Researcher Database
| Murata Shiro |
| Faculty of Veterinary Medicine Veterinary Medicine Disease Control |
| Associate Professor |
Last Updated :2024/05/09
Researcher Profile and Settings
Affiliation
Job Title
J-Global ID
Research Interests
Research Areas
Educational Organization
- Bachelor's degree program School of Veterinary Medicine
- Doctoral (PhD) degree program Graduate School of Infectious Diseases
Academic & Professional Experience
- 2022/10 - Today Hokkaido University Faculty of Veterinary Medicine Associate Profossor
- 2017/04 - Today Hokkaido University Faculty of Veterinary Medicine Assistant Professor
- 2010/03 - 2017/03 Hokkaido University Graduate School of Veterinary Medicine Assistant Professor
- 2008/04 - 2010/02 Ministry of Agriculture, Forestry and Fisheries
Education
- - 2008/03 Hokkaido University Graduate School of Veterinary Medicine
- - 2004/03 Hokkaido University School of Veterinary Medicine
Association Memberships
Research Activities
Published Papers
- 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 MurataVaccines 12 (2) 148 - 148 2024/01/30Poultry 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.
- Yoshinosuke Motai, Shiro Murata, Jumpei Sato, Akihito Nishi, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko OhashiVeterinary sciences 11 (1) 2024/01/20Marek'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.
- 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 OhashiVeterinary research 54 (1) 82 - 82 2023/09/27 [Refereed]
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. - 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 OhashiCancers 15 (11) 2023/06/01 [Refereed]
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. - Wisa Tiyamanee, Satoru Konnai, Tomohiro Okagawa, Yutaro Nojima, Otgontuya Ganbaatar, Naoya Maekawa, Rie Hasebe, Yumiko Kagawa, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiVeterinary immunology and immunopathology 261 110609 - 110609 2023/05/11 [Refereed]
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. - Sotaro Fujisawa, Shiro Murata, Takumi Sato, Eiji Oishi, Akira Taneno, Satoru Konnai, Kazuhiko OhashiParasitology international 95 102754 - 102754 2023/04/22 [Refereed]
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. - Hikari Seo, Shiro Murata, Osamu Ichii, Takashi Namba, Shwe Yee Win, Takumi Sato, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko OhashiThe Journal of veterinary medical science 85 (5) 532 - 535 2023/03/14 [Refereed]
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. - 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 OhashiVaccines 11 (3) 2023/03/01 [Refereed]
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. - Sotaro Fujisawa, Shiro Murata, Masayoshi Isezaki, Shwe Yee Win, Takumi Sato, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko OhashiPoultry science 102 (4) 102532 - 102532 2023/01/26 [Refereed]
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. - Hayato Nakamura, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Yamato Sajiki, Kei Watari, Kana Kamitani, Maya Saito, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiJournal of virology 97 (1) e0143022 2023/01/04 [Refereed]
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. - 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 OhashiPloS one 18 (10) e0291727 2023 [Refereed]
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. - 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 OhashiPloS one 18 (7) e0288565 2023 [Refereed]
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. - 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 OhashiFrontiers in veterinary science 10 1182930 - 1182930 2023 [Refereed]
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. - Mari Ikehata, Satoru Konnai, Tomohiro Okagawa, Kentaro Abe, Mitsuru Honma, Toru Kitamura, Naoya Maekawa, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiFrontiers in veterinary science 10 1145445 - 1145445 2023 [Refereed]
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. - 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 OhashiPloS one 18 (1) e0281143 2023 [Refereed]
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. - Hiroto Takeuchi, Chie Nakajima, Satoru Konnai, Naoya Maekawa, Tomohiro Okagawa, Masaru Usui, Yutaka Tamura, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiPloS one 18 (1) e0281171 2023 [Refereed]
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. - Tomohiro Okagawa, Honami Shimakura, Satoru Konnai, Masumichi Saito, Takahiro Matsudaira, Naganori Nao, Shinji Yamada, Kenji Murakami, Naoya Maekawa, Shiro Murata, Kazuhiko OhashiMicrobiology spectrum 10 (6) e0259522 2022/10/13 [Refereed]
Bovine leukemia virus (BLV), a retrovirus, infects B cells of ruminants and is integrated into the host genome as a provirus for lifelong infection. After a long latent period, 1% to 5% of BLV-infected cattle develop aggressive lymphoma, enzootic bovine leukosis (EBL). Since the clonal expansion of BLV-infected cells is essential for the development of EBL, the clonality of proviral integration sites could be a molecular marker for diagnosis and early prediction of EBL. Recently, we developed Rapid Amplification of the Integration Site without Interference by Genomic DNA Contamination (RAISING) and an analysis software of clonality value (CLOVA) to analyze the clonality of transgene-integrated cells. RAISING-CLOVA is capable of assessing the risk of adult T-cell leukemia/lymphoma development in human T-cell leukemia virus-I-infected individuals through the clonality analysis of proviral integration sites. Thus, we herein examined the performance of RAISING-CLOVA for the clonality analysis of BLV-infected cells and conducted a comprehensive clonality analysis by RAISING-CLOVA in EBL and non-EBL cattle. RAISING-CLOVA targeting BLV was a highly accurate and reproducible method for measuring the clonality value. The comprehensive clonality analysis successfully distinguished EBL from non-EBL specimens with high sensitivity and specificity. A longitudinal clonality analysis in BLV-infected sheep, an experimental model of lymphoma, also confirmed the effectiveness of RAISING-CLOVA for early detection of EBL development. Therefore, our study emphasizes the usefulness of RAISING-CLOVA as a routine clinical test for monitoring virus-related cancers. IMPORTANCE 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. Thus, the identification of high-risk asymptomatic cattle before they develop lymphoma can be effective in reducing the economic damage. Clonal expansion of BLV-infected cells is a promising marker for the development of lymphoma. Recently, we have developed a high-throughput method to amplify random integration sites of transgenes in host genomes and analyze their clonality, named as RAISING-CLOVA. As a new application of our technology, in this study, we demonstrate the value of the RAISING-CLOVA method for the diagnosis and early prediction of lymphoma development by BLV infection in cattle. RAISING-CLOVA is a reliable technology for monitoring the clonality of BLV-infected cells and would contribute to reduce the economic losses by EBL development. - 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 OhashiINFECTION AND IMMUNITY 90 (10) e0021022 0019-9567 2022/09 [Refereed]
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. - 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 OhashiTICKS AND TICK-BORNE DISEASES 13 (4) 101963 - 101963 1877-959X 2022/07 [Refereed]
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. - 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 OhashiScientific reports 12 (1) 9265 - 9265 2022/06/03 [Refereed]
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. - Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Junko Kohara, Atsushi Nitanai, Hirofumi Takahashi, Kentaro Kubota, Hiroshi Takeda, Shiro Murata, Kazuhiko OhashiPLOS ONE 17 (3) e0263660 - e0263660 1932-6203 2022/03/09 [Refereed]
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. - Takuma Ariizumi, Shiro Murata, Sotaro Fujisawa, Masayoshi Isezaki, Takumi Sato, Eiji Oishi, Akira Taneno, Osamu Ichii, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko OhashiPoultry science 101 (3) 101638 - 101638 0032-5791 2022/03 [Refereed]
Poultry red mites (PRMs, Dermanyssus gallinae) are hematophagous ectoparasites that negatively affect egg production, which causes serious economic losses to the poultry industry worldwide. Currently, the emergence of acaricide-resistant PRMs has impeded PRM control in poultry farms. Several alternatives for acaricide use have been described for managing PRM-caused problems. Vaccination is among the methods for controlling PRMs in poultry houses. Currently, several candidates for vaccine antigens have been identified. This study identified a cysteine protease, Deg-CPR-2, which differs from 2 other previously reported cysteine proteases in PRMs, from previously obtained data from RNA-sequencing (RNA-seq) analysis. We investigated the characteristics of Deg-CPR-2 and assessed its efficacy as a vaccine antigen in vitro. Phylogenetic analysis revealed that Deg-CPR-2 belonged to a different cluster from those of other cysteine proteases in PRMs. This cluster also included cathepsin L-like proteases, enzymes thought to be involved in hemoglobin digestion in ticks. Expression analysis revealed Deg-CPR-2 expression in midguts and all the life-stages; however, there were differences in the expression levels across the life-stages. The enzyme activity of recombinant Deg-CPR-2 was inhibited in the presence of a cysteine protease inhibitor, which suggests that Deg-CPR-2 functions as a cysteine protease in PRMs. Finally, there was an in vitro increase in the mortality of PRMs, mainly protonymphs that were artificially fed with plasma from chickens immunized with Deg-CPR-2. These findings suggest that Deg-CPR-2 may contribute to protein digestion in the midgut of PRMs and is crucially involved in physiological processes in PRMs. Additionally, immunization with Deg-CPR-2 may reduce the number of protonymphs, and Deg-CPR-2 should be considered as a candidate antigen for anti-PRM vaccine development. - 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 OhashiVIRUSES-BASEL 14 (2) 382 - 382 2022/02/14 [Refereed]
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. - Kei Watari, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Yamato Sajiki, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiJOURNAL OF VETERINARY MEDICAL SCIENCE 84 (1) 6 - 15 0916-7250 2022/01/07 [Refereed]
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. - Sotaro Fujisawa, Shiro Murata, Masayoshi Isezaki, Takuma Ariizumi, Takumi Sato, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Osamu Ichii, Satoru Konnai, Kazuhiko OhashiParasitology 149 (1) 105 - 115 0031-1820 2022/01 [Refereed]
Poultry red mites (Dermanyssus gallinae, PRM) are dangerous ectoparasites that infest chickens and threaten the poultry industry worldwide. PRMs usually develop resistance to chemical acaricides, necessitating the development of more effective preventive agents, and vaccination could be an alternative strategy for controlling PRMs. The suitability of plasma membrane proteins expressed in the midguts as vaccine antigens was evaluated because these molecules are exposed to antibodies in the ingested blood and the binding of antibodies could potentially induce direct damage to midgut tissue and indirect damage via inhibition of the functions of target molecules. Therefore, in the present study, a copper transporter 1-like molecule (Dg-Ctr1) was identified and its efficacy as a vaccine antigen was assessed in vitro. Dg-Ctr1 mRNA was expressed in the midguts and ovaries and in all the life stages, and flow cytometric analysis indicated that Dg-Ctr1 was expressed on the plasma membrane. Importantly, nymphs fed on plasma derived from chickens immunized with the recombinant protein of the extracellular region of Dg-Ctr1 showed a significant reduction in the survival rate. These data indicate that the application of Dg-Ctr1 as a vaccine antigen could reduce the number of nymphs in the farms, contributing to reduction in the economic losses caused by PRMs in the poultry industry. To establish an effective vaccination strategy, the acaricidal effects of the combined use of Dg-Ctr1 with chemical acaricides or other vaccine antigens must be examined. - Sotaro Fujisawa, Shiro Murata, Masayoshi Isezaki, Takuma Ariizumi, Takumi Sato, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Osamu Ichii, Satoru Konnai, Kazuhiko OhashiVaccines 9 (12) 1472 - 1472 2021/12/13 [Refereed]
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. - 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 OhashiImmunity, inflammation and disease 9 (4) 1573 - 1583 2021/12 [Refereed][Not invited]
INTRODUCTION: Chronic infections lead to the functional exhaustion of T cells. Exhausted T cells are phenotypically differentiated by the surface expression of the immunoinhibitory receptor, such as programmed death-1 (PD-1). The inhibitory signal is produced by the interaction between PD-1 and its PD-ligand 1 (PD-L1) and impairs the effector functions of T cells. However, the expression dynamics of PD-L1 and the immunological functions of the PD-1/PD-L1 pathway in chronic diseases of pigs are still poorly understood. In this study, we first analyzed the expression of PD-L1 in various chronic infections in pigs, and then evaluated the immune activation by the blocking assay targeting the swine PD-1/PD-L1 pathway. METHODS: In the initial experiments, anti-bovine PD-L1 monoclonal antibodies (mAbs) were tested for cross-reactivity with swine PD-L1. Subsequently, immunohistochemical analysis was conducted using the anti-PD-L1 mAb. Finally, we assessed the immune activation of swine peripheral blood mononuclear cells (PBMCs) by the blockade with anti-PD-L1 mAb. RESULTS: Several anti-PD-L1 mAbs tested recognized swine PD-L1-expressing cells. The binding of swine PD-L1 protein to swine PD-1 was inhibited by some of these cross-reactive mAbs. In addition, immunohistochemical analysis revealed that PD-L1 was expressed at the site of infection in chronic infections of pigs. The PD-L1 blockade increased the production of interleukin-2 from swine PBMCs. CONCLUSIONS: These findings suggest that the PD-1/PD-L1 pathway could be also involved in immunosuppression in chronic infections in pigs. This study provides a new perspective on therapeutic strategies for chronic diseases in pigs by targeting immunosuppressive pathways. - Shiro Murata, Eiji Yamamoto, Natsumi Sakashita, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko OhashiPoultry science 100 (11) 101461 - 101461 2021/11 [Refereed][Not invited]
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. - 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 OhashiVaccine 39 (41) 6057 - 6066 2021/10/01 [Refereed][Not invited]
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. - Y. Sajiki, S. Konnai, Y. Ikenaka, T. Okagawa, N. Maekawa, C. Logullo, I. da Silva Vaz, S. Murata, K. OhashiVeterinary Immunology and Immunopathology 236 110238 - 110238 0165-2427 2021/06 [Refereed]
- Takuma Ariizumi, Shiro Murata, Sotaro Fujisawa, Masayoshi Isezaki, Naoya Maekawa, Tomohiro Okagawa, Takumi Sato, Eiji Oishi, Akira Taneno, Satoru Konnai, Kazuhiko OhashiThe Journal of veterinary medical science 83 (4) 558 - 565 2021/04/09 [Refereed][Not invited]
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. - 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 OhashiVeterinary microbiology 254 108976 - 108976 2021/03 [Refereed][Not invited]
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. - 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 OhashiNPJ precision oncology 5 (1) 10 - 10 2021/02/12 [Refereed][Not invited]
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. - Yukiko Taniguchi, Satoru Konnai, Shinichi Sakakibara, Ayano Yamamoto, Tomohiro Okagawa, Naoya Maekawa, Shiro Murata, Kazuhiko OhashiJAPANESE JOURNAL OF VETERINARY RESEARCH 69 (1) 51 - 55 0047-1917 2021/02 [Refereed]
Johne's disease (JD) is caused by infection with Mycobacterium avium subsp. paratuberulosis (MAP). We confirmed the intrauterine infection of MAP in 22 pregnant cattle diagnosed with JD in Hokkaido, Japan. MAP was isolated from the umbilical cord (3/22: 13.6%) or caruncle (6/22: 27.3%) derived from the pregnant dams. Furthermore, dams with MAP from which MAP was isolated were also found to have a high mount of MAP or detected bacterial load in their feces. Fetuses of the tested dams indicated positive polymerase chain reaction (PCR) results for the MAP gene (17/22:77.3%) in several tissues. MAP was isolated from the PCR-positive sites of dams detected with high levels of bacteria (6/ 22: 27.3%). These results indicate that MAP infection in pregnant cattle must be prevented as it is important for JD control. - 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 OhashiJAPANESE JOURNAL OF VETERINARY RESEARCH 69 (1) 57 - 65 0047-1917 2021/02There are several studies that confirm the possibility of developing a vaccine against tick infestations. An immuno-bioinformatics approach was used to identify conserved antigenic regions between Ixodes persulcatus and Rhipicephalus microplus ferritin 2 ( FER2) in order to compose a novel putative vaccine. In addition, R. microplus were fed on blood containing antibodies anti-recombinant FER2 from I. persulcatus (rIp-FER2). The results revealed that anti-ferritin antibodies led to a decrease in the engorgement weight of the R. microplus females. Conservation of the predicted antigenic regions in different tick species suggests that this protein could be useful to develop a vaccine for cross-species protection.
