2008, 日本獣医学会賞　　　　　　　　　　　　　　　
Japan

Genomic characteristics and distinctive genetic polymorphisms of Marek's disease virus strains circulating in Japan.
Shunsuke Yamagami, Jumpei Sato, Yoshinosuke Motai, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi, Shiro Murata
The Journal of veterinary medical science, 03 Apr. 2025, [Domestic magazines]
English, Scientific journal, Marek's disease virus (MDV) is an oncogenic herpesvirus that causes malignant lymphomas in chickens (Marek's disease, MD). Although MD is currently well controlled by vaccination, field strains of MDV tend to increase in virulence, and sporadic outbreaks have been reported worldwide, including in Japan. Previously, we determined the whole-genome sequence of a Japanese MDV strain, Kgs-c1, isolated in 2014, and analyzed its genetic characteristics. However, to date, Kgs-c1 is the only MDV strain that has been subjected to whole-genome sequencing, leaving the possibility that the key genetic features of the Japanese strains remain undiscovered. Therefore, we conducted whole-genome sequencing of two MDV strains, Kgs-c2 and Sg-c1, isolated in 2016 and 2019, respectively, and analyzed their genetic characteristics. Consistent with our previous findings, the genomic sequences of the Japanese strains were classified into the Eurasian cluster, suggesting that the Japanese strains are genetically closer to the European and Chinese strains than they are to the US strains. In contrast, the Japanese strains exhibited genetic features different from those of pathogenic European and Chinese strains. Furthermore, polymorphisms in the meq and infected cell protein 4 (ICP4) genes, which are candidates involved in the enhanced virulence of MDV, were observed among the newly isolated strains and Kgs-c1. Thus, three MDV strains isolated in Japan had different genetic features from those in other countries. These findings emphasize the necessity of periodic monitoring of MDV field strains in order to determine shifts in their characteristics and effectively control MD.

Characterization of a lipocalin-like molecule from Dermanyssus gallinae as a potential vaccine antigen.
Fumiya Horio, Hikari Seo, Shwe Yee Win, Jumpei Sato, Yoshinosuke Motai, Shunsuke Yamagami, Takumi Sato, Eiji Ohishi, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi, Shiro Murata
Veterinary immunology and immunopathology, 283, 110921, 110921, 24 Mar. 2025, [International Magazine]
English, Scientific journal, Poultry red mites (PRMs, Dermanyssus gallinae) are hematophagous ectoparasites of chickens that pose a significant threat to the egg-laying industry. The emergence of acaricide-resistant PRMs raises the demand for alternative control approaches such as vaccination. However, several vaccine antigens have failed to suppress the growth of PRM populations in field trials due to difficulties in maintaining antibody levels. In ticks, the molecules exposed to the host, such as lipocalins, can facilitate antibody production, and are therefore considered advantageous as vaccine antigens. Therefore, we focused on a lipocalin-like molecule (Dg-Lipocalin) identified from an RNA-seq analysis reported by Fujisawa et al. (2020) and analyzed its exposure to the host and potential as a vaccine antigen. Western blotting using 500-fold diluted plasma of chickens from PRM-contaminated farms revealed the presence of antibodies against Dg-Lipocalin, suggesting its exposure to the host. To evaluate its potential as a vaccine antigen, PRMs were artificially fed immune plasma with 32,000- to 64,000-fold antibody titers or plasma from PBS-inoculated control chickens, and their mortality was observed for 7 days. In experiment 1, the immune plasma significantly increased PRM mortality compared to the control plasma. However, these effects were not observed in experiment 2, although the total mortality was significantly increased in immune plasma-fed PRMs. Thus, the efficacy of Dg-Lipocalin appears to be limited; however, its exposure to the host may result in sustained antibody titers. Further investigation is required to evaluate its feasibility.

Performance evaluation of an improved RAISING method for clonality analysis of bovine leukemia virus-infected cells: a collaborative study in Japan
Tomohiro OKAGAWA, Naomi NOJIRI, Hazuka YOSHIDA-FURIHATA, Naganori NAO, Misono TOMINAGA, Junko KOHARA, Satoshi GONDAIRA, Hidetoshi HIGUCHI, Yohei TAKEDA, Haruko OGAWA, Shinji YAMADA, Kenji MURAKAMI, Yasunori SUZUKI, Shinji TAKAI, Masaki MAEZAWA, Hisashi INOKUMA, Kaori SHIMIZU, Yasuo INOSHIMA, Tatsufumi USUI, Michihito TAGAWA, Mari YAMAMOTO, Hirohisa MEKATA, Mana ESAKI, Makoto OZAWA, Takahiro MATSUDAIRA, Naoya MAEKAWA, Shiro MURATA, Kazuhiko OHASHI, Masumichi SAITO, Satoru KONNAI
Journal of Veterinary Medical Science, Japanese Society of Veterinary Science, 2025
Scientific journal

Potential of histamine release factor for the utilization as a universal vaccine antigen against poultry red mites, tropical fowl mites, and northern fowl mites.
Shwe Yee Win, Fumiya Horio, Jumpei Sato, Yoshinosuke Motai, Hikari Seo, Sotaro Fujisawa, Takumi Sato, Eiji Oishi, Lat Lat Htun, Saw Bawm, Tomohiro Okagawa, Naoya Maekawa, Satoru Konnai, Kazuhiko Ohashi, Shiro Murata
The Journal of veterinary medical science, 20 Nov. 2024, [Domestic magazines]
English, Scientific journal, Poultry red mites (PRMs, Dermanyssus gallinae), tropical fowl mites (TFMs, Ornithonyssus bursa), and northern fowl mites (NFMs, Ornithonyssus sylviarum) are hematophagous mites that are distributed worldwide which pose a serious challenge to the poultry industry and negatively impact poultry production and welfare. Vaccines represent a promising approach for controlling avian mites, and the identification of antigens with broad efficacy against multiple avian mite species is advantageous for vaccine control. This study aimed to identify histamine release factor (HRF), which was previously reported as a candidate vaccine antigen against PRMs, from TFMs and NFMs and to analyze its cross-reactivity and acaricidal effects on different avian mite species. The deduced amino acid sequences of the HRFs identified in the TFMs and NFMs were highly homologous to those of the PRMs. We generated recombinant HRF (rHRF) of TFMs, NFMs, and PRMs, and immune plasma against each rHRF was produced by immunization with each antigen. The immune plasma contained antibodies specific to each antigen and showed cross-reactivity with rHRFs from different avian mites. Moreover, PRM nymphs (protonymphs) artificially fed each immune plasma showed higher mortality rates than those fed the control plasma. These results suggest that HRFs can be used as candidate antigens for a universal vaccine with broad efficacy across avian mites.

