,NK細胞の増殖刺激因子として知られるIL-15に対し反応し,増殖することが明らかとなった。免疫系におけるSATB1の機能を直接検討するために,末梢ヘルパーT細胞において強制的に発現するトランスジェニックマウス (E4POK-SATB1Tgマウス) の作製を試みたが,得られたF1マウスには遺伝子が発現しておらず,現在,再び受精卵へのインジェクションを開始した。
• Studies on the molecular mechanisms for Marek's disease virus to increase its virulence in the field

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2008 -2010 

  Author : OHASHI Kazuhiko, OCHIAI Kenji, KONNAI Satoru

   

  To clarify the molecular mechanisms for Marek's disease virus (MDV) to increase its virulence, several viral genes of MDV strains recently isolated from the field were analyzed. Diversities/polymorphisms were identified in one of the MDV genes, meq, that could be potentially related to the increase in MDV virulence. When we analyzed the effects of these diversities/polymorphisms in the meq gene on the functions of the meq gene product (Meq), the amino acid substitutions resulted from the diversities/polymorphisms significantly altered the transactivation and transforming activities of Meq. These findings suggest that the diversities/polymorphisms in the meq gene could contribute to the recent increase in MDV virulence in the field.
• Pathological and molecular biological analysis of nervous lesions caused by glioma-inducing retroviruses

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2006 -2009 

  Author : OCHIAI Kenji, OHASHI Kazuhiko, ONO Etsuro, TOMIOKA Yukiko

   

  In order to clarify the pathogenesis of nervous system disorders due to retrovirus infection, fowl glioma-inducing virus (FGV) belonging to avian leukosis viruses (ALVs), FGV-mutants, and another ALVs isolated from chickens affected with glioma or peripheral nerve tumors were examined by molecular biological analysis and their pathogenicity to nervous system was compared by experimental infections. The main results suggest that env gene may be not a major determinant for the induction of glioma. Also, one transgenic (Tg) chimera chicken (G(0)) carrying FGV-LTR obtained in this study is expected to be useful for analysis of the LTR function.
• Genetic analysis of drug resistant trypanosomes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2006 -2008 

  Author : ONUMA Misao, OHASHI Kazuhiko, INOUE Noboru, KONNAI Satoru

   

  トリパノソーマ原虫は人、家畜および野生動物に感染し睡眠病、ナガナ病、スーラ病などを引き起こし、多大な被害を与えている。本症は、ワクチンによる制御が困難なうえ、近年、原虫薬剤耐性株の出現によって、その被害は深刻化している。そこで原虫遺伝子多型および薬剤耐性機序解明を目的として、ザンビア共和国およびフィリピン共和国における本症の分子疫学調査を行った。さらに、媒介昆虫であるツェツェバエの吸血宿主の同定、すなわち本症のレゼルボア動物の推定のために、トリパノソーマ感染ツェツェバエ由来のDNAサンプルに含まれる哺乳類動物のミトコンドリア遺伝子を検出し、その遺伝子配列解析から吸血宿主を同定した。その結果、ヒトおよびアフリカゾウ、アフリカバッファローなどの野生動物に加え、調査地区集落の主要な家畜であるヤギの遺伝子も検出されたことから、ヤギにおける本症の分子疫学調査も行い、同地区の高率な感染率を報告した。一方、フィリピン共和国において集団流産が認められた家畜の原因病原体の調査も実施するとともに、抗原虫薬剤治療歴を持つ家畜からトリパノソーマ原虫を分離し薬剤感受性ならびに病原性解析を行った。
• 鳥類由来感染症の疫学的研究

  Date (from‐to) : 2008
• Epidemiological study on infectious diseases of wild and domestic birds

  Date (from‐to) : 2008
• 抗糖脂質モノクローナル抗体のイヌ型抗体化とメラノーマ治療への応用

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2006 -2007 

  Author : 大橋 和彦, 今内 覚, 奥村 正裕

   

  糖脂質を標的分子とした新規の抗腫瘍療法を開発することを目的として研究として研究を行った。今年度は、平成18年度に調製し、腫瘍細胞、特にイヌ由来メラノーマ細胞株との反応性を確認した各種糖脂質に対するマウスモノクローナル抗体(MAb)パネルのうち、細胞株に広範に反応した抗GM3 MAb(クローンNo.2-14)について、さらに解析を行った。その結果、このクローンMAbは、非常に多くのメラノーマ細胞株に対して試験管内で細胞傷害性を有していることを見出した。また種々のメラノーマ可移植性細胞株を皮下に移植したヌードマウスを用いた治療試験でも、MAbの連続投与により、移植腫瘍の生育が抑制されることが示された(ただし、対照群と治療群の間に統計学的有意差は検出されなかった)。以上より、in vivoにおいても抗GM3 MAbが抗腫瘍効果を発揮できることがマウスモデルで証明された。 昨年度に、抗GM3 MAb(クローンNo.2-14)の抗原結合部位である超可変領域(抗原特異性決定部位)の塩基配列を決定し、イヌ型抗体化を目指して遺伝子発現ベクターへ挿入するため、遺伝子カセットをH鎖・L鎖それぞれにおいて作成したが、今年度は、さらに組み換えイヌ型抗糖脂質MAbの臨床応用を目的として、作成した遺伝子カセットをH鎖・L鎖のバキュロウイルス発現系を用いた組み換えイヌ型抗GM3 MAbの大量発現系の樹立を試みた。しかしながら、その発現量は、あまり高くないことが判明した。発現したL鎖とH鎖について、そのGM3との結合活性を確認したが、単独では結合せず、組み換えL鎖およびH鎖の会合した分子を調製する必要性が示唆された。今後、spacerなどでL鎖およびH鎖を結合した遺伝子を調製し共発現させる系を検討する。
• ダニの唾液由来免疫抑制因子を用いるダニ媒介性病原体の伝播阻止

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2006 -2007 

  Author : 小沼 操, 大橋 和彦, 今内 覚

   

  ダニの発現遺伝子の網羅的解析のため吸血5日目のフタトゲチマダニ(Haemaphysalis longicornis)唾液腺を用いてEST解析(発現タグ解析)を行った。得られた653配列を米国NCBIのblastXプログラムを用いて蛋白データベースと比較したところ、232配列(36%)で有意な相関を示す類似遺伝子が見つかった。その大半は細胞の生存に必要な、いわゆるハウスキーピング遺伝子であったが、1/3は宿主の生理活性に何らかの影響を与え得る因子であると推察された。今回得られた遺伝子の一つは抗凝固因子であるmadanin1と塩基配列で88%、アミノ酸配列で66%の相同性を示した。そこで本因子の生物活性を解析したところ抗凝固活性が認められた。一方、免疫抑制作用が示唆される1配列について宿主免疫への影響を解析した。その結果、牛末梢血単核球(PBMC)やマウス脾臓細胞の増殖反応を濃度依存的に抑制し、サイトカイン(IL-2、IL-12p40およびTNF-α)の発現も強く抑制した。また、BALB/cマウスへの接種では、脾臓の萎縮が認められ、これらのマウス由来の脾臓細胞は各種Mitogenに対する増殖反応が著しく低下し、Microarray解析では免疫活性化因子の発現抑制も認められた。
• レトロウイルス性グリオーマモデルにおける遺伝子サイレンシングの効果

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2005 -2007 

  Author : 落合 謙爾, 大橋 和彦

   

