Researcher Profile and Settings

Affiliation

• Faculty of Veterinary Medicine　Veterinary Medicine　Disease Control

Job Title

• Professor

URL

http://kaken.nii.ac.jp/d/r/40396304.ja.html

J-Global ID

• 201201080316824797

Research Interests

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

Research Areas

• Life sciences / Veterinary medicine

Educational Organization

•  Bachelor's degree program　School of Veterinary Medicine
•  Doctoral (PhD) degree program　Graduate School of Infectious Diseases

Academic & Professional Experience

• 2022/07 - Today 北海道大学大学院獣医学研究院 先端創薬分野 分野長・教授
• 2022/07 - Today 北海道大学大学院獣医学研究院 病原制御学分野 教授
• 2018/07 - 2022/06 北海道大学大学院獣医学研究院 先端創薬分野 分野長・准教授
• 2017/04 - 2022/06 北海道大学大学院獣医学研究院 病原制御学分野 准教授
• 2008/12 - 2017/03 Hokkaido University Graduate School of Veterinary Medicine
• 2007/04 - 2008/11 Hokkaido University Graduate School of Veterinary Medicine
• 2004/12 - 2007/03 Hokkaido University Graduate School of Veterinary Medicine

Education

• 2003/03 -   Hokkaido University  Graduate School of Veterinary Medicine

Association Memberships

• THE JAPANESE SOCIETY FOR VIROLOGY   大動物臨床研究会   JAPANESE SOCIETY FOR CLINICAL INFECTIOUS DISEASE IN FARM ANIMALS   動物サイトカイン研究会   日本獣医寄生虫学会   動物用ワクチン-バイオ医薬品研究会   北海道獣医師会   JAPAN VETERINARY MEDICAL ASSOCIATION   JAPANESE SOCIETY OF VETERINARY SCIENCE   

Research Activities

Published Papers

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

  Win SY, Seo H, Horio F, Fujisawa S, Sato J, Motai Y, Sato T, Oishi E, Taneno A, Htun LL, Bawn S, Okagawa T, Maekawa N, Konnai S, Ohashi K, Murata S

  Vaccines in press 2024/02 [Refereed][Not invited]
  
• Characterization of a Very Short Meq Protein Isoform in a Marek's Disease Virus Strain in Japan

  Motai Y, Murata S, Sato J, Nishi A, Maekawa N, Okagawa T, Konnai S, Ohashi K

  Vet Sci. 11 (1) 43  2024/01 [Refereed][Not invited]
  
• G6PDH as a key immunometabolic and redox trigger in arthropods

  Moraes B, Martins R, Lopes C, Martins R, Arcanjo A, Nascimento J, Konnai S, da Silva Vaz I Jr, Logullo C

  Front Physiol. 14 1287090  2023/11 [Refereed][Not invited]
  
• An Ixodes persulcatus inhibitor of plasmin and thrombin hinders keratinocyte migration, blood coagulation, and endothelial permeability.

  Berger M, Rosa da Mata S, Pizzolatti NM, Parizi LF, Konnai S, da Silva Vaz I Jr, Seixas A, Tirloni L

  J Invest Dermatol. S0022-202X(23) 03107  2023/11 [Refereed][Not invited]
  
• Safety and clinical efficacy of an anti-PD-L1 antibody (c4G12) in dogs with advanced malignant tumours

  Maekawa N, Konnai S, Hosoya K, Kim S, Kinoshita R, Deguchi T, Owaki R, Tachibana Y, Yokokawa M, Takeuchi H, Kagawa Y, Takagi S, Ohta H, Kato Y, Yamamoto S, Yamamoto K, Suzuk Y, Okagawa T, Murata S, Ohashi K

  PLOS ONE 18 (10) e0291727  2023/10 [Refereed][Not invited]
   

  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.
• Development of a high-affinity anti-bovine PD-1 rabbit–bovine chimeric antibody using an efficient selection and large production system

  Okagawa T, Konnai S, Goto S, Sajiki Y, Ganbaatar O, Watari K, Nakamura H, Wang CX, Tachibana T, Kato Y, Kameda Y, Kohara J, Terasaki N, Kubota M, Takeda A, Takahashi H, Suzuki Y, Maekawa N, Murata S, Ohashi K

  Vet Res. 54 (1) 82 - 82 2023/09 [Refereed][Not invited]
   

  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.
• Characterization of cysteine proteases from poultry red mite, tropical fowl mite, and northern fowl mite to assess the feasibility of developing a broadly efficacious vaccine against multiple mite species

  Win SY, Murata S, Fujisawa S, Seo H, Sato J, Motai Y, Sato T, Oishi E, Taneno A, Htun LL, Bawm S, Okagawa T, Maekawa N, Konnai S, Ohashi K

  PLoS One 18 (7) e0288565  2023/07 [Refereed][Not invited]
   

  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.
• Enhanced systemic antitumour immunity by hypofractionated radiotherapy and anti-PD-L1 therapy in dogs with pulmonary metastatic oral malignant melanoma

  Deguchi T, Maekawa N, Konnai S, Owaki R, Hosoya K, Morishita K, Nakamura M, Okagawa T, Takeuchi H, Kim S, Kinoshita R, Tachibana Y, Yokokawa M, Takagi S, Kato Y, Suzuk Y, Murata S, Ohashi K

  Cancers 15 (11) 3013  2023/06 [Refereed][Not invited]
   

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

  Tiyamanee W, Konnai S, Okagawa T, Nojima Y, Ganbaatar O, Maekawa N, Hasebe R, Kagawa Y, Kato Y, Suzuki Y, Murata S, Ohashi K

  Vet Immunol Immunopathol. 11 (261) 110609 - 110609 2023/05 [Refereed][Not invited]
   

  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.
• Analysis of gene expression in poultry red mite, Dermanyssus gallinae, by RNAscope in situ hybridization

  Seo H, Murata S, Ichii O, Namba T, Win SY, Sato T, Oishi E, Taneno A, Maekawa N, Okagawa T, Konnai S, Ohashi K

  J Vet Med Sci. 85 (5) 532 - 535 2023/05 [Refereed][Not invited]
   

  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.
• Investigation of peripheral blood responses in chickens infested with Dermanyssus gallinae

  Fujisawa S, Murata S, Sato T, Oishi E, Taneno A, Konnai S, Ohashi K

  Parasitol Int. 95 102754 - 102754 2023/04 [Refereed][Not invited]
   

  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.
• Potential of ferritin 2 as an antigen for the development of a universal vaccine for avian mites, poultry red mites, tropical fowl mites, and northern fowl mites

  Win SY, Murata S, Fujisawa S, Seo H, Sato J, Motai Y, Sato T, Oishi E, Taneno A, Htun LL, Bawn S, Okagawa T, Maekawa N, Konnai S, Ohashi K

  Front Vet Sci. 10 1182930 - 1182930 2023/04 [Refereed][Not invited]
   

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

  Ikehata M, Konnai S, Okagawa T, Abe K, Honma M, Kitamura T, Maekawa N, Suzuki Y, Murata S, Ohashi K

  Front Vet Sci. 10 1145445  2023/04 [Refereed][Not invited]
   

  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.
• Chimeric provirus of bovine leukemia virus/ SMAD family member 3 in cattle with enzootic bovine leukosis

  Nao N, Okagawa T, Nojiri N, Konnai S, Shimakura H, Tominaga M, Yoshida-Frihata H, Nishiyama E, Matsudaira T, Maekawa N, Murata S, Muramatsu M, Ohashi K, Saito M

  Archi Virol. in press  2023/03 [Refereed][Not invited]
  
• Enhancement of vaccine-induced T-cell responses by PD-L1 blockade in calves

  Okagawa T, Konnai S, Nakamura H, Ganbaatar O, Sajiki Y, Watari K, Noda H, Honma M, Kato Y, Suzuki Y, Maekawa N, Murata S, Ohashi K

  Vaccines 11 (3) 559  2023/03 [Refereed][Not invited]
   

  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.
• Regulation of programmed death ligand 1 expression by interferon-γ and tumour necrosis factor-α in canine tumour cell lines

  Owaki R, Deguchi T, Konnai S, Maekawa N, Okagawa T, Hosoya K, Kim S, Sunaga T, Okumura M

  Vet Comp Oncol. 1 - 12 2023/02/21 [Refereed][Not invited]
   

  Expression of programmed death ligand 1 (PD-L1) on tumor cells provides an immune evasion mechanism by inducing suppression of cytotoxic T cells. Various regulatory mechanisms of PD-L1 expression have been described in human tumours, however, little is known in canine tumours. To investigate whether inflammatory signaling is involved in PD-L1 regulation in canine tumours, the effects of interferon (IFN)-γ and tumour necrosis factor (TNF)-α treatment were examined in canine malignant melanoma cell lines (CMeC and LMeC) and an osteosarcoma cell line (HMPOS). The protein level of PD-L1 expression was upregulated by IFN-γ and TNF-α stimulation. Upon IFN-γ stimulation, all cell lines showed an increase in expression of PD-L1, signal transducer and activator of transcription (STAT)1, STAT3 and genes regulated by STAT activation. Upregulated expression of these genes was suppressed by the addition of a JAK inhibitor, oclacitinib. Contrastingly, upon TNF-α stimulation, all cell lines exhibited higher gene expression of the nuclear factor kappa B (NF-κB) gene RELA and genes regulated by NF-κB activation, whereas expression of PD-L1 was upregulated in LMeC only. Upregulated expression of these genes was suppressed by the addition of an NF-κB inhibitor, BAY 11-7082. The expression level of cell surface PD-L1 induced by IFN-γ and TNF-α treatment was reduced by oclacitinib and BAY 11-7082, respectively, indicating that upregulation of PD-L1 expression by IFN-γ and TNF-α stimulation is regulated via the JAK-STAT and NF-κB signaling pathways, respectively. These results provide insights into the role of inflammatory signaling in PD-L1 regulation in canine tumours. This article is protected by copyright. All rights reserved.
• Suppressive modulation of host immune responses by Dermanyssus gallinae infestation

  Fujisawa S, Murata S, Isezaki M, Win SY, Sato T, Oishi E, Taneno A, Maekawa N, Okagawa T, Konnai S, Ohashi K

  Poult Sci. 102 (4) 102532 - 102532 2023/02 [Refereed][Not invited]
   

  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.
• Characterization of SpsQ from Staphylococcus pseudintermedius as an affinity chromatography ligand for canine therapeutic antibodies

  Takeuchi H, Nakajima C, Konnai S, Maekawa N, Okagawa T, Usui M, Tamura U, Suzuki Y, Murata S, Ohashi K

  PLOS One 18 (1) e0281171  2023/01 [Refereed][Not invited]
   

  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.
• Molecular characterization of feline immune checkpoint molecules and establishment of PD-L1 immunohistochemistry for feline tumors

  Maekawa N, Konnai S, Asano Y, Otsuka T, Aoki E, Takeuchi H, Kato Y, Kaneko M, Yamada S, Kagawa Y, Nishimura M, Takagi S, Deguchi T, Ohta H, Nakagawa T, Suzuki Y, Okagawa T, Murata S, Ohashi K

  PLOS One 18 (1) e0281143  2023/01 [Refereed][Not invited]
   

  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.
• Combined immune checkpoint blockade enhances antiviral immunity against bovine leukemia virus

  Nakamura H, Konnai S, Okagawa T, Maekawa N, Sajiki Y, Watari K, Kamitani K, Saito M, Kato Y, Suzuki Y, Murata S, Ohashi K

  J Virol. 97 (1) e0143022  2023/01 [Refereed][Not invited]
   

  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.
• Diagnosis and early prediction of lymphoma using high-throughput clonality analysis of bovine leukemia virus-infected cells

  Okagawa T, Shimakura H, Konnai S, Saito M, Matsudaira T, Nao N, Yamada S, Murakami K, Maekawa N, Murata S, Ohashi K

  Microbiol Spectr. 10 (2) e0259522  2022/10 [Refereed][Not invited]
   

  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.
• 乳酸菌Lactobacillus plantarum HOKKAIDO株による免疫制御機序の解明

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

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

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

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

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

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [DI1A - 09] 1347-8621 2022/09
• ダニ媒介性脳炎の治療法開発に向けた血液脳関門透過性分子の研究

  福田 美津紀, 深野 紗代, 小林 進太郎, 前川 直也, 高橋 侑嗣, 平野 港, 苅和 宏明, 今内 覚, 好井 健太朗

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [F1P - 12] 1347-8621 2022/09
• 広範な有効性を示すワクチン開発のための鳥類寄生ダニ由来システインプロテアーゼの特性解析(Characterization of cysteine proteases from avian mites to develop a vaccine with broad efficacy)

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

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

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

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

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

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

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

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

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

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [DI1A - 04] 1347-8621 2022/09
• ダニ媒介性脳炎の治療法開発に向けた血液脳関門透過性分子の研究

  福田 美津紀, 深野 紗代, 小林 進太郎, 前川 直也, 高橋 侑嗣, 平野 港, 苅和 宏明, 今内 覚, 好井 健太朗

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [F1P - 12] 1347-8621 2022/09
• ワクモ(Dermanyssus gallinae)由来シスタチン様分子の抗ワクモワクチン抗原としての評価

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

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

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

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

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

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

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

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [DI1A - 09] 1347-8621 2022/09
• Prostaglandin E2-induced immune suppression viacytotoxic T-lymphocyte antigen-4 in paratuberculosis

  Sajiki Y, Konnai S, Watari K, Okagawa T, Tanaka A, Nagata R, Kawaji S, Maekawa N, Suzuki Y, Kato Y, Murata S, Mori Y, Ohashi K

  Infect Immun. 90 (10) e0021022  2022/09 [Refereed][Not invited]
   

  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.
• RAISING is a high-performance method for identifying random transgene integration sites

  Wada Y, Sato T, Hasegawa H, Matsudaira T, Nao N, Coler-Reilly ALG, Tasaka T, Yamauchi S, Okagawa T, Momose H, Tanio M, Kuramitsu M, Sasaki D, Matsumoto N, Yagishita N, Yamauchi J, Araya N, Tanabe K, Yamagishi M, Nakashima M, Nakahata S, Iha H, Ogata M, Muramatsu M, Imaizumi Y, Uchimaru K, Miyazaki Y, Konnai S, Yanagihara K, Morishita K, Watanabe T, Yamano Y, Saito M

  Commun Biol. 5 (1) 535 - 535 2022/06 [Refereed][Not invited]
   

  Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking clonality via the detection of transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive high throughput sequencing. We analyzed samples from 688 individuals infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL) to model our method. We defined a clonality value identifying ATL patients with 100% sensitivity and 94.8% specificity, and our longitudinal analysis also demonstrates the usefulness of ATL risk assessment. Future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.
• Exploration of serum biomarkers in dogs with malignant melanoma receiving anti-PD-L1 therapy and potential of COX-2 inhibition for combination therapy

  Maekawa N, Konnai S, Asano Y, Sajiki Y, Deguchi T, Okagawa T, Watari K, Takeuchi H, Takagi S, Hosoya K, Kim S, Ohta H, Kato Y, Suzuki Y, Murata S, Ohashi K

  Sci Rep. 12 (1) 9265 - 9265 2022/06 [Refereed][Not invited]
   

  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.
• Suppressive effects of Ixodes persulcatus sialostatin L2 against Borrelia miyamotoi-stimulated immunity

  Sajiki Y, Konnai S, Okagawa T, Maekawa N, Isezaki M, Yamada S, Ito T, Sato K, Kawabata H, Logullo C, da Silva Vaz I Jr, Murata S, Ohashi K

  Ticks Tick Borne Dis. 13 (4) 101963 - 101963 2022/04 [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.
• Estradiol-induced immune suppression via prostaglandin E2 during parturition in bovine leukemia virus-infected cattle

  Sajiki Y, Konnai S, Okagawa T, Maekawa N, Goto S, Kohara J, Nitanai A, Takahashi H, Kubota K, Takeda H, Murata S, Ohashi K

  PLOS One 17 (3) e0263660 - e0263660 2022/03 [Refereed][Not invited]
   

  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.
• Effect of Insertion and Deletion in the Meq Protein Encoded by Highly Oncogenic Marek’s Disease Virus on Transactivation Activity and Virulence

  Sato J, Murata S, Yang Z, Kaufer BB, Fujisawa F, Seo H, Maekawa N, Okagawa T, Konnai S, Osterrider N, Parcells MS, Ohashi K

  Viruses 14 (2) 382  2022/02 [Refereed][Not invited]
  
• Hemangiosarcoma cells induce M2 polarization and PD-L1 expression in macrophages.