子牛における地方病性牛伝染性リンパ腫発症は母牛由来の腫瘍細胞が原因か?　　　　　　　　　　　　　　　
廣瀬 海凪, 今内 覚, 岡川 朋弘, 富永 みその, 松原 幸芽, 前川 直也, 村田 史郎, 大橋 和彦
北海道獣医師会雑誌, 68, 8, 278, 278, (公社)北海道獣医師会, Aug. 2024
Japanese

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, MDPI AG, 30 Jan. 2024
Scientific journal, 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, 20 Jan. 2024, [International Magazine]
English, Scientific journal, 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, 27 Sep. 2023, [International Magazine]
English, Scientific journal, 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, 01 Jun. 2023, [International Magazine]
English, Scientific journal, 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, 11 May 2023, [International Magazine]
English, Scientific journal, 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, 22 Apr. 2023, [International Magazine]
English, Scientific journal, 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, 14 Mar. 2023, [Domestic magazines]
English, Scientific journal, 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, 01 Mar. 2023, [International Magazine]
English, Scientific journal, 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, 26 Jan. 2023, [International Magazine]
English, Scientific journal, 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, 04 Jan. 2023, [International Magazine]
English, Scientific journal, 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, [International Magazine]
English, Scientific journal, 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, [International Magazine]
English, Scientific journal, 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, [International Magazine]
English, Scientific journal, 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, [International Magazine]
English, Scientific journal, 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, [International Magazine]
English, Scientific journal, 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, [International Magazine]
English, Scientific journal, 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, American Society for Microbiology, 13 Oct. 2022
Scientific journal, 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, 14 Sep. 2022, [International Magazine]
English, Scientific journal, 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, 165回, [DI1A, 01], (公社)日本獣医学会, Sep. 2022
Japanese

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, 165回, [DI1A, 04], (公社)日本獣医学会, Sep. 2022
Japanese

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, 165回, [DI1A, 09], (公社)日本獣医学会, Sep. 2022
Japanese

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], (公社)日本獣医学会, Sep. 2022
English

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, 165回, [E3P, 01], (公社)日本獣医学会, Sep. 2022
Japanese

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, 165回, [E3P, 03], (公社)日本獣医学会, Sep. 2022
Japanese

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, 165回, [DI1A, 01], (公社)日本獣医学会, Sep. 2022
Japanese

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, 165回, [DI1A, 04], (公社)日本獣医学会, Sep. 2022
Japanese

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, 165回, [E3P, 01], (公社)日本獣医学会, Sep. 2022
Japanese

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], (公社)日本獣医学会, Sep. 2022
English

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, 165回, [E3P, 03], (公社)日本獣医学会, Sep. 2022
Japanese

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, 165回, [DI1A, 09], (公社)日本獣医学会, Sep. 2022
Japanese

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, Jul. 2022, [International Magazine]
English, Scientific journal, 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, 03 Jun. 2022, [International Magazine]
English, Scientific journal, 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, Public Library of Science (PLoS), 09 Mar. 2022
Scientific journal, 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, Elsevier BV, Mar. 2022
Scientific journal

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, MDPI AG, 14 Feb. 2022
Scientific journal, 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, 07 Jan. 2022, [Domestic magazines]
English, Scientific journal, 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, MDPI AG, 13 Dec. 2021
Scientific journal, 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, Springer Science and Business Media LLC, Dec. 2021, [International Magazine]
English, Scientific journal, 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, Elsevier Inc., 01 Oct. 2021
English, Scientific journal

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, Elsevier BV, Oct. 2021, [International Magazine]
English, Scientific journal, 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, 02 Sep. 2021, [International Magazine]
English, Scientific journal, 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, 164回, [DIO, 4], (公社)日本獣医学会, Sep. 2021
Japanese

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, 164回, [DVO, 5], (公社)日本獣医学会, Sep. 2021
Japanese

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, 164回, [DVO, 25], (公社)日本獣医学会, Sep. 2021
Japanese

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, 164回, [EO, 6], (公社)日本獣医学会, Sep. 2021
Japanese

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, 164回, [DIO, 2], (公社)日本獣医学会, Sep. 2021
Japanese

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], (公社)日本獣医学会, Sep. 2021
English

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, 164回, [DIO, 4], (公社)日本獣医学会, Sep. 2021
Japanese

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, 164回, [DVO, 5], (公社)日本獣医学会, Sep. 2021
Japanese

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, 164回, [DVO, 25], (公社)日本獣医学会, Sep. 2021
Japanese

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, 164回, [EO, 6], (公社)日本獣医学会, Sep. 2021
Japanese

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, Wiley, 20 Aug. 2021
Scientific journal

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, Elsevier BV, Jun. 2021
Scientific journal

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, 09 Apr. 2021, [Domestic magazines]
English, Scientific journal, 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, Elsevier BV, Mar. 2021, [International Magazine]
English, Scientific journal, 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, 12 Feb. 2021, [International Magazine]
English, Scientific journal, 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, Feb. 2021
English, Scientific journal

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, Feb. 2021
English, Scientific journal

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, 2021
Scientific journal

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, [International Magazine]
English, Scientific journal, 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, [International Magazine]
English, Scientific journal, 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, Japanese Society of Veterinary Science, 2021, [Domestic magazines]
English, Scientific journal, 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, Elsevier BV, Jan. 2021, [International Magazine]
English, Scientific journal, 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, Frontiers Media SA, 02 Dec. 2020, [International Magazine]
English, Scientific journal, 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, Springer Science and Business Media LLC, Dec. 2020, [International Magazine]
English, Scientific journal,

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, The American Association of Immunologists, 01 Dec. 2020, [International Magazine]
English, Scientific journal, 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, Springer Science and Business Media LLC, Dec. 2020, [International Magazine]
English, Scientific journal,

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, Public Library of Science (PLoS), 20 Nov. 2020, [International Magazine]
English, Scientific journal, 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, Elsevier BV, Nov. 2020, [International Magazine]
English, Scientific journal, 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, 163回, 221, 221, (公社)日本獣医学会, Oct. 2020
Japanese

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, 163回, 221, 221, (公社)日本獣医学会, Oct. 2020
Japanese

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, 163回, 248, 248, (公社)日本獣医学会, Oct. 2020
Japanese

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, Jun. 2020, [Peer-reviewed]
English, Scientific journal

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., Mar. 2020, [Peer-reviewed]
English, Scientific journal

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, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Jan. 2020, [Peer-reviewed]
English, Scientific journal

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, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, [Peer-reviewed]
English, Scientific journal