- 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 OhashiJOURNAL OF VETERINARY MEDICAL SCIENCE 83 (2) 162 - 166 0916-7250 2021/02/25 [Refereed][Not invited]
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. - Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Hayato Nakamura, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiDevelopmental and comparative immunology 114 103847 - 103847 2021/01 [Refereed][Not invited]
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. - Yamato Sajiki, Satoru Konnai, Yoshinori Ikenaka, Kevin Christian Montecillo Gulay, Atsushi Kobayashi, Luis Fernando Parizi, Benvindo Capela Joao, Kei Watari, Sotaro Fujisawa, Tomohiro Okagawa, Naoya Maekawa, Carlos Logullo, Itabajara da Silva Vaz, Shiro Murata, Kazuhiko OhashiSCIENTIFIC REPORTS 11 (1) 1063 - 1063 2045-2322 2021/01/13 [Refereed][Not invited]
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. - Shiro Murata, Ayaka Taniguchi, Masayoshi Isezaki, Sotaro Fujisawa, Eishi Sakai, Akira Taneno, Osamu Ichii, Takuya Ito, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko OhashiParasite (Paris, France) 28 9 - 9 2021 [Refereed][Not invited]
Poultry red mites (PRMs, Dermanyssus gallinae) are ectoparasites that negatively affect farmed chickens, leading to serious economic losses worldwide. Acaricides have been used to control PRMs in poultry houses. However, some PRMs have developed resistance to acaricides, and therefore different approaches are required to manage the problems caused by PRMs. Vaccination of chickens is one of the methods being considered to reduce the number of PRMs in poultry houses. In a previous study, a cysteine protease, Deg-CPR-1, was identified as a candidate vaccine against PRMs distributed in Europe. In this study, we investigated the characteristics of Deg-CPR-1. A phylogenetic analysis revealed that Deg-CPR-1 is closely related to the digestive cysteine proteases of other mite species, and it was classified into a cluster different from that of chicken cathepsins. Deg-CPR-1 of PRMs in Japan has an amino acid substitution compared with that of PRMs in Europe, but it showed efficacy as a vaccine, consistent with previous findings. Deg-CPR-1 exhibited cathepsin L-like enzyme activity. In addition, the Deg-CPR-1 mRNA was expressed in the midgut and in all stages of PRMs that feed on blood. These results imply that Deg-CPR-1 in the midgut may have important functions in physiological processes, and the inhibition of its expression may contribute to the efficacy of a Deg-CPR-1-based vaccine. Further research is required to fully understand the mechanisms of vaccine efficacy. - Yamato Sajiki, Satoru Konnai, Zimeng Cai, Kensuke Takada, Tomohiro Okagawa, Naoya Maekawa, Sotaro Fujisawa, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiImmunoHorizons 4 (12) 837 - 850 2020/12/21 [Refereed][Not invited]
Combination treatment approaches are increasingly considered to overcome resistance to immunotherapy targeting immunoinhibitory molecules such as programmed death (PD)-1 and PD-ligand 1 (PD-L1). Previous studies have demonstrated that the therapeutic efficacy of anti-PD-L1 Abs is enhanced by combination treatment with cyclooxygenase-2 inhibitors, through downregulation of the immunosuppressive eicosanoid PGE2, although the underlying mechanism remains unclear. In this study, we show that serum PGE2 levels are upregulated after anti-PD-L1 Ab administration in a bovine model of immunotherapy and that PGE2 directly inhibits T cell activation via its receptor E prostanoid (EP) 4. Additionally, anti-PD-L1 Ab induces TNF-α production and TNF-α blockade reduces PGE2 production in the presence of anti-PD-L1 Ab, suggesting that anti-PD-L1 Ab-induced TNF-α impairs T cell activation by PGE2 upregulation. Our studies examining the therapeutic potential of the dual blockade of PD-L1 and EP4 in bovine and murine immune cells reveal that the dual blockade of PD-L1 and EP4 significantly enhances Th1 cytokine production in vitro. Finally, we show that the dual blockade decreases tumor volume and prolongs survival in mice inoculated with the murine lymphoma cell line EG7. Altogether, these results suggest that TNF-α induced by anti-PD-L1 Ab treatment is associated with T cell dysfunction via PGE2/EP4 pathway and that the dual blockade of PD-L1 and EP4 should be considered as a novel immunotherapy for cancer. - Shwe Yee Win, Hla Myet Chel, Myint Myint Hmoon, Lat Lat Htun, Saw Bawm, Mar Mar Win, Shiro Murata, Nariaki Nonaka, Ryo Nakao, Ken KatakuraACTA TROPICA 212 105719 - 105719 0001-706X 2020/12 [Refereed][Not invited]
Village chicken production, a traditional, small-scale, and extensive backyard poultry industry, has been profitable for local farmers in Myanmar. However, there is scanty information available concerning the infection of these chickens with avian pathogens, including haemoprotozoan parasites. In the present study, we provide the first report of microscopic detection and molecular identification of Leucocytozoon and Plasmodium parasites from seven different areas of Myanmar. Leucocytozoon gametocytes were detected in 17.6% (81/461) of the blood smears from village chickens. The nested polymerase chain reaction (PCR) for targeting Leucocytozoon mitochondrial cytochrome b (cyt b) genes had a 17.6% positive rate. Although the positive rate of nested PCR targeting Plasmodium/Haemoproteus cyt b was 34.3%, the PCR protocol was observed to possibly amplify DNA of a certain species of Leucocytozoon. There were no obvious clinical signs in the infected birds. Statistical analysis of the microscopic detection and PCR detection rates using the age and sex of birds as internal factors revealed that the statistical significances differed according to the study area. The sequencing of 32 PCR products obtained from each study area revealed infection by Leucocytozoon caulleryi in three birds, Leucocytozoon sabrazesi in two birds, Leucocytozoon schoutedeni in two birds, Leucocytozoon sp. in eighteen birds, and Plasmodium juxtanucleare in seven birds; however, Haemoproteus infection was not detected. While L. sabrazesi was detected in chickens from the central region of Myanmar, the other haemosporidians were detected in those from different areas. In the haplotype analysis, we detected 17 haemosporidian cyt b haplotypes, including two for L. caulleryi, one for L. sabrazesi, two for L. schoutedeni, nine for Leucocytozoon sp., and three for P. juxtanucleare. Phylogenetic analysis of the cyt b haplotypes revealed a considerably close genetic relationship among chicken haemosporidians detected in Myanmar, Thailand, and Malaysia. These results indicate that well-recognized widespread species of chicken Leucocytozoon and Plasmodium are distributed nationwide in Myanmar, providing new insights into the ecosystem and control strategies of haemosporidian parasites in domesticated chickens in Myanmar. - Andelé M Conradie, Luca D Bertzbach, Jakob Trimpert, Joseph N Patria, Shiro Murata, Mark S Parcells, Benedikt B KauferPLoS pathogens 16 (12) e1009104 2020/12 [Refereed][Not invited]
Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek's disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection. - Shiro Murata, Yuka Machida, Masayoshi Isezaki, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko OhashiVirology journal 17 (1) 186 - 186 2020/11/23 [Refereed][Not invited]
BACKGROUND: Marek's disease virus (MDV) causes malignant lymphomas in chickens (Marek's disease, MD). MD is currently controlled by vaccination; however, MDV strains have a tendency to develop increased virulence. Distinct diversity and point mutations are present in the Meq proteins, the oncoproteins of MDV, suggesting that changes in protein function induced by amino acid substitutions might affect MDV virulence. We previously reported that recent MDV isolates in Japan display distinct mutations in Meq proteins from those observed in traditional MDV isolates in Japan, but similar to those in MDV strains isolated from other countries. METHODS: To further investigate the genetic characteristics in Japanese field strains, we sequenced the whole genome of an MDV strain that was successfully isolated from a chicken with MD in Japan. A phylogenetic analysis of the meq gene was also performed. RESULTS: Phylogenetic analysis revealed that the Meq proteins in most of the Japanese isolates were similar to those of Chinese and European strains, and the genomic sequence of the Japanese strain was classified into the Eurasian cluster. Comparison of coding region sequences among the Japanese strain and MDV strains from other countries revealed that the genetic characteristics of the Japanese strain were similar to those of Chinese and European strains. CONCLUSIONS: The MDV strains distributed in Asian and European countries including Japan seem to be genetically closer to each other than to MDV strains from North America. These findings indicate that the genetic diversities of MDV strains that emerged may have been dependent on the different vaccination-based control approaches. - 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 Jr, Shiro Murata, Kazuhiko OhashiTicks and tick-borne diseases 11 (6) 101547 - 101547 2020/11 [Refereed][Not invited]
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. - Zhiyuan Yang, Shiro Murata, Sotaro Fujisawa, Masaki Takehara, Ken Katakura, Myint Myint Hmoon, Shwe Yee Win, Saw Bawm, Satoru Konnai, Kazuhiko OhashiBMC VETERINARY RESEARCH 16 (1) 453 - 453 2020/11/23 [Refereed][Not invited]
BACKGROUND: Avian infectious laryngotracheitis (ILT) is a highly contagious viral disease that causes severe economic losses to the poultry industry worldwide. In Southeast Asian countries, including Myanmar, poultry farming is a major industry. Although it is known that infectious respiratory pathogens, including infectious laryngotracheitis virus (ILTV), are a major threat to poultry farms, there are no data currently available on the epidemiology of ILTV in Myanmar. Therefore, in this study, we conducted a molecular detection of ILTV in 20 poultry farms in Myanmar. RESULTS: Of the 57 tested oropharyngeal swabs, 10 were positive for ILTV by polymerase chain reaction of a 647 bp region of the thymidine kinase (TK) gene, giving a prevalence of ILTV of 17.5% (10/57). Further sequencing analysis of infected cell protein 4 (ICP4) gene and glycoprotein B, G, and J (gB, gG, and gJ) genes indicated that these isolates were field strains. Phylogenetic analysis revealed that the Myanmar strains clustered together in a single branch and were closely related to other reference strains isolated from Asian countries. CONCLUSIONS: This study demonstrated the presence of ILTV in poultry farms in Myanmar. The genetic characterization analysis performed provides the fundamental data for epidemiological studies that monitor circulating strains of ILTV in Myanmar. - Sotaro Fujisawa, Shiro Murata, Masayoshi Isezaki, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko OhashiParasitology international 78 102156 - 102156 2020/10 [Refereed][Not invited]
- Hiroto Takeuchi, Satoru Konnai, Naoya Maekawa, Erina Minato, Yoshiki Ichikawa, Atsushi Kobayashi, Tomohiro Okagawa, Shiro Murata, Kazuhiko OhashiFRONTIERS IN VETERINARY SCIENCE 7 330 - 330 2020/06 [Refereed][Not invited]
Cancer is one of the most significant causes of death in dogs. Antibody drugs targeting the PD-1/PD-L1 axis represent a promising immunotherapy for both human and canine cancers. However, the regulation mechanisms of PD-L1 expression in canine cancers require further investigation to better understand the resistance mechanisms to anti-PD-L1 therapy. Recent reports have shown that CMTM6 and CMTM4 are critical regulators of PD-L1 protein expression in human cancer cells. By preventing PD-L1 from lysosome-mediated degradation, CMTM6 maintains PD-L1 expression on the cell surface. However, the literature has not reported on CMTM6 and CMTM4 in dogs, and their functions are completely unknown. To reveal a regulation mechanism of PD-L1 in canine cancers, this study firstly identified the gene sequences of CMTM6 and CMTM4. Then, the expression analysis of these proteins was performed by immunohistochemistry. Furthermore, the functions of CMTM6 and CMTM4 in regulating PD-L1 expression were examined by gene knockdown of CMTM6 and CMTM4. Canine CMTM6 and CMTM4 displayed high amino acid sequence identities compared with those of humans and mice. An immunohistochemical analysis using cross-reactive antibodies revealed that canine malignant melanoma and osteosarcoma express CMTM6, CMTM4, and PD-L1 simultaneously. Gene knockdown of CMTM6 and CMTM4 with RNA interference significantly reduced the cell surface expression of PD-L1 in a canine cell line. These results suggest that CMTM6 and CMTM4 are regulators of PD-L1 expression in canine cancers and could serve as potential therapeutic targets to enhance antitumor immunity. - Yamato Sajiki, Satoru Konnai, Akie Ochi, Tomohiro Okagawa, Naftaly Githaka, Masayoshi Isezaki, Shinji Yamada, Takuya Ito, Shuji Ando, Hiroki Kawabata, Carlos Logullo, Itabajara da Silva Vaz Jr, Naoya Maekawa, Shiro Murata, Kazuhiko OhashiTicks and tick-borne diseases 11 (2) 101332 - 101332 2020/03 [Refereed][Not invited]
Tick saliva contains immunosuppressants which are important to obtain a blood meal and enhance the infectivity of tick-borne pathogens. In Japan, Ixodes persulcatus is a major vector for Lyme borreliosis pathogens, such as Borrelia garinii, as well as for those causing relapsing fever, such as B. miyamotoi. To date, little information is available on bioactive salivary molecules, produced by this tick. Thus, in this study, we identified two proteins, I. persulcatus derived sialostatin L1 (Ip-sL1) and sL2 (Ip-sL2), as orthologs of I. scapularis derived sL1 and sL2. cDNA clones of Ip-sL1 and Ip-sL2 shared a high identity with sequences of sL1 and sL2 isolated from the salivary glands of I. scapularis. Semi-quantitative PCR revealed that Ip-sL1 and Ip-sL2 were expressed in the salivary glands throughout the life of the tick. In addition, Ip-sL1 and Ip-sL2 were expressed even before the ticks started feeding, and their expression continued during blood feeding. Recombinant Ip-sL1 and Ip-sL2 were developed to characterize the proteins via biological and immunological analyses. These analyses revealed that both Ip-sL1 and Ip-sL2 had inhibitory effects on cathepsins L and S. Ip-sL1 and Ip-sL2 inhibited the production of IP-10, TNFα, and IL-6 by LPS-stimulated bone-marrow-derived dendritic cells (BMDCs). Additionally, Ip-sL1 significantly impaired BMDC maturation. Taken together, these results suggest that Ip-sL1 and Ip-sL2 confer immunosuppressive functions and appear to be involved in the transmission of pathogens by suppressing host immune responses, such as cytokine production and dendritic cell maturation. Therefore, further studies are warranted to investigate the immunosuppressive functions of Ip-sL1 and Ip-sL2 in detail to clarify their involvement in pathogen transmission via I. persulcatus. - Otgontuya Ganbaatar, Satoru Konnai, Tomohiro Okagawa, Yutaro Nojima, Naoya Maekawa, Erina Minato, Atsushi Kobayashi, Ryo Ando, Nobuya Sasaki, Daisuke Miyakoshi, Osamu Ichii, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiPloS one 15 (11) e0234218 2020 [Refereed][Not invited]
Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on lymphocytes. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. In our previous studies, we have developed anti-bovine PD-L1 monoclonal antibodies (mAbs) and reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Furthermore, we found that blocking antibodies that target PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway for chronic equine diseases, including tumors, remains unclear. In this study, we identified cDNA sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 mAbs against EqPD-L1 using in vitro assays. In addition, we evaluated the expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). The amino acid sequences of EqPD-1 and EqPD-L1 share a considerable identity and similarity with homologs from non-primate species. Two clones of the anti-bovine PD-L1 mAbs recognized EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine PD-1/PD-L1. Of note, immunohistochemistry confirmed the PD-L1 expression in EMM tumor tissues. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These findings showed that our anti-PD-L1 mAbs would be useful for analyzing the equine PD-1/PD-L1 pathway. Further research is warranted to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horses. - Yamato Sajiki, Satoru Konnai, Shinya Goto, Tomohiro Okagawa, Kosuke Ohira, Honami Shimakura, Naoya Maekawa, Satoshi Gondaira, Hidetoshi Higuchi, Motoshi Tajima, Yuki Hirano, Junko Kohara, Shiro Murata, Kazuhiko OhashiFrontiers in veterinary science 7 609443 - 609443 2020 [Refereed][Not invited]
Regulatory T cells (Tregs) regulate immune responses and maintain host immune homeostasis. Tregs contribute to the disease progression of several chronic infections by oversuppressing immune responses via the secretion of immunosuppressive cytokines, such as transforming growth factor (TGF)-β and interleukin-10. In the present study, we examined the association of Tregs with Mycoplasma bovis infection, in which immunosuppression is frequently observed. Compared with uninfected cattle, the percentage of Tregs, CD4+CD25highFoxp3+ T cells, was increased in M. bovis-infected cattle. Additionally, the plasma of M. bovis-infected cattle contained the high concentrations of TGF-β1, and M. bovis infection induced TGF-β1 production from bovine immune cells in in vitro cultures. Finally, we analyzed the immunosuppressive effects of TGF-β1 on bovine immune cells. Treatment with TGF-β1 significantly decreased the expression of CD69, an activation marker, in T cells, and Th1 cytokine production in vitro. These results suggest that the increase in Tregs and TGF-β1 secretion could be one of the immunosuppressive mechanisms and that lead to increased susceptibility to other infections in terms of exacerbation of disease during M. bovis infection. - Shinya Goto, Satoru Konnai, Yuki Hirano, Junko Kohara, Tomohiro Okagawa, Naoya Maekawa, Yamato Sajiki, Kei Watari, Erina Minato, Atsuhi Kobayashi, Satoshi Gondaira, Hidetoshi Higuchi, Masateru Koiwa, Motoshi Tajima, Eiji Taguchi, Ryoko Uemura, Shinji Yamada, Mika K Kaneko, Yukinari Kato, Keiichi Yamamoto, Mikihiro Toda, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiFrontiers in veterinary science 7 12 - 12 2020 [Refereed][Not invited]
Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E2 (PGE2) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE2 in immune dysfunction and the relationship between PGE2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE2 and the PD-1/PD-L1 pathway. - 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 KJpn J Vet Res 68 (2) 77 - 90 0047-1917 2020 [Refereed][Not invited]
Mycoplasma bovis (M. bovis) is a highly contagious pathogen and M. bovis-associated diseases, particularly pneumonia, occur predominantly as herd enzootics, causing considerable economic losses because of calf mortality, weight loss in surviving calves. In our previous studies, the programmed death-1 (PD-1)/ PD-ligand 1 (PD-L1) pathway and prostaglandin E2 (PGE2 ) were shown to be involved in the immunosuppression during chronic infectious diseases in cattle. In this study, the efficacy of dual blockade of the PD-1/PD-L1 pathway and PGE2 in M. bovis infection in vivo was investigated using anti-bovine PDL1 rat-bovine chimeric antibody, Boch4G12, and cyclooxygenase 2 (COX-2) inhibitor, meloxicam. The calves treated with Boch4G12 and meloxicam significantly enhanced M. bovis-specific IFN-γ response after the administration. On the other hand, IFN-γ response was not activated in the controls and cattle treated with meloxicam alone throughout the experimental period. Interestingly, bacterial loads in nasal discharge and bronchoalveolar lavage fluid among calves treated with Boch4G12 with or without meloxicam were significantly decreased. These results suggest that the combination of anti-PD-L1 antibody with a COX-2 inhibitor is a candidate for therapeutic applications in calves infected with M. bovis. - Kei Watari, Satoru Konnai, Naoya Maekawa, Tomohiro Okagawa, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiBMC VETERINARY RESEARCH 15 (1) 380 - 380 2019/10/29 [Refereed][Not invited]
BACKGROUND: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is known as an immune inhibitory receptor that is expressed on activated effector T cells and regulatory T cells. When CTLA-4 binds to CD80 or CD86, immunoinhibitory signals are transmitted to retain a homeostasis of the immune response. Recent studies have reported that CTLA-4 is upregulated in chronic infections and malignant neoplasms, contributing to host immune dysfunction. On the other hand, the blockade of CTLA-4 and CD80 or CD86 binding by antibody restores the immune response against these diseases. In a previous report, we indicated that the expression of CTLA-4 was closely associated with disease progression in cattle infected with the bovine leukemia virus (BLV). In this study, we established an anti-bovine CTLA-4 antibody to confirm its immune enhancing effect. RESULTS: Bovine CTLA-4-Ig binds to bovine CD80 and CD86 expressing cells. Additionally, CD80 and CD86 bind to CTLA-4 expressing cells in an expression-dependent manner. Bovine CTLA-4-Ig significantly inhibited interferon-gamma (IFN-γ) production from bovine peripheral blood mononuclear cells (PBMCs) activated by Staphylococcus enterotoxin B (SEB). An established specific monoclonal antibody (mAb) for bovine CTLA-4 specifically recognized only with bovine CTLA-4, not CD28, and the antibody blocked the binding of CTLA-4-Ig to both CD80 and CD86 in a dose-dependent manner. The bovine CTLA-4 mAb significantly restored the inhibited IFN-γ production from the CTLA-4-Ig treated PBMCs. In addition, the CTLA-4 mAb significantly enhanced IFN-γ production from CTLA-4 expressing PBMCs activated by SEB. Finally, we examined whether a CTLA-4 blockade by CTLA-4 mAb could restore the immune reaction during chronic infection; the blockade assay was performed using PBMCs from BLV-infected cattle. The CTLA-4 blockade enhanced IFN-γ production from the PBMCs in response to BLV-antigens. CONCLUSIONS: Collectively, these results suggest that anti-bovine CTLA-4 antibody can reactivate lymphocyte functions and could be applied for a new therapy against refractory chronic diseases. Further investigation is required for future clinical applications. - Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Naoya Maekawa, Shinya Goto, Kei Watari, Erina Minato, Atsushi Kobayashi, Junko Kohara, Shinji Yamada, Mika K Kaneko, Yukinari Kato, Hirofumi Takahashi, Nobuhiro Terasaki, Akira Takeda, Keiichi Yamamoto, Mikihiro Toda, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiJournal of immunology (Baltimore, Md. : 1950) 203 (5) 1313 - 1324 0022-1767 2019/09/01 [Refereed][Not invited]
Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application. - Inagaki H, Konnai S, Kaburagi H, Murota H, Takabatake N, Watari K, Okagawa T, Maekawa N, Murata S, Ohashi KJpn. J. Vet. Res 67 (3) 235 - 239 0047-1917 2019/08 [Refereed][Not invited]