  研究代表者は「トリのグリオーマ」がA型トリ白血病ウイルス感染症であることを明らかにした。本研究課題ではトリのグリオーマ誘発ウイルス(FGV)を感染させた鶏でのRNA干渉(RNAi)の有効性を検討するためにこれまでin vitroでの効果を検討した。まず,分離されたFGVおよびFGV変異株の分子系統解析に基づき,これら分離株のenv遺伝子内に見出された共通な塩基配列を有する領域3カ所を標的とするsiRNAを作製した。今年度は昨年度までの成績を踏まえ鶏胎子線維芽細胞由来株化細胞DF-1を用いたより単純な実験系で,FGV感染または非感染DF-1に対し新たに4種類のsiRNAトランスフェクション試薬を用いてトランスフェクション効率を上げる条件およびin vitroでの遺伝子抑制効果を検討したが,いずれの条件でも充分な効果は得られなかった。一方,siRNA発現レトロウイルスベクターを用いた実験系を確立するために,遺伝子抑制作用が可視化できる蛍光タンパク質EGFP発現プラスミドを作製し,このプラスミドをDF-1細胞にトランスフェクションして恒常的にEGFPを発現する細胞の確立を検討した。レトロウイルスベクターについてはベクターの遺伝子にsiRNA発現領域を組み込む遺伝子カセットを準備した。本検索により,鶏の細胞でRNAiの有効性を検討することは現状ではきわめて難しいことがわかった。この理由として鶏では培養細胞の種類が少なく電気穿孔法や試薬導入法に使用可能な適切な細胞株が存在しないこと,主に哺乳類細胞用に調整されているトランスフェクション試薬の多くは鶏の細胞では有効ではないことがあげられる。
• Tick vaccine development by using tick testis-derived factors

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2005 -2007 

  Author : ONUMA Misao, OHASHI Kazuhiko, TAJIMA Tomoko, KONNAI Satoru

   

  From the cDNA library constructed from testis/vas deferens, we identified a novel gene of Rhipicephalus appendiculatus (Rappendiculatus) tick, male-specific factor, encoding a secretory component exclusively expressed in the testis/vas deferens. This molecule, tentatively named as Rappendiculatus Mating Factor (RAMF), contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMF mRNA was up-regulated in male ticks during blood feeding. RAMF was detected not only in the testis/vas deferens but also in the postcoitum female ticks by Western blotting, suggesting that this protein could be transferred into the female tick through copulation. The attachment duration had a tendency to prolong in the virgin female ticks microinjected with recombinant RAMF, but there was no obvious effect on blood feeding. These results suggest that RAMF is a male-specific molecule in spermatophore, and other factor(s) might be required for stimulation for female engorgement in R. appendiculatus tick.
• Phylogenetic analysis of avian retroviruses with neuropathogenicity in central nervous system

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2004 -2007 

  Author : OCHIAI Kenji, HOHASHI Kazuhiko

   

  Avian leukosis virus (ALV) mainly causes hematopoietic neoplasms in chicken, whereas fowl glioma-inducing virus (FGV), which belongs to ALV subgroup A, induces astrocytoma in chickens. Fowls kept in zoological garden A in Toyama have been considered as source for the FGV infection in other Japanese zoological gardens. However, the background and process how FGV acquired the unique oncogenicity in nervous system has not been clarified. At first, to detect FGV using nested PCR, primers were designed specific to the 3' untranslated region (UTR) and the PCR-protocol was established. The prevalence of FGV by the nested PCR in 131 Japanese fowls of zoological garden A was 40% and the prevalences in a total of 129 chickens in three other zoological gardens, which were received the eggs or chicks from the zoological garden A, were 26 to 56%. Phylogenetic analysis revealed that 3' UTRs, including the FGV-specific sequence, of the 14 isolated ALVs showed high sequence identity and a close relationship with that of FGV. Secondly, the prevalence in chickens, which were not related by birth to any fowls of the zoological garden A, was examined. Ancestors of Japanese fowl are thought to be brought to Japan in the Edo era from Southeast Asia. Along the propagation path, feather pulps and cloaca swab were collected from 264 chickens in Indonesia, Philippines, South Korea and Japan. Twenty nine chickens, including 9 birds in the foreign countries, were positive for nested PCR and the amplified products of showed 98% nucleotide sequence homology to the corresponding region of FGV. Six strains of FGV were isolated from these chickens in Japan. On the other hand, different ALV strain (Tyms_90) was isolated from brains of layers affected with fowl glioma and subcutaneous mesenchymal neoplasm in Japan. Another two Japanese fowls in Japan showed perineurioma and neurofibroma, respectively and a strain of ALV having endogenous-1(ev-1) locus was isolated from the latter. These results suggest that mutants of FGV spread in Japanese fowls in Japan, that small number of domestic and foreign chickens unrelated to the birds in the zoological garden A is infected with FGV, and that Japanese fowls are infected with other ALVs having ev-1 locus in the genome and showing oncogenicity in nervous system. In conclusion, it is speculated that FGV has appeared outside of the zoological garden A and a part of FGV-infected chickens were brought to this garden and used for breeding.
• Development of new vaccine strategies against Marek's disease virus with increased virulence

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2004 -2006 

  Author : OHASHI Kazuhiko, ONUMA Misao, OCHIAI Kenji, TAKAGI Michihiro

   

  The aim of this research is molecular characterization of recent isolates of Marek's disease virus (MDV) which can cause vaccine breaks in the field, in order to develop new vaccine strategies against theses very virulent MDV (vvMDV) by using a new MDV vaccine with the modified L-meq gene. Strains of vvMDV were isolated from wild birds such as white-fronted geese or from domestic chickens with vaccine breaks, and their nucleotide sequences were compared with those of known vvMDVs. The nucleotide sequences of the meq gene (a candidate oncogene of MDV) of recent isolates of MDVs in Japan showed high homologies with those of known vvMDVs, but some mutations previously unidentified were also found in these isolates. In addition, an additional EcoRI site was identified in the glycoprotein B gene of an isolate, strain Hokkaido, while a small deletion in the glycoprotein L gene, which had been frequently observed in vvMDV, was found in several isolates from wild birds and from chickens. In the next experiment, we analyzed the effects of these mutations on the transcriptional activation by the meq gene product, MEQ. MDV strains with increased virulence showed higher transcriptional activation by their MEQ, and mutations in the transcriptional activation domain of MEQ could be a determinant for the virulence of MDV strains. However, MEQ of strain Hokkaido, with newly identified mutations, did not display higher transcriptional activation by their MEQ. The L-meq gene product (L-MEQ), present in vaccine strains of apathogenic MDV, was shown to suppress both transcriptional activation by MEQ and replication in vitro of vvMDV, suggesting important roles of L-MEQ on the maintenance of latent phase. We also identified a new transcription variant of the meq gene, termed Δmeq, whose expression is up-regulated in Marek's disease tumor cells when apoptosis is induced. Δmeq inhibited the transcriptional activation by MEQ and L-MEQ, showing that Δmeq could play roles as a negative regulator of MEQ on apoptosis or reactivation of MDV from latent phase. Thus, Δmeq can be used to develop a new vaccine which induces programmed cell death only in transformed cells by vvMDV.
• サイトカイン遺伝子多型解析による疾患抵抗性家畜の選抜

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2004 -2005 

  Author : 大橋 和彦, 小沼 操

   

  前年度、腫瘍壊死因子(TNF-α)遺伝子プロモーター領域における多型が牛白血病の病態進行に関与する可能性を示唆した。本年度はTNF-α発現と牛白血病の病態進行との関係を詳細に検討した。白血病発症期あるいは持続性リンパ球増多症を発症した牛では、正常牛あるいは感染無症状キャリアー期の牛に比べ、膜結合型TNF-αの発現が高く、持続性リンパ球増多症由来リンパ球(主にCD5陽性細胞)では、発現したTNF-αが自発性増殖に直接関与していることを示した。またこの自発性増殖リンパ球に抗TNF-α抗体を添加するとその増殖が抑制されることも示した。このことはTNF-α遺伝子プロモーター領域における多型と、それに伴うTNF-α発現増強が牛白血病ウイルス(BLV)感染牛のリンパ球の自発性(腫瘍性)増殖能を少なくとも部分的に規定していることを示し、TNF-α遺伝子プロモーター領域における多型を解析することで、病態進行をある程度予想できることを示唆している。 TNF-αの作用は、細胞表面の2種類のTNF-αレセプター(TNF-RIとTNF-RII)で規定され、TNF-RIは細胞死の誘導に、またTNF-RIIは細胞増殖に作用する。そこでさらにTNF-αと牛白血病の病態進行との関連を検討するため、牛白血病の病態進行に伴うTNF-αレセプターの発現を解析した。その結果、持続性リンパ球増多症由来リンパ球では、非感染牛由来のものに比べてTNF-RI mRNAの発現に違いがないが、TNF-RII mRNAの発現が亢進していた。このことは、牛白血病の病態進行には、TNF-αの発現のみならず、TNF-αレセプター発現のバランスも密接に関与していることを示している。今後、TNF-αレセプターの発現を制御する遺伝子領域における多型の存在の有無や多型とレセプター発現との関連を解析する必要がある。
• Molecular characterization of Marek's disease virus genomes isolated from white-fronted geese