  Gulay KCM, Aoshima K, Maekawa N, Suzuki T, Konnai S, Kobayashi A, Kimura T

  Sci Rep. 12 (1) 2124  2022/02 [Refereed][Not invited]
  
• Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line

  Della Noce B, Martins da Silva R, de Carvalho, Uhl MV, Konnai S, Ohashi K, Calixto C, Arcanjo A, de Abreu LA, de Carvalho SS, da Silva Vaz I Jr, Logullo C

  J Biol Chem. 101599  2022/01 [Refereed][Not invited]
  
• Enhancement of interleukin-2 production by bovine peripheral blood mononuclear cells treated with the combination of anti-programmed death-ligand 1 and cytotoxic T lymphocyte antigen 4 chimeric monoclonal antibodies

  Watari K, Konnai S, Okagawa T, Maekawa N, Sajiki Y, Kato Y, Suzuki Y, Murata S, Ohashi K

  J Vet Med Sci. 84 (1) 6 - 15 2022/01 [Refereed][Not invited]
   

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

  Fujisawa S, Murata S, Isezaki M, Ariizumi T, Sato T, Oishi E, Taneno A, Maekawa N, Okagawa T, Ichii O, Konnai S, Ohashi K

  Parasitology 149 (1) 105 - 115 0031-1820 2022/01 [Refereed][Not invited]
   

  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.
• In vitro evaluation of a cysteine protease from poultry red mites, Demanyssus gallinae, as a vaccine antigen for chickens

  Ariizumi T, Murata S, Fujisawa S, Isezaki M, Sato T, Oishi E, Taneno A, Ichii O, Maekawa N, Okagawa T, Konnai S, Ohashi K

  Poult Sci 101 (3) 101638  2021/12 [Refereed][Not invited]
  
• Characterization of a Novel Cysteine Protease Inhibitor from Poultry Red Mites: Potential Vaccine for Chickens

  Fujisawa S, Murata S, Isezaki M, Ariizumi T, Sato T, Oishi E, Taneno A, Maekawa N, Okagawa T, Ichii O, Konnai S, Ohashi K

  Vaccines (Basel) 9 (12) 1472 - 1472 2021/12 [Refereed][Not invited]
   

  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.
• Programmed death-ligand 1 expression in swine chronic infections and enhancement of interleukin-2 production via programmed death-1/programmed death-ligand 1 blockade

  Ganbaatar O, Konnai S, Okagawa T, Nojima Y, Maekawa N, Ichikawa I, Kobayashi A, Shibahara T, Yanagawa Y, Higuchi H, Kato Y, Suzuki Y, Murata S, Ohashi K

  Immun Inflamm Dis. 9 (4) 1573 - 1583 2021/12 [Refereed][Not invited]
  
• Characterization of S-Meq containing the deletion in Meq protein's transactivation domain in a Marek's disease virus strain in Japan

  Murata S, Yamamoto E, Sakashita N, Maekawa N, Okagawa T, Konnai S, Ohashi K

  Poult Sci. 100 (11) 101461  2021/11 [Refereed][Not invited]
  
• In vitro characterization of adipocyte plasma membrane-associated protein from poultry red mites, Dermanyssus gallinae, as a vaccine antigen for chickens

  Fujisawa S, Murata S, Takehara M, Aoyama J, Morita A, Isezaki M, Win SY, Ariizumi T, Sato T, Oishi E, Taneno A, Maekawa N, Okagawa T, Ichii O, Konnai S, Ohashi K

  Vaccine 39 (41) 6057 - 6066 0264-410X 2021/10 [Refereed][Not invited]
  
• ウシCTLA-4ならびにPD-L1を標的とした抗体併用法による免疫増強効果の検討

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

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

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

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

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

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [DVO - 25] 1347-8621 2021/09
• イヌ腫瘍細胞株におけるProgrammed cell death-ligand 1の発現機序の解析

  大脇 稜, 出口 辰弥, 今内 覚, 前川 直也, 細谷 謙次, 金 尚昊, 須永 隆文, 奥村 正裕

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [HSO - 19] 1347-8621 2021/09
• ワクモ(Dermanyssus gallinae)由来システインプロテアーゼの抗ワクモワクチン抗原としての評価

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

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

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

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

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

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

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

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

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

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

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

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

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

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [EO - 6] 1347-8621 2021/09
• イヌ腫瘍細胞株におけるProgrammed cell death-ligand 1の発現機序の解析

  大脇 稜, 出口 辰弥, 今内 覚, 前川 直也, 細谷 謙次, 金 尚昊, 須永 隆文, 奥村 正裕

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [HSO - 19] 1347-8621 2021/09
• Canine Transforming Growth Factor-β Receptor 2-Ig: A Potential Candidate Biologic for Melanoma Treatment That Reverses Transforming Growth Factor-β1 Immunosuppression

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

  Frontiers in Veterinary Science 8 656715  2021/06/14 [Refereed][Not invited]
   

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

  Mekata H, Okagawa T, Konnai S, Miyazawa T

  Viruses 13 (6) 1017  2021/05 [Refereed][Not invited]
  
• Differential expression of PEPCK isoforms is correlated to Aedes aegypti oogenesis and embryogenesis

  da Silva RM, Vital WO, Martins RS, Moraes J, Gomes H, Calixto C, Konnai S, Ohashi K, da Silva Vaz I Jr, Logullo C

  Comp Biochem Physiol B Biochem Mol Biol. 256 110618  2021/05 [Refereed][Not invited]
  
• Prostaglandin-related immune suppression in cattle

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

  Vet Immunol Immunopathol 236 110238  2021/04 [Refereed][Not invited]
  
• Selection of reference genes for quantitative PCR analysis in poultry red mite (Dermanyssus gallinae)

  Ariizumi T, Murata S, Fujisawa S, Isezaki M, Maekawa N, Okagawa T, Sato T, Oishi E, Taneno A, Konnai S, Ohashi K

  J Vet Med Sci. 83 (4) 558 - 565 2021/04 [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.
• Characterisation of a cysteine protease from poultry red mites and its potential use as a vaccine for chickens.

  Murata S, Taniguchi A, Isezaki M, Fujisawa S, Sakai E, Taneno A, Ichii O, Ito T, Maekawa N, Okagawa T, Konnai S, Ohashi K

  Parasite 28 (9) 9 - 9 2021/02 [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.
• PD-L1 immunohistochemistry for canine cancers and clinical benefit of anti-PD-L1 antibody in dogs with pulmonary metastatic oral malignant melanoma

  Maekawa N, Konnai S, Nishimura M, Kagawa Y, Takagi S, Hosoya K, Ohta H, Kim S, Okagawa T, Izumi Y, Deguchi T, Kato Y, Yamamoto S, Yamamoto K, Toda M, Nakajima C, Suzuki Y, Murata S, Ohashi K

  NPJ Precis Oncol. 5 (1) 10 - 10 2021/02 [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.
• The enhancement of Th1 immune response by anti-PD-L1 antibody in cattle infected with Mycobacterium avium subsp. paratuberculosis

  Sajiki Y, Konnai S, Nagata R, Kawaji S, Nakamura H, Fujisawa S, Okagawa T, Maekawa N, Kato Y, Suzuki Y, Murata S, Mori Y, Ohashi K

  J Vet Med Sci. 83 (2) 162 - 166 2021/02 [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.
• Cross-species reactivity of antibodies against Ixodes persulcatus ferritin 2 to Rhipicephalus microplus

  Xavier AM, Konnai S, Parizi FP, Githaka WN, Isezaki M, Goto S, Fujisawa S, Yamada S, Okagawa T, Maekawa N, Logullo C, Silva Vaz J r I, Murata S, Ohashi K

  Jpn J Vet Res. 69 (1) 57 - 65 0047-1917 2021/02 [Refereed][Not invited]
   

  There are several studies that confirm the possibility of developing a vaccine against tick infestations. An immuno-bioinformatics approach was used to identify conserved antigenic regions between Ixodes persulcatus and Rhipicephalus microplus ferritin 2 ( FER2) in order to compose a novel putative vaccine. In addition, R. microplus were fed on blood containing antibodies anti-recombinant FER2 from I. persulcatus (rIp-FER2). The results revealed that anti-ferritin antibodies led to a decrease in the engorgement weight of the R. microplus females. Conservation of the predicted antigenic regions in different tick species suggests that this protein could be useful to develop a vaccine for cross-species protection.
• Intrauterine infection with Mycobacterium avium subsp. paratuberculosis in pregnant cattle diagnosed with Johne’s disease

  Taniguchi Y, Konnai S, Sakakibara S, Yamamoto A, Okagawa T, Maekawa N, Murata S, Ohashi K

  Jpn J Vet Res 69 (1) 51 - 55 0047-1917 2021/02 [Refereed][Not invited]
   

  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.
• The Role of Saliva in Arthropod-Host-Pathogen Relationships

  Tirloni L, Calvo E, Konnai S, Silva Vaz Jr I

  Front Cell Infect Microbiol 10 630626  2021/01 [Refereed][Invited]
  
• Direct evidence of the preventive effect of milk replacer–based probiotic feeding in calves against severe diarrhea

  Kayasaki F, Okagawa T, Konnai S, Kohara J, Sajiki Y, Watari K, Ganbaatar O, Goto S, Nakamura H, Shimakura H, Minato E, Kobayashi A, Kubota M, Terasaki N, Takeda A, Noda H, Honma M, Maekawa N, Murata S, Ohashi K

  Vet Microbiol. 254 108976. - 108976 0378-1135 2021/01 [Refereed][Not invited]
  
• Tick saliva-induced programmed death-1 and PD-ligand 1 and its related host immunosuppression

  Sajiki Y, Konnai S, Ikenaka Y, Gulay K, Kobayashi A, Parizi L, João B, Watari K, Fujisawa S, Okagawa T, Maekawa N, Logullo C, Silva Vaz Jr I, Murata S, Ohashi K

  Sci Rep. 11 (1) 1063  2021/01 [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.
• A TLR7 agonist activates bovine Th1 response and exerts antiviral activity against bovine leukemia virus

  Sajiki Y, Konnai S, Okagawa T, Maekawa N, Nakamura H, Kato Y, Suzuki Y, Murata S, Ohashi K

  Dev Comp Immunol. 114 103847 - 103847 0145-305X 2021/01 [Refereed][Not invited]
  
• Enhanced immunotherapeutic efficacy of anti-PD-L1 antibody in combination with an EP4 antagonist

  Sajiki Y, Konnai S, Cai Z, Takada K, Okagawa T, Maekawa N, Fujisawa S, Kato Y, Suzuki Y, Murata S, Ohashi K

  ImmunoHorizons 4 (12) 837 - 850 2020/12 [Refereed][Not invited]
  
• The suppression of Th1 response by inducing TGF-β1 from regulatory T cells in bovine mycoplasmosis

  Sajiki Y, Konnai S, Goto S, Okagawa T, Ohira K, Shimakura H, Maekawa N, Gondaira S, Higuchi H, Tajima M, Hirano Y, Kohara J, Murata S, Ohashi K

  Front Vet Sci. 7 609443  2020/12 [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⁺CD25^highFoxp3⁺ T cells, was increased in M. bovis-infected cattle. Additionally, the plasma of M. bovis-infected cattle contained the high concentrations of TGF-β1, and M. bovis infection induced TGF-β1 production from bovine immune cells in in vitro cultures. Finally, we analyzed the immunosuppressive effects of TGF-β1 on bovine immune cells. Treatment with TGF-β1 significantly decreased the expression of CD69, an activation marker, in T cells, and Th1 cytokine production in vitro. These results suggest that the increase in Tregs and TGF-β1 secretion could be one of the immunosuppressive mechanisms and that lead to increased susceptibility to other infections in terms of exacerbation of disease during M. bovis infection.
• Molecular detection and genetic characterization of infectious laryngotracheitis virus in poultry in Myanmar.

  Yang Z, Murata S, Fujisawa S, Takehara M, Katakura K, Hmoon MM, Win SY, Bawm S, Konnai S, Ohashi K

  BMC Vet Res. 16 (1) 453  2020/11 [Refereed][Not invited]
   

  BackgroundAvian 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.ResultsOf the 57 tested oropharyngeal swabs, 10 were positive for ILTV by polymerase chain reaction of a 647bp 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.ConclusionsThis 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.
• Genetic characterization of a Marek's disease virus strain isolated in Japan.

  Murata S, Machida Y, Isezaki M, Maekawa N, Okagawa T, Konnai S, Ohashi K

  Virol J. 17 (1) 186  2020/11 [Refereed][Not invited]
  
• PD-L1 expression in equine malignant melanoma and functional effects of PD-L1 blockade

  Ganbaatar O, Konnai S, Okagawa T, Nojima Y, Maekawa N, Minato E, Kobayashi A, Ando R, Sasaki N, Miyakoshi D, Ichii O, Kato Y, Suzuki Y, Murata S, Ohashi K

  PLOS ONE 15 (11) e0234218  2020/11 [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.
• Identification and functional analysis of ferritin 2 from the Taiga tick Ixodes persulcatus Schulze

  Githaka N, Konnai S, Isezaki M, Goto S, Xavier M, Fujisawa S, Yamada S, Okagawa T, Maekawa N, Logullo C, da Silva Vaz Jr I, Murata S, Ohashi K

  Ticks Tick-Borne Dis 11 (6) 101547 - 101547 1877-959X 2020/11 [Refereed][Not invited]
  
• 牛白血病ウイルス感染症における免疫抑制受容体TIM-3の発現解析および機能解析

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

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

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

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

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

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 163回 248 - 248 1347-8621 2020/10
• Development and Characterization of Anti-Sheep Podoplanin Monoclonal Antibodies PMab-253 and PMab-260.

  Mika K Kaneko, Masato Sano, Junko Takei, Teizo Asano, Yusuke Sayama, Hideki Hosono, Atsushi Kobayashi, Satoru Konnai, Yukinari Kato

  Monoclonal antibodies in immunodiagnosis and immunotherapy 39 (4) 144 - 155 2020/07/16 [Refereed][Not invited]
   

  Anti-podoplanin (PDPN) monoclonal antibodies (mAbs) are needed as markers for lymphatic endothelial cells or type I alveolar cells in immunohistochemical analyses. We have developed anti-PDPN mAbs for many species, including humans, mice, rats, rabbits, dogs, cats, bovines, pigs, Tasmanian devils, alpacas, tigers, whales, goats, horses, and bears. This study develops and characterizes anti-sheep PDPN (sPDPN) mAbs using Cell-Based Immunization and Screening (CBIS) method. A RAP14 tag was added to the N-terminus of sPDPN, and an anti-RAP14 tag mAb (PMab-2) was used to measure the expression level of sPDPN in flow cytometry and Western blots. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. Two of the mAbs, PMab-253 (immunoglobulin M [IgM], kappa) and PMab-260 (IgM, kappa), detected CHO/sPDPN cells specifically using flow cytometry and Western blots. Both PMab-253 and PMab-260 stained the renal glomerulus and Bowman's capsule, lymphatic endothelial cells of the lung and colon, and type I alveolar cells of the lung, suggesting PMab-253 and PMab-260, which were developed by CBIS method, can be applied to functional analyses of sPDPN. We also determined the binding epitope of PMab-253 and PMab-260 using flow cytometry. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-253 and PMab-260 epitope exists between amino acids 110 and 115 of sPDPN. Analysis of sPDPN point mutations revealed that the critical epitope of PMab-253 and PMab-260 includes Thr112 and Ser113 of sPDPN, indicating that the PMab-253 and PMab-260 epitope are independent of the platelet aggregation-stimulating (PLAG) domain or the PLAG-like domain of sPDPN.
• Transcriptome dynamics of blood-fed and starved poultry red mites, Dermanyssus gallinae

  Fujisawa S, Murata S, Isezaki M, Oishi E, Taneno A, Maekawa N, Okagawa T, Konnai S, Ohashi K

  Parasitol Int. 102156 - 102156 2020/06 [Refereed][Not invited]
  
• Expression Analysis of Canine CMTM6 and CMTM4 as Potential Regulators of the PD-L1 Protein in Canine Cancers

  Takeuchi H, Konnai S, Maekawa N, Minato E, Ichikawa Y, Kobayashi A, Okegawa T, Murata S, Ohashi K.