One of the known transmission pathways of bovine leukemia virus (BLV) is bloodsucking insects. Against this background, this study investigated changes in BLV seroconversion in cattle by season on three private farms in Northern Hokkaido. Study results showed that no BLV seroconversion was observed during winter, a season without horse flies, and that all seroconversions occurred during summer. Thus, we collected horse flies which were observed sucking blood on cattle in public grazed fields and performed BLV detection tests in the insects. The tests showed that 75% of the total collected horse flies were BLV-positive. These results suggested the existence of vectors such as horse flies in grazing filed (in summer) was a risk factor of the spread of BLV infection in Northern Hokkaido. - Fujisawa S, Murata S, Takehara M, Katakura K, Hmoon MM, Win SY, Ohashi KBMC veterinary research 15 (1) 261 2019/07 [Refereed][Not invited]
- Masaki Takehara, Shiro Murata, Ken Katakura, Sotaro Fujisawa, Myint Myint Hmoon, Shwe Yee Win, Saw Bawm, Lat Lat Htun, Ye Htut Aung, Mar Mar Win, Masayoshi Isezaki, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko OhashiHELIYON 5 (4) e01544 2019/04 [Refereed][Not invited]
Haematophagous ectoparasites of poultry, such as Ornithonyssus sylviarum, northern fowl mites (NFMs), Dermanyssus gallinae, poultry red mites (PRMs), and Ornithonyssus bursa, tropical fowl mites (TFMs) are prevalent worldwide. Although poultry farming is a major industry in Southeast Asia, there are only a few reports concerning the prevalence of avian mites in this region. In this study, we sampled twenty farms in four major poultry farming areas in Myanmar. We detected the mites on six farms, and they showed morphological similarities to NFMs and TFMs. The nucleotide sequences of cytochrome c oxidase subunit I indicated that some mites were NFMs. This is the first report confirming the presence of NFMs and TFMs among the hematophagous mites infesting chickens on Myanmar poultry farms. - Fujisawa S, Konnai S, Okagawa T, Maekawa N, Tanaka A, Suzuki Y, Murata S, Ohashi KBMC veterinary research 15 (1) 68 - 68 2019/02 [Refereed][Not invited]
BACKGROUND: Refractory diseases, including bacterial infections, are causing huge economic losses in dairy farming. Despite efforts to prevent and treat those diseases in cattle, including the use of antimicrobials, it is not well controlled in the field. Several inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), play important roles in disease progression; thus, blocking these cytokines can attenuate the acute and sever inflammation and may be a novel strategy for treatment. However, biological drugs targeting inflammatory cytokines have not been used in cattle. Therefore, in this study, bovine sTNFR1 and sTNFR2 IgG1 Fc-fusion proteins (TNFR1-Ig and TNFR2-Ig) were produced, and their anti-inflammatory functions were analyzed in vitro, to develop decoy receptors for bovine TNF-α. RESULTS: Both TNFR1-Ig and TNFR2-Ig were shown to bind with TNF-α, and TNFR2-Ig showed higher affinity toward TNF-α than TNFR1-Ig. We next stimulated murine fibroblast-derived cells (L929 cells) with TNF-α to induce cell death and analyzed cell viability in the presence of TNFR-Ig proteins. Both TNFR1-Ig and TNFR2-Ig suppressed TNF-α-induced cell death, significantly improving cell viability. In addition, cell death induced by TNF-α was suppressed, even at low TNFR2-Ig concentrations, suggesting TNFR2-Ig has higher activity to suppress TNF-α functions than TNFR1-Ig. Finally, to examine TNFR2-Ig's anti-inflammatory, we cultured peripheral blood mononuclear cells from cattle with TNF-α in the presence of TNFR2-Ig and analyzed the gene expression and protein production of the inflammatory cytokines IL-1β and TNF-α. TNFR2-Ig significantly reduced the gene expression and protein production of these cytokines. Our results suggest that TNFR2-Ig inhibits inflammatory cytokine kinetics by blocking TNF-α to transmembrane TNFR, thereby attenuating excessive inflammation induced by TNF-α. CONCLUSIONS: Collectively, the findings of this study demonstrated the potential of TNFR2-Ig as a novel therapeutic for inflammatory diseases, such as bovine clinical mastitis. Further investigation is required for future clinical application. - Ochirkhuu N, Konnai S, Odbileg R, Okagawa T, Maekawa N, Murata S, Ohashi KJpn J Vet Res 66 (3) 177 - 192 0047-1917 2018/08 [Refereed][Not invited]
The immuno-inhibitory molecules PD-1, PD-L1, TIM-3, GAL-9, LAG-3, and CTLA-4 from blood samples of Mongolian native cattle and yak were characterized through cloning and sequencing. As these molecules are involved in cell-mediated immune responses, identifying the differences in their reactions against the pathogens found in bovine species may be beneficial. The amino acid sequences of these molecules were predicted for the purpose of characterizing their functional domains, such as the signal peptide, extracellular domain, transmembrane region, and intracellular domain. Amino acid alignment showed that the sequences of these immuno-inhibitory molecules from Mongolian native cattle and yak were highly homologous to those from other bovine species. As a preliminary application of the genetic information, we conducted expression analysis of PD-L1 in bovine viral diarrhea virus (BVDV)-infected yak by using real-time polymerase chain reaction, and PD-L1 mRNA expression in peripheral blood mononuclear cells derived from BVDV-infected yak was significantly upregulated compared to that of uninfected-yak. Further studies are necessary to assess whether these molecules play roles in disease progression during chronic infection of Mongolian native cattle and yak. - Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Junko Kohara, Yasuhiko Suzuki, Shinji Yamada, Yukinari Kato, Shiro Murata, Kazuhiko OhashiVeterinary research 49 (1) 50 - 50 0928-4249 2018/06/19 [Refereed][Not invited]
Bovine leukemia virus (BLV) is a retrovirus that infects B cells in cattle and causes bovine leukosis after a long latent period. Progressive exhaustion of T cell functions is considered to facilitate disease progression of BLV infection. Programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) are immunoinhibitory receptors that contribute to T-cell exhaustion caused by BLV infection in cattle. However, it is unclear whether the cooperation of PD-1 and LAG-3 accelerates disease progression of BLV infection. In this study, multi-color flow cytometric analyses of PD-1- and LAG-3-expressing T cells were performed in BLV-infected cattle at different stages of the disease. The frequencies of PD-1+LAG-3+ heavily exhausted T cells among CD4+ and CD8+ T cells was higher in the blood of cattle with B-cell lymphoma over that of BLV-uninfected and BLV-infected cattle without lymphoma. In addition, blockade assays of peripheral blood mononuclear cells were performed to examine whether inhibition of the interactions between PD-1 and LAG-3 and their ligands by blocking antibodies could restore T-cell function during BLV infection. Single or dual blockade of the PD-1 and LAG-3 pathways reactivated the production of Th1 cytokines, interferon-γ and tumor necrosis factor-α, from BLV-specific T cells of the infected cattle. Taken together, these results indicate that PD-1 and LAG-3 cooperatively mediate the functional exhaustion of CD4+ and CD8+ T cells and are associated with the development of B-cell lymphoma in BLV-infected cattle. - Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Reiko Nagata, Satoko Kawaji, Yumiko Kagawa, Shinji Yamada, Yukinari Kato, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Yasuyuki Mori, Kazuhiko OhashiInfection and immunity 86 (5) 0019-9567 2018/05 [Refereed][Not invited]
Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, is a bovine chronic infection that is endemic in Japan and many other countries. The expression of immunoinhibitory molecules is upregulated in cattle with Johne's disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E2 (PGE2) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE2 were performed to investigate the immunosuppressive effect of PGE2 during Johne's disease. In vitro PGE2 treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 and programmed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) from healthy cattle. PGE2 was upregulated in sera and intestinal lesions of cattle with Johne's disease. In vitro stimulation with Johnin purified protein derivative (J-PPD) induced cyclooxygenase-2 (COX-2) transcription, PGE2 production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with M. avium subsp. paratuberculosis Therefore, Johnin-specific Th1 responses could be limited by the PGE2 pathway in cattle. In contrast, downregulation of PGE2 with a COX-2 inhibitor promoted J-PPD-stimulated CD8+ T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade induced J-PPD-stimulated CD8+ T-cell proliferation and interferon gamma production in vitro Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies enhanced J-PPD-stimulated CD8+ T-cell proliferation in vitro, suggesting that the blockade of both pathways is a potential therapeutic strategy to control Johne's disease. The effects of COX-2 inhibition warrant further study as a novel treatment of Johne's disease. - 柴田明弘, 村田史郎, 中尾哲也, 井関博日本獣医師会雑誌 71 (3) 135‐139 0446-6454 2018/03/20 [Refereed][Not invited]
- Yamato Sajiki, Satoru Konnai, Asami Nishimori, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Masashi Nagano, Junko Kohara, Nana Kitano, Toshihiko Takahashi, Motoshi Tajima, Hirohisa Mekata, Yoichiro Horii, Shiro Murata, Kazuhiko OhashiThe Journal of veterinary medical science 79 (12) 2036 - 2039 0916-7250 2017/12/22 [Refereed][Not invited]
Enzootic bovine leukemia is caused by the bovine leukemia virus (BLV). BLV is transmitted vertically or horizontally through the transfer of infected cells via direct contact, through milk, insect bites and contaminated iatrogenic procedures. However, we lacked direct evidence of intrauterine infection. The purpose of this study was to confirm intrauterine BLV infection in two pregnant dams with high viral load by cesarean delivery. BLV was detected in cord and placental blood, and the BLV in the newborns showed 100% nucleotide identity with the BLV-env sequence from the dams. Notably, a newborn was seropositive for BLV but had no colostral antibodies. In this study, we presented a direct evidence of intrauterine BLV transmission in pregnant dam with a high proviral load. These results could aid the development of BLV control measures targeting viral load. - Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Ayako Nakahara, Yuzumi Chiba, Masaho Ikeda, Shiro Murata, Kazuhiko OhashiClinical and vaccine immunology : CVI 24 (12) 2017/12
- Tumenjargal Sharav, Satoru Konnai, Nyamsuren Ochirkhuu, Erdene T. S. Ochir, Hirohisa Mekata, Yoshihiro Sakoda, Takashi Umemura, Shiro Murata, Tungalag Chultemdorj, Kazuhiko OhashiJOURNAL OF VETERINARY MEDICAL SCIENCE 79 (11) 1884 - 1888 0916-7250 2017/11 [Refereed][Not invited]
The genetic characterization and actual prevalence of EIAV in Mongolian horse in the disease endemic region is currently unknown. Here, 11 of 776 horse serum samples from four Mongolian provinces tested positive on agar gel immunodiffusion test. Genomic DNA extracted from all seropositive samples was subjected to nested PCR assay. Among these, three samples tested positive with nested PCR assay and were identified by sequencing analysis based on long termination repeat and tat gene of the virus. Two of the three sequences were identical, with 94.0% identity with the third. These two independent Mongolian EIAV sequences were retained functional motifs, with no dramatic changes but some variability in the U5 region; they were clustered with genotypes from European countries but not with those from China, U.S.A., or Japan. - Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Ayako Nakahara, Yuzumi Chiba, Masaho Ikeda, Shiro Murata, Kazuhiko OhashiCLINICAL AND VACCINE IMMUNOLOGY 24 (9) 1556-6811 2017/09 [Refereed][Not invited]
Bovine leukemia is classified into two types: enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). EBL is caused by infection with bovine leukemia virus (BLV), which induces persistent lymphocytosis and B-cell lymphoma in cattle after a long latent period. Although it has been demonstrated that BLVassociated lymphoma occurs predominantly in adult cattle of > 3 to 5 years, suspicious cases of EBL onset in juvenile cattle were recently reported in Japan. To investigate the current status of bovine leukemia in Japan, we performed immunophenotypic analysis of samples from 50 cattle that were clinically diagnosed as having bovine leukemia. We classified the samples into five groups on the basis of the analysis and found two different types of EBL: classic EBL (cEBL), which has the familiar phenotype commonly known as EBL, and polyclonal EBL (pEBL), which exhibited neoplastic proliferation of polyclonal B cells. Moreover, there were several atypical EBL cases even in cEBL, including an early onset of EBL in juvenile cattle. A comparison of the cell marker expressions among cEBL, pEBL, and B-cell-type SBL (B-SBL) revealed characteristic patterns in B-cell leukemia, and these patterns could be clearly differentiated from those of healthy phenotypes, whereas it was difficult to discriminate between cEBL, pEBL, and B-SBL only by the expression patterns of cell markers. This study identified novel characteristics of bovine leukemia that should contribute to a better understanding of the mechanism underlying tumor development in BLV infection. - Maekawa N, Konnai S, Balbin MM, Mingala CN, Gicana KRB, Bernando FAEM, Murata S, Ohashi KTicks and tick-borne diseases 9 (2) 266 - 269 1877-959X 2017/09 [Refereed][Not invited]
Canine monocytic ehrlichiosis (CME), caused by a rickettsial bacterium, Ehrlichia canis, is distributed worldwide, particularly in tropical and subtropical regions. Transmission of E. canis is primarily mediated by the vector tick, Rhipicephalus sanguineus sensu lato and the bacteria then infect and replicate in monocytes and macrophages. Many cases are seen in veterinary hospitals and treated routinely; however, the genetic variation of E. canis strains found in the Philippines has been poorly investigated to date. In this study, the 16S rRNA gene and the gp200 gene of E. canis were detected by polymerase chain reaction from an infected dog in the Philippines, and the deduced amino acid sequence of the gp200 gene was subjected to a phylogenetic analysis. The Philippine genotype formed a cluster with the Taiwan genotype, and was somewhat divergent from the USA and Brazil strains. This suggested that E. canis underwent evolution in East and Southeast Asia, confirming the utility of the gp200 gene for the assessment of genetic relationships among strains. - Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Shiro Murata, Kazuhiko OhashiVECTOR-BORNE AND ZOONOTIC DISEASES 17 (8) 539 - 549 1530-3667 2017/08 [Refereed][Not invited]
Anaplasma species are obligate intracellular rickettsial pathogens that cause great economic loss to the animal industry. Few studies on Anaplasma infections in Mongolian livestock have been conducted. This study examined the prevalence of Anaplasma marginale, Anaplasma ovis, Anaplasma phagocytophilum, and Anaplasma bovis by polymerase chain reaction assay in 928 blood samples collected from native cattle and dairy cattle (Bos taurus), yaks (Bos grunniens), sheep (Ovis aries), and goats (Capra aegagrus hircus) in four provinces of Ulaanbaatar city in Mongolia. We genetically characterized positive samples through sequencing analysis based on the heat-shock protein groEL, major surface protein 4 (msp4), and 16S rRNA genes. Only A. ovis was detected in Mongolian livestock (cattle, yaks, sheep, and goats), with 413 animals (44.5%) positive for groEL and 308 animals (33.2%) positive for msp4 genes. In the phylogenetic tree, we separated A. ovis sequences into two distinct clusters based on the groEL gene. One cluster comprised sequences derived mainly from sheep and goats, which was similar to that in A. ovis isolates from other countries. The other divergent cluster comprised sequences derived from cattle and yaks and appeared to be newly branched from that in previously published single isolates in Mongolian cattle. In addition, the msp4 gene of A. ovis using same and different samples with groEL gene of the pathogen demonstrated that all sequences derived from all animal species, except for three sequences derived from cattle and yak, were clustered together, and were identical or similar to those in isolates from other countries. We used 16S rRNA gene sequences to investigate the genetically divergent A. ovis and identified high homology of 99.3-100%. However, the sequences derived from cattle did not match those derived from sheep and goats. The results of this study on the prevalence and molecular characterization of A. ovis in Mongolian livestock can facilitate the control of infectious diseases in livestock. - Naoya Maekawa, Satoru Konnai, Satoshi Takagi, Yumiko Kagawa, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Tatsuya Deguchi, Chie Nakajima, Yukinari Kato, Keiichi Yamamoto, Hidetoshi Uemura, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiSCIENTIFIC REPORTS 7 (1) 8951 2045-2322 2017/08 [Refereed][Not invited]
Immunotherapy targeting immune checkpoint molecules, programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), using therapeutic antibodies has been widely used for some human malignancies in the last 5 years. A costimulatory receptor, PD-1, is expressed on T cells and suppresses effector functions when it binds to its ligand, PD-L1. Aberrant PD-L1 expression is reported in various human cancers and is considered an immune escape mechanism. Antibodies blocking the PD-1/PD-L1 axis induce antitumour responses in patients with malignant melanoma and other cancers. In dogs, no such clinical studies have been performed to date because of the lack of therapeutic antibodies that can be used in dogs. In this study, the immunomodulatory effects of c4G12, a canine-chimerised anti-PD-L1 monoclonal antibody, were evaluated in vitro, demonstrating significantly enhanced cytokine production and proliferation of dog peripheral blood mononuclear cells. A pilot clinical study was performed on seven dogs with oral malignant melanoma (OMM) and two with undifferentiated sarcoma. Objective antitumour responses were observed in one dog with OMM (14.3%, 1/7) and one with undifferentiated sarcoma (50.0%, 1/2) when c4G12 was given at 2 or 5 mg/kg, every 2 weeks. c4G12 could be a safe and effective treatment option for canine cancers. - Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Chie Nakajima, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiFRONTIERS IN IMMUNOLOGY 8 650 1664-3224 2017/06 [Refereed][Not invited]
Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1)/PD-ligand 1 (PD-L1), is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat-bovine chimeric monoclonal antibody 5D2 (Boch5D2) was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV). Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4(+) T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals. - Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Yamato Sajiki, Yasuhiko Suzuki, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Shiro Murata, Kazuhiko OhashiPLOS ONE 12 (4) e0174916 1932-6203 2017/04 [Refereed][Not invited]
Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM(+) B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4(+) T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application. - 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, KImmunity, Inflammation and Disease 10 (3) 1 - 9 2017 [Refereed][Not invited]
INTRODUCTION: Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway in immunosuppression in bovine mycoplasmosis. METHODS: In the initial experiments, we used enzyme-linked immunosorbent assay to measure interferon-γ (IFN-γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis. RESULTS: Expectedly, IFN-γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD-1+ CD4+ and PD-L1+ CD14+ cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD-1+ CD4+ and PD-1+ CD8+ T cells were negatively correlated with IFN-γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD-1/PD-L1 pathway in vitro by anti-bovine PD-1- and anti-bovine PD-L1 antibodies significantly upregulated the production of IFN-γ from anti-mycoplasma-specific cells. CONCLUSIONS: These results suggest that the PD-1/PD-L1 pathway could be involved in immune exhaustion of bovine mycoplasma-specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways. - Yuka Machida, Shiro Murata, Ayumi Matsuyama-Kato, Masayoshi Isezaki, Akira Taneno, Eishi Sakai, Satoru Konnai, Kazuhiko OhashiJOURNAL OF VETERINARY MEDICAL SCIENCE 79 (1) 115 - 122 0916-7250 2017/01 [Refereed][Not invited]
Gallid herpesvirus 2 (GaHV-2) causes malignant lymphomas in chickens (Marek's disease, MD). Although MD is controlled through vaccination efforts, field isolates of GaHV-2 have increased in virulence worldwide and even cause MD in vaccinated chickens. GaHV-2 strains are classified into four categories (mild, virulent, very virulent and very virulent +) based on the virulence exhibited in experimental infection in unvaccinated or MD-vaccinated susceptible chickens. Although MD cases are sporadically reported in Japan, the recent field strains of GaHV-2 in Japan have not been characterized. During isolation of recent field strains by using primary chicken kidney cell cultures, a method classically used for GaHV-2 isolation, vaccine strains were simultaneously isolated. Therefore, it is necessary to separate vaccine strains to characterize the virulence and pathogenicity of the GaHV-2 strains currently distributed in Japan. In this study, we prepared cell suspensions from the spleens of MD-symptomatic chickens, inoculated day old -chicks and isolated GaHV-2 strains by primary chicken kidney cell cultures at 2-3 weeks post inoculation. The isolated strains were passaged several times on chicken embryo fibroblast cells, and PCR analysis revealed that the isolated strains were not contaminated with vaccine strains. Moreover, the contaminant vaccine strains were completely removed by the purification of plaques observed in chicken kidney cells. These procedures are necessary to isolate GaHV-2 field strains from vaccine strains in order to carry out future studies to characterize these strains and glean insights into GaHV-2 virulence and pathogenicity. - Satoru Konnai, Shiro Murata, Kazuhiko OhashiJOURNAL OF VETERINARY MEDICAL SCIENCE 79 (1) 1 - 5 0916-7250 2017/01 [Refereed][Not invited]
Recently, dysfunction of antigen-specific T cells is well documented as T-cell exhaustion and has been defined by the loss of effector functions during chronic infections and cancer in human. The exhausted T cells are characterized phenotypically by the surface expression of immunoinhibitory receptors, such as programmed death 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) and cytotoxic T-lymphocyte antigen 4 (CTLA-4). However, there is still a fundamental lack of knowledge about the immunoinhibitory receptors in the fields of veterinary medicine. In particular, very little is known about mechanism of T cell dysfunction in chronic infection in cattle. Recent our studies have revealed that immunoinhibitory molecules including PD-1/programmed death-ligand 1 (PD-L1) play critical roles in immune exhaustion and disease progression in case of bovine leukemia virus (BLV) infection, Johne's disease and bovine anaplasmosis. This review includes some recent data from us. - Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Shiro Murata, Kazuhiko OhashiJournal of Veterinary Medical Science 79 (12) 2040 - 2042 1347-7439 2017 [Refereed][Not invited]