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2005 

  Author : OHASHI Kazuhiko, ONUMA Misao, TAKAGI Michihiro, ASAKAWA Mitsuhiko

   

  To characterize the genomes of Marek's disease virus serotype 1 (MDV 1) isolated from wild geese, feather tips and blood samples were collected from 200 pintails in Hokkaido, 63 bean geese (Anser fabalis), 15 canada geese (Branta canadensis), and 50 white-fronted geese (Anser Albifrons) in the fareast area of Russia during 2003-2005. Samples were also collected from 147 white-fronted geese at Hokkaido during 2003-2005. Total cellular DNAs were extracted from these samples, and the meq gene-specific nested PCR was performed to detect the MDV 1 genome. The MDV genome was not detected from pintails, but 25-30% of samples from thses geese were positive for the meq gene. These results showed that MDV 1 is present in wild goose populations though no clinical signs were observed in these geese. In the next experiment, nucleotide sequence analysis of the MDV1 genomes obtained from white-fronted geese was done to estimate the pathogenicities of the MDV1 strains. The nucleotide sequences of the meq genes of the MDV1 strains were very similar to those of very virulent MDV1 reported previously. In addition, a deletion in the glycoprotein (g) L gene, which was reported in the very virulent MDV1, was also found in the MDV1 strains from white-fronted geese. These results suggested that the MDV1 strains detected in white-fronted geese are very virulent. However, nucleotide sequence of the gB gene of goose-derived MDV1 was slightly different from that of very virulent MDV1 isolated from chickens in the Hokkaido area, showing that the MDV1 transmission between chickens and geese is not so frequent at the present time. It should be noted that the meq gene of one MDV1 strain was very similar to that of very virulent MDV1 isolated from chickens in the Hokkaido area, and it will be necessary to study larger numbers of samples from goose populations.
• Genetic diversity of bovine lentivirus distributed in south Asian countrirs

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2005 

  Author : ONUMA Misao, OHASHI Kazuhiko

   

  To understand the genetic diversity of bovine lentivirus (BIV), we have examined serological survey of BIV infection in cattle in Hokkaido and found 10% of positive reactors. From the seropositive animals, 3 BIV were isolated and DNA samples were obtained from peripheral blood lymphocytes of these positive animals for the detection of BIV proviral DNA by nested-PCR to detect a part of the pol gene. A 298-bp pol region fragment specific to BIV proviral DNA was amplified. Nucleotide sequences of the amplified fragments from Japanese BIV field isolates had 99% homology with American isolate, R-29. Amplification of the C2, V1, V2 and C3 regions of the env gene of Japanese BIV isolates were carried out by nested PCR. All 3 isolates seemed to be smaller, several base substitutions were observed in the V1 region and deletions were found in the V2 region of the env gene when compared to that of American R-29 isolate. In Cambodia, samples from 544 cattle and 43 water buffalo were obtained and BIV seropositive rate were 26% in cattle and 17% in water buffalo. Five BIV proviruses were obtained from seropositive animals. Nucleotide sequence of pol region was highly conserved when compared to that of American and Japanese BIV. In contrast, env region of Cambodian BIV field isolates seemed to be smaller than those of American isolates. Nucleotide substitutions which alter amino acid sequences were observed in the V2 region of Cambodian isolates. Total 262 serum samples were obtained from cattle in Zambia and seropositive rate was 11.4%. Four BIV proviruses were obtained by nested PCR. Nucleotide sequences of pol region of these proviruses were highly homologous (over 97%) to that of American and Japanese isolates.
• Oncogenesis of retrovirus induced-glioma and the development of animal model

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2005 

  Author : OCHIAI Kenji, OHASHI Kazuhiko, ONO Etsuro

   

  The cause and pathological features of so-called fowl glioma (astrocytoma) had not been determined. We have suggested that the cause is a subgroup A of avian leukosis virus (ALV). The subject of this research project is to clarify the pathogenicity of fowl glioma-inducing virus (FGV) and to analyze the molecular biological role of env gene and LTR in the neuropathogenicity. At first, chickens experimentally infected with FGV were pathologically examined. Whereas other isolated ALVs generally induce hematopoietic neoplasms, FGV was likely to replicate especially in brain and heart and induced nonsuppurative encephalitis and myocarditis, perineurioma, cerebellar hypoplasia as well as astrocytoma in nervous system. Genome sequence analysis suggested FGV to be combined with several virus strains. The U3 region of the LTR had a few deletions and several point mutations compared to that of other ALVs (EMBL, GenBank, and DDBJ nucleotide sequence databases under the accession number AB112960). The promoter activity of LTR also showed subtle differences, which may be related to the induction of glial neoplasm. In addition, histopathology of the 51 affected brains suggested the presence of a histological variant composed of diffuse proliferation of astrocytes and indicated that the disease entity should be named "retrovirus-induced astrocytoma" of chickens. To examine the promoter activity of FGV LTR in vivo, 13 lines of transgenic mice expressing a transgene under the control of the LTR promoter were generated and the tissue specificity of the expression was analyzed by monitoring mRNA in each line using the reverse transcription-polymerase chain reaction. The results indicated that the FGV LTR promoter was capable of driving a stable transgene expression especially in the central nervous system and testis although the LTR acted as a panspecific promoter in vivo.
• Molecluar mechanism of propagation of abnormal prion protein in the cells

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2005 

  Author : HORIUCHI Motohiro, INABA Mutsumi, OHASHI Kazuhiko, MAEDA Akihiko, FURUOKA Hidefumi

   

  In this research, we studied on the molecular mechanism of prion propagation in the cells through analyses of the effect of compounds that inhibit PrP^ formation and identification of host factor(s) and microenvironment that are involved in prion propagation. Four different anti-PrP antibodies that react with PrP^C on the cell surface inhibited PrP^ formation in cells persistently infected with prion. The antibody-PrP^C complex on the cell surface was not internalized efficiently and tended to retain on the cell surface. These results suggest that anti-PrP mAb antagonized PrP^ formation by interfering with the regular PrP^C degradation pathway. In addition, we screened synthesized sulfated glycosides for the inhibition of PrP^ formation and found 4-sulfo-N-acetylglucosamie and 6-sulfo-N-acetylglucosamine inhibited PrPSc formation in prion-infected cells. These sulfated glycosides accelerated the endocytosis of PrP^C and reduced a total amount of PrP^C, while sulfated glycosides that were not inhibited PrP^ formation did not reduce the total amount of PrP^C. These results indicated that sulfated glycosides and glycosaminoglycans inhibited PrP^ formation by facilitating the degradation of PrP^C. We established subclones of Neuro2a (N2a) mouse neuroblastoma cells and distinguished prion-susceptible and non-susceptible subclones. One non-susceptible subclone, N2a-1, expressed PrP^C as the same level as parental N2a and other prion-susceptible subclones. There was no difference in the binding of PrP^ to the susceptible and non-susceptible subclones. Presence of N2a-1, which expresses PrP^C but is resistance to prion propagation, indicated the involvement of host factors other than PrP^C in prion propagation. To identify such host factors, the gene expression profiles between N2a subclones were analyzed by DNA microarray. We selected 36 and 18 genes, which expressed more than two-fold in prion susceptible subclone N2a-5 and prion resistant subclone N2a-1, respectively. We assessed the influence of these genes on prion susceptibility by reducing the gene expression by siRNA technique and found that siRNA against F2, Al, and C5 genes inhibited prion propagation in prion-infected N2a-5 cells.
• 組み換えH鎖抗体を用いた血液脳関門デリバリーによる脳炎の治療