  Front Vet Sci 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.
• Rhipicephalus microplus cystatin as a potential cross-protective tick vaccine against Rhipicephalus appendiculatus

  Parizi L, Rangel C, Sabadina G, Saggin B, Kiio I, Xavier M, Matos R, Camargo-Mathias M, Seixas A, Konnai S, Ohashi K, Githaka N, da Silva Vaz Jr I

  Ticks Tick-Borne Dis 11 (3) 101378  2020/05 [Refereed][Not invited]
  
• Clinical efficacy of the combined treatment of anti-PD-L1 rat-bovine chimeric antibody with a COX-2 inhibitor in calves infected with Mycoplasma bovis

  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, Masaru Ishida, Ryoko Uemura, Shinji Yamada, Mika K. Kaneko, Yukinari Kato, Keiichi Yamamoto, Mikihiro Toda, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  JAPANESE JOURNAL OF VETERINARY RESEARCH 68 (2) 77 - 90 0047-1917 2020/05 [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 E-2 (PGE(2)) 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 PGE(2) in M. bovis infection in vivo was investigated using anti-bovine PDLl 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--gamma response after the administration. On the other hand, IFN-gamma 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.
• Immunosuppressive effects of sialostatin L1 and L2 isolated from the taiga tick Ixodes persulcatus Schulze

  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, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi

  Ticks and Tick-borne Diseases 11 (2) 101332 - 101332 1877-959X 2020/03 [Refereed][Not invited]
  
• Immunosuppression in Cows following Intramammary Infusion of Mycoplasma bovis.

  Satoshi Gondaira, Koji Nishi, Takahiro Tanaka, Takashi Yamamoto, Takanori Nebu, Reina Watanabe, Satoru Konnai, Tomohito Hayashi, Yoshio Kiku, Mariko Okamoto, Kazuya Matsuda, Masateru Koiwa, Hidetomo Iwano, Hajime Nagahata, Hidetoshi Higuchi

  Infection and immunity 88 (3) e00521 - 19 2020/02/20 [Refereed][Not invited]
   

  Mycoplasma bovis is a destructive pathogen that causes large economic losses in rearing cattle for beef and dairy worldwide. M. bovis causes suppression of and evades the host immune response; however, the mechanisms of host immune function involved in M. bovis mastitis have not been elucidated. The purpose of this study was to elucidate the characteristics of the bovine immune response to mycoplasmal mastitis. We evaluated the responsiveness of the bovine mammary gland following infusion of M. bovis Somatic cell counts and bacterial counts in milk from the infected quarter were increased. However, the proliferation of peripheral blood mononuclear cells (blood MNCs) and mononuclear cells isolated from M. bovis-stimulated mammary lymph nodes (lymph node MNCs) did not differ from that in the unstimulated cells. Transcriptome analysis revealed that the mRNA levels of innate immune system-related genes in blood MNCs, complement factor D (CFD), ficolin 1 (FCN1), and tumor necrosis factor superfamily member 13 (TNFSF13) decreased following intramammary infusion of M. bovis The mRNA levels of immune exhaustion-related genes, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) of milk mononuclear cells (milk MNCs) in the infected quarter were increased compared with those before infusion. Increase in immune exhaustion-related gene expression and decrease in innate immune response-related genes of MNCs in quarters from cows were newly characterized by M. bovis-induced mastitis. These results suggested that M. bovis-induced mastitis affected the immune function of bovine MNCs, which is associated with prolonged duration of infection with M. bovis.
• Upregulation of PD-L1 Expression by Prostaglandin E2 and the Enhancement of IFN-γ by Anti-PD-L1 Antibody Combined With a COX-2 Inhibitor in Mycoplasma bovis Infection.

  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 Ohashi

  Frontiers in veterinary science 7 (12) 12 - 12 2020/02 [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.
• PMab-241 Specifically Detects Bear Podoplanin of Lymphatic Endothelial Cells in the Lung of Brown Bear.

  Junko Takei, Shinji Yamada, Satoru Konnai, Tsuyoshi Ishinazaka, Michito Shimozuru, Mika K Kaneko, Yukinari Kato

  Monoclonal antibodies in immunodiagnosis and immunotherapy 38 (6) 282 - 284 2019/12 [Refereed][Not invited]
   

  Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.
• Epitope Mapping of Anti-Bear Podoplanin Monoclonal Antibody PMab-247

  Kato Y, Takei J, Furusawa Y, Sayama Y, Sano M, Konnai S, Kobayashi A, Harada H, Takahashi M, Suzuki H, Yamada S, Kaneko MK

  Monoclon Antib Immunodiagn Immunother. 38 (5) 230 - 233 2019/09 [Refereed][Not invited]
  
• Immune inhibitory function of bovine CTLA-4 and the effects of its blockade in IFN-γ production

  Watari K, Konnai S, Maekawa N, Okagawa T, Suzuki Y, Murata S, Ohashi K

  BMC Vet Res 15 (1) 380  2019/09 [Refereed][Not invited]
  
• ミャンマーの養鶏場におけるマイコプラズマ、鶏伝染性気管支炎ウイルス、鶏伝染性喉頭気管炎ウイルスの分子学的検出と遺伝学的性状解析(Molecular detection and genetic characterization of mycoplasmas, infectious bronchitis virus and infectious laryngotracheitis virus in poultry farms in Myanmar)

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

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

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

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

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

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

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

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

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

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

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

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

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

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 162回 460 - 460 1347-8621 2019/08
• ネコのアクロメガリー関連インスリン抵抗性糖尿病のコントロールを目的とした成長ホルモン受容体/Fc融合タンパク質の作製

  鈴木 理紗, 和泉 雄介, 前川 直也, 今内 覚, 高木 哲, 森下 啓太郎, 細谷 謙次, 滝口 満喜

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 162回 469 - 469 1347-8621 2019/08
• PMab-247 Detects Bear Podoplanin in Immunohistochemical Analysis

  Takei J, Furusawa Y, Yamada S, Nakamura T, Sayama Y, Sano M, Konnai S, Kobayashi A, Harada H, Kaneko MK, Kato Y

  Monoclon Antib Immunodiagn Immunother. 38 (4) 171 - 174 2019/08 [Refereed][Not invited]
   

  Podoplanin (PDPN) is utilized as a specific marker of type I alveolar cells of lung and lymphatic endothelial cells of every tissue. Therefore, sensitive and specific monoclonal antibodies detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for Western blot, flow cytometry, and immunohistochemical analyses. In this study, immunohistochemical analyses showed that PMab-247 strongly detected bPDPN, which is expressed in type I alveolar cells and lymphatic endothelial cells of bear lung and podocytes of bear kidney. These findings suggest that PMab-247 could be useful for pathophysiological analyses using immunohistochemistry.
• Prostaglandin E2 induced immune exhaustion and enhancement of anti-viral effects by anti-PD-L1 antibody combined with COX-2 inhibitor in bovine leukemia virus infection

  Sajiki Y, Konnai S, Okagawa T, Nishimori A, Maekawa N, Goto S, Watari K, Minato E, Kobayashi A, Kohara J, Yamada S, Kaneko M, Kato Y, Takahashi H, Terasaki N, Takeda A, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K

  J. Immunol 203 (5) 1313 - 1324 0022-1767 2019/08 [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.
• Vector transmission of bovine leukemia virus during summer season in Northern Hokkaido

  Inagaki H, Konnai S, Kaburagi H, Murota H, Takabatake N, Watari K, Okagawa T, Maekawa N, Murata S, Ohashi K

  Jpn. J. Vet. Res 67 (3) 235 - 239 0047-1917 2019/08 [Refereed][Not invited]
   

  One of the known transmission pathways of bovine leukemia virus (BLV) is bloodsucking insects. Against this background, this study investigated changes in BLV seroconversion in cattle by season on three private farms in Northern Hokkaido. Study results showed that no BLV seroconversion was observed during winter, a season without horse flies, and that all seroconversions occurred during summer. Thus, we collected horse flies which were observed sucking blood on cattle in public grazed fields and performed BLV detection tests in the insects. The tests showed that 75% of the total collected horse flies were BLV-positive. These results suggested the existence of vectors such as horse flies in grazing filed (in summer) was a risk factor of the spread of BLV infection in Northern Hokkaido.
• Immunohistochemical Analysis of the Harbor Porpoise Using Antipodoplanin Antibody PMab-237.

  Shinji Yamada, Shunsuke Itai, Takuro Nakamura, Junko Takei, Masato Sano, Satoru Konnai, Atsushi Kobayashi, Shotaro Nakagun, Yoshiyasu Kobayashi, Mika K Kaneko, Yukinari Kato

  Monoclonal antibodies in immunodiagnosis and immunotherapy 38 (3) 104 - 107 2019/06 [Refereed][Not invited]
   

  Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolar cells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for immunohistochemical analyses for cetacean tissues.
• Haematophagous mites on poultry farms in the Republic of the Union of Myanmar

  Takehara M, Murata S, Katakura K, Fujisawa S, Hmoon MM, Win SY, Bawn S, Htun LL, Aung YH, Win MM, Isezaki M, Maekawa N, Okagawa T, Konnai S, Ohashi K

  Heliyon 5 (4) e01544  2019/04 [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.
• Carbohydrate Metabolic Compensation Coupled to High Tolerance to Oxidative Stress in Ticks

  Noce BD, Uhl M, Machado J, Waltero C, Abreu L, Silva R, Fonseca R, Barros C, Sabadin G, Konnai S, Silva Vaz Jr. I, Ohashi K, Logullo C

  Scientific Rep. 9 4753  2019/03 [Refereed][Not invited]
  
• Effects of bovine tumor necrosis factor alpha decoy receptors on cell death and inflammatory cytokine kinetics: Potential for bovine inflammation therapy

  Fujisawa S, Konnai S, Okagawa T, Maekawa N, Tanaka A, Suzuki Y, Murata S, Ohashi K

  BMC Vet Res. 15 (1) 68 - 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.
• Anti-Horse Podoplanin Monoclonal Antibody PMab-219 is Useful for Detecting Lymphatic Endothelial Cells by Immunohistochemical Analysis

  Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, Kaneko MK

  Monoclon Antib Immunodiagn Immunother. 37 (6) 272 - 274 2018/12 [Refereed][Not invited]
  
• Detection of Alpaca Podoplanin by Immunohistochemistry Using the Antibovine Podoplanin Monoclonal Antibody PMab-44

  Kato Y, Yamada S, Itai S, Konnai S, Kobayashi A, Kaneko MK

  Monoclon Antib Immunodiagn Immunother. 37 (6) 269 - 271 2018/12 [Refereed][Not invited]
  
• Immunohistochemical Detection of Sheep Podoplanin Using an Antibovine Podoplanin Monoclonal Antibody PMab-44

  Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, Kaneko MK

  Monoclon Antib Immunodiagn Immunother. 37 (6) 265 - 268 2018/12 [Refereed][Not invited]
  
• The mouse-canine chimeric anti-dog podoplanin antibody P38B exerts antitumor activity in mouse xenograft models

  Kato Y, Ohishi T, Kawada M, Maekawa N, Konnai S, Itai S, Yamada S, Kaneko MK

  Biochem Biophys Rep. 17 23 - 26 2018/11 [Refereed][Not invited]
  
• Identification of immuno-inhibitory molecules in Mongolian native cattle and yak

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

  Jpn J Vet Res 66 (3) 177 - 192 0047-1917 2018/08 [Refereed][Not invited]
   

  The immuno-inhibitory molecules PD-1, PD-L1, TIM-3, GAL-9, LAG-3, and CTLA-4 from blood samples of Mongolian native cattle and yak were characterized through cloning and sequencing. As these molecules are involved in cell-mediated immune responses, identifying the differences in their reactions against the pathogens found in bovine species may be beneficial. The amino acid sequences of these molecules were predicted for the purpose of characterizing their functional domains, such as the signal peptide, extracellular domain, transmembrane region, and intracellular domain. Amino acid alignment showed that the sequences of these immuno-inhibitory molecules from Mongolian native cattle and yak were highly homologous to those from other bovine species. As a preliminary application of the genetic information, we conducted expression analysis of PD-L1 in bovine viral diarrhea virus (BVDV)-infected yak by using real-time polymerase chain reaction, and PD-L1 mRNA expression in peripheral blood mononuclear cells derived from BVDV-infected yak was significantly upregulated compared to that of uninfected-yak. Further studies are necessary to assess whether these molecules play roles in disease progression during chronic infection of Mongolian native cattle and yak.
• Cattle with a low bovine leukemia virus proviral load are rarely an infectious source

  Mekata H, Yamamoto M, Hayashi T, Kirino Y, Sekiguchi S, Konnai S, Horii Y, Norimine J

  Jpn J Vet Res 66 (3) 157 - 163 2018/08 [Refereed][Not invited]
  
• Cooperation of PD-1 and LAG-3 in the exhaustion of CD4+ and CD8+ T cells during bovine leukemia virus infection.

  Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Junko Kohara, Yasuhiko Suzuki, Shinji Yamada, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi

  Veterinary research 49 (1) 50 - 50 2018/06/19 [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.
• Epitope mapping of anti-mouse podoplanin monoclonal antibody PMab-1

  Yamada S, Itai S, Kaneko MK, Konnai S, Kato Y

  Biochem Biophys Rep. 15 52 - 56 2018/06 [Refereed][Not invited]
  
• Evaluation of costimulatory molecules in dogs with B cell high grade lymphoma

  Tagawa M, Kurashima C, Takagi S, Maekawa N, Konnai S, Shimbo G, Matsumoto K, Inokuma H, Kawamoto K, Miyahara K

  PLoS One 13 (7) e0201222  2018/06 [Refereed][Not invited]
  
• Prostaglandin E2 Induction Suppresses the Th1 Immune Responses in Cattle with Johne's Disease

  Sajiki Y, Konnai S, Okagawa T, Nishimori A, Maekawa N, Goto S, Ikebuchi R, Nagata R, Kawaji S, Kagawa Y, Yamada S, Kato Y, Nakajima C, Suzuki Y, Murata S, Mori Y, Ohashi K

  Infect Immun. 86 (5) e00910-17  2018/04 [Refereed][Not invited]
  
• Identification of an Atypical Enzootic Bovine Leukosis in Japan by Using a Novel Classification of Bovine Leukemia Based on Immunophenotypic Analysis

  Nishimori A, Konnai S, Okagawa T, Maekawa N, Goto S, Ikebuchi R, Nakahara A, Chiba Y, Ikeda M, Murata S, Ohashi K

  Clin Vaccine Immunol. 24 (12) e00001-18  2018/04 [Refereed][Not invited]
  
• New hematological key for bovine leukemia virus-infected Japanese Black cattle.

  Hirohisa Mekata, Mari Yamamoto, Yumi Kirino, Satoshi Sekiguchi, Satoru Konnai, Yoichiro Horii, Junzo Norimine

  The Journal of veterinary medical science 80 (2) 316 - 319 2018/02/20 [Refereed][Not invited]
   

  The European Community's (EC) Key, which is also called Bendixen's Key, is a well-established bovine leukemia virus (BLV) diagnostic method that classifies cattle according to the absolute lymphocyte count and age. The EC Key was originally designed for dairy cattle and is not necessarily suitable for Japanese Black (JB) beef cattle. This study revealed the lymphocyte counts in the BLV-free and -infected JB cattle were significantly lower than those in the Holstein cattle. Therefore, applying the EC Key to JB cattle could result in a large number of undetected BLV-infected cattle. Our proposed hematological key, which was designed for JB cattle, improves the detection of BLV-infected cattle by approximately 20%. We believe that this study could help promote BLV control.
• Molecular detection and phylogenetic analysis of Ehrlichia canis in a Philippine dog

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

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

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

  Adriana Seixas, Maria Fernanda Alzugaray, Lucas Tirloni, Luis Fernando Parizi, Antonio Frederico Michel Pinto, Naftaly Wang'ombe Githaka, Satoru Konnai, Kazuhiko Ohashi, John R. Yates, Carlos Termignoni, Itabajara da Silva Vaz

  TICKS AND TICK-BORNE DISEASES 9 (1) 72 - 81 1877-959X 2018/01 [Refereed][Not invited]
   

  The vitellogenin receptor (VgR), which belongs to the low-density lipoprotein receptors (LDLR) family, regulates the absorption of yolk protein accumulated in developing oocytes during oogenesis. In the present study, the full sequence of Rhipicephalus microplus VgR (RmVgR) and the partial sequence of Rhipicephalus appendiculatus VgR (RaVgR) ORF were determined and cloned. The RmVgR amino acid sequence contains the five highly conserved structural motifs characteristic of LDLR superfamily members, the same overall structure as observed in other species. Phylogenetic analysis separated VgRs in two major groups, corresponding to receptors from acarines and insects. Consistent with observations from other arthropods, RmVgR was specifically expressed in the ovarian tissue and its peak of expression occurs in females that are detaching from the host. Silencing with RmVgR dsRNA reduced VgR expression, which resulted in reduced fertility, evidenced by a decrease in the number of larvae. The present study confirms RmVgR is a specific receptor involved in yolk protein uptake and oocyte maturation in R. microplus, playing an important role in tick reproduction.
• Intrauterine infection with bovine leukemia virus in pregnant dam with high viral load.