Sheep-associated malignant catarrhal fever (SA-MCF), caused by ovine gammaherpesvirus-2 (OvHV-2), is a fatal disease in all ruminants. The epidemiological survey and molecular characterization of OvHV-2 in Mongolian livestock were performed. Of 928 blood samples, 14 were positive for OvHV-2 in sheep and native cattle from Tsenkher County and in sheep from Lun County. Phylogenetic analyses revealed that the tegument gene of OvHV-2 sequences from Mongolian animals is identical to that in animals from Egypt, India, and Turkey, and is 98.0% similar to that in animals from Germany and Brazil. To our knowledge, this is the first confirmed report of OvHV-2 in Mongolian livestock, and could provide useful information for controlling SA-MCF. - Tomohiro Okagawa, Satoru Konnai, James R. Deringer, Massaro W. Ueti, Glen A. Scoles, Shiro Murata, Kazuhiko Ohashi, Wendy C. BrownINFECTION AND IMMUNITY 84 (10) 2779 - 2790 0019-9567 2016/10 [Refereed][Not invited]
The CD4(+) T-cell response is central for the control of Anaplasma marginale infection in cattle. However, the infection induces a functional exhaustion of antigen-specific CD4(+) T cells in cattle immunized with A. marginale outer membrane proteins or purified outer membranes (OMs), which presumably facilitates the persistence of this rickettsia. In the present study, we hypothesize that T-cell exhaustion following infection is induced by the upregulation of immunoinhibitory receptors on T cells, such as programmed death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3). OM-specific T-cell responses and the kinetics of PD-1-positive (PD-1(+)) LAG-3(+) exhausted T cells were monitored in A. marginale-challenged cattle previously immunized with OMs. Consistent with data from previous studies, OM-specific proliferation of peripheral blood mononuclear cells (PBMCs) and interferon gamma (IFN-gamma) production were significantly suppressed in challenged animals by 5 weeks postinfection (wpi). In addition, bacteremia and anemia also peaked in these animals at 5 wpi. Flow cytometric analysis revealed that the percentage of PD-1(+) LAG-3(+) T cells in the CD4(+), CD8(+), and delta gamma T-cell populations gradually increased and also peaked at 5 wpi. A large increase in the percentage of LAG-3(+) gamma delta T cells was also observed. Importantly, in vitro, the combined blockade of the PD-1 and LAG-3 pathways partially restored OM-specific PBMC proliferation and IFN-gamma production at 5 wpi. Taken together, these results indicate that coexpression of PD-1 and LAG-3 on T cells contributes to the rapid exhaustion of A. marginale-specific T cells following infection and that these immunoinhibitory receptors regulate T-cell responses during bovine anaplasmosis. - Molecular detection and characterization of bovine viral diarrhea virus in Mongolian cattle and yaksNyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Battogtokh Odzaya, Shura Gansukh, Shiro Murata, Kazuhiko OhashiARCHIVES OF VIROLOGY 161 (8) 2279 - 2283 0304-8608 2016/08 [Refereed][Not invited]
Bovine viral diarrhea virus (BVDV) is classified into two species, namely, Bovine viral diarrhea virus 1 and Bovine viral diarrhea virus 2, and affects cattle worldwide, resulting in significant economic loss. The prevalence of BVDV-1 and BVDV-2 infections and its genotypes in Mongolian animals has not been studied. In this study, we surveyed BVDV infection in dairy cattle and yaks from Bornuur and Bulgan counties by RT-PCR, and the average infection rate in the sampling sites was 15.8 % and 20.0 %, respectively. In addition, molecular features of the 5'-UTR region of the BVDV genome in Mongolian cattle and yaks were identified as belonging to the subtypes BVDV-1a and BVDV-2a, respectively. Determining the prevalence, geographical distribution, and molecular diversity of BVDV-1 and BVDV-2 in various host species in Mongolia is important for further studies and process control programs. - Naoya Maekawa, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Satoshi Takagi, Yumiko Kagawa, Chie Nakajima, Yasuhiko Suzuki, Yukinari Kato, Shiro Murata, Kazuhiko OhashiPLOS ONE 11 (6) e0157176 1932-6203 2016/06 [Refereed][Not invited]
Spontaneous cancers are common diseases in dogs. Among these, some malignant cancers such as oral melanoma, osteosarcoma, hemangiosarcoma, and mast cell tumor are often recognized as clinical problems because, despite their high frequencies, current treatments for these cancers may not always achieve satisfying outcomes. The absence of effective systemic therapies against these cancers leads researchers to investigate novel therapeutic modalities, including immunotherapy. Programmed death 1 (PD-1) is a costimulatory receptor with immunosuppressive function. When it binds its ligands, PD-ligand 1 (PD-L1) or PD-L2, PD-1 on T cells negatively regulates activating signals from the T cell receptor, resulting in the inhibition of the effector function of cytotoxic T lymphocytes. Aberrant PD-L1 expression has been reported in many human cancers and is considered an immune escape mechanism for cancers. In clinical trials, anti-PD-1 or anti-PD-L1 antibodies induced tumor regression for several malignancies, including advanced melanoma, non-small cell lung carcinoma, and renal cell carcinoma. In this study, to assess the potential of the PD-1/PD-L1 axis as a novel therapeutic target for canine cancer immunotherapy, immunohistochemical analysis of PD-L1 expression in various malignant cancers of dogs was performed. Here, we show that dog oral melanoma, osteosarcoma, hemangiosarcoma, mast cell tumor, mammary adenocarcinoma, and prostate adenocarcinoma expressed PD-L1, whereas some other types of cancer did not. In addition, PD-1 was highly expressed on tumor-infiltrating lymphocytes obtained from oral melanoma, showing that lymphocytes in this cancer type might have been functionally exhausted. These results strongly encourage the clinical application of PD-1/PD-L1 inhibitors as novel therapeutic agents against these cancers in dogs. - Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Ayako Nakahara, Shiro Murata, Kazuhiko OhashiJOURNAL OF VETERINARY MEDICAL SCIENCE 78 (5) 791 - 796 0916-7250 2016/05 [Refereed][Not invited]
Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective therapy or vaccine; thus, the diagnosis and elimination of BLV-infected cattle are the most effective method to eradicate the infection. Clinical veterinarians need a simpler and more rapid method of diagnosing infection, because both nested polymerase chain reaction (PCR) and real-time PCR are labor intensive, time-consuming, and require specialized molecular biology techniques and expensive equipment. In this study, we describe a novel PCR method for amplifying the BLV provirus from whole blood, thus eliminating the need for DNA extraction. Although the sensitivity of PCR directly from whole blood (PCR-DB) samples as measured in bovine blood containing BLV-infected cell lines was lower than that of nested PCR, the PCR-DB technique showed high specificity and reproducibility. Among 225 clinical samples, 49 samples were positive by nested PCR, and 37 samples were positive by PCR-DB. There were no false positive samples; thus, PCR-DB sensitivity and specificity were 75.51% and 100%, respectively. However, the provirus loads of the samples detected by nested PCR and not PCR-DB were quite low. Moreover, PCR-DB also stably amplified the BLV provirus from tumor tissue samples. PCR-DB method exhibited good reproducibility and excellent specificity and is suitable for screening of thousands of cattle, thus serving as a viable alternative to nested PCR and real-time PCR. - Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Asami Nishimori, Tomohiro Okagawa, Shiro Murata, Kazuhiko OhashiARCHIVES OF VIROLOGY 161 (4) 985 - 991 0304-8608 2016/04 [Refereed][Not invited]
Epidemiological studies have indicated that bovine leukemia virus (BLV) infection is globally distributed. However, no information regarding the disease and genetic diversity of the virus in the cattle of Mongolia is currently available. In this study, the prevalence of BLV was assessed using PCR, and the genetic diversity was analyzed through DNA sequencing. Of the 517 samples tested, 20 positives were identified. Phylogenetic analysis showed that six, one, and four isolates were classified into genotype 4, 7, and 1, respectively. Most isolates were clustered with isolates from Eastern Europe and Russia. This study is the first to investigate the BLV genotype in Mongolia. - Ohira K, Nakahara A, Konnai S, Okagawa T, Nishimori A, Maekawa N, Ikebuchi R, Kohara J, Murata S, Ohashi KImmunity, inflammation and disease 4 (1) 52 - 63 2016/03 [Refereed][Not invited]
CD4(+)CD25(high)Foxp3(+) T cells suppress excess immune responses that lead to autoimmune and/or inflammatory diseases, and maintain host immune homeostasis. However, CD4(+)CD25(high)Foxp3(+) T cells reportedly contribute to disease progression by over suppressing immune responses in some chronic infections. In this study, kinetic and functional analyses of CD4(+)CD25(high)Foxp3(+) T cells were performed in cattle with bovine leukemia virus (BLV) infections, which have reported immunosuppressive characteristics. In initial experiments, production of the Th1 cytokines IFN-γ and TNF-α was reduced in BLV-infected cattle compared with uninfected cattle, and numbers of IFN-γ or TNF-α producing CD4(+) T cells decreased with disease progression. In contrast, IFN-γ production by NK cells was inversely correlated with BLV proviral loads in infected cattle. Additionally, during persistent lymphocytosis disease stages, NK cytotoxicity was depressed as indicated by low expression of the cytolytic protein perforin. Concomitantly, total CD4(+)CD25(high)Foxp3(+) T cell numbers and percentages of TGF-β(+) cells were increased, suggesting that TGF-β plays a role in the functional declines of CD4(+) T cells and NK cells. In further experiments, recombinant bovine TGF-β suppressed IFN-γ and TNF-α production by CD4(+) T cells and NK cytotoxicity in cultured cells. These data suggest that TGF-β from CD4(+)CD25(high)Foxp3(+) T cells is immunosuppressive and contributes to disease progression and the development of opportunistic infections during BLV infection. - Kochi Toyomane, Satoru Konnai, Ayano Niwa, Naftaly Githaka, Masayoshi Isezaki, Shinji Yamada, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Shiro Murata, Kazuhiko OhashiTICKS AND TICK-BORNE DISEASES 7 (1) 119 - 125 1877-959X 2016 [Refereed][Not invited]
Ixodes ricinus immunosuppressor (Iris) is a tick salivary gland protein derived from I. ricinus. In this study, Iris homolog was identified in the salivary glands of Ixodes persulcatus, which is the specific vector of the Lyme disease agent in Japan. The homolog was named Ipis-1. To investigate the function of Ipis-1, we prepared a recombinant Ipis-1 expressed in COS-7 cells as a rabbit IgG Fc-fused protein (Ipis-1-Ig). Cell proliferation assay and IFN-gamma ELISA showed that Ipis-1-Ig inhibits the proliferation and IFN-gamma production of bovine peripheral blood mononuclear cells (PBMCs). Notably, Ipis-1-Ig inhibited the cell proliferation and production of IFN-gamma in bovine PBMCs even when CD14(+) cells were depleted, suggesting that Ipis could directly interact with T cells and inhibit their functions. In conclusion, Ipis could contribute to the establishment of environments suitable for tick blood feeding and pathogen transmission by suppressing the function of immune cells. (C) 2015 Elsevier GmbH. All rights reserved. - Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Ryoyo Ikebuchi, Seiko Mizorogi, Reiko Nagata, Satoko Kawaji, Shogo Tanaka, Yumiko Kagawa, Shiro Murata, Yasuyuki Mori, Kazuhiko OhashiINFECTION AND IMMUNITY 84 (1) 77 - 89 0019-9567 2016/01 [Refereed][Not invited]
Johne's disease (paratuberculosis) is a chronic enteritis in cattle that is caused by intracellular infection with Mycobacterium avium subsp. paratuberculosis. This infection is characterized by the functional exhaustion of T-cell responses to M. avium subsp. paratuberculosis antigens during late subclinical and clinical stages, presumably facilitating the persistence of this bacterium and the formation of clinical lesions. However, the mechanisms underlying T-cell exhaustion in Johne's disease are poorly understood. Thus, we performed expression and functional analyses of the immunoinhibitory molecules programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) and lymphocyte activation gene 3 (LAG-3)/major histocompatibility complex class II (MHC-II) in M. avium subsp. paratuberculosis-infected cattle during the late subclinical stage. Flow cytometric analyses revealed the upregulation of PD-1 and LAG-3 in T cells in infected animals, which suffered progressive suppression of interferon gamma (IFN-gamma) responses to the M. avium subsp. paratuberculosis antigen. In addition, PD-L1 and MHC-II were expressed on macrophages from infected animals, consistent with PD-1 and LAG-3 pathways contributing to the suppression of IFN-gamma responses during the subclinical stages of M. avium subsp. paratuberculosis infection. Furthermore, dual blockade of PD-L1 and LAG-3 enhanced M. avium subsp. paratuberculosis-specific IFN-gamma responses in blood from infected animals, and in vitro LAG-3 blockade enhanced IFN-gamma production from M. avium subsp. paratuberculosis-specific CD4(+) and CD8(+) T cells. Taken together, the present data indicate that M. avium subsp. paratuberculosis-specific T-cell exhaustion is in part mediated by PD-1/PD-L1 and LAG-3/MHC-II interactions and that LAG-3 is a molecular target for the control of M. avium subsp. paratuberculosis-specific T-cell responses. - Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Shiro Murata, Kazuhiko OhashiJAPANESE JOURNAL OF VETERINARY RESEARCH 63 (4) 191 - 194 0047-1917 2015/11 [Refereed][Not invited]
Johne's disease is a chronic infection with Mycobacterium avium susp. paratuberculosis (MAP), which causes huge economic losses to cattle industry. The seroprevalence of MAP in cattle of Mongolian was estimated by an ELISA assay using 356 serum samples which were collected from eleven provinces and Ulaanbaatar city. Out of these samples, 3 (0.84%) were found to be seropositive for MAP, originating from Tsenkher sum of Arkhangai province, Murun sum of khuvsgul province, and Bornuur sum of Tuv province in Mongolia. This study represents first conformation of Johne's disease in Mongolian cattle. These findings provide vital information that can be used for the planning and execution of control measures for Johne's disease in the Mongolian cattle industry. - Nyamsuren Ochirkhuu, Satoru Konnai, Claro N. Mingala, Tomohiro Okagawa, Marvin Villanueva, Flor Marie Immanuelle R. Pilapil, Shiro Murata, Kazuhiko OhashiVETERINARY PARASITOLOGY 212 (3-4) 161 - 167 0304-4017 2015/09 [Refereed][Not invited]
In the Philippines, vector-borne disease is one of the important problems in the livestock industry. To elucidate the epidemiology of vector-borne diseases in cattle on Luzon Island, the Philippines, the prevalence of five protozoan agents was assessed by polymerase chain reaction. Out of the 339 samples, 324 (95.5%), 154 (45.4%), 209 (61.6%), 140 (41.3%), and 2 (0.6%) were positive for Anaplasma marginale, Babesia bigemina, Babesia bovis, Theileria spp., and Trypanosoma evansi infections, respectively. Mixed infections were detected in 290 (85.5%) samples, of which 115 (33.9%) had two pathogens, 144 (42.5%) had three pathogens, and 31 (9.1%) had four kinds of pathogens. 16S rRNA gene was 100% identical in A. marginale compared with the same lineage across the world. B. bovis RAP-1 and B. bigemina AMA-1 genes were identical with 92.27%-100% and 97.07%-100% sequences, respectively, in the database (Asian isolates). MPSP genes of Theileria spp. were 83.51%-100% identical with the one another. Phylogenetic analysis showed that they belong to the groups of T. sergenti and T. buffeli. Positive rates of the tick-borne pathogens were extremely high in this area. These findings provide vital information that can be used for the planning and execution of effective control measures for vector-borne diseases in the Philippine cattle industry. (C) 2015 Elsevier B.V. All rights reserved. - Hirohisa Mekata, Shiro Murata, Claro Niegos Mingala, Kazuhiko Ohashi, Satoru KonnaiJOURNAL OF VETERINARY MEDICAL SCIENCE 77 (8) 1017 - 1019 0916-7250 2015/08 [Refereed][Not invited]
Trypanosoma evansi causes wasting disease in many livestock. T. evansi infection gives rise to inflammatory immune responses, which contribute to the development of inflammation-associated tissue injury. We previously reported that regulatory dendritic cells (DCs), which act as potential regulators of inflammation, were activated in infected mice and transfer of regulatory DCs to infected mice prolonged their survival. However, the kinetics of regulatory DCs in cattle, which are natural hosts of T. evansi, remained unclear. In this study, we report that the expressions of CCL8 and IL-10, which promote the development of regulatory DCs, were up-regulated in cattle experimentally infected with T. evansi. This finding is potentially useful for studying the control strategy of T. evansi infection in cattle. - Saori Suzuki, Satoru Konnai, Tomohiro Okagawa, Ryoyo Ikebuchi, Asami Nishimori, Junko Kohara, Claro N. Mingala, Shiro Murata, Kazuhiko OhashiVETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 163 (3-4) 115 - 124 0165-2427 2015/02 [Refereed][Not invited]
Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4(+)CD25(+)Foxp3(+) T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3(+)CD4(+) cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-gamma expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CfLA-4(+) cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4+ T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4(+) and CD25(+) T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4(+) cells in the CD4(+) T cell subpopulation was positively correlated with TGF-beta mRNA expression, suggesting that CD4(+)CTLA-4(+) T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-gamma mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection. (C) 2014 Elsevier B.V. All rights reserved. - Murase Y, Konnai S, Yamada S, Githaka N, Isezaki M, Ito T, Takano A, Ando S, Kawabata H, Murata S, Ohashi KInsect Biochemistry and Molecular Biology 60 59 - 67 2015 [Refereed][Not invited]
- Influence of PD-L1 cross-linking on cell death in PD-L1-expressing cell lines and bovine lymphocytesRyoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiIMMUNOLOGY 142 (4) 551 - 561 0019-2805 2014/08 [Refereed][Not invited]
Programmed death-ligand 1 (PD-L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death-1 (PD-1). However, the mechanism of PD-L1-mediated inhibitory signalling after PD-L1 cross-linking by anti-PD-L1 monoclonal antibody (mAb) or PD-1-immunogloblin fusion protein (PD-1-Ig) is still unknown, although it may induce cell death of PD-L1(+) cells required for regular immune reactions. In this study, PD-1-Ig or anti-PD-L1 mAb treatment was tested in cell lines that expressed PD-L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD-L1-mediated cell death. PD-L1 cross-linking by PD-1-Ig or anti-PD-L1 mAb primarily increased the number of dead cells in PD-L1(high) cells, but not in PD-L1(low) cells; these cells were prepared from Cos-7 cells in which bovine PD-L1 expression was induced by transfection. The PD-L1-mediated cell death also occurred in Cos-7 and HeLa cells transfected with vectors only encoding the extracellular region of PD-L1. In bovine lymphocytes, the anti-PD-L1 mAb treatment up-regulated interferon-gamma (IFN-gamma) production, whereas PD-1-Ig treatment decreased this cytokine production and cell proliferation. The IFN-gamma production in B-cell-depleted peripheral blood mononuclear cells was not reduced by PD-1-Ig treatment and the percentages of dead cells in PD-L1(+) B cells were increased by PD-1-Ig treatment, indicating that PD-1-Ig-induced immunosuppression in bovine lymphocytes could be caused by PD-L1-mediated B-cell death. This study provides novel information for the understanding of signalling through PD-L1. - Ayumi Matsuyama-Kato, Shiro Murata, Masayoshi Isezaki, Sarah Takasaki, Rika Kano, Satoru Konnai, Kazuhiko OhashiARCHIVES OF VIROLOGY 159 (8) 2123 - 2126 0304-8608 2014/08 [Refereed][Not invited]
PD-L2 is a ligand of the immunoinhibitory receptor PD-1. Here, we report functional and expression analyses of PD-L2 in tumor lesions and spleens from chickens infected with gallid herpesvirus 2 (GaHV-2, Marek's disease virus), which induces malignant lymphomas in chickens. We show that the expression of IFN-gamma protein was decreased in PBMCs and splenocytes co-cultured with PD-L2-expressing cells and that the expression of PD-L2 mRNA was significantly higher in the spleens of infected chickens in the latent phase and in tumor lesions caused by GaHV-2. These results suggest that chicken PD-L2 has an immunoinhibitory function and is involved in the establishment of latency and tumor formation by GaHV-2. - Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Ayako Nakahara, Shiro Murata, Kazuhiko OhashiJOURNAL OF GENERAL VIROLOGY 95 (Pt 8) 1832 - 1842 0022-1317 2014/08 [Refereed][Not invited]
Bovine leukemia virus (BLV) induces abnormal B-cell proliferation and B-cell lymphoma in cattle, where the BLV provirus is integrated into the host genome. BLV-infected B-cells rarely express viral proteins in vivo, but short-term cultivation augments BLV expression in some, but not all, BLV-infected B-cells. This observation suggests that two subsets, i.e. BLV-silencing cells and BLV-expressing cells, are present among BLV-infected B-cells, although the mechanisms of viral expression have not been determined. In this study, we examined B-cell markers and viral antigen expression in B-cells from BLV-infected cattle to identify markers that may discriminate BLV, expressing cells from BLV-silencing cells. The proportions of IgM(high) B-cells were increased in blood lymphocytes from BLV-infected cattle. IgM(high) B-cells mainly expressed BLV antigens, whereas IgM(low) B-cells did not, although the provirus load was equivalent in both subsets. Several parameters were investigated in these two subsets to characterize their cellular behaviour. Real-time PCR and microarray analyses detected higher expression levels of some proto-oncogenes (e.g. Maf, Jun and Fos) in IgM(low) B-cells than those in IgM(high) B-cells. Moreover, lymphoma cells obtained from the lymph nodes of 14 BLV-infected cattle contained IgM(low) or IgM(-) B-cells but no IgM(high) B-cells. To our knowledge, this is the first study to demonstrate that IgM(high) B-cells mainly comprise BLV-expressing cells, whereas IgM(low) B-cells comprise a high proportion of BLV-silencing B-cells in BLV-infected cattle. - Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Chie Nakajima, Yasuhiko Suzuki, Claro N. Mingala, Shiro Murata, Kazuhiko OhashiMICROBIOLOGY AND IMMUNOLOGY 58 (7) 388 - 397 0385-5600 2014/07 [Refereed][Not invited]