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2003 -2004 

  Author : 小沼 操, 杉本 千尋, 大橋 和彦

   

  抗体と毒素を人工的に結合させたイムノトキシンは癌治療に注目されている。そこで単クローン抗体産生細胞より抗体H鎖およびL鎖超可変領域のCD領域をクローニングし、遺伝子工学的に一本鎖抗体を構築した。一方、トキシンとしては緑膿菌から毒素遺伝子をクローニングし、組み換え緑膿菌毒素(PE38KDEL)を構築して一本鎖抗体と結合させイムノトキシンを作出した。このイムノトキシン遺伝子を大腸菌発現系を用い組み換え蛋白として発現させた。今回用いた単クローン抗体は鶏マレック病ウイルス(MDV)gBに対するものであり、MDVgB遺伝子発現細胞に対する作用を調べた。その結果、組み換えイムノトキシンはMDVgB発現細胞と特異的に結合し、緑膿菌毒素により細胞障害作用を示した。また組み換えイムノトキシンをMDVに処理するとMDVによるプラーク形成が顕著に阻害された。この成績はイムノトキシンが抗ウイルス活性のあることを示している。今後一本鎖抗体に結合させたイムノトキシンの抗腫瘍活性についても検討する予定である。 一方、リャマとラクダの免疫系を知る目的でリャマとラクダのサイトカイン遺伝子の解析を行った。方法は、末梢血リンパ球のmRNAから作成したcDNAより、牛・豚等の既知サイトカイン遺伝子の蛋白質コード領域の塩基配列をもとにプライマーを設計し、PCR法で増幅する方法である。この方法でこれまでにIL-1,2,4,6,8,10,12の他、IFNγ、TNFαなど多くのサイトカイン遺伝子を明らかにした。またReal-time PCRによる定量法を確立しラクダでのブルセラ菌感染に対するサイトカインプロファイルの変化を明らかにした。すなわちブルセラ菌生ワクチン接種ラクダではTh1系サイトカインの顕著な活性化がみられた。
• Search for biological active molecules from tick salivary glands

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2004 

  Author : ONUMA Misao, OHASHI Kazuhiko

   

  To obtain biological active molecules from tick, a cDNA library was constructed from semi-fed female Haemaphysalis longicornis salivary glands. Randomly selected clones were sequenced and total of 653 sequences were analyzed by bioinformatic programs including NCBI blastX. The sequences were assembled into 321 non-redundant clusters. About 35 % of the sequences showed significant similarity to known genes in homology search against non-redundant protein database and suggested as function predictable genes whereas rest of the 65 % had no homologs. Two thirds of the function predictable sequences were suggested to be so-called house keeping genes. Among non-house keeping genes, function annotated genes which we found were as follows ; several protease inhibitors, two metalloproteases, anti-thrombin type coagulation inhibitors previously identified from H. longicpornis and a homolog of D.andersoni derived predicted immunosuppressant protein (Da-p36). Recombinant of homolog protein of Da-p36 showed an immunosuppressive activity against bovine lymphocytes. These proteins may play important roles during tick feeding and are expected to be novel tick vaccine antigen candidates.
• Molecular mimicry of carbohydrate receptors and its application for the control of infectious diseases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2001 -2003 

  Author : OHASHI Kazuhiko, TAKAGI Michihiro, SUGIMOTO Chihiro, ONUMA Misao

   

  Many kinds of infectious agents, such as viruses and bacteria, invade target cells through their binding to carbohydrate receptors expressed on the target cell surfaces. Since their specificities to these carbohydrate receptors do not change even after these infectious agents undergo antigenic shift or mutations, it would be possible to use these carbohydrate receptors for the protection from these infectious diseases. However, the preparation of a large amount of carbohydrate receptors is technically difficult. Thus, in this study, using the New castle disease virus (NDV)-carbohydrate receptor (sialic acid) interaction as a model, a search for peptide surrogates which can mimic the carbohydrate structure of the receptor was performed, and their protective effcicacies were evaluated against NDV infection. Three individual peptide sequences, EVSHPKVG, WVTTSNQW and SGGSNRSP, which have potentials to bind to hemagglutinin-neuraminidase of NDV were identified by the biopanning method using phage display system. The binding specificities of these peptides presented on phages were confirmed by the ELISA competition assay using chicken antiserum to NDV. The synthetic peptides designed based on the results did not inhibit the hemagglutination by inactivated NDV, but partially neutralized the infection of NDV in vitro. In the future, it will be necessary to modify these peptides sequences, for example substitution of amino acids, to improve their abilities to bind to NDV and to inhibit NDV infection. It is also important to examine the protective efficacies of them against NDV in vivo for clinical application.
• Development of anti-tick vaccine

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2003 

  Author : ONUMA Misao, SUGIMOTO Chihiro, OHASHI Kazuhiko, HATAKEYAMA Hideki

   

  Maintenance of mammalian homeostatic balance is a multi-faced process involving an interaction of many proteins, however the need for a balance and coordinated interaction of many proteins, however the need for a balance and coordinated interaction between members of the serine proteinase super family and their inhibitors, known as serpins occupy a central role. In mammalian systems where extensive data exists, both serine proteinases and serpins are known to regulate such important functions as the blood coagulation cascade, fibrinolysis, food digestion, fertilization, inflammatory and immune responses. Imbalances or deficiencies in function of either serine proteinases or serpins have resulted in diseases. Ticks encode proteinases or serpins to disrupt the host homeostatic balance to their advantage. For this reason, we examined the molecular cloning of tick-encoded serpins as a candidate for tick vaccine. By using rapid amplification of the cDNA (RACE method), two genes (HLS l and HLS 2) from H. longicornis were cloned. Both recombinant HLS l and 2 showed the anti-coagulant activities. Vaccinaion of rabbits with either recombinant HLS 1 or 2 expressed in Escherichia coli resulted in significant anti-tick immunity. At present, we have characterized 4 tick salivary gland proteins whose recombinant products were effective against tick. We are planning to use these 4 cocktail vaccine for prevent the transmission of pathogens.
• Analysis of tick-encoded immunoregulatory serine proteinase inhibitors (serpins)

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2001 -2002 

  Author : ONUMA Misao, YAMASAKI Masahiro, OHASHI Kazuhiko

   

  Ticks are obligate ectoparasites that infest mammals, birds, repltiles, and amphibians and are found in many region of the world where they are the major vectors transmitting a great number of pathogens of human and animals. In Japan and other East Asian countries, Haemaphisalis longicornis, a tick commonly infesting cattle and dogs is a major vector of Theileria sergenti/buffeli, which are economically important disease of cattle. In the livestock industry ticks, the importance of ticks is their role as vectors of disease pathogens of livestock. Suppression of the tick vector population is considered as the method of choice in the absence of vaccines to target specific diseases transmitted by ticks. Several disadvantages associated with the use of acaricide which is the current method of choice to control ticks have necessitated the need to develop alternative methods for the tick control. Host immunization against ticks is one of the most promising method whose success is dependent on the identification and characterization of tick vaccine candidates Serpins may represent one of the most interesting target antigens for tick vaccine development because of their role in regulation of several physiological functions such as the blood clotting cascade, clot resolution, the inflammatory response and complement activation. In humans, dysfunctions or deficiencies of serpins have resulted in diseases. Given the critical role that serpins play in physiology of several organisms, we proposed that ticks are likely to use serpins to disrupt defensive host processes and therefore could be considered as potential candidates far anti-tick vaccines. We have previously characterized two tick salivary gland proteins whose recombinant products were effective against tick. The focus of our research is to characterize additional protective antigens which can be used in cocktail with our previously characterized antigens. Recently, we succeed to the molecular cloning of H. longicomis serpin gene and the recombinant protein suggest the possible use of the tick vaccine.
• THE CHARACTERISTIC OF SO-CALLED FOWL GLIOMA, WHICH IS A UNIQUE CENTRAL NERVOUS SYSTEM NEOPLASM IN ANIMALS CAUSED BY A RETROVIRUS

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2001 

  Author : OCHIAI Kenji, OHASHI Kazuhiko

   