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, Springer Science and Business Media LLC, Dec. 2019, [International Magazine]
English, Scientific journal, 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, Sep. 2019, [Peer-reviewed]
English, Scientific journal

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, Sep. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, (公社)日本獣医学会, Aug. 2019
English

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, 162回, 380, 380, (公社)日本獣医学会, Aug. 2019
Japanese

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, 162回, 380, 380, (公社)日本獣医学会, Aug. 2019
Japanese

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, 162回, 381, 381, (公社)日本獣医学会, Aug. 2019
Japanese

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, 162回, 381, 381, (公社)日本獣医学会, Aug. 2019
Japanese

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, 162回, 412, 412, (公社)日本獣医学会, Aug. 2019
Japanese

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, 162回, 460, 460, (公社)日本獣医学会, Aug. 2019
Japanese

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, Aug. 2019, [Peer-reviewed]
English, Scientific journal

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, Apr. 2019, [Peer-reviewed]
English, Scientific journal

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, Mar. 2019, [Peer-reviewed]
English, Scientific journal

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, Feb. 2019, [Peer-reviewed]
English, Scientific journal

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, Aug. 2018, [Peer-reviewed]
English, Scientific journal

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, Jun. 2018, [Peer-reviewed]
English, Scientific journal

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, Apr. 2018, [Peer-reviewed]
English, Scientific journal

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, American Society for Microbiology, Apr. 2018, [Peer-reviewed]
English, Scientific journal

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, Feb. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Jan. 2018, [Peer-reviewed]
English, Scientific journal

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, Dec. 2017
Scientific journal

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., 79, 12, 2040, 2042, JAPANESE SOCIETY OF VETERINARY SCIENCE, Dec. 2017, [Peer-reviewed]
English, Scientific journal,

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, Dec. 2017, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, 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., 79, 11, 1884, 1888, JAPANESE SOCIETY OF VETERINARY SCIENCE, Nov. 2017, [Peer-reviewed]
English, Scientific journal,

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, Sep. 2017, [Peer-reviewed]
English, Scientific journal

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, Aug. 2017, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Aug. 2017, [Peer-reviewed]
English, Scientific journal

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, Jun. 2017, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Apr. 2017, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Mar. 2017, [Peer-reviewed]
English, Scientific journal

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, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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., 79, 1, 115, 122, JAPANESE SOCIETY OF VETERINARY SCIENCE, Jan. 2017, [Peer-reviewed]
English, Scientific journal,

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, Japanese Society of Veterinary Science, Jan. 2017, [Peer-reviewed], [Invited]
English, Scientific journal

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, Jan. 2017, [Peer-reviewed]
English, Scientific journal

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, Sep. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Aug. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Jun. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Apr. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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., 78, 5, 791, 6, JAPANESE SOCIETY OF VETERINARY SCIENCE, Jan. 2016, [Peer-reviewed]
English, Scientific journal, 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, Jan. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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., 63, 4, 191, 4, Graduate School of Veterinary Medicine, Hokkaido University, Nov. 2015, [Peer-reviewed]
English, Scientific journal, 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, Oct. 2015, [Peer-reviewed]
English, Scientific journal

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, Sep. 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Aug. 2015, [Peer-reviewed]
English, Scientific journal

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, Jul. 2015, [Peer-reviewed]
English, Scientific journal

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, Feb. 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Feb. 2015, [Peer-reviewed]
English, Scientific journal

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, Jan. 2015, [Peer-reviewed]
English, Scientific journal

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, [Peer-reviewed]
English, Scientific journal

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, Aug. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Aug. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Aug. 2014, [Peer-reviewed]
English, Scientific journal

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, Aug. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Aug. 2014, [Peer-reviewed]
English, Scientific journal

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, Jul. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Jun. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Mar. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 2014, [Peer-reviewed]
English, Scientific journal

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, Dec. 2013, [Peer-reviewed]
English, Scientific journal

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, Nov. 2013, [Peer-reviewed]
English, Scientific journal

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, Oct. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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., Oct. 2013, [Peer-reviewed]
English, Scientific journal

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, Aug. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Jul. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Jun. 2013, [Peer-reviewed]

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, 16, 137, 43, Elsevier Science Bv, Jun. 2013, [Peer-reviewed]
English, Scientific journal, 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, Jun. 2013, [Invited]
Japanese

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, May 2013, [Peer-reviewed]
English, Scientific journal

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, Apr. 2013, [Peer-reviewed]
English, Scientific journal

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, Mar. 2013, [Peer-reviewed]
English, Scientific journal

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, Feb. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Jan. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 75, 2, 215, 218, JAPANESE SOCIETY OF VETERINARY SCIENCE, 2013, [Peer-reviewed]
English, 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, Nov. 2012, [Peer-reviewed]
English, Scientific journal

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, Oct. 2012, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Aug. 2012, [Peer-reviewed]
English, Scientific journal

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., 192, 3, 538, 40, Elsevier, Jun. 2012, [Peer-reviewed]
English, Scientific journal, 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, Jun. 2012, [Peer-reviewed]
English, Scientific journal

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, May 2012, [Peer-reviewed]
English, Scientific journal

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, May 2012, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Apr. 2012, [Peer-reviewed]
English, Scientific journal

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, [Peer-reviewed]
English, Scientific journal

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, Dec. 2011, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Sep. 2011, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Aug. 2011, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Elsevier BV, May 2011
Scientific journal

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, Elsevier BV, Feb. 2011, [International Magazine]
English, Scientific journal, 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, Elsevier BV, Jan. 2011
Scientific journal

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, Elsevier BV, Jan. 2011
Scientific journal

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, 70, 9, 923, 928, JAPANESE SOCIETY OF VETERINARY SCIENCE, 2008
English, 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, 52, 432, 432, ECOLOGICAL SOCIETY OF JAPAN, 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, Elsevier, 2004
English, Scientific journal

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, (公社)日本獣医学会, Mar. 2003
English

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, [Peer-reviewed]
English, International conference proceedings

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, Nov. 2002
English, Scientific journal

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, 133回, 95, 95, (公社)日本獣医学会, Mar. 2002
Japanese

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, 2002
English, Scientific journal

Host Immune Responses in the Course of Bovine Leukemia Virus Infection (Immunology)
KABEYA Hidenori, OHASHI Kazuhiko, ONUMA Misao
The journal of veterinary medical science, 63, 7, 703, 708, s・iii, Japanese Society of Veterinary Science, Jul. 2001
English, 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, May 1999, [Peer-reviewed]
English, Scientific journal

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, 60, 1, 1, 8, JAPANESE SOCIETY OF VETERINARY SCIENCE, 1998
English, 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.