  Yamato Sajiki, Satoru Konnai, Asami Nishimori, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Masashi Nagano, Junko Kohara, Nana Kitano, Toshihiko Takahashi, Motoshi Tajima, Hirohisa Mekata, Yoichiro Horii, Shiro Murata, Kazuhiko Ohashi

  The Journal of veterinary medical science 79 (12) 2036 - 2039 2017/12/22 [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.
• Expression of Cat Podoplanin in Feline Squamous Cell Carcinomas

  Shunsuke Itai, Shinji Yamada, Mika K. Kaneko, Hiroyuki Harada, Yumiko Kagawa, Satoru Konnai, Yukinari Kato

  Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 36 (6) 243 - 250 2167-9436 2017/12/01 [Refereed][Not invited]
   

  Oral squamous cell carcinoma is an aggressive tumor in cats however, molecular-targeted therapies against this tumor, including antibody therapy, have not been developed. Sensitive and specific monoclonal antibodies (mAbs) against highly expressed membrane proteins are needed to develop antibody therapies. Podoplanin, a type I transmembrane glycoprotein, is expressed in many human malignant tumors, including brain tumor, esophageal cancer, lung cancer, mesothelioma, and oral cancer. Podoplanin binds to C-type lectin-like receptor-2 (CLEC-2) and activates platelet aggregation, which is involved in cancer metastasis. Until now, we have established several mAbs against podoplanin in humans, mice, rats, rabbits, dogs, cattle, and cats. We have reported podoplanin expression in canine melanoma and squamous cell carcinomas using an anti-dog podoplanin mAb PMab-38. In this study, we investigated podoplanin expression in 40 feline squamous cell carcinomas (14 cases of mouth floor, 13 of skin, 9 of ear, and 4 of tongue) by immunohistochemical analysis using an anti-cat podoplanin mAb PMab-52, which we recently developed by cell-based immunization and screening (CBIS) method. Of the total 40 cases, 38 (95%) showed positive staining for PMab-52. In particular, 12 cases (30%) showed a strong membrane-staining pattern of squamous cell carcinoma cells. PMab-52 can be useful for antibody therapy against feline podoplanin-expressing squamous cell carcinomas.
• Detection and molecular characterization of equine infectious anemia virus in Mongolian horses

  Tumenjargal Sharav, Satoru Konnai, Nyamsuren Ochirkhuu, Erdene T. S. Ochir, Hirohisa Mekata, Yoshihiro Sakoda, Takashi Umemura, Shiro Murata, Tungalag Chultemdorj, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 79 (11) 1884 - 1888 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.
• PMab-52: Specific and Sensitive Monoclonal Antibody Against Cat Podoplanin for Immunohistochemistry

  Shinji Yamada, Shunsuke Itai, Takuro Nakamura, Miyuki Yanaka, Noriko Saidoh, Yao-Wen Chang, Saori Handa, Hiroyuki Harada, Yumiko Kagawa, Osamu Ichii, Satoru Konnai, Mika K. Kaneko, Yukinari Kato

  Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 36 (5) 224 - 230 2167-9436 2017/10/01 [Refereed][Not invited]
   

  Podoplanin (PDPN) is expressed in several normal tissues, such as lymphatic endothelial cells, podocytes of renal glomerulus, and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. Although monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, dog PDPN, and bovine PDPN have been established, anticat PDPN (cPDPN) mAbs have not been developed. In this study, we immunized mice with Chinese hamster ovary (CHO)-K1 cell lines expressing cPDPN, and developed anti-cPDPN mAbs. One of the clones, PMab-52 (IgM, kappa), detected cPDPN specifically in flow cytometry and Western blot analysis. PMab-52 is also useful for detecting feline squamous cell carcinoma cells in immunohistochemical analysis. PMab-52 is expected to be useful for investigating the function of cPDPN in feline carcinomas.
• Increase of cells expressing PD-1 and PD-L1 and enhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine mycoplasmosis

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

  Immun Inflamm Dis. 5 (3) 355 - 363 2017/09 [Refereed][Not invited]
   

  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.
• Molecular Epidemiological Survey and Genetic Characterization of Anaplasma Species in Mongolian Livestock

  Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Shiro Murata, Kazuhiko Ohashi

  VECTOR-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.
• A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma

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

  Sci Rep. 7 (1) 8951 - 8951 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.
• Characterization of the Anti-Bovine Podoplanin Monoclonal Antibody PMab-44

  Shinji Yamada, Ryusuke Honma, Mika K. Kaneko, Takuro Nakamura, Miyuki Yanaka, Noriko Saidoh, Michiaki Takagi, Satoru Konnai, Yukinari Kato

  Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 36 (3) 129 - 134 2167-9436 2017/06/01 [Refereed][Not invited]
   

  A type I transmembrane sialoglycoprotein podoplanin (PDPN) is expressed in several normal cells, including podocytes of the kidney, type I alveolar cells of the lung, and lymphatic endothelial cells. We recently produced an anti-bovine PDPN (bovPDPN) monoclonal antibody (mAb), PMab-44, by immunizing mice with recombinant proteins of bovPDPN. In this study, we determined the critical epitope of PMab-44 for the recognition of bovPDPN using many deletion mutants and point mutants of bovPDPN. Flow cytometric analyses revealed that the epitope of PMab-44 was Glu46-Thr50, which corresponds to platelet aggregation-stimulating (PLAG) domain-3. The important amino acids in the PMab-44 epitope were determined to be Glu46, Tyr48, and Thr50. Western blot analysis also confirmed these results, indicating that the PLAG domain of bovPDPN is also important in immunogenicity for producing useful anti-PDPN mAbs.
• Anti-Bovine Programmed Death-1 Rat-Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle

  Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Chie Nakajima, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  FRONTIERS IN IMMUNOLOGY 8 650  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.
• Molecular characterization of the lymphocyte activation gene-3 (LAG-3, CD223) of swamp- and riverine-type water buffaloes (Bubalus bubalis)

  Shanemae M. Rivera, Ryan Bismark C. Padiernos, Evaristo A. Abella, Satoru Konnai, Claro N. Mingala

  JAPANESE JOURNAL OF VETERINARY RESEARCH 65 (2) 65 - 74 0047-1917 2017/05 [Refereed][Not invited]
   

  The present study was conducted to characterize LAG-3 of swamp- and riverine-type water buffaloes by DNA sequencing, homology and phylogenetic analysis. Bubaline LAG-3 sequence contained an open reading frame of 1551 nucleotide, encoding a polypeptide of 516 amino acids. Nucleotide and amino acid sequence homology of LAG-3 revealed 76-96% and 61-94% identity in water buffalo to that of other mammals, respectively. LAG-3 protein sequence of water buffalo contained four extracellular domains, a transmembrane domain and different conserved regions. There were three N-glycosylation sites, two sequence motifs: 'RGD' and 'WXC' motif and five cysteine residues located at different positions of extracellular region. Likewise, the possible serine phosphorylation site and the 'KTGELE' inhibitory motif were found in the intracellular region of bubaline LAG-3. However, one highly conserved cysteine residue in mammalian LAG-3 was replaced by tyrosine in both swamp- and riverine-type water buffaloes. Phylogenetic analysis generated high bootstrap value between the two types of water buffalo which further confirmed the degree of relationship between bubaline species. This was the first report that describe the genetic characteristic of LAG-3 in swamp- and riverine-type water bufffaloes.
• Clinical and subclinical bovine leukemia virus infection in a dairy cattle herd in Zambia

  Girja S. Pandey, Edgar Simulundu, Danstan Mwiinga, Kenny L. Samui, Aaron S. Mweene, Masahiro Kajihara, Alfred Mangani, Racheal Mwenda, Joseph Ndebe, Satoru Konnai, Ayato Takada

  ARCHIVES OF VIROLOGY 162 (4) 1051 - 1056 0304-8608 2017/04 [Refereed][Not invited]
   

  Bovine leukemia virus (BLV) causes enzootic bovine leucosis (EBL) and is responsible for substantial economic losses in cattle globally. However, information in Africa on the disease is limited. Here, based on clinical, hematological, pathological and molecular analyses, two clinical cases of EBL were confirmed in a dairy cattle herd in Zambia. In contrast, proviral DNA was detected by PCR in five apparently healthy cows from the same herd, suggesting subclinical BLV infection. Phylogenetic analysis of the env gene showed that the identified BLV clustered with Eurasian genotype 4 strains. This is the first report of confirmed EBL in Zambia.
• Development of mPMab-1, a Mouse-Rat Chimeric Antibody Against Mouse Podoplanin

  Shinji Yamada, Mika K. Kaneko, Takuro Nakamura, Osamu Ichii, Satoru Konnai, Yukinari Kato

  Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 36 (2) 77 - 79 2167-9436 2017/04/01 [Refereed][Not invited]
   

  Podoplanin (PDPN), the ligand of C-type lectin-like receptor-2, is used as a lymphatic endothelial marker. We previously established clone PMab-1 of rat IgG2a as a specific monoclonal antibody (mAb) against mouse PDPN. PMab-1 is also very sensitive in immunohistochemical analysis however, rat mAbs seem to be unfavorable for pathologists because anti-mouse IgG and anti-rabbit IgG are usually used as secondary antibodies in commercially available kits for immunohistochemical analysis. In this study, we develop a mouse-rat chimeric antibody, mPMab-1 of mouse IgG2a, which was derived from rat PMab-1 mAb. Immunohistochemical analysis shows that mPMab-1 detects podocytes of the kidney, lymphatic endothelial cells of the colon, and type I alveolar cells of the lung. Importantly, mPMab-1 is more sensitive than PMab-1. This conversion strategy from rat mAb to mouse mAb could be applicable to other mAbs.
• In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) ratbovine chimeric antibody against bovine leukemia virus infection

  Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Yamato Sajiki, Yasuhiko Suzuki, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi

  PLOS ONE 12 (4) e0174916  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.
• Isolation and purification of Gallid herpesvirus 2 strains currently distributed in Japan

  Yuka Machida, Shiro Murata, Ayumi Matsuyama-Kato, Masayoshi Isezaki, Akira Taneno, Eishi Sakai, Satoru Konnai, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 79 (1) 115 - 122 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.
• Immune exhaustion during chronic infections in cattle

  Satoru Konnai, Shiro Murata, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 79 (1) 1 - 5 0916-7250 2017/01 [Refereed][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.
• A novel mechanism of functional cooperativity regulation by thiol redox status in a dimeric inorganic pyrophosphatase

  Evenilton P. Costa, Arnoldo R. Facanha, Criscila S. Cruz, Jhenifer N. Silva, Josias A. Machado, Gabriel M. Carvalho, Mariana R. Fernandes, Renato Martins, Eldo Campos, Nelilma C. Romeiro, Naftaly W. Githaka, Satoru Konnai, Kazuhiko Ohashi, Itabajara S. Vaz, Carlos Logullo

  BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1861 (1) 2922 - 2933 0304-4165 2017/01 [Refereed][Not invited]
   

  Background: Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase from R. microplus embryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status. Methods: In this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface. Results: Cysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PPi), probably by inducing disulfide bridge formation. Conclusions: Our results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface. General significance: This study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status. (C) 2016 Elsevier B.V. All rights reserved.
• Molecular and structural characterization of novel cystatins from the taiga tick Ixodes persulcatus

  Carolina K. Rangel, Luis F. Parizi, Gabriela A. Sabadin, Evenilton P. Costa, Nelilma C. Romeiro, Masayoshi Isezaki, Naftaly W. Githaka, Adriana Seixas, Carlos Logullo, Satoru Konnai, Kazuhiko Ohashi, Itabajara da Silva Vaz

  TICKS AND TICK-BORNE DISEASES 8 (3) 432 - 441 1877-959X 2017 [Refereed][Not invited]
   

  Cystatins are cysteine peptidase inhibitors that in ticks mediate processes such as blood feeding and digestion. The ixodid tick Ixodes persulcatus is endemic to the Eurasia, where it is the principal vector of Lyme borreliosis. To date, no I. persulcatus cystatin has been characterized. In the present work, we describe three novel cystatins from I. persulcatus, named JpIpcys2a, JpIpcys2b and JpIpcys2c. In addition, the potential of tick cystatins as cross-protective antigens was evaluated by vaccination of hamsters using BrBmcys2c, a cystatin from Rhipicephalus microplus, against I. persulcatus infestation. Sequence analysis showed that motifs that are characteristic of cystatins type 2 are fully conserved in JpIpcys2b, while mutations are present in both JpIpcys2a and JpIpcys2c. Protein-protein docking simulations further revealed that JpIpcys2a, JpIpcys2b and JpIpcys2c showed conserved binding sites to human cathepsins L, all of them covering the active site cleft. Cystatin transcripts were detected in different I. persulcatus tissues and instars, showing their ubiquitous expression during I. persulcatus development. Serological analysis showed that although hamsters immunized with BrBmcys2c developed a humoral immune response, this response was not adequate to protect against a heterologous challenge with I. persulcatus adult ticks. The lack of cross-protection provided by BrBmcys2c immunization is perhaps linked to the fact that cystatins cluster into multigene protein families that are expressed differentially and exhibit functional redundancy. How to target such small proteins that are secreted in low quantities remains a challenge in the development of suitable anti-tick vaccine antigens. (C) 2017 Elsevier GmbH. All rights reserved.
• Molecular epidemiological survey and genetic characterization of ovine gammaherpesvirus-2 in Mongolian livestock

  Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Shiro Murata, Kazuhiko Ohashi

  Journal 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.
• Podoplanin Expression in Canine Melanoma

  Satoshi Ogasawara, Ryusuke Honma, Mika K. Kaneko, Yuki Fujii, Yumiko Kagawa, Satoru Konnai, Yukinari Kato

  Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 35 (6) 304 - 306 2167-9436 2016/12/01 [Refereed][Not invited]
   

  A type I transmembrane protein, podoplanin (PDPN), is expressed in several normal cells such as lymphatic endothelial cells or pulmonary type I alveolar cells. We recently demonstrated that anticanine PDPN monoclonal antibody (mAb), PMab-38, recognizes canine PDPN of squamous cell carcinomas, but does not react with lymphatic endothelial cells. Herein, we investigated whether PMab-38 reacts with canine melanoma. PMab-38 reacted with 90% of melanoma cells (9/10 cases) using immunohistochemistry. Of interest, PMab-38 stained the lymphatic endothelial cells and cancer-associated fibroblasts in melanoma tissues, although it did not stain any lymphatic endothelial cells in normal tissues. PMab-38 could be useful for uncovering the function of PDPN in canine melanomas.
• Cooperation of PD-1 and LAG-3 Contributes to T-Cell Exhaustion in Anaplasma marginale-Infected Cattle

  Tomohiro Okagawa, Satoru Konnai, James R. Deringer, Massaro W. Ueti, Glen A. Scoles, Shiro Murata, Kazuhiko Ohashi, Wendy C. Brown

  INFECTION AND IMMUNITY 84 (10) 2779 - 2790 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.
• PMab-38 Recognizes Canine Podoplanin of Squamous Cell Carcinomas

  Mika K. Kaneko, Ryusuke Honma, Satoshi Ogasawara, Yuki Fujii, Takuro Nakamura, Noriko Saidoh, Michiaki Takagi, Yumiko Kagawa, Satoru Konnai, Yukinari Kato

  Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 35 (5) 263 - 266 2167-9436 2016/10/01 [Refereed][Not invited]
   

  Podoplanin, a type I transmembrane protein, is expressed in lymphatic endothelial cells. Although we previously developed an anticanine podoplanin monoclonal antibody (mAb), PMab-38, immunohistochemistry (IHC) showed that it did not react with canine lymphatic endothelial cells. Here, we determined whether PMab-38 recognizes canine podoplanin of squamous cell carcinomas (SCCs) and clarified its epitope. In IHC, PMab-38 reacted with 83% of SCCs (15/18 cases). Flow cytometry showed that the epitope of PMab-38 was different from that of the platelet aggregation-stimulating domain of the N-terminus, which was detected by almost all antipodoplanin mAbs such as D2-40 or NZ-1. PMab-38 is expected to be useful for investigating the function of podoplanin in canine tumors.
• Molecular detection and characterization of bovine viral diarrhea virus in Mongolian cattle and yaks

  Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Battogtokh Odzaya, Shura Gansukh, Shiro Murata, Kazuhiko Ohashi

  ARCHIVES 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.
• Specific Detection of Dog Podoplanin Expressed in Renal Glomerulus by a Novel Monoclonal Antibody PMab-38 in Immunohistochemistry

  Ryusuke Honma, Mika K. Kaneko, Satoshi Ogasawara, Yuki Fujii, Satoru Konnai, Michiaki Takagi, Yukinari Kato

  Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 35 (4) 212 - 216 2167-9436 2016/08/01 [Refereed][Not invited]
   

  Podoplanin (PDPN) is expressed in several normal tissues including podocytes of renal glomerulus, lymphatic endothelial cells (LECs), and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets. Many monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, and bovine PDPN have been established antidog PDPN (dPDPN) mAbs have not been developed. Herein, we immunized mice with the recombinant proteins of dPDPN and developed anti-dPDPN mAbs. One of the clones, PMab-38, is useful for detecting podocytes in immunohistochemical analysis in contrast, it did not react with LECs or type I alveolar cells. PMab-38 also detected dPDPN specifically in flow cytometry and Western blot analysis. PMab-38 is expected to be useful for investigating the function of dPDPN, which is expressed in podocytes.
• PMab-44 Detects Bovine Podoplanin in Immunohistochemistry

  Honma R, Ogasawara S, Kaneko MK, Fujii Y, Oki H, Nakamura T, Takagi M, Konnai S, Kato Y

  Monoclon Antib Immunodiagn Immunother. 2016/06 [Refereed][Not invited]
  
• Immunohistochemical Analysis of PD-L1 Expression in Canine Malignant Cancers and PD-1 Expression on Lymphocytes in Canine Oral Melanoma

  Naoya Maekawa, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Satoshi Takagi, Yumiko Kagawa, Chie Nakajima, Yasuhiko Suzuki, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi

  PLOS ONE 11 (6) e0157176  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.
• Direct polymerase chain reaction from blood and tissue samples for rapid diagnosis of bovine leukemia virus infection

  Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Ayako Nakahara, Shiro Murata, Kazuhiko Ohashi

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

  Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Asami Nishimori, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi

  ARCHIVES 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.
• Critical Epitope of Anti-Rabbit Podoplanin Monoclonal Antibodies for Immunohistochemical Analysis

  Ryusuke Honma, Yuki Fujii, Satoshi Ogasawara, Hiroharu Oki, Satoru Konnai, Yumiko Kagawa, Michiaki Takagi, Mika K. Kaneko, Yukinari Kato

  Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 35 (2) 65 - 72 2167-9436 2016/04/01 [Refereed][Not invited]
   

  Podoplanin (PDPN) is a type I transmembrane sialoglycoprotein, which is expressed in several normal cells, including lymphatic endothelial cells throughout the body, podocytes of the kidney, and lung type I alveolar cells of the lung. We have established many monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, and rat PDPN. In addition, we recently produced an anti-rabbit PDPN mAb, PMab-32, which was established by immunizing mice with recombinant proteins of rabbit PDPN. Herein, we compared the reactivity of PMab-32 with that of newly established anti-rabbit PDPN mAbs, PMab-33 and PMab-21, against normal tissues in immunohistochemistry. PMab-32 reacted with podocytes, type I alveolar cells, and lymphatic endothelial cells, whereas PMab-33 detected only podocytes and type I alveolar cells but not lymphatic endothelial cells. PMab-21 was not useful in immunohistochemistry. We identified the epitope of PMab-32 and PMab-33 as Ser61-Ala68 of rabbit PDPN using western blot and flow cytometric analyses. In contrast, the epitope of PMab-21 was identified as Leu44-Glu48, which is corresponding to platelet aggregation-stimulating (PLAG) domain, indicating that Ser61-Ala68 of rabbit PDPN is a more appropriate epitope for immunohistochemistry compared with PLAG domain. PMab-32 could be useful for uncovering the function of rabbit PDPN.
• Evaluation of Costimulatory Molecules in Peripheral Blood Lymphocytes of Canine Patients with Histiocytic Sarcoma

  Michihito Tagawa, Naoya Maekawa, Satoru Konnai, Satoshi Takagi

  PLOS ONE 11 (2) e0150030  1932-6203 2016/02 [Refereed][Not invited]
   

  Histiocytic sarcoma is a rapidly progressive and fatal neoplastic disease in dogs. It is unclear whether costimulatory molecules, including CD28, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and programmed death-1 (PD-1), are expressed on peripheral blood lymphocytes (PBLs) of canine patients with histiocytic sarcoma. The objective of this study was to evaluate the expression of CD28, CTLA-4, and PD-1 molecules on PBLs of patients with histiocytic sarcoma, patients with other tumors, and healthy controls. Twenty-six dogs were included in the study, with eight, ten, and eight dogs in the histiocytic sarcoma, other tumor, and healthy control groups, respectively. PBLs and serum were prospectively obtained from patients diagnosed histopathologically with histiocytic sarcoma, other tumors and healthy controls. The surface expression of CTLA-4, CD28, and PD-1 on T lymphocytes was examined using flow cytometric analysis. Serum samples were frozen at -30 degrees C until serum interferon-gamma (IFN-gamma) was measured by enzyme-linked immunosorbent assay. The expression level of CTLA-4 on CD4+ lymphocytes was significantly higher in the histiocytic sarcoma group than in the control group. The expression of CTLA-4 on CD8+ lymphocytes was significantly higher in the histiocytic sarcoma group than in the other two groups. In addition, the expression of PD-1 on CD8+ lymphocytes was significantly higher in the histiocytic sarcoma group than in the control group. However, no significant differences in CD28 expressions and serum IFN-gamma levels were observed. The present results provided evidence showing that the expression levels of CTLA-4 on both CD4+ and CD8+ lymphocytes and PD-1 on CD8+ lymphocytes in peripheral blood obtained from dogs with histiocytic sarcoma were upregulated. The overexpressions of CTLA 4 and PD-1 suggested that antitumor immunity may be suppressed in dogs with histiocytic sarcoma.
• Identification and the preliminary in vitro characterization of IRIS homologue from salivary glands of Ixodes persulcatus Schulze

  Kochi Toyomane, Satoru Konnai, Ayano Niwa, Naftaly Githaka, Masayoshi Isezaki, Shinji Yamada, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Shiro Murata, Kazuhiko Ohashi

  TICKS AND TICK-BORNE DISEASES 7 (1) 119 - 125 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.
• Bovine leukemia virus reduces anti-viral cytokine activities and NK cytotoxicity by inducing TGF-β secretion from regulatory T cells

  Ohira K, Nakahara A, Konnai S, Okagawa T, Nishimori A, Maekawa N, Ikebuchi R, Kohara J, Murata S, Ohashi K

  Immun Inflamm Dis. 4 (1) 52 - 63 2016/01 [Refereed][Not invited]
   

  CD4(+)CD25(high)Foxp3(+) T cells suppress excess immune responses that lead to autoimmune and/or inflammatory diseases, and maintain host immune homeostasis. However, CD4(+)CD25(high)Foxp3(+) T cells reportedly contribute to disease progression by over suppressing immune responses in some chronic infections. In this study, kinetic and functional analyses of CD4(+)CD25(high)Foxp3(+) T cells were performed in cattle with bovine leukemia virus (BLV) infections, which have reported immunosuppressive characteristics. In initial experiments, production of the Th1 cytokines IFN-γ and TNF-α was reduced in BLV-infected cattle compared with uninfected cattle, and numbers of IFN-γ or TNF-α producing CD4(+) T cells decreased with disease progression. In contrast, IFN-γ production by NK cells was inversely correlated with BLV proviral loads in infected cattle. Additionally, during persistent lymphocytosis disease stages, NK cytotoxicity was depressed as indicated by low expression of the cytolytic protein perforin. Concomitantly, total CD4(+)CD25(high)Foxp3(+) T cell numbers and percentages of TGF-β(+) cells were increased, suggesting that TGF-β plays a role in the functional declines of CD4(+) T cells and NK cells. In further experiments, recombinant bovine TGF-β suppressed IFN-γ and TNF-α production by CD4(+) T cells and NK cytotoxicity in cultured cells. These data suggest that TGF-β from CD4(+)CD25(high)Foxp3(+) T cells is immunosuppressive and contributes to disease progression and the development of opportunistic infections during BLV infection.
• Molecular characterization of T-cell immunoglobulin mucin domain-3 and Galectin-9 genes of swamp- and riverine-type water buffaloes

  P. L. H. Duran, R. B. C. Padiernos, E. A. Abella, S. Konnai, C. N. Mingala

  INTERNATIONAL JOURNAL OF IMMUNOGENETICS 42 (6) 469 - 478 1744-3121 2015/12 [Refereed][Not invited]
   

  Molecular characterization of T-cell immunoglobulin mucin domain-3 (TIM-3) and Galectin-9 (GAL-9) genes of swamp-and riverine-type water buffaloes was conducted to compare these genes with other species; determine the unique characteristic specific in water buffalo; and provide baseline information for the assessment of disease progression in buffalo species. TIM-3 and GAL-9 genes were amplified, purified, sequenced and characterized. The sequence result of TIM-3 in both types of water buffaloes contained 843 nucleotides encoding to 280 amino acids while GAL-9 of swamp-type and riverine-type water buffaloes contained 1023 and 972 nucleotides encoding to 340 and 323 amino acids, respectively. Meanwhile, the nucleotide and amino sequence of TIM-3 in water buffalo were 83-98% and 94-97% identical with other artiodactyl species, respectively. On the other hand, GAL-9 nucleotide and amino acid sequence in water buffalo were 85-98% and 76-96% identical with other artiodactyl species. The tyrosine-kinase phosphorylation motif and potential glycosylation sites were conserved within the tribe Bovinae. It is imperative to have further studies in the assessment of the role of these genes in disease progression in water buffalo during chronic infection. The study is the first report that describes the genetic characteristic of TIM-3 and GAL-9 genes in water buffalo.
• A preliminary survey of the seroprevalence of Mycobacterium avium subspecies paratuberculosis in Mongolian cattle

  Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Shiro Murata, Kazuhiko Ohashi

  JAPANESE 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.
• Metal extent in blood of livestock from Dandora dumping site, Kenya: Source identification of Pb exposure by stable isotope analysis

  Hokuto Nakata, Shouta M. M. Nakayama, Yoshinori Ikenaka, Hazuki Mizukawa, Chihiro Ishii, Yared B. Yohannes, Satoru Konnai, Wageh Sobhy Darwish, Mayumi Ishizuka

  ENVIRONMENTAL POLLUTION 205 8 - 15 0269-7491 2015/10 [Refereed][Not invited]
   

  Nairobi city in Kenya produces 2000 tons/day of garbage, and most of it is dumped onto the Dandora dumping site, home to a quarter-million residents. This study was conducted (1) to assess the contamination levels of nine metals and a metalloid (arsenic) in the blood of pigs, goats, sheep and cattle from Dandora, and (2) to identify a possible source of lead (Pb) pollution. Cadmium (Cd, 0.17-435 mu g/kg, dry-wt) and Pb (90-2710 mu g/kg) levels in blood were generally high, suggesting human exposure to Cd through livestock consumption and Pb poisoning among pigs (2600 mu g/kg) and cattle (354 mu g/kg). Results of Pb isotope ratios indicated that the major exposure route might differ among species. Our results also suggested a possibility that the residents in Dandora have been exposed to the metals through livestock consumption. (C) 2015 Elsevier Ltd. All rights reserved.
• Horizontal transmission and phylogenetic analysis of bovine leukemia virus in two districts of Miyazaki, Japan

  Hirohisa Mekata, Satoshi Sekiguchi, Satoru Konnai, Yumi Kirino, Yoichiro Horii, Junzo Norimine

  JOURNAL OF VETERINARY MEDICAL SCIENCE 77 (9) 1115 - 1120 0916-7250 2015/09 [Refereed][Not invited]
   

  Horizontal transmission is recognized as a major infection route for bovine leukemia virus (BLV), and cattle with high viral loads are considered to be a major infectious source in a herd. However, a correlation between viral loads and the risk of infection has been insufficient to use as a foundation for BLV control strategies. In this report, we examined the epidemiology of BLV infection and the infectious source in a local area. In 2013-2014, BLV infection was investigated in 1,823 cattle from 117 farms in two adjacent districts, Miyazaki, Japan. Seropositive samples for BLV were detected with 88 cattle and in 14 farms. Phylogenetic analysis revealed that 94% of the isolates clustered into genotype I and the remaining isolate into genotype III. Among genotype I, genetically distinct strains were spread at each farm, and cattle infected with less than 3 copies/100 cells did not transmit BLV to other cattle for more than thirty months. This is the first report of concrete data of viral load in relation to viral horizontal transmission under the field condition. The data facilitate farmers and veterinarians understanding the status of BLV infected cattle. This research contributes to BLV infection control and the development of effective BLV eradication programs.
• Molecular epidemiological survey and genetic analysis of vector-borne infections of cattle in Luzon Island, the Philippines

  Nyamsuren Ochirkhuu, Satoru Konnai, Claro N. Mingala, Tomohiro Okagawa, Marvin Villanueva, Flor Marie Immanuelle R. Pilapil, Shiro Murata, Kazuhiko Ohashi

  VETERINARY PARASITOLOGY 212 (3-4) 161 - 167 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.
• 組織球性肉腫罹患犬における末梢血中免疫状態の解析

  田川 道人, 前川 直也, 今内 覚, 高木 哲

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 158回 407 - 407 1347-8621 2015/08
• Expression of regulatory dendritic cell-related cytokines in cattle experimentally infected with Trypanosoma evansi

  Hirohisa Mekata, Shiro Murata, Claro Niegos Mingala, Kazuhiko Ohashi, Satoru Konnai

  JOURNAL 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.
• Vaccination with cyclin-dependent kinase tick antigen confers protection against Ixodes infestation

  Helga Gomes, Jorge Moraes, Naftaly Githaka, Renato Martins, Masayoshi Isezaki, Itabajara da Silva Vaz, Carlos Logullo, Satoru Konnai, Kazuhiko Ohashi

  VETERINARY PARASITOLOGY 211 (3-4) 266 - 273 0304-4017 2015/07 [Refereed][Not invited]
   

  Among arthropods, ticks lead as vectors of animal diseases and rank second to mosquitoes in transmitting human pathogens. Cyclin-dependent kinases (CDK) participate in cell cycle control in eukaryotes. CDKs are serine/threonine protein kinases and these catalytic subunits are activated or inactivated at specific stages of the cell cycle. To determine the potential of using CDKs as anti-tick vaccine antigens, hamsters were immunized with recombinant lxodes persulcatus CDK10, followed by a homologous tick challenge. Though it was not exactly unexpected, IpCDK10 vaccination significantly impaired tick blood feeding and fecundity, which manifested as low engorgement weights, poor oviposition, and a reduction in 80% of hatching rates. These findings may underpin the development of more efficacious anti-tick vaccines based on the targeting of cell cycle control proteins. (C) 2015 Elsevier B.V. All rights reserved.
• CELLULAR IMMUNE RESPONSE TO INTERFERON-tau IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF JAPANESE BLACK CATTLE WITH BOVINE LEUKEMIA VIRUS INFECTION

  Kakinuma Sei-Ichi, Izawa Tomohiro, Matsuda Kei-Ichi, Konnai Satoru, Maeda Yosuke, Ohtsuka Hiromichi

  ACTA VETERINARIA-BEOGRAD 65 (2) 287 - 296 0567-8315 2015/06 [Refereed][Not invited]
   

  IFN-tau is a type I interferon, and it is known to be non-virus inducible in ruminants. IFN-tau reduced syncytium formation by PBMC obtained from BLV infected cattle in vitro. In order to clarify the effects of IFN-tau on cellular immune function in Japanese Black (JB) cattle with bovine leukemia virus (BLV) infection, immune related factors of peripheral blood mononuclear cells (PBMC) were analyzed using IFN-tau as a stimulator. Thirty-two JB cattle were used in this investigation, and these cattle were divided into three groups: cattle with enzootic bovine leucosis (EBL) (EBL Group, N=7), clinically healthy cattle with BLV infection (Carrier Group, N=13), and clinically healthy cattle without BLV infection (non-Carrier Group, N=12). A number of mRNA expressions of interleukin-12 and interferon (IFN)-gamma as immune cell activating cytokines, perforin and granulysin as cytotoxic factors, and myxovirus resistance protein (MX)-1 and MX-2 as anti-virus factors of PBMC were analyzed after culturing cells with phytohemagglutinin (PHA) or IFN-tau. The basal mRNA levels of perforin and granulysin in the Carrier Group were significantly higher than those in the non-Carrier Group. Also, significantly higher basal mRNA levels of MX-1 and MX-2 in the EBL Group were detected compared with the non-Carrier Group. The mRNA expressions of perforin and granulysin in PBMC stimulated with PHA were higher in the Carrier Group than those in the non-Carrier Group. There were significantly higher mRNA levels of MX-1 and MX-2 in PBMC stimulated with IFN-tau in the EBL Group compared with those in the non-Carrier Group. These results suggest an enhanced sensitivity of anti-virus reaction in PBMC by IFN-tau treatment in JB cattle with EBL.
• An investigation of binding ability of Ixodes persulcatus Schulze Salp15 with Lyme disease spirochetes