  So-called fowl glioma is characterized by multiple nodular gliomatous growths associated with disseminated nonsuppurative encephalitis. The causal agent is still unknown. The purpose of the present study was to examine the relationship between so-called fowl glioma and an avian leukosis virus (ALV) as the causative candidate. One-day-old chicks of Japanese bantams and specific pathogen-free (SPF) chickens were intracerebrally inoculated with the brain homogenate or culture supernatant from the affected bantam. Most of birds in the inoculated roups showed nonsuppurative encephalitis, and the 18 bantams (82%) and five chickens (28%) developed multiple nodules consisting of aggregations of astrocytes in the cerebrum. These astrocytes immunohistochemically had ALV antigen. By Southern blot analysis, the ALV proviral sequence was detected both in DNA from the brains of the inoculated birds and in DNA from the inoculum. Tadpole-shaped particles, approximately 100 nm in diameter, were detected in the inoculum, and budding of the particles was noted on the cell surface of the infeced CEF. The env gene sequence of the DNA from the CEF was most similar (> 92% identity) to the env gene of ALV-B subgroup. Nonsuppurative myocarditis was also observed in the inoculated SPF chickens. The gliomatous nodules at the initial stage ultrastructurally consisted of neoplastic proliferation of astrocytes and mild desmoplastic reaction of the adventitial fibroblasts. Perineurioma-like neoplasm, which were also suspected to be related with the ALV, was rarely observed in the peripheral nerves of the birds. The present study demonstrated that so-called fowl glioma is a viral disease caused by a subgroup A or B of ALV. Our results suggest that the infected birds reveal nonsuppurative encephalitis and myocarditis immediately after the infection and most of these birds develop glioma/gliomesenchymal mixed tumor or peripheral nerve neoplasm since 4 months of age.
• Molecular mechanisms of diversity of theileria parasite genes distributed in middle and South America

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2001 

  Author : ONUMA Misao, TSUJI Masayoshi, IWANAGA Toshihiko, SUGIMOTO Chihiro, KAKUDA Tsutomu, OHASHI Kazuhiko

   

  Theileria sergenti is a tick-borne protozoa of cattle. Benign bovine theileria parasites, which are presumed to be of the T. sergenti / buffeli / orientalis group are distributed in Asian countries. The parasites have the major piroplasm surface protein (MPSP) gene which is one of the means of parasite evasion of host immunity. Ticks represent one of the most important groups of anthropods, and transmit theileria parasites. To compare the tick salivary gland derived molecules among ticks distributed all over the world, molecular cloning of saliva derived molecules, such as calreticulin, a Ca<++>-binding multifunctional protein showing anti-complement and anti-thrombin antivities. In this study, the calreticulin gene from a tick species Rhipicephalus appendiculatus, which is a major vector of T. parva causing East coast fever in Africa was clearified. A calreticulin cDNA clone was obtained from a R. appendiculatus cDNA library by the 3' and 5' RACE methods. The mucleotide and deduced amino acid sequences of the calreticulin genes showed high homology with those of known calreticulin genes of other tick species, Amblyoma americanum and Boopiylus microplus. The expression of this gene is now under investigation. Next, we examined the molecular cloning of serine proteinase inhibitors (serpins). Serpins are known to regulate important functions in maintenance of homeostasis, such as the blood coagulation cascade, fibrinolysis, food digestion and fertilization. Three and 4 serpin cDNAs were obtained from H. longicornis and R. appendiculatus cDNA libraries, respectively. These clones showed high homology with known serpin genes of other ticks or mammalian species. Expression of these genes in E. coli and examination of biological activities are now under investigation.
• Preparation of the recombinant Marek's disease virus which can induce apoptosis in tumor cells and its application for an anti-tumor vaccine.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1999 -2000 

  Author : OHASHI Kazuhiko, TAKAGI Michihiro, SUGIMOTO Chihiro

   

  Marek's disease virus (MDV) causes malignant lymphomas in chickens, but the molecular mechanism of the transformation remains unknown. Strains of MDV are classified into 3 serotypes based on their pathogenicity : only serotype 1 strains of MDV (MDV1), except for attenuated vaccine strains (i.e. CVI988), are oncogenic. Although Marek's disease (MD) is effecivecontrolled by vaccination, the protection mechanism by live vaccines is not known, and emergence of highly virulent MDV which can cause vaccine breaks has been reported. For this reason, a recombinant MDV which can induce tumor-specific apoptosis was constructed for the development of a newly invented anti-tumor vaccine in this study. In addition, differencein the structure of the meq gene has been identified between oncogenic and attenuated MDV1. Since the meq gene is known as an MDV oncogene, detailedanalysis on the change in the viral genome was done to obtain the insight into the role of the meq gene on the transformation by MDV. A gene cassette to express an apoptosis-inducing gene, the VP3 gene cloned from chicken infectious anemia virus, under the control of the meq promoter was constructed. The meq promoter was chosen since the meq gene has been known to be expressed only in tumor or MDV latently-infectedcells. When this gene cassette was introduced into an MD tumor-derived cell line, MSB1, viability of MSB1 was decreased within 24 hours, and DNA fragmentation was also detected in total cellular DNA prepared from MSB1. However, no change in the cell viability was observed in chick embryo fibroblast (CEF) transfected with this cassette. These results suggest that this gene cassette could be used for the tumor-specific induction of apoptosis. To study genetic differences between oncogenic and nononcogenic MDV1, polymerase chain reaction (PCR) was performed to amplify the meq gene in the viral genome. In addition to a PCR product including the native meq open reading frame (ORF), a 1.2-kb product was amplified from the DNA sample prepared from CEF infected with CVI988, but not with oncogenic strains, RB1B and Md5. Sequence analysis showed that a 178-bp sequence was inserted to the meq ORF of CVI988 (L-meq gene). This L-meq gene was also detected in an oncogenic MDV1, strain JM, which had been passaged for more than 70 times in vitro. Since this insertion could potentially cause a frame shift in the meq ORF, the resultant ORF could encode for the Meq protein with a different transacti vator domain. The L-meq gene was also detected in the MDV genome prepared from MD tumor-derived cell lines. The biological significance and function of this long meq gene is not yet elucidated, but this insertion may also contribute to the changes in the biological properties of different MDV1 strains.
• Molecular approach to analyze strategies of pathogens in wildlife for acuierement of new host

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1999 -2000 

  Author : SUGIMOTO Chihiro, INOUE Noboru, FUISAKI Kozo, NAGASAWA Hideyuki, FUKISHI Hideto, TAKAI Shinji

   

  Some of pathogens are communicable among humans, domesticated and wild animals and cause various diseases. In this study, we investigated protozoan, bacterial and vial pathogens from wild or domestic animals and their environment in order to determine whether they can acquire new host range by crossing biological and environmental barriers. Theileria parasite in ruminants are devided into highly pathogenic and apathogenic species. Wildlife harbor parasites of both categories, but they are not closely related to those from domestic animals on the basis of ribosomal RNA gene analysis. Rhodococcus equi with virulent plasmid are isolated from soil in horse farms, but the plasmid types are similar to those isolated in isolate from European horses. Therefore, pathogenic strains of R. equi may have been brought from Europe and colonized in soil in Africa. Herpesviruses were demonstrated in blood samples on 5 species of antelopes and rhinocerous. The isolate from impala is different from those isolated from domestic ruminants. Taken together, it is concluded that pathogens investigated in this study have been associated and coevolved with their original hosts, and that they are not easily able to acquire new host even under environments where wildlife and domestic animals are in close contact with each other.
• Molecular mechanisms of diversity of theileria parasite genes and control of the disease

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1999 -2000 

  Author : ONUMA Misao, OHASHI Kazuhiko, SUGIMOTO Chihiro

   