Effect of polymorphisms in the meq gene of Marek’s disease virus distributed in Japan on the pathogenicity and transcriptional regulation
もたい吉之介, 村田史郎, 村田史郎, 佐藤純平, 山上竣裕, SHWE Win Yee, 堀尾文彌, 前川直也, 岡川朋弘, 今内覚, 今内覚, 今内覚, 大橋和彦, 大橋和彦, 大橋和彦, 日本ウイルス学会学術集会プログラム・予稿集(Web), 71st, 2024

Effect of polymorphisms in the basic region of Meq protein of Marek’s disease, an avian oncogenic herpesvirus, on virulence
佐藤純平, 村田史郎, 村田史郎, もたい吉之介, 山上竣裕, 堀尾文彌, SHWE Win Yee, 前川直也, 岡川朋弘, 今内覚, 今内覚, 今内覚, 大橋和彦, 大橋和彦, 大橋和彦, 日本ウイルス学会学術集会プログラム・予稿集(Web), 71st, 2024

非典型的な地方病性牛伝染性リンパ腫症例のRAISING法を用いた性状解析
松原幸芽, 岡川朋弘, 斎藤益満, 松平崇弘, 直亨則, 神谷可菜, 齋藤麻矢, 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, 167th, 2024

抗PD-L1抗体により治療した悪性黒色腫の犬における血清バイオマーカーの探索
飯高早紀子, 前川直也, 今内覚, 出口辰弥, 岡川朋弘, 高木哲, 高木哲, 鈴木定彦, 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, 167th, 2024

プロバイオティクスによる子牛のワクチン効果増強作用の検証
池端麻里, 岡川朋弘, 中村隼人, 前川直也, 村田史郎, 大橋和彦, 今内覚, 今内覚, 日本獣医学会学術集会講演要旨集, 167th, 2024

日本に分布するマレック病ウイルスmeq遺伝子による病原性への影響
もたい吉之介, 村田史郎, 佐藤純平, 山上竣裕, WIN Shwe Yee, 堀尾文彌, 前川直也, 岡川朋弘, 今内覚, 今内覚, 大橋和彦, 日本獣医学会学術集会講演要旨集, 167th, 2024

ワクモ曝露型抗原による抗ワクモワクチン抗原としての評価
堀尾文彌, 村田史郎, ウィン シュウェ・イー, 佐藤純平, もたい吉之介, 山上竣裕, 佐藤匠, 大石英司, 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, 167th, 2024

マレック病ウイルスmeq遺伝子の転写活性化調節ドメインの多型と病原性との関連
山上竣裕, 村田史郎, 佐藤純平, もたい吉之介, WIN Shwe Yee, 堀尾文彌, 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, 167th, 2024

抗ワクモワクチンを用いたワクモの制御法の開発(1)
大橋和彦, 村田史郎, 鶏の研究, 99, 6, 2024

抗ワクモワクチンを用いたワクモの制御法の開発(2)
大橋和彦, 村田史郎, 鶏の研究, 99, 8, 2024

抗ワクモワクチンを用いたワクモの制御法の開発(3)
大橋和彦, 村田史郎, 鶏の研究, 99, 10, 2024

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, Feb. 2020
日本動物薬事協会, Japanese

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, Dec. 2019, [Invited]
Japanese, Report scientific journal

Tick factors associated pathogen transmission ~Tick immunosuppressants in saliva~　　　　　　　　　　　　　　　
Satoru Konnal, Med. Entomol. Zool., 70, 4, 189, 197, Dec. 2019, [Peer-reviewed], [Invited]
Japanese, Introduction scientific journal

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, 09 Aug. 2019
Japanese

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, 09 Aug. 2019
Japanese

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, Mar. 2019, [Invited]
(株)緑書房, Japanese, Introduction scientific journal

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, Oct. 2018, [Invited]
Japanese, Introduction scientific journal

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, Sep. 2018, [Invited]
Japanese, Introduction scientific journal

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, 24 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 21 Aug. 2018
Japanese

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, 25 Jun. 2018
Japanese

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, Apr. 2018, [Invited]
Japanese, Introduction scientific journal

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, 30 Aug. 2017
Japanese

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, 30 Aug. 2017
Japanese

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, 30 Aug. 2017
Japanese

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, 30 Aug. 2017
Japanese

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, 30 Aug. 2017
Japanese

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, 30 Aug. 2017
Japanese

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, 30 Aug. 2017
Japanese

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, 30 Aug. 2017
Japanese

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, Aug. 2017
(公社)北海道獣医師会, Japanese

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
Japanese

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, 30 Aug. 2016
(公社)日本獣医学会, Japanese

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, 30 Aug. 2016
Japanese

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, 30 Aug. 2016
Japanese

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, 30 Aug. 2016
Japanese

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, 30 Aug. 2016
Japanese

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, 30 Aug. 2016
Japanese

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, Jul. 2016, [Invited]
日本畜産振興会, Japanese, Introduction scientific journal

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, Feb. 2016, [Invited]
北海道農業改良普及協会, Japanese, Introduction scientific journal

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, 30 Aug. 2015
Japanese

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, 30 Aug. 2015
Japanese

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, 30 Aug. 2015
Japanese

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, 30 Aug. 2015
Japanese

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, 30 Aug. 2015
Japanese

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, 30 Aug. 2015
Japanese

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, 30 Aug. 2015
Japanese

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, 30 Aug. 2015
Japanese

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, 25 Feb. 2015
Japanese

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, 11 Aug. 2014
Japanese

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, 11 Aug. 2014
Japanese

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, 11 Aug. 2014
Japanese

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, 11 Aug. 2014
Japanese

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, 01 Aug. 2014
Japanese

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, 15 Dec. 2013
Japanese

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, Dec. 2013, [Invited]
Japanese, Introduction scientific journal

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, Sep. 2013, [Invited]
Japanese, Introduction scientific journal

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, 30 Aug. 2013
Japanese

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, 30 Aug. 2013
Japanese

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, 30 Aug. 2013
Japanese

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, 30 Aug. 2013
Japanese

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, 30 Aug. 2013
Japanese

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, 30 Aug. 2013
Japanese

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, 30 Aug. 2013
Japanese

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, 30 Aug. 2013
Japanese

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, 15 Jun. 2013
Japanese

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, 04 Mar. 2013
Japanese

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, 04 Mar. 2013
Japanese

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, 15 Dec. 2012
Japanese

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, Nov. 2012, [Invited]
Japanese, Introduction scientific journal

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, Oct. 2012, [Invited]
Japanese, Introduction scientific journal

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, Sep. 2012, [Invited]
Japanese, Introduction scientific journal