Previous reports from this group have indicated that the immunoinhibitory programmed death (PD)-1 receptor and its ligand, PD-L1, are involved in the mechanism of immune evasion of bovine chronic infection. However, no functional analysis of bovine PD-L2 in cattle has been reported. Thus, in this study, the molecular function of bovine PD-L2 was analyzed in vitro. Recombinant PD-L2 (PD-L2-Ig), which comprises an extracellular domain of bovine PD-L2 fused to the Fc portion of rabbit IgG1, was prepared based on the cloned cDNA sequence for bovine PD-L2. Bovine PD-L2-Ig bound to bovine PD-1-expressing cells and addition of soluble bovine PD-1-Ig clearly inhibited the binding of PD-L2-Ig to membrane PD-1 in a dose-dependent manner. Cell proliferation and IFN-gamma production were significantly enhanced in the presence of PD-L2-Ig in peripheral blood mononuclear cells (PBMCs) from cattle. Moreover, PD-L2-Ig significantly enhanced IFN-gamma production from virus envelope peptides-stimulated PBMCs derived from bovine leukemia virus-infected cattle. Interestingly, PD-L2-Ig-induced IFN-gamma production was further enhanced by treatment with anti-bovine PD-1 antibody. These data suggest potential applications of bovine PD-L2-Ig as a therapy for bovine diseases. - Naoya Maekawa, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Mami Adachi, Satoshi Takagi, Yumiko Kagawa, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiPLOS ONE 9 (6) e98415 1932-6203 2014/06 [Refereed][Not invited]
Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together induce the "exhausted'' status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells. In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1-expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-gamma production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity in vivo, and its therapeutic potential has to be further discussed. - Naftaly Githaka, Satoru Konnai, Richard Bishop, David Odongo, Isaac Lekolool, Edward Kariuki, Francis Gakuya, Lucy Kamau, Masayoshi Isezaki, Shiro Murata, Kazuhiko OhashiVETERINARY PARASITOLOGY 202 (3-4) 180 - 193 0304-4017 2014/05 [Refereed][Not invited]
Waterbuck (Kobus defassa), an ungulate species endemic to the Eastern African savannah, is suspected of being a wildlife reservoir for tick-transmitted parasites infective to livestock. Waterbuck is infested by large numbers of Rhipicephalus appendiculatus, the tick vector for Theileria parva, and previous data suggests that the species may be a source of T. parva transmission to cattle. In the present study, a total of 86 cattle and 26 waterbuck blood samples were obtained from Marula, a site in Kenya endemic for East Coast fever (ECF) where the primary wildlife reservoir of T. parva the Cape buffalo (Syncerus coffer) is also common. To investigate for the presence of cattle-infective Theileria parasites, DNA specimens extracted from the blood samples were subjected to two diagnostic assays; a nested PCR based on the p104 gene that is specific for T. parva, and a reverse line blot (RLB) incorporating 13 oligonucleotide probes including all of the Theileria spp. so far described from livestock and wildlife in Kenya. Neither assay provided evidence of T. parva or Theileria sp. (buffalo) infection in the waterbuck DNA samples. By contrast, majority of the cattle samples (67.4%) were positive for T. parva using a nested PCR assay. The RLB assay, including a generic probe for the genus Theileria, indicated that 25/26 (96%) of the waterbuck samples were positive for Theileria, while none of the 11 Theileria species-specific probes hybridized with the waterbuck-derived PCR products. Phylogenetic analysis of 18S ribosomal RNA (18S rRNA) and internal transcribed spacer (ITS) sequences within the RLB-positive waterbuck samples revealed the occurrence of three Theileria genotypes of unknown identity designated A, B and C. Group A clustered with Theileria equi, a pathogenic Theileria species and a causative agent of equine piroplasmosis in domestic equids. However, DNA from this group failed to hybridize with the T. equi oligonucleotide present on the RLB filter probe, suggesting the occurrence of novel taxa in these animals. This was confirmed by DNA sequencing that revealed heterogeneity between the waterbuck isolates and previously reported T. equi genotypes. Group B parasites clustered closely with Theileria luwenshuni, a highly pathogenic parasite of sheep and goats reported from China. Group C was closely related to Theileria ovis, an apparently benign parasite of sheep. Together, these findings provided no evidence that waterbuck plays a role in the transmission of T. parva. However, novel Theileria genotypes detected in this bovid species may be of veterinary importance. (C) 2014 Elsevier B.V. All rights reserved. - Hidano A, Konnai S, Yamada S, Githaka N, Isezaki M, Higuchi H, Nagahata H, Ito T, Takano A, Ando S, Kawabata H, Murata S, Ohashi KInsect Molecular Biology 23 (4) 466 - 474 0962-1075 2014 [Refereed][Not invited]
Salp16, a 16-kDa tick salivary gland protein, is known to be the molecule involved in the transmission of Anaplasma phagocytophilum, an obligate intracellular pathogen causing zoonotic anaplasmosis, from its mammalian hosts to Ixodes scapularis. Recently, the presence of A. phagocytophilum was documented in Japan and Ixodes persulcatus was identified as one of its vectors. The purpose of this study was to identify Salp16 genes in I. persulcatus and characterize their function. Two cDNA clones encoding the Salp16-like sequences were obtained from the salivary glands of fed female I. persulcatus ticks and designated Salp16 Iper1 and Iper2. Gene expression analyses showed that the Salp16 Iper genes were expressed specifically in the salivary glands and were up-regulated by blood feeding. These proteins attenuated the oxidative burst of activated bovine neutrophils and inhibited their migration induced by the chemoattractant interleukin-8 (IL-8). These results demonstrate that Salp16 Iper proteins contribute to the establishment of blood feeding as an immunosuppressant of neutrophil, an essential factor in innate host immunity. Further examination of the role of Salp16 Iper in the transmission of pathogens, including A. phagocytophilum, will increase our understanding of the tick-host-pathogen interface. © 2014 The Royal Entomological Society. - Ooshiro M, Konnai S, Katagiri Y, Afuso M, Arakaki N, Tsuha O, Murata S, Ohashi KVet Rec. 173 (21) 527 2013/11 [Refereed][Not invited]
- Naftaly Githaka, Satoru Konnai, Robert Skilton, Edward Kariuki, Esther Kanduma, Shiro Murata, Kazuhiko OhashiParasitology International 62 (5) 448 - 453 1383-5769 2013/10 [Refereed][Not invited]
Recently, mortalities among giraffes, attributed to infection with unique species of piroplasms were reported in South Africa. Although haemoparasites are known to occur in giraffes of Kenya, the prevalence, genetic diversity and pathogenicity of these parasites have not been investigated.In this study, blood samples from 13 giraffes in Kenya were investigated microscopically and genomic DNA extracted. PCR amplicons of the hyper-variable region 4 (V4) of Theileria spp. small subunit ribosomal RNA (18S rRNA) gene were hybridized to a panel of genus- and species-specific oligonucleotide probes by reverse line blot (RLB). Two newly designed oligonucleotide probes specific for previously identified Theileria spp. of giraffes found single infections in eight of the specimens and mixed infections in the remaining five samples.Partial 18S rRNA genes were successfully amplified from 9 samples and the PCR amplicons were cloned. A total of 28 plasmid clones representing the Kenyan isolates were analyzed in the present study and compared with those of closely-related organisms retrieved from GenBank. In agreement with RLB results, the nucleotide sequence alignment indicated the presence of mixed infections in the giraffes. In addition, sequence alignment with the obtained 18S rRNA gene sequences revealed extensive microheterogeneities within and between isolates, characterized by indels in the V4 regions and point mutations outside this region. Phylogeny with 18S rRNA gene sequences from the detected parasites and those of related organisms places Theileria of giraffes into two major groups, within which are numerous clades that include the isolates reported in South Africa. Collectively, these data suggest the existence of at least two distinct Theileria species among giraffes, and extensive genetic diversity within the two parasite groups. © 2013 Elsevier Ireland Ltd. - Saori Suzuki, Satoru Konnai, Tomohiro Okagawa, Ryoyo Ikebuchi, Tatsuya Shirai, Yuji Sunden, Claro N. Mingala, Shiro Murata, Kazuhiko OhashiMICROBIOLOGY AND IMMUNOLOGY 57 (8) 600 - 604 0385-5600 2013/08 [Refereed][Not invited]
In the present study, we monitored Foxp3+ T cells in bovine leukemia virus (BLV)-infected cattle. By flow cytometric analysis, the proportion of Foxp3+CD4+ cells from persistent lymphocytotic cattle was significantly increased compared to control and AL cattle. Interestingly, the proportion of Foxp3+CD4+ cells correlated positively with the increased number of lymphocytes, virus titer and virus load, whereas it inversely correlated with IFN-γ mRNA expression, suggesting that Foxp3+CD4+ T cells in cattle have a potentially immunosuppressive function. Further studies are necessary to elucidate the detailed mechanism behind the increased Treg during BLV infection. © 2013 The Societies and Wiley Publishing Asia Pty Ltd. - Shiro Murata, Tomoyuki Hashiguchi, Yuko Hayashi, Yuki Yamamoto, Ayumi Matsuyama-Kato, Sarah Takasaki, Masayoshi Isezaki, Misao Onuma, Satoru Konnai, Kazuhiko OhashiINFECTION GENETICS AND EVOLUTION 16 137 - 143 1567-1348 2013/06 [Refereed][Not invited]
Serotype 1 strains of Marek's disease virus (MDV-1) cause malignant lymphomas in chickens (Marek's disease; MD). Although MD has been controlled by vaccination, field isolates of MDV-1 have tended to increase in virulence and cause MD even in vaccinated chickens. Meq, a putative MDV-1 oncoprotein, resembles the Jun/Fos family of basic leucine zipper (bZIP) transcription factors and can regulate the expression of viral and cellular genes as a homodimer or as a heterodimer with a variety of bZIP family proteins. Sequencing analysis of some of the viral genes of various MDV-1 strains revealed a distinct diversity of and point mutations in Meq, which may contribute to changes in the transcriptional activities of Meq and, consequently, to increases in MDV-1 oncogenicity. However, few reports have characterized MDV-1 strains isolated in Japan. In this study, we established the amino acid sequences of MDV-1 field isolates from Japan in order to determine whether they display a distinct diversity of and point mutations in Meq. In addition, we analyzed the transactivation activities of the Meq proteins in order to evaluate whether the observed mutations affect their functions. Japanese MDV-1 isolates displayed the distinct mutations in basic region 2 (BR2) and proline-rich repeats (PRRs) of the Meq proteins as well as some unique mutations. Reporter assays revealed that the amino acid substitutions in BR2 and the PRRs affected the Meq transactivation activity. These results suggest that the distinct mutations are also present in the Meq proteins of MDV-1 isolates from Japan and affect their transactivation activities. (C) 2013 Elsevier B.V. All rights reserved. - Suzuki S, Konnai S, Okagawa T, Ikebuchi R, Shirai T, Sunden Y, Mingala CN, Murata S, Ohashi KMicrobiology and immunology 0385-5600 2013/06 [Refereed][Not invited]
- Saiki Imamura, Mari Nakamizo, Michiko Kawanishi, Nao Nakajima, Kinya Yamamoto, Mariko Uchiyama, Fumiya Hirano, Hidetaka Nagai, Mayumi Kijima, Ryoyo Ikebuchi, Hirohisa Mekata, Shiro Murata, Satoru Konnai, Kazuhiko OhashiVETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 153 (1-2) 153 - 158 0165-2427 2013/05 [Refereed][Not invited]
In order to analyze bovine immune reactions against the Gram-negative bacterial vaccine, bovine whole-blood culture was used to investigate the pro-inflammatory cytokine responses stimulated with lipopolysaccharides (LPS) extracted from Escherichia colt, Salmonella enterica, Pseudomonas aeruginosa, and Klebsiella pneumoniae. We also examined the interaction between LPS and aluminum hydroxide gel for endotoxin activity and pro-inflammatory cytokine responses of whole bovine blood. Alteration in the mRNA concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and IL-10 in whole-blood culture at 4 h after stimulation with different doses of LPS was observed and determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The mRNA concentrations of TNF-alpha and IL-1 beta changed in a dose-dependent manner and differed depending on the type of LPS. Limulus test revealed that endotoxin activity was remarkably reduced when aluminum hydroxide gel was added to LPS. In contrast, the mRNA concentration of TNF-alpha in whole bovine blood was enhanced by LPS mixed with aluminum hydroxide gel. These results suggest that bovine whole-blood culture can be utilized to detect endotoxin activity of Gram-negative bacterial vaccines. In addition, whole-blood culture offers several advantages, such as ease of performance, few preparation artifacts, and a physiological cell environment, for investigating bovine immune response compared with the Limulus test. (C) 2013 Elsevier B.V. All rights reserved. - Hirohisa Mekata, Satoru Konnai, Claro N. Mingala, Nancy S. Abes, Charito A. Gutierrez, Alan P. Dargantes, William H. Witola, Noboru Inoue, Misao Onuma, Shiro Murata, Kazuhiko OhashiPARASITOLOGY RESEARCH 112 (4) 1513 - 1521 0932-0113 2013/04 [Refereed][Not invited]
In recent years, the emergence of highly pathogenic Trypanosoma evansi strains in the Philippines has resulted in substantial losses in livestock production. In this study, we isolated T. evansi from infected-water buffaloes in the Philippines and analyzed their virulence using mice and cattle. A total of 10 strains of T. evansi were isolated. Evaluation of the virulence of each strain using mice depicted significant differences among the strains in the prepatent period, the level of parasitemia, and the survival time of the infected animals. In mice infected with the highly pathogenic T. evansi, signs of excessive inflammation such as marked splenomegaly and increase more than 6-fold in the number of leukocytes were observed at 8 days post-infection. To study the virulence of the parasite strains in cattle (which are the common T. evansi hosts in Philippines), cattle were infected with the T. evansi isolates that showed high and low virulence in mice. The rate of parasite growth and the length of the prepatent periods were found to be similar to those observed in mice for the respective strains. The cattle infected with the highly pathogenic strain developed anemia and a marked decrease in leukocyte counts. To determine the cause of the pathological changes, we analyzed the expression levels of inflammatory cytokines and observed up-regulation of tumor necrosis factor-alpha in anemic infected cattle. Our findings suggest that the epidemic of T. evansi in the Philippines is characterized by T. evansi strains with varying virulences from low to very high pathogenicity in cattle. - Yusuke Murase, Satoru Konnai, Naftaly Githaka, Arata Hidano, Kyle Taylor, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Toshio Tsubota, Shiro Murata, Kazuhiko OhashiJOURNAL OF VETERINARY MEDICAL SCIENCE 75 (2) 215 - 218 0916-7250 2013/02 [Refereed][Not invited]
In this study, the prevalence of Borrelia infections in Ixodes ticks from a site in Hokkaido, Japan, with confirmed cases of Lyme disease was determined by a PCR method capable of detecting and differentiating between strains of pathogenic Borrelia, with particular emphasis on Borrelia garinii (B. garinii) and Borrelia afzelli (B. afzelli), using tick-derived DNA extracts as template. A total of 338 ticks, inclusive of 284 Ixodes persulcatus (I. persulcatus), were collected by flagging vegetation in mid-spring. Ninety-eight (34.5%) of I. persukatus tested positive for Borrelia species DNA, whereas the overall prevalence of Borrelia species in Ixodes ovatus and Haemaphysalis longicornis ticks was 19.5 and 7.7%, respectively. PCR-RFLP and sequence analysis of Borrelia rrj(5S)-rr/(23S) intergenic spacer DNA amplicons indicated that they originated from three different Borrelia species namely, B. garinii, B. afzelii and B. japonica. Among the I. persulcatus species, which is a known vector of human borreliosis, 86 were mono-infected with B. garinii, 2 ticks were mono-infected with B. afzelii and whereas 12 ticks had dual infections. Most significant, 11 of the I. persukatus ticks were coinfected with Anaplasma phagocytophilum and B. garinii. The difference between the number of obtained and expected co-infections was significant (chi(2)=4.32, P=0.038). - Satoru Konnai, Saori Suzuki, Tatsuya Shirai, Ryoyo Ikebuchi, Tomohiro Okagawa, Yuji Sunden, Claro N. Mingala, Misao Onuma, Shiro Murata, Kazuhiko OhashiCOMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 36 (1) 63 - 69 0147-9571 2013/01 [Refereed][Not invited]
An immunoinhibitory receptor, lymphocyte activation gene-3 (LAG-3), which is mainly expressed in T-cells, is involved in the immune evasion of several pathogens causing chronic infections and tumors. However, unlike human or mouse LAG-3, no functional analysis of LAG-3 has been reported in domestic animals. Thus, in this study, bovine LAG-3 expression was analyzed in bovine leukemia virus (BLV)-infected cattle. In persistent lymphocytotic (PL) cattle, the numbers of LAG-3(+)CD4(+) cells and LAG-3(+)CD8(+) cells were conserved whilst the number of MHC class II+ cells was remarkably higher than in the control animals. In contrast, the mean fluorescence intensity (MFI) for LAG-3 on PBMCs from PL cattle was significantly increased compared to control and asymptomatic (AL) cattle. Specifically, the LAG-3 expression level was significantly increased in both CD4(+) and CD8(+) T cells from PL cattle. LAG-3 expression correlated positively with increased numbers of lymphocytes and MHC class II+ cells in infected animals. Preliminary results from PD-L1 and LAG-3 blockade assay revealed that IFN-gamma and IL-2 expressions were significantly up-regulated by addition of anti- PD-L1 and LAG-3 antibodies in PBMCs from PL cattle. These findings suggest that LAG-3 might be involved in the inhibition of T-cell function through its binding and signaling on MHC class II molecule during BLV infection. (c) 2012 Elsevier Ltd. All rights reserved. - Saiki Imamura, Satoru Konnai, Shinji Yamada, Luis F. Parizi, Naftaly Githaka, Itabajara da S. Vaz, Shiro Murata, Kazuhiko OhashiTICKS AND TICK-BORNE DISEASES 4 (1-2) 138 - 144 1877-959X 2013 [Refereed][Not invited]
Vaccines are among the alternative tick control methods expected to replace at least in part the volumes of chemical acaricides currently used worldwide. However, a vaccination approach depends on a host immune response against proteins that are essential to tick physiology. The cystatin family is a protein class recently investigated to compose an effective antigen in a tick vaccine. In this study, a cDNA from Rhipicephalus appendiculatus with high sequence similarity to cystatins type 2 was identified by random sequencing analysis and called R. appendiculatus cystatin 1 (Ra-cyst-1). DNA sequence analysis showed that the cloned Ra-cyst-1 has a 423-bp open reading frame and codified to a 140-amino acid polypeptide. The putative mature protein consists of 115 amino acid residues with a deduced molecular weight of 12.8 kDa. The highly conserved G (P-I), QxVxG (P-II), and PW (P-III) type 2 cystatins motifs are present in Ra-cyst-1 cDNA. RT-PCR analysis showed that the Ra-cyst-1 gene is expressed in nymph, male, and female midgut following blood feeding, but not in the salivary glands of fed females. In addition, Western blot revealed that recombinant Ra-cyst-1 was not recognized by sera derived from rabbits infested with ticks, suggesting that this cystatin is not secreted into the host during infestation. We hypothesize that Ra-cyst-1 may play a role in the tick feeding process and could be a concealed antigen candidate in further anti-tick vaccination trials. (C) 2012 Elsevier GmbH. All rights reserved. - Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko OhashiVeterinary Research 44 (1) 59 0928-4249 2013 [Refereed][Not invited]
Programmed death-1 (PD-1) is a known immunoinhibitory receptor that contributes to immune evasion of various tumor cells and pathogens causing chronic infection, such as bovine leukemia virus (BLV) infection. First, in this study, to establish a method for the expression and functional analysis of bovine PD-1, hybridomas producing monoclonal antibodies (mAb) specific for bovine PD-1 were established. Treatment with these anti-PD-1 mAb enhanced interferon-gamma (IFN-γ) production of bovine peripheral blood mononuclear cells (PBMC). Next, to examine whether PD-1 blockade by anti-PD-1 mAb could upregulate the immune reaction during chronic infection, the expression and functional analysis of PD-1 in PBMC isolated from BLV-infected cattle with or without lymphoma were performed using anti-PD-1 mAb. The frequencies of both PD-1+ CD4+ T cells in blood and lymph node and PD-1 + CD8+ T cells in lymph node were higher in BLV-infected cattle with lymphoma than those without lymphoma or control uninfected cattle. PD-1 blockade enhanced IFN-γ production and proliferation and reduced BLV-gp51 expression and B-cell activation in PBMC from BLV-infected cattle in response to BLV-gp51 peptide mixture. These data show that anti-bovine PD-1 mAb could provide a new therapy to control BLV infection via upregulation of immune response.© 2013 Ikebuchi et al. licensee BioMed Central Ltd. - Naftaly Githaka, Satoru Konnai, Edward Kariuki, Esther Kanduma, Shiro Murata, Kazuhiko OhashiACTA TROPICA 124 (1) 71 - 78 0001-706X 2012/10 [Refereed][Not invited]
Piroplasms frequently infect domestic and wild carnivores. At present, there is limited information on the occurrence and molecular identity of these tick-borne parasites in wild felids in Kenya. In 2009, a pair of captive lions (Panthare leo) was diagnosed with suspected babesiosis and mineral deficiency at an animal orphanage on the outskirts of Nairobi, Kenya. Blood smears indicated presences of haemoparasites in the erythrocytes, however, no further investigations were conducted to identify the infecting agent. The animals recovered completely following diet supplementation and treatment with anti-parasite drug. In this report, we extracted and detected parasite DNA from the two lions and seven other asymptomatic feline samples; two leopards (Panthera pardus) and five cheetahs (Acinonyx jubatus). Reverse line blot with probes specific for Babesia spp. of felines indicated the presence of new Babesia species or genotypes in the lions and leopards, and unknown Theileria sp. in the cheetahs. Phylogenetic analyses using partial sequences of 185 ribosomal RNA (185 rRNA) gene showed that the parasite infecting the lions belong to the Babesia canis complex, and the parasite variant detected in the leopards clusters in a clade bearing other Babesia spp. reported in wild felids from Africa. The cheetah isolates falls in the Theileria sensu stricto group. Our findings indicate the occurrence of potentially new species or genotypes of piroplams in all three feline species. (C) 2012 Elsevier B.V. All rights reserved. - Tomohiro Okagawa, Satoru Konnai, Hirohisa Mekata, Naftaly Githaka, Saori Suzuki, Edward Kariuki, Francis Gakuya, Esther Kanduma, Tatsuya Shirai, Ryoyo Ikebuchi, Yoshinori Ikenaka, Mayumi Ishizuka, Shiro Murata, Kazuhiko OhashiVETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 148 (3-4) 373 - 379 0165-2427 2012/08 [Refereed][Not invited]
Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus coffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo. (c) 2012 Elsevier B.V. All rights reserved. - Kinetics of regulatory dendritic cells in inflammatory responses during Trypanosoma evansi infectionH. Mekata, S. Konnai, C. N. Mingala, N. S. Abes, C. A. Gutierrez, A. P. Dargantes, W. H. Witola, N. Inoue, M. Onuma, S. Murata, K. OhashiPARASITE IMMUNOLOGY 34 (6) 318 - 329 0141-9838 2012/06 [Refereed][Not invited]
Trypanosoma evansi (T.similar to evansi) causes a wasting disease in almost all mammals. Trypanosoma evansi infection gives rise to the inflammatory responses that contribute to the development of inflammation-associated tissue injury. To determine what kinds of inflammatory molecules play roles in the pathogenicity of T.similar to evansi infection, polymerase chain reaction array analysis was performed on samples from the infected and uninfected mice. The inflammatory cytokine and chemokine storm, caused mainly by macrophages, was observed. On the other hand, the expression levels of Ccl8 and Il10 in splenocytes were also markedly increased. These results suggested an augmentation in the number and activity of regulatory dendritic cells (DCs). Therefore, the kinetics of regulatory DCs in T.similar to evansi-infected mice were investigated. During T.similar to evansi infection, the regulatory DCs became prevalent, with reducing the amount of inflammatory DCs. Interestingly, when the regulatory DCs were implanted into T.similar to evansi-infected mice, the survival was prolonged, and the expression levels of inflammatory molecules were suppressed. Taken together, these results showed that a subset of regulatory DCs acted as a potential regulator of the inflammatory responses. - Shiro Murata, Yuko Hayashi, Ayumi Kato, Masayoshi Isezaki, Sarah Takasaki, Misao Onuma, Yuichi Osa, Mitsuhiko Asakawa, Satoru Konnai, Kazuhiko OhashiVETERINARY JOURNAL 192 (3) 538 - 540 1090-0233 2012/06 [Refereed][Not invited]
Marek's disease virus serotype 1 (MDV-1) strains cause malignant lymphoma in chickens. MDV-1 has been previously reported to be widespread in white-fronted geese (Anser albifrons); however, the prevalence of MDV-1 in other wild birds has not been determined. In this study, we investigated the prevalence of MDV-1 in various wild birds in Hokkaido, Japan. The MDV-1 genome was widespread in geese and ducks, but was not detected in other birds. MDV-1 was detected in both sedentary and migratory species. These results suggest that, in Japan. MDV-1 is widespread in wild goose and duck populations, and that resident ducks may be significant carriers and reservoirs of MDV-1. (C) 2011 Elsevier Ltd. All rights reserved. - Tomohiro Okagawa, Satoru Konnai, Ryoyo Ikebuchi, Saori Suzuki, Tatsuya Shirai, Yuji Sunden, Misao Onuma, Shiro Murata, Kazuhiko OhashiVETERINARY RESEARCH 43 45 0928-4249 2012/05 [Refereed][Not invited]
The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4(+) and CD8(+) cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4(+) and CD8(+) cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-gamma and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-gamma mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection. - Ayumi Matsuyama-Kato, Shiro Murata, Masayoshi Isezaki, Rika Kano, Sara Takasaki, Osamu Ichii, Satoru Konnai, Kazuhiko OhashiVIROLOGY JOURNAL 9 94 1743-422X 2012/05 [Refereed][Not invited]
Background: An immunoinhibitory receptor, programmed death-1 (PD-1), and its ligand, programmed death-ligand 1 (PD-L1), are involved in immune evasion mechanisms for several pathogens causing chronic infections and for neoplastic diseases. However, little has been reported for the functions of these molecules in chickens. Thus, in this study, their expressions and roles were analyzed in chickens infected with Marek's disease virus (MDV), which induces immunosuppression in infected chickens. Results: A chicken T cell line, Lee1, which constitutively produces IFN-gamma was co-cultured with DF-1 cells, which is a spontaneously immortalized chicken fibroblast cell line, transiently expressing PD-L1, and the IFN-gamma expression level was analyzed in the cell line by real-time RT-PCR. The IFN-gamma expression was significantly decreased in Lee1 cells co-cultured with DF-1 cells expressing PD-L1. The expression level of PD-1 was increased in chickens at the early cytolytic phase of the MDV infection, while the PD-L1 expression level was increased at the latent phase. In addition, the expression levels of PD-1 and PD-L1 were increased at tumor lesions found in MDV-challenged chickens. The expressions levels of PD-1 and PD-L1 were also increased in the spleens and tumors derived from MDV-infected chickens in the field. Conclusions: We demonstrated that the chicken PD-1/PD-L1 pathway has immunoinhibitory functions, and PD-1 may be involved in MD pathogenesis at the early cytolytic phase of the MDV infection, whereas PD-L1 could contribute to the establishment and maintenance of MDV latency. We also observed the increased expressions of PD-1 and PD-L1 in tumors from MDV-infected chickens, suggesting that tumor cells transformed by MDV highly express PD-1 and PD-L1 and thereby could evade from immune responses of the host. - Molecular cloning and characterization of Th1 and Th2 cytokines of African buffalo (Syncerus caffer)S. Suzuki, S. Konnai, T. Okagawa, N. W. Githaka, E. Kariuki, F. Gakuya, E. Kanduma, T. Shirai, R. Ikebuchi, Y. Ikenaka, M. Ishizuka, S. Murata, K. OhashiINTERNATIONAL JOURNAL OF IMMUNOGENETICS 39 (2) 170 - 182 1744-3121 2012/04 [Refereed][Not invited]
The African buffalo (Syncerus caffer) has been implicated as the reservoir of several bovine infectious agents. However, there is insufficient information on the protective immune responses in the African buffalo, particularly in infected animals. In this study, we analysed Th1 cytokines IL-2 and IFN-?, and Th2 cytokines IL-4 and IL-10. The cloned cDNA of IL-2, IL-4, IL-10 and IFN-? contained an open reading frame of 468, 501, 408 and 540 nucleotides, encoding polypeptides of 155, 166, 135 and 179 amino acids, respectively. Nucleotide sequence homology of IL-2, IFN-? and IL-4 was more than 98% between the African buffalo and cattle, which resulted in identical polypeptides. Meanwhile, IL-10 gene of African buffalo and cattle had 95% homology in nucleotide sequence, corresponding to thirteen amino acid residues substitution. Cysteine residues and potential glycosylation sites were conserved within the family Bovinae. Phylogenetic analyses including cytokines of the African buffalo placed them within a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle, water buffalo, sheep, goat, pig and artiodactyl wildlife. A deeper understanding of the structure of these cytokines will shed light on their protective role in the disease-resistant African buffalo in comparison with other closely related species. - Satoru Konnai, Shinji Yamada, Saiki Imamura, Hideto Nishikado, Naftaly Githaka, Takuya Ito, Ai Takano, Hiroki Kawabata, Shiro Murata, Kazuhiko OhashiTICKS AND TICK-BORNE DISEASES 3 (2) 75 - 77 1877-959X 2012 [Refereed][Not invited]
The tick receptor for outer surface protein A (TROSPA) is an Ixodes scapularis (I. scapularis) receptor for Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease in North America. The blockade of TROSPA has been shown to reduce B. burgdorferi adherence to the I. scapularis gut in vivo. Thus. TROSPA is one of the potential targets for the development of vector-antigen-based vaccines to prevent the transmission of B. burgdorferi. The aim of this study is to identify the TROSPA gene in I. persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan. The cDNA clone encoding the TROSPA-like sequence with 483 nucleotides was obtained from whole-body homogenates of fed nymphs of I. persulcatus. The putative amino acid sequence of I. persulcatus TROSPA was 88.2% and 87.8% identical to that of I. scapularis and I. ricinus, respectively. This finding will facilitate investigations on the role of I. persulcatus TROSPA and its interaction with Borrelia spp. and will have important implications on endeavors to develop a tick vaccine. (C) 2012 Elsevier GmbH. All rights reserved. - Tatsuya Shirai, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Saori Suzuki, Yuji Sunden, Misao Onuma, Shiro Murata, Kazuhiko OhashiVETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 144 (3-4) 462 - 467 0165-2427 2011/12 [Refereed][Not invited]
Lymphocyte activation gene-3 (LAG-3), a major histocompatibility complex (MHC) class II binding CD4 homologue has recently been shown as one of the mechanisms for down-regulating immune responses during chronic disease progression. For the first time, we cloned LAG-3 from two breeds of cattle (Holstein and Japanese Black), and analyzed its expression levels in cattle infected with bovine leukemia virus (BLV), a chronic viral infection that leads to immuno-suppression. The cloned cDNA of bovine LAG-3 have an open reading frame of 1551 nucleotides, encoding a polypeptide of 515 amino acids in length. Similar to the swine LAG-3, the bovine LAG-3 protein sequence consisted of four extracellular domains, a transmembrane domain and an inhibitory motif, KTGELE. We found that the bovine LAG-3 mRNA transcripts were expressed predominantly on T-cells such as CD4(+) and CD8(+) cells, among peripheral blood mononuclear cells. In subsequent expression analysis, LAG-3 mRNA expression on CD4(+) T-cells from BLV-infected cattle was upregulated compared to that in normal cattle. Comparable results were obtained with CD8(+) T-cells from cattle infected with BLV. We further observed strong upregualtion of MHC class II molecule, the ligand for LAG-3 in BLV-infected cattle. These findings indicate an important role for inhibitory receptor molecules such as LAG-3 in chronic bovine infections and future studies will elucidate the specific role of LAG-3 in bovine diseases. (C) 2011 Elsevier B.V. All rights reserved. - Ryoyo Ikebuchi, Satoru Konnai, Tatsuya Shirai, Yuji Sunden, Shiro Murata, Misao Onuma, Kazuhiko OhashiVETERINARY RESEARCH 42 103 0928-4249 2011/09 [Refereed][Not invited]
The inhibitory receptor programmed death-1 (PD-1) and its ligand, programmed death-ligand 1 (PD-L1) are involved in immune evasion mechanisms for several pathogens causing chronic infections. Blockade of the PD-1/PD-L1 pathway restores anti-virus immune responses, with concomitant reduction in viral load. In a previous report, we showed that, in bovine leukemia virus (BLV) infection, the expression of bovine PD-1 is closely associated with disease progression. However, the functions of bovine PD-L1 are still unknown. To investigate the role of PD-L1 in BLV infection, we identified the bovine PD-L1 gene, and examined PD-L1 expression in BLV-infected cattle in comparison with uninfected cattle. The deduced amino acid sequence of bovine PD-L1 shows high homology to the human and mouse PD-L1. The proportion of PD-L1 positive cells, especially among B cells, was upregulated in cattle with the late stage of the disease compared to cattle at the aleukemic infection stage or uninfected cattle. The proportion of PD-L1 positive cells correlated positively with prediction markers for the progression of the disease such as leukocyte number, virus load and virus titer whilst on the contrary, it inversely correlated with the degree of interferon-gamma expression. Blockade of the PD-1/PD-L1 pathway in vitro by PD-L1-specific antibody upregulated the production of interleukin-2 and interferon-gamma, and correspondingly, downregulated the BLV provirus load and the proportion of BLV-gp51 expressing cells. These data suggest that PD-L1 induces immunoinhibition in disease progressed cattle during chronic BLV infection. Therefore, PD-L1 would be a potential target for developing immunotherapies against BLV infection. - Shiro Murata, Tsukasa Okada, Rika Kano, Yuko Hayashi, Tomoyuki Hashiguchi, Misao Onuma, Satoru Konnai, Kazuhiko OhashiVIRUS GENES 43 (1) 66 - 71 0920-8569 2011/08 [Refereed][Not invited]
Marek's disease virus (MDV) is an oncogenic herpesvirus that causes malignant lymphomas in chickens. Recent field isolates of MDV have tended to exhibit increasing virulence, and MDV strains are currently classified into four categories based on their relative virulence. Meq, a putative MDV oncoprotein, resembles the Jun/Fos family of basic leucine zipper (bZIP) transcription factors and can regulate the expression of viral and cellular genes as a homodimer or as a heterodimer with a variety of bZIP family proteins. MDV isolates display distinct diversity and point mutations in Meq, which may contribute to changes in the transcriptional activities of Meq and subsequently, to observed increases in MDV oncogenicity. In this study, we introduced mutations into the meq gene and used dual luciferase reporter assays to analyze the transcriptional activities of the resulting Meq proteins to determine whether distinct mutations in Meq could be responsible for differences in transcriptional activity among MDV strains. A proline-to-alanine substitution at position 217, the second position of one of the proline direct repeats in the transactivation domain, enhanced the transactivation activity of Meq. In addition, we found that two substitutions at positions 283 and 320 affected transactivation activity. These results suggest that the distinct diversity of and point mutations in the Meq proteins are responsible for differences in transactivation activity among MDV strains. - 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 OhashiVETERINARY MICROBIOLOGY 149 (3-4) 504 - 507 0378-1135 2011/05 [Refereed][Not invited]
The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmaposis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals. Crown Copyright (C) 2010 Published by Elsevier B.V. All rights reserved. - Satoru Konnai, Hideto Nishikado, Shinji Yamada, Saiki Imamura, Takuya Ito, Misao Onuma, Shiro Murata, Kazuhiko OhashiEXPERIMENTAL PARASITOLOGY 127 (2) 467 - 474 0014-4894 2011/02 [Refereed][Not invited]
Lipocalins have been known for their several biological activities in blood-sucking arthropods. Recently, the identification and characterization of lipocalins from Ixodes ricinus (LIRs) have been reported and functions of lipocalins are well documented. In this study, we have characterized four Ixodes persulcatus lipocalins that were discovered while analyzing I. persulcatus tick salivary gland EST library. We show that the four I. persulcatus lipocalins, here after named LIPERs (lipocalin from I. persulcatus) are 28.8-94.4% identical to LIRs from I. ricinus. Reverse transcriptase-PCR analysis revealed that lipocalin genes were expressed specifically in the salivary glands throughout life cycle stages of the ticks and were up-regulated by blood feeding. The specific expressions were also confirmed by Western blotting analysis. Furthermore, to investigate whether native lipocalins are secreted into the host during tick feeding, the reactivity of anti-serum raised against saliva of adult ticks to recombinant lipocalins was tested by Western blotting. The lipocalins are potentially secreted into the host during tick feeding as revealed by specific reactivity of recombinant lipocalins with mouse antibodies to I. persulcatus tick saliva. Preliminary vaccination of mice with recombinant lipocalins elicited that period to reach engorgement was significantly delayed and the engorgement weight was significantly reduced as compared to the control. Further elucidation of the biological functions of LIPERs are required to fully understand the pathways involved in the modulation of host immune responses. (C) 2010 Elsevier Inc. All rights, reserved. - Takehiro Murao, Yoshitaka Omata, Rika Kano, Shiro Murata, Tsukasa Okada, Satoru Konnai, Mitsuhiko Asakawa, Kazuhiko Ohashi, Misao OnumaJOURNAL OF PARASITOLOGY 94 (4) 830 - 833 0022-3395 2008/08 [Refereed][Not invited]
Antibodies to Toxoplasma gondii were assayed by ELISA in 22 experimentally inoculated domestic ducks. In addition a serological assay was carried out at Obihiro, Hokkaido, Japan, in 2004 and 2005, on 221 wild ducks of 5 species: Anas platyrhynchus (n = 111); A. poecilorhyncha (n = 27); A. acute (n = 58); A.penelope (n = 16); and A. crecca (n = 9). Assays were also conducted using sera from 197 wild geese of 2 species, i.e., Anser albifrons (n = 162) and Ans. fabalis (n = 35). Birds were collected between 2003 and 2005 from 3 different areas: Lake Miyajima-numa, Hokkaido, Japan, regions around Anadyr city of Chukotka antonomous okrug, and Lake Makobetukoa, Kamchatka oblast, Russia. The ELISA cutoff value (OD) was >= 0.395 based on results from uninfected ducks; the final dilution ratio recognized as positive was represented by the end titer. The end titer in the experimentally induced ducks ranged from 1:400 to 1:3,200. Antibodies to T. gondii were found in 49 of the 221 wild duck samples from Japan: A. platyrhynchus (22/74); A. poecilorhyncha (2/15); and A. poecilorhyncha (5/12). Thirty-two of 197 wild goose samples were seropositive, i.e., Ans. albifrons (7/51) in 2004 and (11/72) in 2005 in Miyajima-numa, Japan and 9/39 in Chukotka. Russia as well as in Ans. fabalis (5/35) in Kamchatka, for which the end titer ranged from 1:100 to 1:3,200. In immunoblotting, the A.platyrhynchus samples showed specific IgG antibody binding to several antigens in the T. gondii lane, i.e., at 30 and 43 kDa, but not in the Neospora caninum lane. No specific bands were noted in samples for which antibody activity was not detected. These results suggest that wild waterfowl inhabiting Hokkaido, Chukotka, and Kamchatka may be exposed to T. gondii. - Shiro Murata, Kyung-Soo Chang, Sung-Il Lee, Satoru Konnai, Misao Onuma, Kazuhiko OhashiJOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION 19 (5) 471 - 478 1040-6387 2007/09 [Refereed][Not invited]
For the easy survey of Marek's disease virus (MDV), feather tip-derived DNA from MDV-infected chickens can be used because feather tips are easy to collect and feather follicle epithelium is known to be the only site of productive replication of cell-free MDV. To develop a diagnostic method to differentiate highly virulent strains of MDV from the attenuated MDV vaccine strain, CV1988, which is widely used, nested polymerase chain reaction (PCR) was performed to detect a segment of the meq gene in feather tip samples of chickens experimentally infected with MDV. In chickens infected with Md5, a strain of oncogenic MDV, the meq gene was consistently detected, whereas the L-meq gene, in which a 180-base pair (180-bp) sequence is inserted into the meq gene, was detected in CV1988-infected chickens. Moreover, the meq gene was mainly detected even in chickens co-infected with both Md5 and CV1988. These results suggest that this method is appropriate for the surveillance of the highly virulent MDV infection in the field. - S. Murata, K.-S. Chang, Y. Yamamoto, T. Okada, S.-I. Lee, S. Konnai, M. Onuma, Y. Osa, M. Asakawa, K. OhashiARCHIVES OF VIROLOGY 152 (8) 1523 - 1526 0304-8608 2007/08 [Refereed][Not invited]
Marek's disease (MD) virus (MDV) is known to cause malignant lymphomas in chickens. In 2001, we first reported an MD case in a white-fronted goose (Anser albifrons) in Japan. Therefore, the prevalence of MDV in the wild geese was surveyed by nested PCR using feather-tip samples in Japan and the Far East region of Russia, breeding habitats of geese migrating to Japan. MDV was detected in about 30% of analyzed white-fronted geese. Furthermore, by nucleotide sequence analysis, we confirmed that this MDV shows high homology to very virulent MDV, suggesting that highly virulent MDV is widespread in white-fronted geese migrating between Japan and Far East region of Russia. - Tsukasa Okada, Michihiro Takagi, Shiro Murata, Misao Onuma, Kazuhiko OhashiJOURNAL OF GENERAL VIROLOGY 88 (8) 2111 - 2120 0022-1317 2007/08 [Refereed][Not invited]
In tumour cell lines established from Marek's disease (MID) lymphomas L-meq is consistently expressed. It contains a 180 bp insertion encoding additional copies of the proline-rich repeat in the meq open reading frame and its product may contribute to the maintenance of MID virus (MDV) latency. In this study, we identified a novel spliced form of the meq transcript in MD-derived lymphoblastoid cell lines and in MDV-infected cells. This transcript, termed Delta meq, encodes an N-terminal 98 aa of the Meq protein and lacks part of the basic leucine zipper (bZIP) and transactivation domains. In MID cell lines, transcription of L-meq was significantly downreguiated, while that of the Delta meq transcript was upregulated during apoptosis. These observations were also confirmed at the protein expression level. Reporter assays using meq- and interleukin-2 (IL-2)-promoter-driven luciferase vectors revealed that AMeq suppressed transactivation by L-Meq or Meq in a dose-dependent manner. Immunoprecipitation confirmed that AMeq was associated with L-Meq or Meq physically. These results suggest that AMeq could be involved in 2006 apoptosis in MID cell lines as it works as a negative regulator of L-Meq and Meq by direct interaction. - HM Pham, S Konnai, T Usui, KS Chang, S Murata, M Mase, K Ohashi, M OnumaARCHIVES OF VIROLOGY 150 (12) 2429 - 2438 0304-8608 2005/12 [Refereed][Not invited]
In order to rapidly detect and differentiate Newcastle disease virus (NDV) isolates, a method based on real-time PCR SYBR Green I melting-curve analysis of the fusion (F) protein gene was developed. The detection limit of real-time PCR was 9 x 10(2). plasmid copies and was 100 times more sensitive than conventional PCR. Thirty eight reference NDV strains were rapidly identified by their distinctive melting temperatures (T(m)s): 89.23 +/- 0.27 degrees C for velogenic strains, 90.17 +/- 0.35 degrees C for pigeon mesogenic strains, 91.25 +/- 0.14 degrees C for two lentogenic strains (B1 and Ishii). No amplification was detected from unrelated RNA samples by this method. This real-time PCR directly detected NDV from infected tissues and eliminated the gel electrophoretic step for analyzing PCR product using ethidium bromide. The total time for a PCR run was less than 1 hour. The results obtained in this study showed that the real-time PCR presented here is a good screening test for the identification of NDV.
Books etc
- 感染症の生態学長雄一, 大橋和彦, 村田史郎 (Joint work第20章 鳥インフルエンザ)
共立出版 2016/03 - Ducks: Habitat, Behavior, and DiseasesAsakawa, M, Nakade, T, Murata, S, Ohashi, K, Osa, Y, Taniyama, H (Joint workRecent viral disease of Japanese Anatid with a fatal case of Marek’s disease in an endangered species, white-fronted goose (Anser albifrons).)