  Theileria sergenti is a tick-borne protozoa of cattle. Benign bovine theileria parasites, which are presumed to be of the T.sergenti/buffeli/orientalis group are distributed in Asian countries. The parasites have the major piroplasm surface protein (MPSP) gene which is one of the means of parasite evasion of host immunity. T.sergenti has 4 chromosomes similar to T.parva.Most of genes used for molecular karyotype showed a conserved synteny between T.sergenti and T.parva. To underatand the genome mapping of theileria parasites, characterization of bovine genes expressed in T.parva transformed T-lymphoblastoid cell lines was performed by using expressed sequence tag (EST) sequencing approach of random cDNA clones from libraries. Single pass sequencing was done on 394 cDNA clones and the sequences search for significant homolgies in GeneBank, EBML and DDBJ databases. About 141 clones showed significant homology to genes in the databases. The majority of these ESTs (47) showed homology to human sequences and only a few (19) matched bovine sequence. 126 clones did not match database sequences and may represent novel transcripts. The EST sequencing approach has recently contributed tremendously to discover of new genes in several parasites. Thus, we performed the sequencing of 461 cDNA clones from the schizont stage of T.parva. The cDNA colnes were mapped to the 4-T.parva chromosomes and 20 of these clones were mapped to the 33 Sfil fragments of the T.parva genome. EST sequencing demonstrated its potential in the identification of new genes in this stage of the parasite and has contributed greatly to the addition of ESTs into the T.parva database. The sequence data reported is invaluable for T.parva genome mapping and in the comparative genome mapping of theileria parasites.
• Molecular analysis of intraerythrocytic parasite population dinamics and acquirement of biological diversity

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -2000 

  Author : SUGIMOTO Chihiro, IGARASHI Ikuo, FUJISAKI Kozo, NAGASAWA Hideyuki, INOUE Noboru

   

  During the course of Theileria orientalis infection in cattle, parasite population bearing different major piroplasm surface protein (MPSP) genes appear by turns, which may be one of immune evasion mechanisms of the parasite. Therefore, analysis of parasite population is important in understanding pathogenesis and developing control methods. In this study, degenerated gel electrophoresis methods for the analysis of parasite subpopulations at genetic level has been developed. By using this method, mutations within MPSP gene of C-type allele could be detected. From blood samples from cattle and buffalo, which were possibly infected with different Theileria, species was analyzed by rRNA gene-targeted PCR analysis and revealed mixed infection with 4 different species. The expressed sequence tag analysis of T. parva and EST mapping on chromosomes was carried out and 461 ESTs have been mapped. We further focused on tick saliva and salivary glands where tick, mammals and Theileria interact. Several proteins and their genes including cement proteins, proteinase inhibitors have been characterized. Probably, some of these tick proteins affect host protective responses and help parasite to invade into mammalian hosts.
• PLANT EXPRESSIONS OF CYTOKINES AND IIST APPILICATIONS FOR ANIMAL DISEASE CONTROL

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -1999 

  Author : SUGIMOTO Chihiro, UYEDA Ichiro, OHASHI Kazuhiko, ONUMA Masao, MATSUMURA Takeshi

   

  The aim of this study is to express bioactive animal proteins in plants and to apply the recombinant proteins for animal disease control. We used transgenic plant and plant viral vector stems for expression of animal genes. By Agrobacterium-mediated transformation, we introduced cDNAs of two subtypes of human interferon α (IFNα2b and IFNα8). Expression of the introduced genes was confirmed by northern blot analysis and enzyme immunoassay. Ten to 500 IU/g of IFN8 were detected in transgenic potatoes. In bovine leukemia virus infection, tumor necrosis factor α was revealed to be involved in pathogenesis, especially control of viral burden in infected animals. We also clones genes for a tick salivary grand protein, tick serine proteases, and a piroplasm surface antigen that are expected to be expressed in plant and used for method of disease controls. A plant expression system based on clover yellow vein virus has been developed by combining its cDNA and 35S promoter as pC1YVV. Expression of green fluorescence protein by this virus vector was confirmed. INF8 cDNA was introduced into this vector and its expression was tested. However, plant infected with this recombinant virus did not express any product as tested by enzyme immunoassay, provably because the viral vector had not retained the cDNA. Further investigation will be required for stable expression of animal genes in this system.
• Molecular evolution of Theileria parasites in Southern Asia

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -1999 

  Author : ONUMA Misao, OHASHI Kazuhiko, TUJI Masayoshi, SUGIMOTO Chihiro, TAKAHASHI Eiji

   

  Bovine piropurasmosis due to Theileria sergenti is a major cause of economic loss in grazing cattle in Japan. T. sergentii / buffeli / orientalis grop is referred to as bengin theileria parasites of cattle and is known to be distributed all over the world. Previously, we developed the allele-specific polymerase chain reaction (PCR) which specifically amplifies lach allelic from using 3 sets of olgonucleotide primers designed from nucleotide sequences of major Immunodominant primers designed from nucleotide sequences of major Immunodominant piroplasm surface protein (p32) genes of two T. sergenti stocks, Ikeda (1) and Chitose c and one t, buffeli (B) stok, Warwick, and appled the method to the field survey of benign theileria parasites in southern asian countries. During 2 years, we have collected blood samples from the following countries : Korea (Cheju island), Thailand, Cambodia, India, Paklstan, Vietnam and China. Parasites DNA was extracted and the DNA was analyzed by using allele-specific PCR. In Korea (Cheju island), all the isolates contained I type parasites as well as C type parasites. However In Thailand and Cambodia, Major parasites distributed there countries were Thai type parasites, different from T. sergentii /buffeli / orientalis. C type parasites and T, buffeli type parasites were also isolated from Thailand and Cambodia. In India and Pakistan, major theileria parasites are known as T. annulata. We expected to isolate benign theileria parasites, however, we could not isolate benign theileria parasites. The present data showed that benign theileria parasites in east and Southern asian countries were mixed parasites populations.
• マレック病腫瘍化におけるアポトーシス関連及び癌抑制遺伝子の発現動態の解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1998 -1999 

  Author : 大橋 和彦

   

  マレック病ウイルス(MDV)の腫瘍化における発癌抑制遺伝子やアポトーシス関連遺伝子の役割を解析するため、特にbcl-2、bcl-x遺伝子に関して検討した。そしてこれまで、1)マレック病腫瘍株化細胞、MSB1、MTB1やMDV感染鶏由来CD4^+T細胞においてはアポトーシス抑制遺伝子であるbcl-2遺伝子の発現が抑制されていること、2)しかしbcl-2同様にアポトーシスを抑制するbcl-xLが代償的にMSB1.MTB1では発現していることが判明した。これらのことはMDVによる腫瘍化(少なくとも腫瘍化の維持)にはbcl-2よりもbcl-xL遺伝子の発現がより大きく寄与していることを示唆している。しかしながら、可移植性マレック病腫瘍株化細胞、RP1、HP1等ではbcl-2遺伝子の発現が認められ、bcl-2遺伝子が腫瘍の悪性化(腫瘍株の可移植性を指標として)に関与していることも示唆された。そこでbcl-2遺伝子の可移植性に対する役割を検討した。PCR法にて鶏bcl-2α遺伝子cDNAをクローニングし、ネオマイシン耐性マーカーを持つ真核細胞発現ベクター(pCIneo)に組み込み、bcl-2遺伝子を発現していないMSB1細胞に遺伝子導入してbcl-2遺伝子発現MSB1細胞を樹立した。bcl-2遺伝子の発現はノーザンブロット法にて確認した。そして現在、この細胞をマレック病感受性鶏の翼下皮下に接種して可移植性が付与するかどうかを検討している。 さらにMDV感染鶏由来CD4^*T細胞における他のアポトーシス関連遺伝子、DAD1、ITA、p58等の発現をノーザンブロット法で検討したが、対照鶏との間に有意な差は認められなかった。今後さらに腫瘍株化細胞における発現を検討する。
• Immunological modulation and control of infections diseases by cytokaines

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1997 -1999 

  Author : ONUMA Misao, KIRISAWA Rikio, WATARAI Sinobu, SUGIMOTO Chihiro, OHASHI Kazuhiko, YASHIRO Masahiko

   