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, 31 Aug. 2012
Japanese

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, 31 Aug. 2012
Japanese

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, 31 Aug. 2012
Japanese

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, 31 Aug. 2012
Japanese

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, 31 Aug. 2012
Japanese

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, 31 Aug. 2012
Japanese

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, 31 Aug. 2012
Japanese

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, Jul. 2012, [Invited]
Japanese, Introduction scientific journal

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, Jun. 2012, [Invited]
Japanese, Introduction scientific journal

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, 01 Mar. 2012
Japanese

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, Mar. 2012
Japanese, Report scientific journal

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, 01 Mar. 2012
Japanese

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, 01 Mar. 2012
Japanese

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, 20 Jan. 2012
Japanese

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, Dec. 2011, [Invited]
Japanese, Introduction scientific journal

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, 01 Dec. 2011
Japanese

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, Dec. 2011, [Invited]
Japanese, Introduction scientific journal

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, 01 Nov. 2011
Japanese

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, Nov. 2011, [Invited]
Japanese, Introduction scientific journal

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, Oct. 2011, [Invited]
Japanese, Introduction scientific journal

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, 01 Sep. 2011
Japanese

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, Sep. 2011, [Invited]
Japanese, Introduction scientific journal

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, 31 Aug. 2011
Japanese

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, 31 Aug. 2011
Japanese

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, 31 Aug. 2011
Japanese

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, 31 Aug. 2011
Japanese

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, 31 Aug. 2011
Japanese

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, 31 Aug. 2011
Japanese

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, 15 Jun. 2011
Japanese

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, 01 Jun. 2011
Japanese

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, Jun. 2011, [Invited]
Japanese, Introduction scientific journal

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, May 2011
English

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, 01 Mar. 2011
Japanese

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, 01 Mar. 2011
Japanese

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, 01 Mar. 2011
Japanese

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, Feb. 2011, [Invited]
Japanese, Introduction scientific journal

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, Feb. 2011, [Invited]
Japanese, Introduction scientific journal

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, Feb. 2011
English

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, 01 Jan. 2011
Japanese

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, Jan. 2011, [Invited]
Japanese, Introduction scientific journal

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, Jan. 2011
English

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, Jan. 2011
English

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, 01 Nov. 2010
Japanese

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, 01 Sep. 2010
Japanese

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, 01 Sep. 2010
Japanese

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, 01 Sep. 2010
Japanese

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, 01 Sep. 2010
Japanese

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, 01 Sep. 2010
Japanese

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, 01 Sep. 2010
Japanese

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, 01 Sep. 2010
Japanese

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, 01 Sep. 2010
Japanese

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, 01 Sep. 2010
Japanese

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, 10 Aug. 2010
Japanese

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, Jun. 2010, [Invited]
Japanese, Introduction scientific journal

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, Jun. 2010, [Invited]
Japanese Society of Large Animal Clinics / Large Animal Clinic Research Association, Japanese, Introduction scientific journal

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, Jun. 2010
English

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, May 2010
English

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, Apr. 2010, [Invited]
全国農業共済協会, Japanese, Introduction scientific journal

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
Japanese

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, Jan. 2010, [Peer-reviewed], [Invited]
Japanese, Introduction scientific journal

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, Dec. 2009
English

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, Nov. 2009
English

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, Oct. 2009
English

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, Oct. 2009
English

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, Oct. 2009
English

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, Oct. 2009
English

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, Aug. 2009
English

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, Aug. 2009
English

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, Jul. 2009
English

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, May 2009

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, May 2009
English

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, May 2009
English

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, May 2009
English

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, Apr. 2009
English

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, Feb. 2009
English

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, [Invited]
Japanese, Introduction scientific journal

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, Jan. 2009
English

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, Dec. 2008
English

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, 05 Sep. 2008
Japanese

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, 05 Sep. 2008
Japanese

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, 05 Sep. 2008
Japanese

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, Sep. 2008
English

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, Aug. 2008
English

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, Aug. 2008
English

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, Aug. 2008
English

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, May 2008
English

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, 07 Mar. 2008
Japanese

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, Mar. 2008
English

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, Feb. 2008
English

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
Japanese

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, Jan. 2008
English

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
English

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, 06 Sep. 2007
Japanese

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, Sep. 2007
English

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, 27 Aug. 2007
Japanese

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, 27 Aug. 2007
Japanese

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, 27 Aug. 2007
Japanese

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, 27 Aug. 2007
Japanese

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, 27 Aug. 2007
Japanese

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, 27 Aug. 2007
Japanese

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, 27 Aug. 2007
Japanese

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, Aug. 2007
English

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, Aug. 2007
English

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, Jun. 2007
English

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, Apr. 2007
English, Summary international conference

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, Apr. 2007
English, Summary international conference

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, Mar. 2007
English

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, Jan. 2007
English

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, Oct. 2006
English

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, Sep. 2006
English

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, 31 Aug. 2006
Japanese

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, 31 Aug. 2006
Japanese

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, 31 Aug. 2006
Japanese

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, Aug. 2006
English

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, Aug. 2006
English

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, Aug. 2006
English

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, Jul. 2006
English

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, Jun. 2006
English

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, Jun. 2006
English

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, May 2006
English

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, Mar. 2006
English

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, Feb. 2006
English

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, Dec. 2005
English

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, Dec. 2005
English

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, Dec. 2005
English

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, Dec. 2005
English

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, Nov. 2005
English

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, Nov. 2005
English

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, Nov. 2005
English

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, Sep. 2005
English

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, Sep. 2005
English

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, 31 Aug. 2005
Japanese

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, 31 Aug. 2005
Japanese

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, 31 Aug. 2005
Japanese

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, 31 Aug. 2005
Japanese

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, 31 Aug. 2005
Japanese

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, Aug. 2005
English

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, Aug. 2005
English

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, Aug. 2005
English

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, Apr. 2005
English

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, Apr. 2005
English

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, Mar. 2005
English

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, Mar. 2005
English

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, Feb. 2005
English

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, Jan. 2005
English

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, Jan. 2005
English

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, Jan. 2005
English

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, +, Nov. 2004
English, Others

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, 01 Aug. 2004
Japanese

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, Aug. 2004
English

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, 01 Jul. 2004
Japanese

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, Jun. 2004
English

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, May 2004
English

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, May 2004
English

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, 01 Mar. 2004
Japanese

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, Mar. 2004
English

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
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。, 日本獸医師会, Japanese