  Yusuke Murase, Satoru Konnai, Shinji Yamada, Naftaly Githaka, Masayoshi Isezaki, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Siro Murata, Kazuhko Ohashi

  INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 60 59 - 67 0965-1748 2015/05 [Refereed][Not invited]
   

  Salp15, a 15-kDa tick salivary gland protein, has several suppressive modes of activity against host immunity and plays a critical role in the transmission of Lyme disease spirochetes in Ixodes scapularis and Ixodes ricinus, major vectors of Lyme disease in North America and Western Europe. Salp15 adheres to Borrelia burgdorferi and specifically interacts with its outer surface protein C (OspC), protecting the spirochete from antibody-mediated cytotoxicity and facilitating infection in the mice. Recently, we identified two Salp15 homologues, IperSalp15-1 and IperSalp15-2, in Ixodes persulcatus, a vector for Lyme disease in Japan. Here we describe the function of IperSalp15 in the transmission of Lyme borreliosis. To investigate the function of IperSalp15, recombinant IperSalp15-1 and IperSalp15-2 were prepared in bacterial and insect cells. Both were identified in the sera of tick-immunized hamsters, indicating that these are secretory proteins in exposed host animals. Solid-phase overlay and indirect fluorescence assays showed that IperSalp15 binds to OspC from B. burgdorferi, Borrelia garinii, and Borrelia afzelii. Importantly, this binding likely protected the spirochete from antibody-mediated cytotoxicity in vitro. In addition, IperSalp15 tended to facilitate infection in mice. Thus, further characterization of tick molecules, including IperSalp15, could lead to the development of new strategies to prevent the transmission of tick-borne diseases. (C) 2015 Elsevier Ltd. All rights reserved.
• Cytokine mRNA profiling and the proliferative response of bovine peripheral blood mononuclear cells to Mycoplasma bovis

  Satoshi Gondaira, Hidetoshi Higuchi, Hidetomo Iwano, Keiichi Nakajima, Kazuhiro Kawai, Shuhei Hashiguchi, Satoru Konnai, Hajime Nagahata

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 165 (1-2) 45 - 53 0165-2427 2015/05 [Refereed][Not invited]
   

  Mycoplasma bovis is known as a significant pathogen and cause of large economic losses in beef and dairy calves worldwide. Numerous factors appear to play an important role in the development of disease during infection with M. bovis, e.g., inhibition of immune cell proliferation and induction of lymphocyte apoptosis. However, the mechanisms involved in M. bovis infections have not been explored and remain incompletely understood. We investigated the major cytokine mRNA expression in bovine PBMC stimulated with M. bovis, for comparison, Staphylococcus aureus and Escherichia coli, which are the representative mastitis-causing pathogens. Here we demonstrated that live M. bovis significantly induced tumor necrosis factor alpha (TNF-alpha), interleukin 12p40 (IL-12), and interferon gamma (IFN-gamma) mRNA expression in bovine peripheral blood mononuclear cells (PBMC) at a multiplicity of infection (MOI) of 1000 but not at an MOI of 10 and 100. Live M. bovis at MOIs of 1, 10, and 100 induced significant bovine PBMC proliferative responses compared with unstimulated bovine PBMC. Furthermore, we showed that the cultural supernatant of M. bovis induced a significant increase in TNF-alpha, IL-6, and IL-10 mRNA expression in bovine PBMC. Our results suggest that M. bovis weakly affects the cellular integrity of bovine PBMC and induces clear proliferative responses and associated cytokine production in them. However, large numbers of live M. bovis are required to induce an immune response in bovine PBMC. (C) 2015 Elsevier B.V. All rights reserved.
• Evaluation of the natural perinatal transmission of bovine leukaemia virus

  Hirohisa Mekata, Satoshi Sekiguchi, Satoru Konnai, Yumi Kirino, Kazuyuki Honkawa, Nariaki Nonaka, Yoichiro Horii, Junzo Norimine

  VETERINARY RECORD 176 (10) 254 - + 0042-4900 2015/03 [Refereed][Not invited]
   

  The perinatal transmission of bovine leukaemia virus (BLV) plays a critical role in the spread and persistence of BLV infection in cattle herds. The purpose of this study was to examine the frequency of perinatal infections in an area in Japan and investigate some risk factors associated with infection. Altogether, 129 calves born to BLV-infected cows in a herd in Japan were tested for infection immediately after birth and again at one month of age using nested PCR. Twenty-four calves (18.6 per cent) were infected with BLV, of which 14 (10.8 per cent) and 10 (7.7 per cent) calves were infected via the transplacental and the birth canal routes, respectively. Maternal viral loads, breed, the presence or absence of assistance during parturition and the number of births per dam were evaluated to investigate risk factors associated with infection. Maternal viral load was significantly correlated with the frequency of perinatal infection, and more than 40 per cent of newborn calves born to dams with high viral loads were infected with BLV. The results of this study could contribute towards developing effective eradication programmes by providing necessary data for replacement of breeding cow in the field.
• Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection

  Saori Suzuki, Satoru Konnai, Tomohiro Okagawa, Ryoyo Ikebuchi, Asami Nishimori, Junko Kohara, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 163 (3-4) 115 - 124 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.
• Rhipicephalus microplus and Ixodes ovatus cystatins in tick blood digestion and evasion of host immune response

  Luis Fernando Parizi, Gabriela Alves Sabadin, Mara Fernanda Alzugaray, Adriana Seixas, Carlos Logullo, Satoru Konnai, Kazuhiko Ohashi, Aoi Masuda, Itabajara da Silva Vaz

  PARASITES & VECTORS 8 122  1756-3305 2015/02 [Refereed][Not invited]
   

  Background: Cystatins are a group of cysteine protease inhibitors responsible for physiological proteolysis regulation and present in a wide range of organisms. Studies about this class of inhibitors in parasites have contributed to clarify their roles in important physiological processes, like blood digestion and modulation of host immune response during blood feeding. Thus, cystatins are a subject of research on the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to find potential targets in multi-species control methods. However, cystatin functions in ticks remain undetermined, especially in Rhipicephalus microplus and Ixodes ovatus, two species that affect livestock and human health, respectively. Methods: Here we report the inhibitory profile of two R. microplus (BrBmcys2b and BrBmcys2c) and one I. ovatus (JpIocys2a) cystatins to commercial cathepsins B, C, and L. The presence of native cystatins in R. microplus tissues was analyzed using sera against recombinant BrBmcys2b and BrBmcys2c. Also, a peptide from JpIocys2a was synthesized for rabbit immunization, and this serum was used to analyze the cross antigenicity between R. microplus and I. ovatus cystatins. Results: Enzymatic inhibition profile of tick cystatins shows a distinct modulation for cathepsins related to tick blood digestion and evasion of host immune response. Furthermore, BrBmcys2b was detected in saliva and different tissues along tick stages, while BrBmcys2c was detected mainly in gut from partially engorged R. microplus females, demonstrating a distinct pattern of cystatin expression, secretion and traffic between tick tissues. Moreover, phylogenetic analysis suggests that JpIocys2a belongs to the group of tick gut secreted cystatins. Finally, cross-antigenicity assays revealed that antibodies against the JpIocys2a peptide recognize native and recombinant R. microplus cystatins. Conclusion: The presence of these proteins in different tissues and their ability to differentially inhibit cathepsins suggest distinct roles for JpIocys2a, BrBmcys2b, and BrBmcys2c in blood digestion, egg and larvae development, and modulation of host immune response in tick physiology. The cross-antigenicity between native and recombinant cystatins supports further experiments using JpIocys2a, BrBmcys2b, and BrBmcys2c as vaccine antigens.
• Non-Classical Gluconeogenesis-Dependent Glucose Metabolism in Rhipicephalus microplus Embryonic Cell Line BME26

  Renato Martins da Silva, Barbara Della Noce, Camila Fernanda Waltero, Evenilton Pessoa Costa, Leonardo Araujo de Abreu, Naftaly Wang'ombe Githaka, Jorge Moraes, Helga Fernandes Gomes, Satoru Konnai, Itabajara da Silva Vaz, Kazuhiko Ohashi, Carlos Logullo

  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 16 (1) 1821 - 1839 1422-0067 2015/01 [Refereed][Not invited]
   

  In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells.
• Bovine immunoinhibitory receptors contribute to suppression of Mycobacterium avium subsp. paratuberculosis-specific T-cell responses

  Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Ryoyo Ikebuchi, Seiko Mizorogi, Reiko Nagata, Satoko Kawaji, Shogo Tanaka, Yumiko Kagawa, Shiro Murata, Yasuyuki Mori, Kazuhiko Ohashi

  Infection and Immunity 84 (1) 77 - 89 1098-5522 2015 [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-γ) 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-γ 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-γ responses in blood from infected animals, and in vitro LAG-3 blockade enhanced IFN-γ 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.
• 北海道のsika deerおよびHaemaphysalis属ダニより検出されたRF Borrelia属(A RF Borrelia sp. found among wild sika deer and Haemaphysalis ticks in Hokkaido)

  李 景利, 高野 愛, Taylor Kyle, 佐鹿 万里子, 下鶴 倫人, 今内 覚, 川端 寛樹, 坪田 敏男

  日本獣医学会学術集会講演要旨集 157回 548 - 548 1347-8621 2014/08
• Influence of PD-L1 cross-linking on cell death in PD-L1-expressing cell lines and bovine lymphocytes

  Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  IMMUNOLOGY 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.
• Expression analysis of programmed death ligand 2 in tumors caused by the avian oncovirus Marek's disease virus

  Ayumi Matsuyama-Kato, Shiro Murata, Masayoshi Isezaki, Sarah Takasaki, Rika Kano, Satoru Konnai, Kazuhiko Ohashi

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

  Abid Ali, Lucas Tirloni, Masayoshi Isezaki, Adriana Seixas, Satoru Konnai, Kazuhiko Ohashi, Itabajara da Silva Vaz Junior, Carlos Termignoni

  EXPERIMENTAL AND APPLIED ACAROLOGY 63 (4) 559 - 578 0168-8162 2014/08 [Refereed][Not invited]
   

  Metalloproteases (MPs) have been considered essential for blood feeding and other physiological functions in several hematophagous animals, including ticks. We report the characterization of MP sequences of three important ticks from Asia, Africa and America: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) and R. microplus (BrRm-MPs). Amino acid sequence identity between R. microplus and R. sanguineus MPs ranged from 76 to 100 %, and identities among I. persulcatus, I. ricinus and I. scapularis MP sequences ranged from 88 to 97 %. This high sequence identity and typical functional motifs show that all sequences are MPs. The presence of a zinc binding site, a Met-turn and cysteine rich domain at the C-terminal region indicates that these proteins belong to the reproplysin family of MPs. Differences in amino acid sequences of BrRm-MP1, BrRm-MP2, BrRm-MP4 and BrRm-MP5 (from Porto Alegre strain ticks) were 6, 2, 7 and 5 %, respectively, when compared with sequences deposited in GenBank for the same genes from other R. microplus isolates. Analyses of MPs predicted that they have various highly antigenic regions. Semi-quantitative RT-PCR analysis revealed the presence of transcripts in salivary glands of partially and fully fed female ticks. None of these transcripts were observed in males (except BrRm-MP4) and eggs. These enzymes may be functional components required during tick feeding to manipulate host defenses and support tick hematophagy.
• Suppressive effects of neutrophil by Salp16-like salivary gland proteins from Ixodes persulcatus Schulze tick

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

  INSECT MOLECULAR BIOLOGY 23 (4) 466 - 474 0962-1075 2014/08 [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.
• Differences in cellular function and viral protein expression between IgM(high) and IgM(low) B-cells in bovine leukemia virus-infected cattle

  Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Ayako Nakahara, Shiro Murata, Kazuhiko Ohashi

  JOURNAL 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.
• Human Infections with Borrelia miyamotoi, Japan

  Sato, Kozue, Takano, Ai, Konnai, Satoru, Nakao, Minoru, Ito, Takuya, Koyama, Kojiro, Kaneko, Minoru, Ohnishi, Makoto, Kawabata, Hiroki

  EMERGING INFECTIOUS DISEASES 20 (8) 1391 - 1393 1080-6040 2014/08 [Refereed][Not invited]
   

  We confirmed infection of 2 patients with Borrelia miyamotoi in Japan by retrospective surveillance of Lyme disease patients and detection of B. miyamotoi DNA in serum samples. One patient also showed seroconversion for antibody against recombinant glycerophosphodiester phosphodiesterase of B. miyamotoi. Indigenous relapsing fever should be considered a health concern in Japan.
• Tick Surveillance for Relapsing Fever Spirochete Borrelia miyamotoi in Hokkaido, Japan

  Takano, Ai, Toyomane, Kochi, Konnai, Satoru, Ohashi, Kazuhiko, Nakao, Minoru, Ito, Takuya, Andoh, Masako, Maeda, Ken, Watarai, Masahisa, Sato, Kozue, Kawabata, Hiroki

  PLOS ONE 9 (8) e104532  1932-6203 2014/08 [Refereed][Not invited]
   

  During 2012-2013, a total of 4325 host-seeking adult ticks belonging to the genus Ixodes were collected from various localities of Hokkaido, the northernmost island of Japan. Tick lysates were subjected to real-time PCR assay to detect borrelial infection. The assay was designed for specific detection of the Relapsing fever spirochete Borrelia miyamotoi and for unspecific detection of Lyme disease-related spirochetes. Overall prevalence of B. miyamotoi was 2% (71/3532) in Ixodes persulcatus, 4.3% (5/117) in Ixodes pavlovskyi and 0.1% (1/676) in Ixodes ovatus. The prevalence in I. persulcatus and I. pavlovskyi ticks were significantly higher than in I. ovatus. Co-infections with Lyme disease-related spirochetes were found in all of the tick species. During this investigation, we obtained 6 isolates of B. miyamotoi from I. persulcatus and I. pavlovskyi by culture in BSK-M medium. Phylogenetic trees of B. miyamotoi inferred from each of 3 housekeeping genes (glpQ, 16S rDNA, and flaB) demonstrated that the Hokkaido isolates were clustered with Russian B. miyamotoi, but were distinguishable from North American and European B. miyamotoi. A multilocus sequence analysis using 8 genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) suggested that all Japanese B. miyamotoi isolates, including past isolates, were genetically clonal, although these were isolated from different tick and vertebrate sources. From these results, B. miyamotoi-infected ticks are widely distributed throughout Hokkaido. Female I. persulcatus are responsible for most human tick-bites, thereby I. persulcatus is likely the most important vector of indigenous relapsing fever from tick bites in Hokkaido.
• Identification and characterization of bovine programmed death-ligand 2

  Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Chie Nakajima, Yasuhiko Suzuki, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi

  MICROBIOLOGY AND IMMUNOLOGY 58 (7) 388 - 397 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.
• Expression of PD-L1 on Canine Tumor Cells and Enhancement of IFN-gamma Production from Tumor-Infiltrating Cells by PD-L1 Blockade

  Naoya Maekawa, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Mami Adachi, Satoshi Takagi, Yumiko Kagawa, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  PLOS 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.
• 牛白血病ウイルスに感染した黒毛和種牛の臨床例における免疫状態

  伊澤 智宏, 田中 健一郎, 柿沼 清市, 杉山 由夏, 松田 敬一, 小比類巻 正幸, 今内 覚, 大塚 浩通

  日本獣医師会雑誌 (公社)日本獣医師会 67 (5) 328 - 332 0446-6454 2014/05
• Identification and sequence characterization of novel Theileria genotypes from the waterbuck (Kobus defassa) in a Theileria parva-endemic area in Kenya

  Naftaly Githaka, Satoru Konnai, Richard Bishop, David Odongo, Isaac Lekolool, Edward Kariuki, Francis Gakuya, Lucy Kamau, Masayoshi Isezaki, Shiro Murata, Kazuhiko Ohashi

  VETERINARY PARASITOLOGY 202 (3-4) 180 - 193 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.
• Bovine Leukemia virus titer and leukocyte population associated with mastitis in periparturient dairy cows

  Seiichi Kakinuma, Yousuke Maeda, Hiromichi Ohtsuka, Satoru Konnai, Masa-aki Oikawa