  In order to understand the role of cytokines in protective immune responses against bovine leukemia virus (BLV) infection, 12 sheep were inoculated with BLV and the levels of cytokine mRNA expressions in the peripheral blood mononuclear cells (PBMCs) were analyzed. The mRNA expressions of IFN γ, IL2, IL4 and IL10 in PBMCs were downregulated after BLV infection in all sheep. However, the expressions of tumor necrosis factor α (TNF α) mRNA was up-regulated only in BLV-resistant sheep in which BLV were eliminated, though down-regulated in -susceptible sheep in which BLV propagated. This may suggest that TNF α play an important role in the protection against BLV infection during the early phase of infection. Additionally, the mRNA expressions of TNF α receptors type I(TNFR I) and type II (TNFR II) were investigated in BLV-resistant and -susceptible sheep PBMCs. Interestingly, the mRNA expression of TNFR I was significantly downregulated only in the BLV-susceptible sheep. By the use of in vitro stimulation with TNF α, higher stimulation index was observed for PBMCs from BLV-susceptible than -resistant sheep. In addition, flowcytometric analysis showed that membrane form of TNF α, which is thought to be one of the ligands which induce B-cell activation, was expressed more on PBMCs from BLV-susceptible than those from -resistant sheep. These results suggest that TNFα and its receptors are involved in the development of BLV-induced B-cell proliferation in sheep.
• GENOME ANALYSIS OF THEILERIA PARASITES

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1997 -1998 

  Author : SUGIMOTO Chihiro, NENE Vishnash, BISHOP Richard, MORZARIA Subhash, OHASHI Kazuhiko, ONUMA Misao

   

  This project was carried out to analyze genomes of Theileria parasites Eight hundred cDNA clones of Theileria parva was partially sequences to use as expressed sequence tags (ESTs). Some of them were mapped on chromosomes of T.parva. A bacterial artificial chromosome (BAC) library, each of which clones contained 50-200 kilobase pairs of T.parva chromosome fragments, was constructed. The library consisted of 3600 clones that theoretically covered entire T.parva genome. BAC clones that contained gene(s) essential for parasite growth including sporozoite surface protein were identified, which will be useful to analyze genome structures flanking these genes. 4 chromosomes of T.sergenti were identified. Size of each chromosome and total genome size of this species was almost same as those of T.parva. Comparative mapping analysis of several homologous genes on T.parva and T.sergenti chromosomes revealed that syntenies among some of the genes are not conserved, which indicated difference of genome structures between these two Theileria species. Although further studies are required to determine whole chromosome structure of T.parva, the results of this project provide a basal information about genome structures of Theileria parasite by establishing BAC library, obtaining ESTs, and comparing two theilerial genomes.
• Molecular escape mechanisms of Theileria parasites

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1995 -1998 

  Author : ONUMA Misao, OHASHI Kazuhiko, SUGIMOTO Chihiro

   

  Theileria sergenti is a tick-borne protozoa of cattle. T.sergenti infected calves show persistent infection, possibly over their life span, but the mechanisms by which the parasites evade host defense mechanisms are not clearly understood. The complete life-cycle of T.sergenti in the mammalian host and tick vector remains unclear, the parasite may undergo a sexual stage in tick gut. To study the population changes during persistent infection in cattle and vector ticks, we have inoculated T.sergenti which consist of 2 parasite populations with C and I type allele of p32 and analyzed the population changes by allele-specific PCR to differentiate between individual types of parasites. Changes in a dominant parasite population were demonstrated during transmission from calves to the tick vector and from infected ticks to calves. The antigenic diversity which may be produced at the sexual stage in tick vector. The analysis of chromosome provide us the basic information to study the generation of genetic and antigenic polymorphism. Chromosome analysis of T.sergenti showed 4 chromosomes. From the results of Southern blot analysis, p32 gene was located on the 1st chromosome, p23, heat shock protein 70 (HSP 70) and cystein proteinase genes were on the chromosome 2 and ribosomal RNA gene was on chromosomes 1 and 3/4.
• ANALYSIS OF INTRACELLULAR ADAPTATION MECHANISMS OF PROTOZOAN PARASITES AND HOST ELIMINATION

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1996 -1997 

  Author : SUGIMOTO Chihiro, OHASHI Kazuhiko

   

  This project was aiming at clarifying mechanisms of parasite adaptation to host cell environment which will lead us to establish control methods of intracellular parasite infections. We cloned a gene encoding small subunit of ribonucleotide reductase (R2) which is involved denovo synthesis of nucleotides. To clone the gene from Theileria sergenti, oligonucleotide primers for polymerase chain reaction (PCR) were designed based on the corresponding gene sequence of T.annulata. As a reverse transcription-PCR product contained a partial fragment of the gene, 5'-3' RACE (rapid amplification of cDNA ends) were carried out to obtain a full length mRNA sequence. A genomic clone containing R2 gene was also obtained by screening of T.sergenti genomic DNA library with cDNA probe. Sequence analysis of the genomic DNA clone revealed that the R2 gene contains at least 2 introns. Predicted amino acid sequence of R2 C-terminal portion showed significant difference from that of host R2, indicating that inhibitory peptides specific for the parasite enzyme would be designed. Molecules expressed in erythrocytic stage of Babesia caballi and B.equi were also characterized. Libraries of cDNAs from B.caballi and B.equi merozoites were screened with infected horse sera. From B.equi cDNA library, two genes encoding a glutamic acid-rich protein (GARP) and ribosomal protein (L10) were obtained. GARP contained motifs rich in glutamic acid which showed structural similarity to surface molecules expressed in intraerythrocytic stage of malaria parasites. A gene for B.caballi rhoptry-associated protein-l (RAP-1) , one of parasite proteins which plays a central role in parasite invasion into host erythrocytes was also cloned. N-terminal amino acid sequences of veil proteins of T.sergenti were determined and compared to sequences of known erythrocyte proteins. As no homologous poplypeptides were found in data base search, veil proteins were concluded to be parasite-origin.
• Molecular evolution of Theileria parasites in Pacific area

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1996 -1997 

  Author : ONUMA Misao, OHASHI Kazuhiko, TSUJI Masayoshi, SUGIMOTO Chihiro

   

  Bovine pirolasmosis caused by Theileria sergenti cause of economic loss in grazing cattle in Japan. We found parasite stocks and isolates consist of genetically and antigenically mixed populations. We have developed allele-specific polymerase chain reaction (PCR) using specific primers to differentiate parasite populations. From entire major piroplasm surface protein gene sequences of two T.sergenti Stocks (C and I) and one Warwick stock of T.buffeli (B), 3 sets of oligonucleotide primers were designed to amplify either of the 3 alleles by PCR.Amplified products by B-specific primers showed 2 different restriction enzyme patterns, thus B-type was further divided into the Bl-(T.buffeli type) and B2-types (parasites isolated from Japanese cattle but not from Australian cattle). By using this allele-spesific PCR,we found that in Japan the majority of bovines infected with benign Theileria parasites herbored mixed parasite population. For further analysis of parasite populations of T.sergenti/T.buffeli/T.orientalis, field isolates were collected from East Asian countries (Korea, China, Taiwan) where antigenically and genetically similar parasites to T.sergenti were distributed. Parasites were also collected from Australia, Thailand, USA and Italy. Parasite DNA was extracted from parasitized erythrocytes and tested by allele-specific PCR.All 5 isolates from China contained C-type parasites and one isolate was a mixture of I,C and B2-type parasites. The C-type parasites were detected in all l5 isolates from Taiwan and 4 of these also contained the B1-type. The I-type parasites were commonly found in Korean isolates and 28 out of 33 isolates were mixtures of I-and C-type parasite. Parasites isolated from Australian cattle showed mixed patterns of B1-type (T.buffeli) and C-type of T.sergenti. Theileria parasites isolated from Thailand were classified into C- type, and B1-type but unknown type (T.sp.) was also found. The Australian isolates and an Italian benign Theileria isolates were found to be a mixture of Bl and C-type of T.sergenti. In our preliminary experiments, apathogenic Theileria species collected from Thailand, China and USA seem to be unique and "yet-unidentified" Theileria parasite (T.sp.). The MPSP genes of these 3 T.sp.were amplified by the same specific primers. Nucleotide sequences of MPSP genes of these 3 T.sp.are quite similar but differ from that of T.sergenti/buffeli/orienta1is.
• ディファレンシャルデスプレイ法による鶏免疫関連分子の解析と病態変化への応用