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

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, Dec. 2003
English

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, Nov. 2003
English

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, 10 Sep. 2003
Japanese

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, Aug. 2003
English

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, Jul. 2003
English

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, Jun. 2003
English

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, May 2003
English

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, Apr. 2003
English

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, Feb. 2003
English

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, Feb. 2003
English

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, Dec. 2002
English

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, Dec. 2002
English

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, Nov. 2002
English, Others

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, Aug. 2002
English

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, May 2002
English

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, May 2002
English

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, Apr. 2002
English

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, Mar. 2002
English

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

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

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, Jan. 2002
English

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, 01 Dec. 2001
日本野生動物医学会, Japanese

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, Oct. 2001
English

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, Aug. 2001
English

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, Aug. 2001
English

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, Jul. 2001
English

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, Jun. 2001
English

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, Apr. 2001
English

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
English

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
English

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
English

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
English

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, Jul. 2000
English

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, Jun. 2000
English

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, Mar. 2000
English

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, Mar. 2000
English

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

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

Expression of biologically active molecules in transgenic potatoes.　　　　　　　　　　　　　　　
Proceedings of the International Veterinary Cytokine and Vaccine Conference, 116, 119, 2000

Tick saliva proteins interacting with the host immune response factors.　　　　　　　　　　　　　　　
Proceedings of the International Veterinary Cytokine and Vaccine Conference, 93, 96, 2000

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, Jul. 1999
English

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, Jul. 1999
English

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, May 1999
English

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, May 1999
English

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, May 1999
English

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, May 1999
English

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, Apr. 1999
English

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, Apr. 1999
English

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, Mar. 1999
English

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
English

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
English

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, Nov. 1998
English

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, Aug. 1998
English

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, Jul. 1998
English

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, May 1998
English

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, Jan. 1998
English

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, 28 Nov. 1997
Hokkaido University, English

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, 25 Nov. 1997
学会出版センタ-, Japanese

Apoptosis in peripheral CD4＋ T cells and thymocytes by Marek's disease virus-infection.　　　　　　　　　　　　　　　
Leukemia, 11 Supplement 3, 206, 208, 1997

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, Dec. 1996
English

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, Dec. 1996
English

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, Aug. 1996
English

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, May 1996
English

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
English

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
English

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
English

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, Jan. 1996
English

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
English

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, Oct. 1995
English

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
Microbiology Society, English

Possible mechanisms of immuno-suppression in chickens induced by Marek’s disease virus.　　　　　　　　　　　　　　　
日仏獣医学雑誌, 6, 89, 99, 1995

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, Mar. 1994
English

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, Feb. 1994
English, Introduction scientific journal

cDNA clones derived from the Marek's disease tumor cell line MDCC-CU41.　　　　　　　　　　　　　　　
Proceedings of 19th World's Poultry Congress, 54, 57, 1992

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, Jan. 1991
English

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

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
English

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, Nov. 1986
English

Detection of cell-surface antigens on MDCC-MSB1 cells using monoclonal antibodies.　　　　　　　　　　　　　　　
2nd International Symposium on Marek's Disease, 214, 235, 1985

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
English

Development of cocktail vaccines against poultry red mites using the recombinant turkey herpesvirus
Grants-in-Aid for Scientific Research
01 Apr. 2023 - 31 Mar. 2027
村田 史郎, 今内 覚, 大橋 和彦
鳥類の外部寄生虫であるワクモによる吸血被害は、世界中の養鶏場で深刻な問題を引き起こしており、最近では薬剤に耐性を示すワクモの出現がその防除をさらに困難にさせている。そのため薬剤に変わる新たな防除法の開発研究が行われているが、その中でもワクチンによる防除法開発が注目されている。本ワクチンは、ワクモの生理機能に重要な分子を抗原として免疫し、血液に含まれる抗体を吸血したワクモに致死的影響を与えることを作用機序とする。しかし、単一の抗原による効果には限界があり、効果増強のためには複数抗原を用いたカクテルワクチンの開発が求められる。また実用化に際しては、従来のワクチンプログラムに抗ワクモワクチンを追加することになり、飼養者の負担が増えることになる。そこで本研究では、家禽の監視伝染病であるマレック病の予防に使用される七面鳥ヘルペスウイルス（HVT）をベクターとして用い、ウイルスゲノムに複数の抗原遺伝子を挿入することで、組換えHVT抗ワクモカクテルワクチンを開発することを目的とした。本年度は、ウイルスベクターとして用いるHVTの感染性クローンの作製に必要なベクター作製と、カクテルワクチンの作製に用いるワクチン抗原の評価を行なった。感染性クローン作製にはpBeloBac11ベクターを用い、ウイルスゲノム中のUS2遺伝子と相同組換えにより置換させることで行う。そのため、HVTのUS2遺伝子の両端の領域をpBeloBac11にクローニングした。さらに薬剤による組換えHVTの選抜のため、ecogpt遺伝子の発現カセットをクローニングした。カクテルワクチンの作製に用いるワクチン抗原として、複数の抗原候補の効果をin vitroにて評価した。その際には、ワクモと形態が類似するその他の家禽の吸血性ダニへの効果も期待される、ユニバーサルワクチンとしての応用の可能性を持つ抗原を選抜した。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23K27063

Development of cocktail vaccines against poultry red mites using the recombinant turkey herpesvirus
Grants-in-Aid for Scientific Research
01 Apr. 2023 - 31 Mar. 2027
村田 史郎, 今内 覚, 大橋 和彦
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23H02370

The roles of polymorphism in Meq on the pathogenicity of Marek's disease virus and their application for the vaccine development
Grants-in-Aid for Scientific Research
01 Apr. 2023 - 31 Mar. 2026
大橋 和彦, 村田 史郎, 今内 覚
マレック病ウイルス(MDV)の病原性進化の分子機構を明らかにして新規防除法を確立するために、MDVの病原性に最も重要なMeqについて、挿入や欠損を含むMeqアイソフォームを導入した組換えMDVを作出して、MDVの病原性発現機構を明らかにすることを目的として実施した。
これまでMeqにおける挿入配列が病原性を増強することを明らかにしたが、その詳細について転写活性化能やmeq遺伝子をL-meq遺伝子に置換した組換えMDV（rMDV_L-Meq）を用いた感染実験等で検討した。L-MeqはMeqに比べ高い転写活性化能を示し、rMDV_Meq感染鶏群と比較して、rMDV_L-Meq感染鶏群ではがん関連因子の発現や末梢血におけるCD4+T細胞や制御性T細胞の増加が早期に認められた。一方で腫瘍病変部での発現遺伝子には両群に有意な差は認められなかった。以上よりMeqの挿入配列は、転写活性化能を増強させ早期に病態を形成させることが示唆された。
当研究室では、野外株で欠損配列を持つmeq遺伝子（S-meq遺伝子）の同定・性状解析を報告したが、2022年に過去に報告例のない欠損配列を持つmeq遺伝子が同定されたため、その性状について解析を行なった。検出されたS-meq遺伝子よりも小さく（VS-meq遺伝子）、meq遺伝子と比べて転写活性化調節領域内に192bpの欠損が認められた。またMDV強毒株であるRB-1B株のMeqの配列と比べて、欠損以外に2箇所のアミノ酸配列の相違が認められた。RB-1B MeqとVS-Meqの転写活性化能に差は認められなかったが、欠損以外のアミノ酸配列をそろえたところ、VS-Meqの転写活性化能はRB-1B Meqより有意に高く、VS-Meqの欠損配列が転写活性化能を増加させることが示された。今後、組換えMDVを用いた感染実験により病原性への影響を調査する必要がある。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23K27068