Nova Science Publishers 2013
Conference Activities & Talks
- Effects of an Insertion and a Deletion in the Marek’s Disease Virus Oncogene Meq on Transactivation Activity and Virulence [Not invited]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 Ohashi46th annual international herpesvirus workshop 2022/07
- Transcriptome analysis in blood-fed and starved poultry red mites, Dermanyssus gallinae [Not invited]Fujisawa, S, Murata, S, Takehara, M, Isezaki, M, Ogawa, R, Uno, Y, Taneno, A, Okagawa, T, Maekawa, N, Konnai, S, Ohashi, KThe 4th International Symposium on Parasite Infections in Poultry, Vieena 2019/06
- Cysteine protease and ferritin 2 as vaccine antigens to control poultry red mites, Dermanyssus gallinae [Not invited]S. Murata, M. Isezaki, A. Taniguchi, S. Fujisawa, A. Taneno, E. Sakai, Y. Uno, R. Ogawa, O. Ichii, T. Ito, M. Takehara, A. Morita, N. Maekawa, T. Okagawa, S. Konnai, K. OhashiThe 4th International Symposium on Parasite Infections in Poultry, Vienna 2019/06
- マレック病に関する最近の知見について ―日本に分布するマレック病ウイルスの性状 [Invited]村田史郎, 今内覚, 大橋和彦鶏病研究会佐賀県支部鶏病技術研修会 2019/06
- 抗ワクモワクチン開発に向けた試み [Invited]村田 史郎第２８４回鶏病事例検討会 2018/09
- Molecular detection of Marek’s disease virus in poultry farms in Myanmar [Not invited]Shiro Murata, Masaki Takehara, Ken Katakura, Myint Myint Hmoon, Shwe Yee Win, Masayoshi Isezaki, Satoru Konnai, Kazuhiko OhashiThe 12th International Symposium on Marek's Disease and Avian Herpesviruses 2018/07
- Enhanced immune responses after vaccination with a gallid alphaherpesvirus 2 (Marek’s disease virus, MDV) expressing PD-1 [Not invited]Shiro Murata, Benedikt B Kaufer, Nikolaus Osterrieder, Masayoshi Isezaki, Satoru Konnai, Kazuhiko Ohashi42nd International Herpesvirus Workshop 2017/07
- The Role of Meq Mutations on Marek’s Disease Virus Pathogenesis [Not invited]Nirajan Bhandari, Phaedra Tavlarides-Hontz, Upendra Katneni, Benedikt B. Kaufer, Shiro Murata, Mark S. ParcellsThe 11th International Symposium on Marek's Disease and Avian herpesviruses 2016/07
- Sequence analysis of a Marek’s disease virus strain isolated in Japan [Not invited]Shiro Murata, Yuka Machida, Masayoshi Isezaki, Satoru Konnai, Kazuhiko OhashiThe 11th International Symposium on Marek's Disease and Avian herpesviruses 2016/07
- Expression analysis of programmed death ligand 2 in tumors caused by the avian oncovirus Marek’s disease virus [Not invited]Shiro Murata, Ayumi Matsuyama-Kato, Masayoshi Isezaki, Satoru Konnai, Kazuhiko OhashiThe 39th Annual International Herpesvirus Workshop 2014/07
- Characterization of Meq proteins from field isolates of Marek’s disease virus in Japan [Not invited]Shiro Murata, Tomoyuki Hashiguchi, Yuko Hayashi, Yuki Yamamoto, Ayumi Kato, Sarah Takasaki, Masayoshi Isezaki, Satoru Konnai, Kazuhiko OhashiThe 9th International Symposium on Marek’s disease and Avian Herpesviruses 2012/06
- Characterization of immunoinhibitory factors PD-1/PD-L1 in chickens infected with Marek’s disease virus [Not invited]Ayumi Matsuyama-Kato, Shiro Murata, Masayoshi Isezaki, Rika Kano, Sarah Takasaki, Osamu Ichii, Satoru Konnai, Kazuhiko OhashiThe 9th International Symposium on Marek’s disease and Avian Herpesviruses 2012/06
- Amino acid substitutions or insertion in the Meq proteins could affect their transactivation and transformation abilities [Not invited]Shiro Murata, Tomoyuki Hashiguchi, Tsukasa Okada, Rika Kano, Misao Onuma, Satoru Konnai, Kazuhiko OhashiInternational Union of Microbiological Societies 2011 Congress 2011/09
- Regulation of the interferon gamma expression by an oncoprotein of Marek’s disease virus in wild waterfowls and chickens [Not invited]Ayako Matsubara, Shiro Murata, Rika Kano, Tomoyuki Hashiguchi, Masayoshi Isezaki, Ayumi Kato, Misao Onuma, Satoru Konnai, Kazuhiko OhashiThe 9th International Veterinary Immunology Symposium 2010/08
- Analysis of transcriptional activities by the Meq proteins with highly virulent Marek’s disease virus strains [Not invited]Shiro MURATA, Tsukasa OKADA, Rika KANO, Yuko HAYASHI, Satoru KONNAI, Misao ONUMA, Kazuhiko OHASHI4th International Workshop of the Molecular Pathogenesis of Marek's disease Virus 2006/08
MISC
- 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
- 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
- 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
- 村田史郎 日本獣医学会学術集会講演要旨集 166th- 2023
- 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
- 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
- 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
- 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
- 青木絵理, 今内覚, 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, 村田史郎, 大橋和彦, 大橋和彦, 大橋和彦 日本獣医学会学術集会講演要旨集 165th (CD-ROM)- 2022
- 瀬尾光里, 村田史郎, 村田史郎, WIN Shwe Yee, 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, 市居修, 前川直也, 岡川朋弘, 今内覚, 今内覚, 大橋和彦, 大橋和彦, 大橋和彦 日本獣医学会学術集会講演要旨集 165th (CD-ROM)- 2022
- 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 165th (CD-ROM)- 2022
- 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
- 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
- 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
- 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
- 前川 直也, 今内 覚, 岡川 朋弘, 村田 史郎, 大橋 和彦 Veterinary Oncology 7- (2) 89 -94 2020/04
- 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- (46) 31 -33 2020/03 [Not refereed][Invited]
Mycoplasma bovis(M.bovis)による牛マイコプラズマ感染症は肺炎や乳房炎などを呈する伝染性疾病である。本症は慢性に経過し、極めて難治性に至る。このような病態の形成には免疫抑制の関与が示唆されるが、未だ解明に至っていない。そこで、感染牛の末梢単核球(PBMC)を採取し、解析を行った結果、免疫チェックポイント因子Programmed death-1(PD-1)およびPD-ligand 1(PD-L1)の発現が増加しており、本経路が牛マイコプラズマ感染症の免疫疲弊化に関与していることが示唆された。(著者抄録) - 今内 覚, 岡川 朋弘, 前川 直也, 中島 千絵, 鈴木 定彦, 山本 啓一, 戸田 幹洋, 村田 史郎, 大橋 和彦 JVPA digest : 日本動物用医薬品協会会報 (67) 1 -10 2020/02
- 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
- 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- 221 -221 2020
- 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- 221 -221 2020
- 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- 248 -248 2020
- 今内覚, 今内覚, 岡川朋弘, 前川直也, 村田史郎, 村田史郎, 大橋和彦, 大橋和彦 衛生動物 70- (4) 189 -197 2019/12マダニの唾液の役割と、唾液に含まれる免疫抑制因子を中心に取り上げ、同定され始めた病原体伝播に関わるマダニ因子について紹介した。ダニの唾液または唾液を産生する唾液腺を用いた研究から、マダニは宿主免疫を撹乱したり抑制したりすることが報告されており、唾液は直接的または間接的に病原体の伝播を補助・促進することも明らかとなっている。同定された免疫抑制因子のうちSalp15は宿主CD4+T細胞のCD4に結合し、IL-2産生の抑制によるCD4+T細胞の増殖および活性化を阻害する。さらに近年になって、マダニの吸血補助や維持に関わる因子の他に、ダニ体内において病原体の伝播や定着に関与する特異的な分子の存在が明らかとなってきた。
- 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 2019/08
- イヌ腫瘍由来細胞株におけるCKLF-like MARVEL transmembrane domain containing protein 6(CMTM6)および4(CMTM4)の発現解析竹内 寛人, 今内 覚, 岡川 朋弘, 前川 直也, 村田 史郎, 大橋 和彦 日本獣医学会学術集会講演要旨集 162回- 380 -380 2019/08
- BLV感染症に対するCOX-2阻害剤と抗PD-L1抗体併用法の抗ウイルス効果の検討佐治木 大和, 今内 覚, 岡川 朋弘, 前川 直也, 後藤 伸也, 小原 潤子, 山田 慎二, 加藤 幸成, 鈴木 定彦, 村田 史郎, 大橋 和彦 日本獣医学会学術集会講演要旨集 162回- 381 -381 2019/08
- 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 2019/08
- 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 2019/08
- RNA-Seqを用いたワクモ(Dermanyssus gallinae)の吸血状態別における遺伝子発現解析藤澤 宗太郎, 村田 史郎, 竹原 昌生, 伊勢崎 政美, 小川 遼, 宇野 有紀子, 種子野 章, 前川 直也, 岡川 朋弘, 今内 覚, 大橋 和彦 日本獣医学会学術集会講演要旨集 162回- 412 -412 2019/08
- COX-2阻害剤の併用による抗PD-L1抗体を用いたイヌ腫瘍免疫療法の効果増強に向けた基礎的検討前川 直也, 今内 覚, 浅野 裕美恵, 岡川 朋弘, 高木 哲, 村田 史郎, 大橋 和彦 日本獣医学会学術集会講演要旨集 162回- 460 -460 2019/08
- 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 -343 2019/08 [Not refereed][Not invited]
- 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 -302 2019/08 [Not refereed][Not invited]
- 今内覚, 岡川朋弘, 前川直也, 中島千絵, 中島千絵, 鈴木定彦, 鈴木定彦, 山本啓一, 山本啓一, 戸田幹洋, 戸田幹洋, 村田史郎, 大橋和彦 臨床獣医 37- (3) 37 -41 2019/03 [Not refereed][Not invited]
- 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
- 今内 覚, 岡川朋弘, 前川直也, 中島千絵, 鈴木定彦, 山本啓一, 戸田幹洋, 村田史郎, 大橋和彦 MPアグロジャーナル 35- (10) 22 -25 2018/10 [Not refereed][Invited]
- 前川直也, 今内覚, 村田史郎, 大橋和彦, 高木哲, 高木哲 獣医畜産新報 71- (9) 659 -663 2018/09 [Not refereed][Not invited]
近年、医療では、免疫抑制受容体programmed cell death 1(PD-1)およびそのリガンドPD-ligand 1(PD-L1)を標的とした抗体医薬に代表される免疫チェックポイント阻害薬が、種々の悪性腫瘍に対し著効を示し、一部の進行癌において標準治療としての地位を確立しつつある。著者らの研究グループは犬のPD-L1に対する抗体薬を作製し、北海道大学動物医療センターに来院した悪性腫瘍罹患犬に対し臨床応用研究を行っている。本稿ではその作用機序やこれまでに得られた知見を、臨床応用研究の成績を中心に概説する。(著者抄録) - 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 -282 2018/08 [Not refereed][Not invited]
- 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 161回- 216 -216 2018/08 [Not refereed][Not invited]
- 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 161回- 233 -233 2018/08 [Not refereed][Not invited]
- 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 161回- 202 -202 2018/08 [Not refereed][Not invited]
- 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 161回- 353 -353 2018/08 [Not refereed][Not invited]
- 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 161回- 341 -341 2018/08 [Not refereed][Not invited]
- 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 161回- 340 -340 2018/08 [Not refereed][Not invited]
- 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 161回- 429 -429 2018/08 [Not refereed][Not invited]
- 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 161回- 430 -430 2018/08 [Not refereed][Not invited]
- 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 161回- 342 -342 2018/08 [Not refereed][Not invited]
- 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 161回- 351 -351 2018/08 [Not refereed][Not invited]
- 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 161回- 375 -375 2018/08 [Not refereed][Not invited]
- 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 -103 2018/06 [Not refereed][Not invited]
- 村田史郎, 種子野章, 酒井英史, 町田柚香, 松山あゆ美, 伊勢崎政美, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 160th- 405 2017/08/30 [Not refereed][Not invited]
- 青山珠里愛, 村田史郎, 伊勢崎政美, 種子野章, 酒井英史, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 160th- 405 2017/08/30 [Not refereed][Not invited]
- 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 -370 2017/08/30 [Not refereed][Not invited]
- 今内覚, 岡山朋弘, 山田慎二, SIMUUNZA Martin, CONNELLEY Timothy, MORRISON Ivan, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 160th- 338 2017/08/30 [Not refereed][Not invited]
- 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 -460 2017/08/30 [Not refereed][Not invited]
- 佐治木大和, 今内覚, 西森朝美, 岡川朋弘, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 160th- 370 2017/08/30 [Not refereed][Not invited]
- 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 -370 2017/08/30 [Not refereed][Not invited]
- 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 -371 2017/08/30 [Not refereed][Not invited]
- 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 2017/08/10 [Not refereed][Not invited]
- 今内覚, 伊東拓也, 高野愛, 川端寛樹, 安藤秀二, 村田史郎, 大橋和彦 日本細菌学雑誌(Web) 72- (1) 35(J‐STAGE) 2017 [Not refereed][Not invited]
- 渡慧, 今内覚, 前川直也, 岡川朋弘, 西森朝美, 鈴木定彦, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 159th- 365 -365 2016/08/30 [Not refereed][Not invited]
- 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 -364 2016/08/30 [Not refereed][Not invited]
- 越智晶絵, 今内覚, 伊東拓也, 川端寛樹, 高野愛, 安藤秀二, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 159th- 339 2016/08/30 [Not refereed][Not invited]
- 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 -364 2016/08/30 [Not refereed][Not invited]
- 村田史郎, 町田柚香, 伊勢崎政美, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 159th- 399 2016/08/30 [Not refereed][Not invited]
- 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 159回- 399 -399 2016/08 [Not refereed][Not invited]
- 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 -324 2015/08/30 [Not refereed][Not invited]
- 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 -405 2015/08/30 [Not refereed][Not invited]
- 町田柚香, 村田史郎, 伊勢崎政美, 酒井英史, 種子野章, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 158th- 354 2015/08/30 [Not refereed][Not invited]
- 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 -301 2015/08/30 [Not refereed][Not invited]
- 西森朝美, 今内覚, 池渕良洋, 岡川朋弘, 中原綾子, 千葉由純, 戸塚知恵, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 158th- 336 2015/08/30 [Not refereed][Not invited]
- 北條巧, 谷口綾香, 村田史郎, 伊勢崎政美, 酒井英史, 矢吹卓也, 寺田晴菜, 種子野章, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 158th- 353 2015/08/30 [Not refereed][Not invited]
- 村田史郎, 山本英次, 坂下奈津美, 松山あゆ美, 伊勢崎政美, 町田柚香, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 158th- 354 2015/08/30 [Not refereed][Not invited]
- 村瀬優介, 今内覚, 伊東拓也, 高野愛, 川端寛樹, 安藤秀二, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 158th- 301 2015/08/30 [Not refereed][Not invited]
- 高木道浩, 村田史郎, 今内覚, 大橋和彦 鶏病研究会報 50- (4) 199 -205 2015/02/25 [Not refereed][Not invited]
マレック病(MD)は鶏のウイルス感染症であり，家畜伝染病予防法で届出伝染病に指定されている。MDは翼麻痺や脚麻痺などの神経症状を示し，末梢神経の腫大，悪性リンパ腫形成(CD4+T細胞を標的とする)が見られ，死亡率は様々である。原因ウイルスであるMDウイルス(MDV)は直鎖状の二本鎖DNAウイルスでヘルペスウイルス科(Herpesviridae)アルファヘルペスウイルス亜科(Alphaherpesvirinae)，マルディウイルス(Mardivirus)に属する。これまではその病原性に基づきMDVは血清型1から3型(MDV1～3)に分類されていたが，最近，MDV1はGallid herpesvirus 2(GaHV-2)，MDV2はGallid herpesvirus 3(GaHV-3)およびMDV3はMeleagrid herpesvirus 1(MeHV-1)と新たに区分された。MDはワクチンによって効果的にコントロールできる疾病であり，ワクチンとしてすべての血清型があり，GaHV-2では弱毒株が用いられている。しかし，国内ではワクチンブレークによる発生，国外では強毒株の発生が見られている。また，ワクチンの作用機序およびMDVの腫瘍化機序も未だに解明されていない。ここではMDVの癌遺伝子と考えられているmeqについて述べると共に最近のMDV腫瘍化機序の知見について述べる。 - 岡川朋弘, 今中覚, 池渕良洋, 西森朝美, 溝呂木聖子, 永田礼子, 川治聡子, 田中省吾, 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 157th- 385 2014/08/11 [Not refereed][Not invited]
- 中原綾子, 今内覚, 池渕良洋, 岡川朋弘, 西森朝美, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 157th- 388 2014/08/11 [Not refereed][Not invited]
- 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 -388 2014/08/11 [Not refereed][Not invited]
- 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 -483 2014/08/11 [Not refereed][Not invited]
- 西森朝美, 今内覚, 池渕良洋, 岡川朋弘, 大平浩輔, 中原綾子, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 157th- 424 2014/08/11 [Not refereed][Not invited]
- 長雄一, 大越安吾, 平井綱雄, 藤井啓, 大橋和彦, 村田史郎, 遠藤大二, 金子正美, 田中克佳, 浅川満彦 酪農ジャーナル 67- (8) 29 -31 2014/08/01 [Not refereed][Not invited]
- 長雄一, 藤井啓, 大越安吾, 大橋和彦, 村田史郎, 千嶋淳, 久保清司 日本野生動物医学会大会・講演要旨集 20th- 42 2014 [Not refereed][Not invited]
- 今内覚, 伊東拓也, 川端寛樹, 高野愛, 安藤秀二, 村田史郎, 大橋和彦 大原綜合病院年報 53- 65 2013/12/15 [Not refereed][Not invited]
- 免疫抑制受容体PD-1のリガンドPD-L2の機能的特徴と臨床応用研究西森朝美, 今内覚, 池渕良洋, 岡川朋弘, 村田史郎, 大橋和彦 動物ワクチン-バイオ医薬品研究会ニュースレター 8- 18 -19 2013/12 [Not refereed][Invited]
- 今内覚, 村田史郎, 大橋和彦 Dairyman 63- (9) 42 -43 2013/09/01 [Not refereed][Not invited]
- 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 -257 2013/08/30 [Not refereed][Not invited]
- 池渕良洋, 今内覚, 岡川朋弘, 西森朝美, 中原綾子, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 156th- 271 2013/08/30 [Not refereed][Not invited]
- 松山あゆ美, 村田史郎, 伊勢崎政美, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 156th- 257 2013/08/30 [Not refereed][Not invited]
- 豊間根耕地, 今内覚, 伊東拓也, 川端寛樹, 高野愛, 安藤秀二, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 156th- 235 2013/08/30 [Not refereed][Not invited]
- 今村彩貴, 中溝万里, 川西路子, 池渕良洋, 目堅博久, 村田史郎, 今内覚, 大橋和彦, 中島奈緒, 山本欣也, 内山万利小, 平野文哉, 永井英貴, 木島まゆみ 日本獣医学会学術集会講演要旨集 156th- 260 2013/08/30 [Not refereed][Not invited]
- 西森朝美, 今内覚, 池渕良洋, 岡川朋弘, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 156th- 257 2013/08/30 [Not refereed][Not invited]
- 今内覚, 豊間根耕地, 小池菜々子, 伊東拓也, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 156th- 231 2013/08/30 [Not refereed][Not invited]
- 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 156回- 304 -304 2013/08 [Not refereed][Not invited]
- 今内覚, 川端寛樹, 伊東拓也, 高野愛, 安藤秀二, 村田史郎, 大橋和彦 衛生動物 64- (2) 112 2013/06/15 [Not refereed][Not invited]
- 目堅博久, 今内覚, MINGALA Claro N, 井上昇, 村田史郎, 小沼操, 大橋和彦 日本獣医学会学術集会講演要旨集 155th- 81 2013/03/04 [Not refereed][Not invited]
- 松山智慧麿, 遠藤大二, 大橋和彦, 今内覚, 村田史郎, 有川二郎, 吉松組子, 水谷哲也, 林正信 日本獣医学会学術集会講演要旨集 155th- 229 2013/03/04 [Not refereed][Not invited]
- 今内覚, 川端寛樹, 伊東拓也, 高野愛, 安藤秀二, 村田史郎, 大橋和彦 大原綜合病院年報 52- 105 -106 2012/12/15 [Not refereed][Not invited]
- 大橋和彦, 村田史郎, 今内覚 臨床獣医 30- (11) 50 -52 2012/11/01 [Not refereed][Not invited]
- 伊勢崎政美, 村田史郎, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 154th- 203 2012/08/31 [Not refereed][Not invited]