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1996 -1996 

  Author : 大橋 和彦

   

  活性化された鶏胸腺細胞に発現するマーカーやサイトカイン及びその受容体遺伝子をディファレンシャルデスプレイ法により同定し、分子クローニングにより遺伝子構造を解析するとともに、その生物活性を保持した組み換え蛋白質の大量調製法を確立し、将来的にはワクチンとは異なった免疫賦活作用による家畜の感染防御法の開発に応用することを目的として行った。 マレック病ウイルス感染あるいは非感染の若齢鶏(0日〜14日齢)の胸腺を細切して浮遊細胞を調製してPMA及びIonomycin存在下で培養を行った。この操作で胸腺細胞の活性化あるいはアポトーシスを誘導することができる。なお薬剤存在下の培養時間は予備試験を行い12〜24時間と決定した。そして処理及び未処理の胸腺細胞より全RNAを抽出(Trisolを用いる)した後オリゴdTスピンカラムを用いてmRNAを精製した。 RNA map Kit (GenHunter社)を用いて、4群のオリゴdTプライマー(各塩基+dT)を用いて逆転写反応を行いcDNAクローニングを得た後、任意に設計された20種類の10merランダムプライマーと4群のオリゴdTプライマーを用いて標識dATP存在下でPCRを行った。この反応により理論的に相当種類(数千〜数万)のmRNAを増幅することができる。得られたDNAはポリアクリルアミドゲル電気泳動法後オートラジオグラフィーにより解析したが、薬剤未処理のものと処理のもので異なったパターンを示すDNA断片を数十個同定し、ゲルより切断後さらにDNAを任意のプライマーを用いて増幅した。現在これらのクローンが薬剤処理前後で発現パターンが変化するかどうかをノーザンブロットで検討している。
• Molecular epidemiology of Theileria infections in Africa : Characterization of parasites of wild and domestic animals.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1995 -1996 

  Author : SUGIMOTO Chihiro, OHASHI Kazuhiko, ONUMA Misao

   

  Lymphoblastoid cell lines infected with Theileria parva schizonts were established from cattle in Central Province of Zambia. The parasites were genetically and antigenically different from buffalo-derived T.parva (T.parva lawrencei) and also from cattle-derived parasites (T.parva parva) in Eastern Province of Zambia, as revealed by Southern blot analysis and indirect fluorescent assay by using a panel of anti-schizont monoclonal antibodies. Polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis of major piroplasm and shizont protein genes of Zambian isolates also confirmed the above-mentioned results. DNA samples were prepared from Theileria-infected African buffalo in South Africa and analyzed for the detection of Theileria by PCR based on small subunit ribosomal RNA (srRNA) gemes. They contained several species of Theileria, including T.buffeli (or sergenti) and other parasites unique in this animal species. Possible transmission of the former species among cattle and buffaloes are suggested. A major piroplasm surface antigen gene of Theileria parasite isolated from a sable antelope was amplified by PCR and its nucleotide sequence was determined. The parasite was phylogenically distinct from other Theileria species isolated from cattle and African buffalo. Babesia-like piroplasms were detected in erythrocytes of zebra in South Africa, and successfully cultured in vitro. They were morphologically similar to Babesia equi in horses. Further molecular characterizations of the zebra-derived parasites are now underway based on EMA-1 gene analysis. Several other piroplasms (Babesia or Theileria) were also detected in blood smears from other wild animal species including kudu, cheetah, giraffe, lion.caracol (wild cat in Africa). Some of the parasites have not been classified so that further molecular characterizations of the parasites based on srRNA genes are essential to clarify taxonomical and phylogenical relationships among parasites from wld animals and those from domestic animals.
• 鶏CD4:CD8遺伝子のクローニングによるマレック病ウイルスの癌化標的細胞の解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1995 -1995 

  Author : 大橋 和彦

   

  鶏CD8遺伝子ついては研究期間中に別のグループによりα、β鎖の塩基配列が決定された(Tregaskes et al.,J.Immunol.154:4485-4494,1995)。そこでこの塩基配列より、α、β鎖のORFを増幅するようなプライマーをデザインして既に調製しておいた鶏脾臓由来cDNAを用いてPCRを行い増幅された断片を直接クローニングした。クローニングした断片は制限酵素による切断試験でCD8 αおよびβ鎖であることを確認した。そしてこのCD8 αおよびβ鎖遺伝子をプローブとしてマレック病ウイルス(MDV)感染鶏から得られた脾臓や末梢血リンパ球中のCD8^+T細胞におけるCD8遺伝子の転写レベルを測定した。その結果、非感染対照鶏に比べMDV感染鶏ではCD8遺伝子の転写が著しく低下していることが示された。なお、CD8分子の発現低下はモノクローナル抗体を用いたフローサイトメトリーでも示されておりMDV感染鶏におけるCD8分子のDown-regulationは転写レベルで起こっていることが示された(Morimura et al.,J.Gen.Virol.in press,1995;Morimura et al.,国際獣医免疫シンポジウム、Davis,USA,1995)。現在はCD8 αおよびβ鎖遺伝子を各々大腸菌で発現させて得られた融合蛋白質を用いて種々の抗CD8モノクローナル抗体が認識する鎖を決定中である。 CD4遺伝子については未だ塩基配列の報告がないので現在までクローニングされた人、マウス、サル、犬等のCD4遺伝子間で相同性が高い部分からプライマーをデザインしてPCRを行い増幅されたDNA断片を直接クローニングしてその塩基配列を決定した。現在まで数種のクローンの塩基配列を決定したが他の動物CD4遺伝子と高い相同性を示すようなクローンは得られていない。今後この方法を継続すると同時にcDNAクローンを大腸菌または培養細胞で発現させ抗CD4モノクローナル抗体を用いてクローン選択する発現クローニングを行う予定である。
• 鶏Fas抗原遺伝子のフローニングによるマレック病ワクチンの作用機序の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1994 -1994 

  Author : 大橋 和彦

   

  現在鶏Fas遺伝子のクローニングを進めている。2日齢鶏の胸腺よりフェノール・グアニジン法により調製したRNAを用いてcDNAライブラリーを調製した。ライブラリーの作成には発現ファージベクターであるλEX/loxを用いた。このライブラリをジゴキシゲン(DIG)標識したマウスFas cDNAを用いてプラークハイブリダイゼーションによりスクリーニングして(約400,000プラーク)鶏Fas遺伝子cDNAのクローニングを試みた。ハイブリダイゼーションの条件はFas遺伝子が既にクローニングされているヒト、マウス、ラットにおいてもお互いの相同性約65%と低く、鶏ではさらに相同性が低いことが予想され、約55%の相同性を想定した。スクリーニングの結果、数種類の陽性クローンが得られ、2次および3次クローニングを行い、最終的に5個のcDNAクローンを得た。 また同時に上記の方法で鶏胸腺の他、脾臓、マレック病腫瘍由来株化細胞(MSB1)よりcDNAを調製して、ヒト、ラット、マウスFas遺伝子で相同性の高い塩基配列よりデザインしたプライマー(5′-GACCCAGAATACCAAGT-3′及び5′-TTCTGCTGTGTCTTGGA-3′)を用いてPCR法により鶏Fas遺伝子の増幅を試みた。その結果、サイズの異なる3種類の特異的バンド(約700〜300塩基対)が得られ、これらをpGEM-Tベクターに組み込んだ。 今後、これらの塩基配列を決定するとともに、これらのクローンをプローブとしてFas遺伝子の完全長を含むcDNAクローンをライブラリーよりスクリーニングする予定である。さらにゲノムを用いたPCR法によるクローニングも行っている。
• マレック病ウイルスによる腫瘍化の分子メカニズムの解析、ワクチン作用機序の解明

  Date (from‐to) : 1992
• 動物感染症に対する免疫応答の分子機序の解明

  Date (from‐to) : 1992
• Characterization of molecular mechanisms of immune responses against infectious diseases in animals

  Date (from‐to) : 1992