The roles of polymorphism in Meq on the pathogenicity of Marek's disease virus and their application for the vaccine development
Grants-in-Aid for Scientific Research
01 Apr. 2023 - 31 Mar. 2026
大橋 和彦, 村田 史郎, 今内 覚
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23H02375

Development of evolving therapeutic technologies to address unmet medical needs for intractable diseases in animals
Grants-in-Aid for Scientific Research
01 Apr. 2022 - 31 Mar. 2025
今内 覚, 村田 史郎, 大橋 和彦
アンメット・メディカルニーズとは、未だ有効な治療方法や薬剤がない医療へのニーズを示す。獣医療においてもアンメット・メディカルニーズは難治性疾病を中心に存在する。そこでこれまで開発を進めてきた動物用免疫チェックポイント阻害薬を基盤とした副作用が小さく、より高い奏効率を発揮する発展的な新規治療・予防技術の構築を行い将来的な社会実装・実用化に向けた臨床知見を収集する。牛の慢性感染症では、免疫チェックポイント分子PD-1/PD-L1がT細胞の疲弊を引き起こし、病気の進行に関与している。我々は、ウシのPD-1/PD-L1の結合を特異的に阻害するモノクローナル抗体（mAbs）を確立したが、従来の抗PD-1mAbsは抗原との結合親和性が低いため、治療薬としては適さない。そこで今年度は、高い結合親和性を示す抗ウシPD-1ウサギmAbおよびキメラ抗体を樹立した。抗ウシPD-1ウサギmAbは、従来の抗PD-1 mAbと比較してウシのPD-1に強く結合し、PD-1/PD-L1相互作用に対して顕著な阻害活性を示した。また、キメラ抗体はIFN-γ産生の増強などの免疫活性化を示すことも確認された。さらに、牛伝染性リンパ腫ウイルス感染牛にキメラ抗体を投与し、抗ウイルス効果を調べた結果、抗ウイルス効果が確認された。一方、抗PD-L1抗体による治療を行った肺転移のある口腔内悪性黒色腫のイヌについて、低分割放射線療法と抗PD-L1抗体療法の組み合わせが奏効に及ぼす影響を検討した。その結果、抗PD-L1抗体治療を開始する直前(8 週間以内)に放射線治療を受けていたイヌで肺転移病変に対する奏効率が高く、また免疫療法における生存期間も長くなる傾向が認められた。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23K23768

Development of evolving therapeutic technologies to address unmet medical needs for intractable diseases in animals
Grants-in-Aid for Scientific Research
01 Apr. 2022 - 31 Mar. 2025
今内 覚, 村田 史郎, 大橋 和彦
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 22H02503

The search for the candidate molecule for the vaccine to control a wide range of ectoparasite species of chickens
Grants-in-Aid for Scientific Research
01 Apr. 2020 - 31 Mar. 2023
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。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 20H03137

Experimental study for practical application of cross-sectional cancer therapy in companion animals
Grants-in-Aid for Scientific Research
01 Apr. 2019 - 31 Mar. 2022
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 19H03114

Development of a method to control poultry red mites using recombinant Marek's disease virus that secretes a vaccine antigen
Grants-in-Aid for Scientific Research
01 Apr. 2018 - 31 Mar. 2021
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 18H02332

Analysis of the molecular mechanism for the increase in the virulence of Marek's disease virus
Grants-in-Aid for Scientific Research
01 Apr. 2017 - 31 Mar. 2020
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 17H03911

Analysis of genetic polymorphism of poultry red mites in the world and search for candidate molecules of anti-mite vaccines
Grants-in-Aid for Scientific Research
01 Apr. 2016 - 31 Mar. 2019
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 16H05804

Comprehensive analysis of molecules expressed on the midgut of poultry red mites for the development of new anti-mite vaccines
Grants-in-Aid for Scientific Research
01 Apr. 2016 - 31 Mar. 2018
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 16K15043

Empirical research for bovine leukemia control strategy
Grants-in-Aid for Scientific Research
01 Apr. 2014 - 31 Mar. 2018
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 26252051

Studies on the molecular mechanisms for the increase in MDV virulence in the field
Grants-in-Aid for Scientific Research
01 Apr. 2014 - 31 Mar. 2017
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 26292147

Pathologic analysis of malignant infectious diseases in foreign livestock and its applications for establishment of novel control strategy.
Grants-in-Aid for Scientific Research
01 Apr. 2013 - 31 Mar. 2017
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 25257415

Establishment of control measure for bovine leukemia based on risk analysis of virus transmission
Grants-in-Aid for Scientific Research
18 Nov. 2011 - 31 Mar. 2015
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 23248051

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
科学研究費助成事業
01 Apr. 2012 - 31 Mar. 2014
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。
日本学術振興会, 基盤研究(B), 北海道大学, 24405042

Studies on the immunoinhibitory mechanisms of Marek's disease virus and its application for adjuvant
Grants-in-Aid for Scientific Research
01 Apr. 2011 - 31 Mar. 2014
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23380176

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
科学研究費助成事業
01 Apr. 2010 - 31 Mar. 2014
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。
日本学術振興会, 基盤研究(B), 北海道大学, 22380162

Elucidation of the evasion mechanism of cytokine storm in disease resistant native livestock
Grants-in-Aid for Scientific Research
2010 - 2012
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 22405036

Identification and characterization of factors involved in the resistance to Marek' s disease in chickens and wild waterfowls
Grants-in-Aid for Scientific Research
2009 - 2011
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 21405034

The origin and genome diversity of retroviruses causing neurological disorders
Grants-in-Aid for Scientific Research
2008 - 2011
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 20405039

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
科学研究費助成事業
2008 - 2010
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。
日本学術振興会, 基盤研究(B), 北里大学, 20380160

Studies on the molecular mechanisms for Marek's disease virus to increase its virulence in the field
Grants-in-Aid for Scientific Research
2008 - 2010
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 20380157