- 今内覚, 目堅博久, CLARO Mingala, NANCY Abes, ALAN Dargantes, 井上昇, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 154th- 213 2012/08/31 [Not refereed][Not invited]
- 高崎紗蘭, 村田史郎, 松山あゆ美, 伊勢崎政美, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 154th- 234 2012/08/31 [Not refereed][Not invited]
- 岡川朋弘, 今内覚, 池渕良洋, 鈴木紗織, 寸田祐嗣, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 154th- 233 2012/08/31 [Not refereed][Not invited]
- 高田春奈, 今内覚, NAFTALY Githaka, 伊勢崎政美, 伊東拓也, 安藤秀二, 川端寛樹, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 154th- 204 2012/08/31 [Not refereed][Not invited]
- 池渕良洋, 今内覚, 岡川朋弘, 横山和正, 中島千絵, 鈴木定彦, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 154th- 233 2012/08/31 [Not refereed][Not invited]
- 感染症と免疫の基礎 免疫とは何か大橋 和彦, 村田史郎, 今内 覚 臨床獣医 8- 60 -62 2012/08 [Not refereed][Invited]
- 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 154回- 234 -234 2012/08 [Not refereed][Not invited]
- 今内覚, 村田史郎, 大橋和彦 北海道獣医師会雑誌 56- (7) 245 -251 2012/07/10 [Not refereed][Not invited]
- ウシ難治性疾病に対する多機能型新規治療法の開発岡川朋弘, 今内覚, 池渕良洋, 鈴木紗織, 村田史郎, 大橋和彦 動物ワクチン-バイオ医薬品研究会ニュースレター 5- 21 -23 2012/06 [Not refereed][Invited]
- A comparative analysis of cytokine profile of Theileria parva infection in BovidaeOkagawa T, Konnai S, Githaka N, Mekata H, Suzuki S, Kariuki E, Skilton R, Ishizuka M, Murata S, Ohashi K Japanese Journal of Veterinary Parasitology 10- (1-2) 37 2012/03 [Not refereed][Not invited]
- 児玉道, 荻原喜久美, 納谷裕子, 村田史郎, 今内覚, 大橋和彦, 岸川正剛 日本獣医学会学術集会講演要旨集 153rd- 223 2012/03/01 [Not refereed][Not invited]
- 今内覚, 池渕良洋, 岡川朋弘, 鈴木紗織, 白井達哉, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 153rd- 117 2012/03/01 [Not refereed][Not invited]
- 池渕良洋, 今内覚, 村田史郎, 大橋和彦 日本獣医師会獣医学術学会年次大会講演要旨集 2011- 45 2012/01/20 [Not refereed][Not invited]
- 今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 Dairy Jpn 56- (15) 63 -65 2011/12/01 [Not refereed][Not invited]
- ウシTim-3の発現・機能解析と新規疾病制御法への応用展開岡川朋弘, 今内覚, 池渕良洋, 鈴木紗織, 村田史郎, 大橋和彦 動物ワクチン-バイオ医薬品研究会ニュースレター 4- 27 -29 2011/12 [Not refereed][Invited]
- 牛白血病の感染予防対策 —診療・生産現場からの声に対して—今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 Dairy Japan 12- 62 -64 2011/12 [Not refereed][Invited]
- 今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 Dairy Jpn 56- (14) 38 -40 2011/11/01 [Not refereed][Not invited]
- 牛白血病の感染予防対策 牛白血病に対する風評被害と間違った知識とは？今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 Dairy Japan 11- 57 -58 2011/11 [Not refereed][Invited]
- 今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 Dairy Jpn 56- (12) 34 -36 2011/10/01 [Not refereed][Not invited]
- 今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 Dairy Jpn 56- (11) 53 -55 2011/09/01 [Not refereed][Not invited]
- 牛白血病の感染予防対策 —牛白血病はなぜ増えたのか？—今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 Dairy Japan 9- 37 -39 2011/09 [Not refereed][Invited]
- 丹羽彩乃, 今内覚, GITHAKA Naftary, 伊東拓也, 川端寛樹, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 152nd- 207 2011/08/31 [Not refereed][Not invited]
- 村田史郎, 伊勢崎政美, 松山あゆ美, 高崎紗蘭, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 152nd- 241 2011/08/31 [Not refereed][Not invited]
- 岡川朋弘, 今内覚, 池渕良洋, 鈴木紗織, 寸田祐嗣, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 152nd- 224 2011/08/31 [Not refereed][Not invited]
- 鈴木紗織, 今内覚, 池渕良洋, 岡川朋弘, 寸田祐嗣, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 152nd- 224 2011/08/31 [Not refereed][Not invited]
- 目堅博久, 今内覚, 井上昇, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 152nd- 205 2011/08/31 [Not refereed][Not invited]
- 今内覚, 岡川朋弘, NAFTALY Githaka, 目堅博久, 鈴木紗織, EDWARD Kariuki, ROBERT Skilton, 石塚真由美, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 152nd- 204 2011/08/31 [Not refereed][Not invited]
- 今内覚, 村瀬優介, 伊東拓也, 高野愛, 川端寛樹, 村田史郎, 大橋和彦 衛生動物 62- (2) 133 2011/06/15 [Not refereed][Not invited]
- 今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 Dairyman 61- (6) 30 -31 2011/06/01 [Not refereed][Not invited]
- ウイルスを原因とする牛白血病の防疫（感染牛を把握し、伝播リスクを抑える）今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 DAIRYMAN 61- 30 -31 2011/06 [Not refereed][Invited]
- 今内覚, 池渕良洋, 寸田祐嗣, 小沼操, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 151st- 120 2011/03/01 [Not refereed][Not invited]
- 児玉道, 荻原喜久美, 納屋裕子, 久保正法, 榊原伸一, 村田史郎, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 151st- 217 2011/03/01 [Not refereed][Not invited]
- 山田慎二, 今内覚, 今村彩貴, SIMMUNZA Martin, NANBOTA Andrew, 小沼操, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 151st- 79 2011/03/01 [Not refereed][Not invited]
- 牛白血病の感染防御対策今内 覚, 田島誉士, 小沼 操, 村田史郎, 大橋和彦 北海道しゃくなげ会会報 50- 10 -12 2011/02 [Not refereed][Invited]
- 国内の牛白血病の現状と今後の対策について今内 覚, 田島 誉士, 村田 史郎, 大橋 和彦 牛臨床寄生虫研究会誌 2- 2 -3 2011/02 [Not refereed][Invited]
- 大橋和彦, 松原綾子, 村田史郎, 今内覚 獣医畜産新報 64- (1074) 21 -26 2011/01/01 [Not refereed][Not invited]
- 牛白血病の正体と予防を考察する今内 覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 HOLSTEIN MAGAZINE 505- 4 -7 2011/01 [Not refereed][Invited]
- 今内覚, 田島誉士, 小沼操, 村田史郎, 大橋和彦 産業動物臨床医学雑誌 1- (2) 110 -114 2010/11/05 [Not refereed][Not invited]
- 池渕良洋, 今内覚, 寸田祐輔, 小沼操, 村田史郎, 大橋和彦 日生研たより 56- (6) 77 -81 2010/11/01 [Not refereed][Not invited]
- 目堅博久, 今内覚, MINGALA Claro, DARGANTES Alan, 井上昇, 小沼操, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 150th- 191 2010/09/01 [Not refereed][Not invited]
- 村瀬優介, 今内覚, 伊藤拓也, 高野愛, 川端寛樹, 小沼操, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 150th- 193 2010/09/01 [Not refereed][Not invited]
- 岡川朋弘, 鈴木紗織, 今内覚, GITHAKA Naftali, KARIUKI Edward, GAKUYA Francis, ESTHER Kanduma, 石塚真由美, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 150th- 212 2010/09/01 [Not refereed][Not invited]
- 河東あゆ美, 村田史郎, 加納里佳, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 150th- 214 2010/09/01 [Not refereed][Not invited]
- 池渕良洋, 今内覚, 白井達哉, 寸田祐嗣, 小沼操, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 150th- 214 2010/09/01 [Not refereed][Not invited]
- 今内覚, 肥田野新, 村瀬優介, 伊東拓也, 川端寛樹, 樋口豪起, 小沼操, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 150th- 193 2010/09/01 [Not refereed][Not invited]
- 白井達哉, 今内覚, 池渕良洋, 寸田祐嗣, 小沼操, 村田史郎, 大橋和彦 日本獣医学会学術集会講演要旨集 150th- 214 2010/09/01 [Not refereed][Not invited]
- 伊勢崎政美, 村田史郎, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 150th- 193 2010/09/01 [Not refereed][Not invited]
- 松原綾子, 村田史郎, 加納里佳, 橋口知幸, 伊勢崎政美, 河東あゆ美, 小沼操, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 150th- 215 2010/09/01 [Not refereed][Not invited]
- 今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦 北海道獣医師会雑誌 54- (8) 378 2010/08/10 [Not refereed][Not invited]
- 難治性慢性疾病における免疫疲弊化機序の解明と新規治療法への応用〜獣医医療への応用に向けて〜池渕良洋, 今内 覚, 寸田祐嗣, 小沼 操, 村田史郎, 大橋和彦 NIBS Letter 56- (6) 7 -11 2010/06 [Not refereed][Invited]
- The detection and characterization of the meq variants of Marek's diseases virusOhashi K, Murata S, Okada T, Konnai S Animal Viruses 16- 117 -132 2010/01 [Refereed][Invited]
- 大橋和彦, 松原綾子, 村田史郎, 今内覚 日本獣医学会学術集会講演要旨集 149th- 198 2010 [Not refereed][Not invited]
- 橋口知幸, 村田史郎, 岡田宰, 加納里佳, 今内覚, 小沼操, 大橋和彦 日本獣医学会学術集会講演要旨集 146th- 189 2008/09/05 [Not refereed][Not invited]
- 耕崎志乃, 村田和子, 大西剛直, 福本光孝, 小川恭弘, 前田博教, 笹栗史郎, 松本学, 弘井誠 Abstracts. Annual Symposium. Japanese Society for the Advancement of Women’s Imaging 9th (Web)- 2008
- 加納里佳, 橋口知幸, 岡田宰, 村田史郎, 今内覚, 小沼操, 大橋和彦 日本ウイルス学会北海道支部夏季シンポジウム講演抄録 42nd- 14 2008 [Not refereed][Not invited]
- 村尾岳洋, 加納里佳, 村田史郎, 今内覚, 大橋和彦, 小沼操, 前田龍一郎, 小俣吉孝 日本野生動物医学会大会・講演要旨集 13th- 88 2007/09/06 [Not refereed][Not invited]
- 村田史郎, 岡田宰, 加納里佳, 小沼操, 今内覚, 大橋和彦 日本獣医学会学術集会講演要旨集 144th- 83 2007/08/27 [Not refereed][Not invited]
- 加納里佳, 村田史郎, 岡田宰, 今内覚, 小沼操, 大橋和彦 日本獣医学会学術集会講演要旨集 144th- 77 2007/08/27 [Not refereed][Not invited]
- 村尾岳洋, 加納里佳, 村田史郎, 今内覚, 大橋和彦, 小沼操, 前田龍一郎, 小俣吉孝 日本獣医学会学術集会講演要旨集 144th- 65 2007/08/27 [Not refereed][Not invited]
- 岡田宰, 村田史郎, 加納里佳, 高木道浩, 今内覚, 小沼操, 大橋和彦 日本獣医学会学術集会講演要旨集 144th- 83 2007/08/27 [Not refereed][Not invited]
- 岡田宰, 高木道浩, 村田史郎, 加納里佳, 林裕子, 今内覚, 小沼操, 大橋和彦 日本獣医学会学術集会講演要旨集 142nd- 80 2006/08/31 [Not refereed][Not invited]
- 村田史郎, 岡田宰, 加納里佳, 林裕子, 今内覚, 大橋和彦, 小沼操 日本獣医学会学術集会講演要旨集 142nd- 80 2006/08/31 [Not refereed][Not invited]
- 村田史郎, 山本裕己, 岡田宰, 浅川満彦, 長雄一, 池内俊雄, 今内覚, 大橋和彦, 小沼操 日本獣医学会学術集会講演要旨集 140th- 108 2005/08/31 [Not refereed][Not invited]
- 大橋和彦, CHANG K‐S, LEE S‐I, 村田史郎, 高木道浩, 小沼操 獣医畜産新報 (996) 594 -595 2004/07/01 [Not refereed][Not invited]
- 大橋和彦, CHANG K‐S, LEE S‐I, 村田史郎, 高木道浩, 小沼操 日本獣医学会学術集会講演要旨集 137th- 86 2004/03/01 [Not refereed][Not invited]
- 村田史郎, CHANG K‐S, LEE S‐I, 大橋和彦, 小沼操 日本獣医学会学術集会講演要旨集 136th- 135 2003/09/10 [Not refereed][Not invited]
- 張しゅ, 大橋和彦, 李成一, 村田史郎, 浅川満彦, 小沼操 日本獣医学会学術集会講演要旨集 134th- 134 2002/08/20 [Not refereed][Not invited]
- 山口均, 村田厚夫, 三島史郎, 山口芳裕, 四蔵勇一, 高橋由典, 須田健二, 松田博青, 島崎修次 日本救急医学会雑誌 11- (10) 2000
- 山本明彦, 村田厚夫, 田中秀治, 山口芳裕, 菊地充, 三島史郎, 後藤英明, 小川道雄, 島崎修次 日本救急医学会雑誌 11- (10) 2000
Industrial Property Rights
- 鈴木 定彦, 中川 美樹, 亀田 弥生, 今内 覚, 岡川 朋弘, 前川 直也, 後藤 伸也, 佐治木 大和, 大橋 和彦, 村田 史郎, 北原 譲, 山本 啓一 国立大学法人北海道大学, 扶桑薬品工業株式会社 202303011844880950
- 特開2022-174692:抗ワクモワクチン組成物及びその使用 2022/11/24村田 史郎, 大橋 和彦, 藤澤 宗太郎, 有泉 拓馬, 今内 覚, 岡川 朋弘, 前川 直也, 大石 英司, 佐藤 匠 国立大学法人北海道大学, ワクチノーバ株式会社 202203006614155970
- 特開2022-169389:イヌTGF-β受容体II 2022/11/09今内 覚, 前川 直也, 岡川 朋弘, 大橋 和彦, 村田 史郎, 鈴木 定彦, 竹内 寛人 国立大学法人北海道大学 202203018896478340
- 今内 覚, 大橋 和彦, 村田 史郎, 岡川 朋弘, 前川 直也, 西森 朝美, 後藤 伸也, 鈴木 定彦, 中島 千絵, 佐治木 大和 国立大学法人北海道大学, 扶桑薬品工業株式会社 202203000928000549
- 今内 覚, 大橋 和彦, 村田 史郎, 岡川 朋弘, 西森 朝美, 前川 直也, 鈴木 定彦, 中島 千絵 国立大学法人北海道大学, 扶桑薬品工業株式会社 202203006529697836
- 今内 覚, 大橋 和彦, 村田 史郎, 岡川 朋弘, 西森 朝美, 前川 直也, 高木 哲, 賀川 由美子, 鈴木 定彦, 中島 千絵 国立大学法人北海道大学, 扶桑薬品工業株式会社 202203010523660792
- 今内 覚, 大橋 和彦, 村田 史郎, 岡川 朋弘, 西森 朝美, 前川 直也, 鈴木 定彦, 中島 千絵 国立大学法人北海道大学, 扶桑薬品工業株式会社 202103001370378735
- 今内 覚, 大橋 和彦, 村田 史郎, 岡川 朋弘, 西森 朝美, 前川 直也, 鈴木 定彦, 中島 千絵 国立大学法人北海道大学, 扶桑薬品工業株式会社 202103010624578216
- 大橋 和彦, 村田 史郎, 伊勢崎 政美, 今内 覚, 谷口 綾香, 北條 巧 国立大学法人北海道大学 202003006944716043
- WO2019-225372:新規ベクターおよびその利用 2019/11/28鈴木 定彦, 中川 美樹, 亀田 弥生, 今内 覚, 岡川 朋弘, 前川 直也, 後藤 伸也, 佐治木 大和, 大橋 和彦, 村田 史郎, 北原 譲, 山本 啓一 国立大学法人北海道大学, 扶桑薬品工業株式会社 202103019221572843
Awards & Honors
- 2023/09 The Japanese Society of Veterinary Science Veterinary Science Award
Studies on the epidemiological surveys, pathogenesis and strategies for the control of avian diseases
Research Grants & Projects
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2023/04 -2027/03Author : 村田 史郎, 今内 覚, 大橋 和彦
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2023/04 -2026/03Author : 大橋 和彦, 村田 史郎, 今内 覚
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)Date (from‐to) : 2022/04 -2025/03Author : 今内 覚, 村田 史郎, 大橋 和彦
- 家禽の吸血性外部寄生虫ワクモに由来する鶏免疫応答調節因子の解明一般財団法人旗影会：2023 年度 旗影会研究助成Date (from‐to) : 2023/04 -2024/03Author : 村田史郎
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)Date (from‐to) : 2020/07 -2023/03Author : 村田 史郎ワクモ（鳥類の外部寄生虫）による吸血被害は、世界中の養鶏場で深刻な問題となっている。最近では薬剤に耐性を示すワクモの出現により、現行の薬剤による防除では制御が困難になりつつある。薬剤に変わる新規防除法としてワクチンが注目されている。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。そのため腸管の細胞膜上に発現するタンパク質は最も影響を受けやすいと考えられ、これまでに腸管細胞の細胞膜上に発現が予想される分子を複数同定してきた。しかしそれらの多くは、複数回膜貫通型の分子や、大きな細胞膜領域を持つ分子であり、組換えタンパク質の作製には困難が予想され、ワクチン抗原としての利用には工夫が必要とされる。そこで本研究では、ウイルスベクターを用いて、ワクチン抗原を組換えウイルス感染細胞に細胞膜タンパク質として発現させることで、ワクチン効果に有効な免疫応答を誘導すること目的とした。本年度は組換えウイルスゲノムに挿入するためのワクチン抗原の選抜を行った。以前に実施したRNA sequencingのデータより、細胞膜上に発現が予想される分子を抽出し、本研究の趣旨に合致する、組換えタンパク質としての発現に困難が予想される立体構造を持つ抗原を選抜した。複数の抗原候補の中において、細胞膜上に発現が予想されるadipocyte plasma membrane-associated protein (APMAP) は、一回膜貫通型の大きな細胞外領域を持つ構造であると予想された。また、APMAPは腸管に発現することを確認することができた。そのためAPMAPは腸管細胞の細胞膜上に発現が予想されることから、APMAPを組換えウイルスゲノムに挿入する抗原として用いることとした。
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)Date (from‐to) : 2020/04 -2023/03Author : 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、次世代シークエンサーを用いた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、最初にワクモにおいて、飢餓および吸血後のワクモより試料を調製して次世代シークエンサーを用いたRNA-SEQを行い、発現する遺伝子群の網羅的解析を行った。その結果、吸血後のワクモでは、細胞内の代謝やエネルギー合成に関わる遺伝子群の発現が上昇し、飢餓ワクモでは、脂肪酸酸化や糖質合成など関わる遺伝子群の発現が顕著であった。 新規“ユニバーサル”ワクチン候補分子を同定する際には、候補分子の発育ステージ毎あるいは組織別での詳細な発現解析を行う必要があるが、ワクモなどでは相対的発現量に計測に必須のreference遺伝子の情報が乏しい。そこで適当な２つのreference遺伝子を同定して、定量的real-time PCR法を樹立した。
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)Date (from‐to) : 2019/04 -2022/03Author : KONNAI SATORUOur 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)Date (from‐to) : 2018/04 -2021/03Author : Murata ShiroPoultry 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)Date (from‐to) : 2017/04 -2020/03Author : Ohashi KazuhikoMarek’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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)Date (from‐to) : 2016/04 -2019/03Author : OHASHI KazuhikoPoultry 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)Date (from‐to) : 2016/04 -2018/03Author : Shiro MurataThe poultry red mite (PRM), is one of the major hematophagous ectoparasites of poultry farming, and the decreased productivity due to blood-sucking by PRMs is a severe problem for the poultry industry. The use of acaricides often leads to the spread of acaricide-resistant PRMs. Therefore, alternative strategies are required to reduce economic losses in poultry farming. Here, we focus on preventive methods such as vaccination to control PRMs, and we have already identified several vaccine candidates. In order to prepare an easy-to-use anti-PRM vaccine, the recombinant Marek's disease (MD) virus (rMDV) expressing a vaccine antigen was reconstituted. Reconstituted rMDV showed no significant differences in the growth kinetics compared to the parental strain, and the vaccine antigen was expressed in the infected cells. Thus, the rMDV live vaccine expressing a vaccine antigen against PRMs may be applicable as a candidate dual vaccine that provides protection against both PRMs and MD.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory ResearchDate (from‐to) : 2016/04 -2018/03Author : OHASHI KazuhikoFor 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)Date (from‐to) : 2014/04 -2018/03Author : KONNAI SATORUBovine 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)Date (from‐to) : 2014/04 -2017/03Author : Kazuhiko OhashiMarek’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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)Date (from‐to) : 2013/04 -2017/03Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)Date (from‐to) : 2013/04 -2015/03Author : MURATA ShiroField isolates of Marek’s disease virus (MDV) have tended to increase in virulence, and therefore, the development of more effective vaccines would be desirable. To develop a novel vaccine, we focused on the PD1/PD-L1 pathway that contributes to the immune suppression, and reconstituted the recombinant MDVs that carry the soluble-PD1 gene to competitively inhibit the immunosuppression regulated by the endogenous PD1. The recombinant vaccine expressing soluble PD1 induced higher production of interferon γ in the immune cells from the vaccinated chickens, indicating that this vaccine potentially possesses an ability to induce more effective cell-mediated immunity in chickens.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)Date (from‐to) : 2011/11 -2015/03Author : KONNAI SATORU, OHASHI Kazuhiko, MURATA ShiroBovine 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.
- Studies on the immunoinhibitory mechanisms of Marek's disease virus and its application for adjuvantJapan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)Date (from‐to) : 2011/04 -2014/03Author : OHASHI KAZUHIKO, KONNAI Satoru, MURATA ShiroThe 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.
- 秋山記念生命科学振興財団研究助成秋山記念生命科学振興財団：Date (from‐to) : 2010 -2010Author : 村田史郎
Educational Activities
Teaching Experience
- Advanced Lecture on Immunology開講年度 : 2021課程区分 : 博士後期課程開講学部 : 獣医学院
- Advanced Lecture on Immunology開講年度 : 2021課程区分 : 博士後期課程開講学部 : 国際感染症学院
- Seminar in Veterinary Ecology開講年度 : 2021課程区分 : 学士課程開講学部 : 獣医学部キーワード : 疫学研究手法、原因論、リスク分析、統計学
- Practice in Infectious Diseases開講年度 : 2021課程区分 : 学士課程開講学部 : 獣医学部キーワード : 細菌、ウイルス、原虫、同定、診断
- Seminar in Veterinary Epidemiology開講年度 : 2021課程区分 : 学士課程開講学部 : 獣医学部キーワード : 疫学研究手法、原因論、リスク分析、統計学
- Practice in Animal Hygiene開講年度 : 2021課程区分 : 学士課程開講学部 : 獣医学部キーワード : 飼養衛生、衛生管理、疾病予防、ワクチン、消毒、アニマルウェルフェア