Pathological and molecular biological analysis of nervous lesions caused by glioma-inducing retroviruses
Grants-in-Aid for Scientific Research
2006 - 2009
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 18380179

Genetic analysis of drug resistant trypanosomes
Grants-in-Aid for Scientific Research
2006 - 2008
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。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 18405037

鳥類由来感染症の疫学的研究　　　　　　　　　　　　　　　
2008
Competitive research funding

Epidemiological study on infectious diseases of wild and domestic birds　　　　　　　　　　　　　　　
2008
Competitive research funding

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
科学研究費助成事業
2006 - 2007
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。
日本学術振興会, 萌芽研究, 北海道大学, 18658123

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
科学研究費助成事業
2006 - 2007
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。
日本学術振興会, 萌芽研究, 北海道大学, 18658117

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
科学研究費助成事業
2005 - 2007
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。
日本学術振興会, 萌芽研究, 北海道大学, 17658134

Tick vaccine development by using tick testis-derived factors
Grants-in-Aid for Scientific Research
2005 - 2007
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 17380177

Phylogenetic analysis of avian retroviruses with neuropathogenicity in central nervous system
Grants-in-Aid for Scientific Research
2004 - 2007
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 16405033

Development of new vaccine strategies against Marek's disease virus with increased virulence
Grants-in-Aid for Scientific Research
2004 - 2006
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 16380203

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
科学研究費助成事業
2004 - 2005
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。
日本学術振興会, 萌芽研究, 北海道大学, 16658112

Molecular characterization of Marek's disease virus genomes isolated from white-fronted geese
Grants-in-Aid for Scientific Research
2003 - 2005
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 15405038

Genetic diversity of bovine lentivirus distributed in south Asian countrirs
Grants-in-Aid for Scientific Research
2003 - 2005
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 15405035

Oncogenesis of retrovirus induced-glioma and the development of animal model
Grants-in-Aid for Scientific Research
2003 - 2005
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 15380207

Molecluar mechanism of propagation of abnormal prion protein in the cells
Grants-in-Aid for Scientific Research
2003 - 2005
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), HOKKAIDO UNIVERSITY, 15208029

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
科学研究費助成事業
2003 - 2004
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。
日本学術振興会, 萌芽研究, 北海道大学, 15658095

Search for biological active molecules from tick salivary glands
Grants-in-Aid for Scientific Research
2003 - 2004
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 15380206

Molecular mimicry of carbohydrate receptors and its application for the control of infectious diseases
Grants-in-Aid for Scientific Research
2001 - 2003
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 13556045

Development of anti-tick vaccine
Grants-in-Aid for Scientific Research
2000 - 2003
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 12356008

Analysis of tick-encoded immunoregulatory serine proteinase inhibitors (serpins)
Grants-in-Aid for Scientific Research
2001 - 2002
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 13460128

THE CHARACTERISTIC OF SO-CALLED FOWL GLIOMA, WHICH IS A UNIQUE CENTRAL NERVOUS SYSTEM NEOPLASM IN ANIMALS CAUSED BY A RETROVIRUS
Grants-in-Aid for Scientific Research
2000 - 2001
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), HOKKAIDO UNIVERSITY, 12660280

Molecular mechanisms of diversity of theileria parasite genes distributed in middle and South America
Grants-in-Aid for Scientific Research
2000 - 2001
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 12575027

Preparation of the recombinant Marek's disease virus which can induce apoptosis in tumor cells and its application for an anti-tumor vaccine.
Grants-in-Aid for Scientific Research
1999 - 2000
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), HOKKAIDO UNIVERSITY, 11836001

Molecular approach to analyze strategies of pathogens in wildlife for acuierement of new host
Grants-in-Aid for Scientific Research
1999 - 2000
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), 11691164

Molecular mechanisms of diversity of theileria parasite genes and control of the disease
Grants-in-Aid for Scientific Research
1999 - 2000
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B)., HOKKAIDO UNIVERSITY, 11460141

Molecular analysis of intraerythrocytic parasite population dinamics and acquirement of biological diversity
Grants-in-Aid for Scientific Research
1998 - 2000
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), 10460126

PLANT EXPRESSIONS OF CYTOKINES AND IIST APPILICATIONS FOR ANIMAL DISEASE CONTROL
Grants-in-Aid for Scientific Research
1998 - 1999
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 10556067

Molecular evolution of Theileria parasites in Southern Asia
Grants-in-Aid for Scientific Research
1998 - 1999
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A)., HOKKAIDO UNIVERSITY, 10041150

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
科学研究費助成事業
1998 - 1999
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。
日本学術振興会, 奨励研究(A), 北海道大学, 09760262

Immunological modulation and control of infections diseases by cytokaines
Grants-in-Aid for Scientific Research
1997 - 1999
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), HOKKAIDO UNIVERSITY, 09356009

GENOME ANALYSIS OF THEILERIA PARASITES
Grants-in-Aid for Scientific Research
1997 - 1998
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.
Japan Society for the Promotion of Science, Grant-in-Aid for international Scientific Research, Hokkaido University, 09044199

Molecular escape mechanisms of Theileria parasites
Grants-in-Aid for Scientific Research
1995 - 1998
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 07406019

ANALYSIS OF INTRACELLULAR ADAPTATION MECHANISMS OF PROTOZOAN PARASITES AND HOST ELIMINATION
Grants-in-Aid for Scientific Research
1996 - 1997
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 08456143

Molecular evolution of Theileria parasites in Pacific area
Grants-in-Aid for Scientific Research
1996 - 1997
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.
Japan Society for the Promotion of Science, Grant-in-Aid for international Scientific Research, Hokkaido University, 08041130

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
科学研究費助成事業
1996 - 1996
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。
日本学術振興会, 奨励研究(A), 北海道大学, 08760268

Molecular epidemiology of Theileria infections in Africa : Characterization of parasites of wild and domestic animals.
Grants-in-Aid for Scientific Research
1995 - 1996
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.
Japan Society for the Promotion of Science, Grant-in-Aid for international Scientific Research, Hokkaido University, 07041119

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
科学研究費助成事業
1995 - 1995
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。
日本学術振興会, 奨励研究(A), 北海道大学, 07760271

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
科学研究費助成事業
1994 - 1994
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。
日本学術振興会, 奨励研究(A), 北海道大学, 06760252

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　　　　　　　　　　　　　　　
1992
Competitive research funding

動物感染症に対する免疫応答の分子機序の解明　　　　　　　　　　　　　　　
1992
Competitive research funding

Characterization of molecular mechanisms of immune responses against infectious diseases in animals　　　　　　　　　　　　　　　
1992
Competitive research funding