  INTERNATIONAL JOURNAL OF APPLIED RESEARCH IN VETERINARY MEDICINE 12 (3) 239 - 244 1542-2666 2014 

  We examined the association of postpartum mastitis with virus titer and leukocyte population of Bovine leukemia virus (BLV) infected cows monitoring sixteen BLV infected cows for 3 months after calving. Five cows developed mastitis (Mastitis Group) and 11 cows without mastitis served as a control group (Control Group). Flow cytometric analysis of peripheral blood leukocyte population exhibited higher number of MHC class-II+CD14- cells in both groups without significant difference between groups. CD3(+), CD4(+), CDS+, and CD335(+) cell numbers in Mastitis Group were lower than the Control Group around the time of calving, but significant difference was observed only for CD335(+) cell number. Virus titer in blood significantly increased in Mastitis Group after calving compared to pre-calving titer. These results suggested that lower number of peripheral blood T cells in Mastitis Group cows might have heightened susceptibility to mastitis. In addition, Mastitis Group cows had higher peripheral blood BLV, which might have spread to surroundings.
• A relapsing fever group Borrelia sp similar to Borrelia lonestari found among wild sika deer (Cervus nippon yesoensis) and Haemaphysalis spp. ticks in Hokkaido, Japan

  Kyunglee Lee, Ai Takano, Kyle Taylor, Mariko Sashika, Michito Shimozuru, Satoru Konnai, Hiroki Kawabata, Toshio Tsubota

  TICKS AND TICK-BORNE DISEASES 5 (6) 841 - 847 1877-959X 2014 [Refereed][Not invited]
   

  A relapsing fever Borrelia sp. similar to Borrelia lonestari (herein referred to as B. lonestari-like) was detected from wild sika deer (Cervus nippon yesoensis) and Haemaphysalis ticks in the eastern part of Hokkaido, Japan. The total prevalence of this Borrelia sp. in tested deer blood samples was 10.6% using conventional PCR and real-time PCR. The prevalence was significantly higher in deer fawns compared to adults (21.9% and 9.4%, respectively). Additionally, there was significant regional difference between our two sampling areas, Shiretoko and Shibetsu with 17% and 2.8% prevalence, respectively. Regional differences were also found in tick species collected from field and on deer. In the Shiretoko region, Haemaphysalis spp. were more abundant than Ixodes spp., while in Shibetsu, Ixodes spp. were more abundant. Using real-time PCR analysis, B. lonestari-like was detected from 2 out of 290 adult Haemaphysalis spp. ticks and 4 out of 76 pools of nymphs. This is the first report of a B. lonestari-like organism in Haemaphysalis spp. ticks, and the first phylogenetic analysis of this B. lonestari-like organism in Asia. Based on our results, Haemaphysalis spp. are the most likely candidates to act as a vector for B. lonestari-like; furthermore, regional variation of B. lonestari-like prevalence in sika deer may be dependent on the population distribution of these ticks. (C) 2014 Elsevier GmbH. All rights reserved.
• Sequence characterization and immunogenicity of cystatins from the cattle tick Rhipicephalus (Boophilus) microplus

  Luís F. Parizi, Naftaly W. Githaka, Carolina Acevedo, Uruguaysito Benavides, Adriana Seixas, Carlos Logullo, Satoru Konnai, Kazuhiko Ohashi, Aoi Masuda, Itabajara Da Silva Vaz

  Ticks and Tick-borne Diseases 4 (6) 492 - 499 1877-959X 2013/12 [Refereed][Not invited]
   

  Various classes of endopeptidases and their inhibitors facilitate blood feeding and digestion in ticks. Cystatins, a family of tight-binding and reversible inhibitors of cysteine endopeptidases, have recently been found in several tick tissues. Moreover, vaccine trials using tick cystatins have been found to induce protective immune responses against tick infestation. However, the mode of action of tick cystatins is still poorly understood, limiting the elucidation of their physiological role. Against this background, we have investigated sequence characteristics and immunogenic properties of 5 putative cystatins from Rhipicephalus (Boophilus) microplus from Brazil and Uruguay. The similarity of the deduced amino acid sequences among cystatins from the Brazilian tick strain was 27-42%, all of which had a secretory signal peptide. The cystatin motif (QxVxG), a glycine in the N-terminal region, and the PW motif in the second hairpin loop in the C-terminal region are highly conserved in all 5 cystatins identified in this study. Four cysteine residues in the C terminus characteristic of type 2 cystatins are also present. qRT-PCR revealed differential expression patterns among the 5 cystatins identified, as well as variation in mRNA transcripts present in egg, larva, gut, salivary glands, ovary, and fat body tissues. One R. microplus cystatin showed 97-100% amino acid similarity between Brazilian and Uruguayan isolates. Furthermore, by in silico analysis, antigenic amino acid regions from R. microplus cystatins showed high degrees of homology (54-92%) among Rhipicephalus spp. cystatins. Three Brazilian R. microplus cystatins were expressed in Escherichia coli, and immunogenicity of the recombinant proteins were determined by vaccinating mice. Western blotting using mice sera indicated cross-reactivity between the cystatins, suggesting shared epitopes. The present characterization of Rhipicephalus spp. cystatins represents an empirical approach in an effort to evaluate the physiological role of cystatins in a larger context of targeting them for use in future tick control strategies. © 2013 Elsevier GmbH.
• Short communication: Horizontal transmission of bovine leukemia virus from lymphocytotic cattle, and beneficial effects of insect vector control

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

  Veterinary Record 173 (21) 527  0042-4900 2013/11/30 [Refereed][Not invited]
  
• Identification and Structural-Functional Analysis of Cyclin-Dependent Kinases of the Cattle Tick Rhipicephalus (Boophilus) microplus

  Helga Gomes, Nelilma C. Romeiro, Gloria R. C. Braz, Eduardo Alves Gamosa de Oliveira, Camilla Rodrigues, Rodrigo Nunes da Fonseca, Naftaly Githaka, Masayoshi Isezaki, Satoru Konnai, Kazuhiko Ohashi, Itabajara da Silva Vaz Jr., Carlos Logullo, Jorge Moraes

  PLoS ONE 8 (10) e76128.  1932-6203 2013/10/11 [Refereed][Not invited]
   

  Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases essential for cell cycle progression. Herein, we describe the participation of CDKs in the physiology of Rhipicephalus microplus, the southern cattle tick and an important disease vector. Firstly, amino acid sequences homologous with CDKs of other organisms were identified from a R. microplus transcriptome database in silico. The analysis of the deduced amino acid sequences of CDK1 and CDK10 from R. microplus showed that both have caspase-3/7 cleavage motifs despite their differences in motif position and length of encoded proteins. CDK1 has two motifs (DKRGD and SAKDA) located opposite to the ATP binding site while CDK10 has only one motif (SLLDN) for caspase 3-7 near the ATP binding site. Roscovitine (Rosco), a purine derivative that inhibits CDK/cyclin complexes by binding to the catalytic domain of the CDK molecule at the ATP binding site, which prevents the transfer of ATP's γphosphoryl group to the substrate. To determine the effect of Rosco on tick CDKs, BME26 cells derived from R. microplus embryo cells were utilized in vitro inhibition assays. Cell viability decreased in the Rosco-treated groups after 24 hours of incubation in a concentration-dependent manner and this was observed up to 48 hours following incubation. To our knowledge, this is the first report on characterization of a cell cycle protein in arachnids, and the sensitivity of BME26 tick cell line to Rosco treatment suggests that CDKs are potential targets for novel drug design to control tick infestation. © 2013 Gomes et al.
• Genotypic variations in field isolates of Theileria species infecting giraffes (Giraffa camelopardalis tippelskirchi and Giraffa camelopardalis reticulata) in Kenya

  Naftaly Githaka, Satoru Konnai, Robert Skilton, Edward Kariuki, Esther Kanduma, Shiro Murata, Kazuhiko Ohashi

  Parasitology 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.
• Expression analysis of Foxp3 in T cells from bovine leukemia virus infected cattle

  Saori Suzuki, Satoru Konnai, Tomohiro Okagawa, Ryoyo Ikebuchi, Tatsuya Shirai, Yuji Sunden, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi

  Microbiology and Immunology 57 (8) 600 - 604 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.
• Expression analysis of Foxp3 in T-cells from bovine leukemia virus infected cattle.

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

  Microbiology and immunology 0385-5600 2013/06 [Refereed][Not invited]
  
• 北海道の野生下におけるボレリア種の維持、小型哺乳類とダニの関係について(On the maintenance of Borrelia spp. among wildlife in Hokkaido: the relationship between small mammals and ticks)

  カイル・テイラー, 川端 寛樹, 今内 覚, 下鶴 倫人, 高野 愛, 坪田 敏男

  衛生動物 64 (2) 112 - 112 0424-7086 2013/06
• Characterization of Meq proteins from field isolates of Marek's disease virus in Japan

  Shiro Murata, Tomoyuki Hashiguchi, Yuko Hayashi, Yuki Yamamoto, Ayumi Matsuyama-Kato, Sarah Takasaki, Masayoshi Isezaki, Misao Onuma, Satoru Konnai, Kazuhiko Ohashi

  INFECTION GENETICS AND EVOLUTION 16 137 - 143 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.
• Differential Tick Burdens May Explain Differential Borrelia afzelii and Borrelia garinii Infection Rates among Four, Wild, Rodent Species in Hokkaido, Japan

  Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (6) 785 - 790 0916-7250 2013/06 [Refereed][Not invited]
   

  The ecologies of Lyme disease Borrelia spp. are very specific to location, as they are dependent upon the spirochete species and genotypes, the vectors and the host vertebrates present. In Hokkaido, Japan, where two human pathogenic, Lyme disease Borrelia spp. are present, and human cases are reported annually, the ecologies have been poorly studied. Our goal was to determine whether variation in borrelial infection rates among rodent species sharing an environment, is due to immunological or ecological differences. To this end, we examined the relationships between tick burden and borrelial infection, by including examination of agreement between nested PCR, as a test for infection, and serology, as a test for exposure. We collected 868 rodents, comprised of four species commonly found in Hokkaido, and tested for infection rates with Borrelia spp. using PCR for the borrelial flaB gene, seroprevalence of Borrelia afzelii and Borrelia garinii using ELISA, and attachment of ticks by direct counts. We noted a correlation between differential nymph and larval burdens and the borrelial infection rates found among the four rodent species. Furthermore, there was significant correlation between infection and seroprevalence of B. afzelii and B. garinii (P<0.01), between infection and Ixodes persulcatus nymph burden (P<0.01), and between seroprevalence and I. persulcatus nymph burden (P<0.01). The close agreement among rodent species seroprevalences with infection rates and tick burdens suggest the differences in infection rates of Borrelia spp. may largely be a direct consequence of differential exposure to vectors.
• 免疫抑制受容体PD-1のリガンドPD-L2の機能的特徴と臨床応用研究

  西森朝美, 今内 覚, 池渕良洋, 岡川朋弘, 村田史郎, 大橋和彦

  動物用ワクチンーバイオ医薬品研究会ニュースレター 7 19 - 20 2013/06 [Not refereed][Invited]
  
• Bovine whole-blood culture as a tool for the measurement of endotoxin activities in Gram-negative bacterial vaccines

  Saiki Imamura, Mari Nakamizo, Michiko Kawanishi, Nao Nakajima, Kinya Yamamoto, Mariko Uchiyama, Fumiya Hirano, Hidetaka Nagai, Mayumi Kijima, Ryoyo Ikebuchi, Hirohisa Mekata, Shiro Murata, Satoru Konnai, Kazuhiko Ohashi

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 153 (1-2) 153 - 158 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.
• Isolation, cloning, and pathologic analysis of Trypanosoma evansi field isolates

  Hirohisa Mekata, Satoru Konnai, Claro N. Mingala, Nancy S. Abes, Charito A. Gutierrez, Alan P. Dargantes, William H. Witola, Noboru Inoue, Misao Onuma, Shiro Murata, Kazuhiko Ohashi

  PARASITOLOGY RESEARCH 112 (4) 1513 - 1521 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.
• The conserved role of the AKT/GSK3 axis in cell survival and glycogen metabolism in Rhipicephalus (Boophilus) microplus embryo tick cell line BME26

  Leonardo Araujo de Abreu, Christiano Calixto, Camila Fernanda Waltero, Barbara Pitta Della Noce, Naftaly Wang'ombe Githaka, Adriana Seixas, Luis Fernando Parizi, Satoru Konnai, Itabajara da Silva Vaz, Kazuhiko Ohashi, Carlos Logullo

  BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1830 (3) 2574 - 2582 0304-4165 2013/03 [Refereed][Not invited]
   

  Background: Tick embryogenesis is a metabolically intensive process developed under tightly controlled conditions and whose components are poorly understood. Methods: In order to characterize the role of AKT (protein kinase B) in glycogen metabolism and cell viability, glycogen determination, identification and cloning of an AKT from Rhipicephalus microplus were carried out, in parallel with experiments using RNA interference (RNAi) and chemical inhibition. Results: A decrease in glycogen content was observed when AKT was chemically inhibited by 10-DEBC treatment, while GSK3 inhibition by alsterpaullone had an opposing effect. RmAKT ORF is 1584-bp long and encodes a polypeptide chain of 60.1 kDa. Phylogenetic and sequence analyses showed significant differences between vertebrate and tick AKTs. Either AKT or GSK3 knocked down cells showed a 70% reduction in target transcript levels, but decrease in AKT also reduced glycogen content, cell viability and altered cell membrane permeability. However, the GSK3 reduction promoted an increase in glycogen content. Additionally, either GSK3 inhibition or gene silencing had a protective effect on BME26 viability after exposure to ultraviolet radiation. R. microplus AKT and GSK3 were widely expressed during embryo development. Taken together, our data support an antagonistic role for AKT and GSK3, and strongly suggest that such a signaling axis is conserved in tick embryos, with AKT located upstream of GSK3. General significance: The AKT/GSK3 axis is conserved in tick in a way that integrates glycogen metabolism and cell survival, and exhibits phylogenic differences that could be important for the development of novel control methods. (C) 2013 Elsevier B.V. All rights reserved.
• Prevalence of Lyme Borrelia in Ixodes persulcatus Ticks from an Area with a Confirmed Case of Lyme Disease

  Yusuke Murase, Satoru Konnai, Naftaly Githaka, Arata Hidano, Kyle Taylor, Takuya Ito, Ai Takano, Shuji Ando, Hiroki Kawabata, Toshio Tsubota, Shiro Murata, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (2) 215 - 218 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).
• Borrelia miyamotoi Infections among Wild Rodents Show Age and Month Independence and Correlation with Ixodes persulcatus Larval Attachment in Hokkaido, Japan

  Kyle R. Taylor, Ai Takano, Satoru Konnai, Michito Shimozuru, Hiroki Kawabata, Toshio Tsubota

  VECTOR-BORNE AND ZOONOTIC DISEASES 13 (2) 92 - 97 1530-3667 2013/02 [Refereed][Not invited]
   

  To clarify how Borrelia miyamotoi is maintained in the environment in Hokkaido, we examined Ixodes persulcatus for its prevalence among wild rodents and its tick vector by detecting a portion of the borrelial flaB gene in rodent urinary bladder and blood samples, and from whole ticks. We compared B. miyamotoi infection rates to Borrelia garinii and Borrelia afzelii, which are human Lyme disease pathogens also carried by wild rodents, and which are transmitted by the same vector tick. Whereas B. garinii and B. afzelii showed age dependence of infection rates among wild rodents (18.4% and 9.9% among adults and 6.0% and 3.4% among sub-adults, respectively) when looking at urinary bladder samples, B. miyamotoi infection rates were not age dependent for either blood (4.2% among adults, and 7.9% among sub-adults) or urinary bladder samples (1.0% among adults, and 1.7% among sub-adults). Moreover, while B. garinii and B. afzelii infection rates showed increases across months (June, July [p < 0.05] and August [p < 0.01] had higher rates than in May for adult rodents with B. garinii, and July and August had higher rates than in May [p < 0.01] for adult rodents with B. afzelii), B. miyamotoi infection rates did not show significant month dependence. These differences in month and age dependence led us to suspect that B. miyamotoi may not develop persistent infections in wild rodents, as B. garinii and B. afzelii are thought to. Furthermore, we examined the extent of rodent exposure to I. persulcatus nymphs and larvae throughout most of the tick's active season (May through September), and determined that B. miyamotoi infection rates in sub-adult rodents were correlated with larval burden (p < 0.01), suggesting that larvae may be very important in transmission of B. miyamotoi to wild rodents.
• Enhanced expression of LAG-3 on lymphocyte subpopulations from persistently lymphocytotic cattle infected with bovine leukemia virus

  Satoru Konnai, Saori Suzuki, Tatsuya Shirai, Ryoyo Ikebuchi, Tomohiro Okagawa, Yuji Sunden, Claro N. Mingala, Misao Onuma, Shiro Murata, Kazuhiko Ohashi

  COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 36 (1) 63 - 69 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.
• Identification and partial characterization of a gut Rhipicephalus appendiculatus cystatin

  Saiki Imamura, Satoru Konnai, Shinji Yamada, Luis F. Parizi, Naftaly Githaka, Itabajara da S. Vaz, Shiro Murata, Kazuhiko Ohashi

  TICKS AND TICK-BORNE DISEASES 4 (1-2) 138 - 144 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.
• Blockade of bovine PD-1 increases T cell function and inhibits bovine leukemia virus expression in B cells in vitro

  Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

  Veterinary 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.
• The quest for a universal vaccine against ticks: Cross-immunity insights

  Luis F. Parizi, Naftaly W. Githaka, Carlos Logullo, Satoru Konnai, Aoi Masuda, Kazuhiko Ohashi, Itabajara da Silva Vaz

  VETERINARY JOURNAL 194 (2) 158 - 165 1090-0233 2012/11 [Refereed][Not invited]
   

  As blood-sucking parasites, ticks inflict great damage to animals and humans in many parts of the world. The continued use of chemical acaricides is not sustainable due to increasing tick resistance, growing public concern over drug residues in food and in the environment, and the high cost of developing new acaricides. Therefore, an alternative control strategy is urgently needed. Vaccines against ticks have been shown to be functionally feasible, as highlighted by the success of Bm86 vaccines against Rhipicephalus (Boophilus) microplus and closely related tick species. However, a limited number of tick antigens with cross-protective epitopes have been characterized so far, limiting widespread deployment of the available vaccines, including those derived from Bm86. Therefore, identifying tick antigens with potential broad-spectrum protection against multiple tick species is subject of vigorous research at present. In this paper, progress towards effective anti-tick vaccines is reviewed in the light of emerging data from studies including heterologous tick challenge. Taken together, these studies indicate that the decades-long search for a universal tick vaccine is making progress, with such a vaccine likely to be based on multiple cross-reactive antigens. (c) 2012 Elsevier Ltd. All rights reserved.
• Molecular detection and characterization of potentially new Babesia and Theileria species/variants in wild felids from Kenya

  Naftaly Githaka, Satoru Konnai, Edward Kariuki, Esther Kanduma, Shiro Murata, Kazuhiko Ohashi

  ACTA 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.
• EFFECTS OF ENVIRONMENTAL LEAD CONTAMINATION ON CATTLE IN A LEAD/ZINC MINING AREA: CHANGES IN CATTLE IMMUNE SYSTEMS ON EXPOSURE TO LEAD IN VIVO AND IN VITRO

  Yoshinori Ikenaka, Shouta M. M. Nakayama, Taro Muroya, John Yabe, Satoru Konnai, Wageh Sobhy Darwish, Kaampwe Muzandu, Kennedy Choongo, Geoffrey Mainda, Hiroki Teraoka, Takashi Umemura, Mayumi Ishizuka

  ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 31 (10) 2300 - 2305 0730-7268 2012/10 [Refereed][Not invited]
   

  The Republic of Zambia is rich in mineral resources, such as zinc (Zn) and lead (Pb), and mining is a key industry in Zambia. A previous study of Pb pollution in Kabwe, one of the main mining areas, found that soil was contaminated with high levels of toxic metals over a substantial area. In the present study, the authors focus on toxic metal pollution in cattle, one of the most important domestic animals in Zambia. Blood samples from cattle in Kabwe and a control area (Lusaka) were tested for toxic metal content. They also measured mRNA expression of metal-responsive proteins and cytokines in white blood cells using real-time reverse transcriptase polymerase chain reaction. In the present in vitro study, The authors cultured peripheral blood mononuclear cells (PBMCs) from cattle, exposing them to Pb acetate for 24?h and analyzing mRNA expression of metal-responsive proteins and selected cytokines. Lead concentrations in cattle blood from Kabwe were significantly greater than those from Lusaka, as were the mRNA expressions of metallothionein-2 (MT-2), tumor necrosis factor-a (TNF-a), interferon-? (IFN-?), interleukin-1 beta (IL-1 beta), IL-6, and inducible nitric oxide synthase (iNOS). The present in vitro study demonstrated that Pb exposure led to an increase in the expressions of MT-2, TNF-a, IL-1 beta, and iNOS, similar to those found in vivo. These results indicate the possibility of immune system modulations in cattle from the Kabwe area. Environ. Toxicol. Chem. 2012; 31: 23002305. (c) 2012 SETAC
• Transcriptional profiling of inflammatory cytokine genes in African, buffaloes (Syncerus caffer) infected with Theileria parva

  Tomohiro Okagawa, Satoru Konnai, Hirohisa Mekata, Naftaly Githaka, Saori Suzuki, Edward Kariuki, Francis Gakuya, Esther Kanduma, Tatsuya Shirai, Ryoyo Ikebuchi, Yoshinori Ikenaka, Mayumi Ishizuka, Shiro Murata, Kazuhiko Ohashi

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 148 (3-4) 373 - 379 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.
• Effects of interferon-tau on cattle persistently infected with bovine viral diarrhea virus

  Junko Kohara, Yumiko Nishikura, Satoru Konnai, Motoshi Tajima, Misao Onuma

  JAPANESE JOURNAL OF VETERINARY RESEARCH 60 (2-3) 63 - 70 0047-1917 2012/08 [Refereed][Not invited]
   

  In this study, the antiviral effects of bovine interferon-tau (boIFN-tau) on bovine viral diarrhea virus (BVDV) were examined in vitro and in vivo. In the in vitro experiments, the replication of cytopathic and non-cytopathic BVDV was inhibited in the bovine cells treated with boIFN-tau. The replication of BVDV was completely suppressed by boIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the effect of boIFN-tau on virus propagation in cattle persistently infected (PI) with non-cytopathic BVDV, boIFN-tau was subcutaneously administered to PI cattle at 10(5) U/kg or 10(6) U/kg body weight 5 times per week for 2 weeks. No physical abnormality such as depression was observed in the cattle during the experiment. The mean BVDV titers in the serum of the PI cattle decreased slightly during the boIFN-tau administration period with the dose of 10(6) U/kg. However, the BVDV titers in the serum returned to the pre-administration level after the final boIFN-tau administration. These results suggest that boIFN-tau demonstrates an anti-BVDV effect, reducing the BVDV level in serum transiently when injected into PI cattle.
• Surveillance of Marek's disease virus in migratory and sedentary birds in Hokkaido, Japan

  Shiro Murata, Yuko Hayashi, Ayumi Kato, Masayoshi Isezaki, Sarah Takasaki, Misao Onuma, Yuichi Osa, Mitsuhiko Asakawa, Satoru Konnai, Kazuhiko Ohashi

  VETERINARY JOURNAL 192 (3) 538 - 540 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.
• Kinetics of regulatory dendritic cells in inflammatory responses during Trypanosoma evansi infection

  H. Mekata, S. Konnai, C. N. Mingala, N. S. Abes, C. A. Gutierrez, A. P. Dargantes, W. H. Witola, N. Inoue, M. Onuma, S. Murata, K. Ohashi

  PARASITE IMMUNOLOGY 34 (6) 318 - 329 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.
• Molecular characterization of immunoinhibitory factors PD-1/PD-L1 in chickens infected with Marek's disease virus

  Ayumi Matsuyama-Kato, Shiro Murata, Masayoshi Isezaki, Rika Kano, Sara Takasaki, Osamu Ichii, Satoru Konnai, Kazuhiko Ohashi

  VIROLOGY 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.
• Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection

  Tomohiro Okagawa, Satoru Konnai, Ryoyo Ikebuchi, Saori Suzuki, Tatsuya Shirai, Yuji Sunden, Misao Onuma, Shiro Murata, Kazuhiko Ohashi

  VETERINARY 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.
• 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. Ohashi

  INTERNATIONAL 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.
• The Leukocyte Population in the Peripheral Blood and the Colostrum of Cows Infected with Bovine Leukemia Virus is Skewed Towards Humoral Immunity

  Seiichi Kakinuma, Yousuke Maeda, Hiromichi Ohtsuka, Kaori Ohmae, Kyouko Ayabe, Satoru Konnai, Masa-aki Oikawa

  INTERNATIONAL JOURNAL OF APPLIED RESEARCH IN VETERINARY MEDICINE 10 (4) 323 - 327 1542-2666 2012 [Refereed][Not invited]
   

  Infection of cows with Bovine leukemia virus (BLV) occurs throughout the world, and is especially prevalent in dairy cows. Thus, the current study determined the immune status of cows infected with BLV by examining the leukocyte population and the cytokine mRNA expression of mononuclear cells in the peripheral blood and colostrum of clinically healthy BLV-positive cows. All samples were obtained on the day of calving within 12 hr after calving. Eighteen cows were seropositive for BLV infection (BLV+ group) and 31 cows tested negative (BLV- group), as determined by the agar gel immunodiffusion assay. The number of major histocompatibility complex class II (MHC-II)+ cluster of differentiation (CD)14- B-cells was significantly higher in BLV seropositive cows in both the peripheral blood and the colostrum. Interleukin (IL)-4 mRNA expression levels in the peripheral blood and the colostrum of the BLV+ group were significantly higher than those of the BLV- group. These results suggest that the immune status of BLV infected cows was skewed toward humoral immunity shortly after calving.
• Identification of TROSPA homologue in Ixodes persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan

  Satoru Konnai, Shinji Yamada, Saiki Imamura, Hideto Nishikado, Naftaly Githaka, Takuya Ito, Ai Takano, Hiroki Kawabata, Shiro Murata, Kazuhiko Ohashi

  TICKS AND TICK-BORNE DISEASES 3 (2) 75 - 77 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.
• A novel relapsing fever Borrelia sp infects the salivary glands of the molted hard tick, Amblyomma geoemydae

  Takano, Ai, Sugimori, Chieko, Fujita, Hiromi, Kadosaka, Teruki, Taylor, Kyle R., Tsubota, Toshio, Konnai, Satoru, Tajima, Tomoko, Sato, Kozue, Watanabe, Haruo, Ohnishi, Makoto, Kawabata, Hiroki

  TICKS AND TICK-BORNE DISEASES 3 (4) 259 - 261 1877-959X 2012 [Refereed][Not invited]
   

  A novel relapsing fever Borrelia sp. was found in Amblyomma geoemydae in Japan. The novel Borrelia sp. was phylogenetically related to the hard (ixodid) tick-borne relapsing fever Borrelia app. Borrelia miyamotoi and B. lonestari. The novel relapsing fever Borrelia sp. was detected in 39 A. geoemydae (39/274: 14.2%), of which 14(14/274: 5.1%) were co-infected with the novel relapsing fever Borrelia sp. and Borrelia sp. tAG, one of the reptile-associated borreliae. Transstadial transmission of the novel relapsing fever Borrelia sp. occurred in the tick midgut and the salivary glands, although Borrelia sp. tAG was only detected in the tick midgut. The difference of the borrelial niche in molted ticks might be associated with borrelial characterization. (c) 2012 Published by Elsevier GmbH.
• Molecular cloning of bovine lymphocyte activation gene-3 and its expression characteristics in bovine leukemia virus-infected cattle

  Tatsuya Shirai, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Saori Suzuki, Yuji Sunden, Misao Onuma, Shiro Murata, Kazuhiko Ohashi

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 144 (3-4) 462 - 467 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.
• Characterization of reptile-associated Borrelia sp in the vector tick, Amblyomma geoemydae, and its association with Lyme disease and relapsing fever Borrelia spp.

  Ai Takano, Hiromi Fujita, Teruki Kadosaka, Satoru Konnai, Tomoko Tajima, Haruo Watanabe, Makoto Ohnishi, Hiroki Kawabata

  ENVIRONMENTAL MICROBIOLOGY REPORTS 3 (5) 632 - 637 1758-2229 2011/10 [Refereed][Not invited]
   

  The genus Borrelia is arthropod-borne infectious agents in vertebrates, and is classified into Lyme disease (LD) Borrelia spp. and Relapsing fever (RF) Borrelia spp. In addition to these Borrelia groups, we recently reported reptile-associated (REP) Borrelia spp. from reptiles and from hard-bodied ticks, which probably transmitted the REP Borrelia spp. In this study, we investigated the presence of REP Borrelia sp. in moulted ticks, and found that trans-stadial transmission of REP Borrelia sp. occurred in the midgut, while it was observed that REP Borrelia sp. entered the salivary gland during blood-feeding. This characteristic is also found in LD Borrelia spp., which are also transmitted by hard-bodied ticks. Although phylogenetic analysis demonstrated that REP Borrelia spp. are similar to RF Borrelia spp., the ecology of the spirochaetes within the vector ticks is different for REP Borrelia spp. and RF Borrelia spp. Elucidation of the evolutionary history of the genus Borrelia and its adaptation to ticks promises to be of great interest to researchers of vector-borne microorganisms.
• Increase of cells expressing PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via PD-L1 blockade

  Ryoyo Ikebuchi, Satoru Konnai, Tatsuya Shirai, Yuji Sunden, Shiro Murata, Misao Onuma, Kazuhiko Ohashi

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

  Shiro Murata, Tsukasa Okada, Rika Kano, Yuko Hayashi, Tomoyuki Hashiguchi, Misao Onuma, Satoru Konnai, Kazuhiko Ohashi

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

  Yusuke Murase, Satoru Konnai, Arata Hidano, Naftali W. Githaka, Takuya Ito, Ai Takano, Hiroki Kawabata, Manabu Ato, Tomoko Tajima, Motoshi Tajima, Misao Onuma, Shiro Murata, Kazuhiko Ohashi

  VETERINARY MICROBIOLOGY 149 (3-4) 504 - 507 0378-1135 2011/05 [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.
• Multilocus Sequence Typing Implicates Rodents as the Main Reservoir Host of Human-Pathogenic Borrelia garinii in Japan

  Ai Takano, Minoru Nakao, Toshiyuki Masuzawa, Nobuhiro Takada, Yasuhiro Yano, Fubito Ishiguro, Hiromi Fujita, Takuya Ito, Xiaohang Ma, Yozaburo Oikawa, Fumihiko Kawamori, Kunihiko Kumagai, Toshiyuki Mikami, Nozomu Hanaoka, Shuji Ando, Naoko Honda, Kyle Taylor, Toshio Tsubota, Satoru Konnai, Haruo Watanabe, Makoto Ohnishi, Hiroki Kawabata

  JOURNAL OF CLINICAL MICROBIOLOGY 49 (5) 2035 - 2039 0095-1137 2011/05 [Refereed][Not invited]
   

  Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.
• Molecular identification and expression analysis of lipocalins from blood feeding taiga tick, Ixodes persulcatus Schulze

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

  EXPERIMENTAL PARASITOLOGY 127 (2) 467 - 474 0014-4894 2011/02 [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.
• Peripheral Blood Leukocyte Populations in Dairy Cows Infected with Bovine Leukemia Virus

  Seiichi KAKINUMA, Hiromichi OHTSUKA, Kaori OHMAE, Kyouko AYABE, Motoharu KAKINUMA, Satoru KONNAI, Masaaki OIKAWA

  Journal of the Japan Veterinary Medical Association 64 (5) 375 - 380 0446-6454 2011 [Refereed][Not invited]
  
• Characterization of CTLA-4, PD-1 and PDL-1 of swamp and riverine type water buffaloes

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

  COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 34 (1) 55 - 63 0147-9571 2011/01 [Refereed][Not invited]
   

  Characterization of CTLA-4, PD-1 and PDL-1 genes from swamp and riverine type water buffaloes was done by molecular cloning, sequencing and phylogenetic analysis. The cloned cDNA of CTLA-4, PD-1 and PDL-1 contained an open reading frame of 666, 849 and 870 nucleotides, encoding a polypeptide of 221, 282 and 298 amino acids, respectively. Nucleotide sequence homology of both CTLA-4 and PDL-1 had 99.8% in swamp and riverine type, which gives the identical polypeptide. Meanwhile, PD-1 genes of swamp and riverine type water buffaloes had 99.2% of homology in nucleotide sequence, which has substitution of two amino acid residues. The hexapeptide motif, phosphatidylinositol 3'-kinase and potential glycosylation sites were conserved within the tribe Bovinae. Phylogenetic analysis confirmed the degree of relationship between the bubaline species and justify the distinctness of each breeds by the bootstrap value generated. (C) 2010 Elsevier Ltd. All rights reserved.
• Two novel Salp15-like immunosuppressant genes from salivary glands of Ixodes persulcatus Schulze tick

  A. Mori, S. Konnai, S. Yamada, A. Hidano, Y. Murase, T. Ito, A. Takano, H. Kawabata, M. Onuma, K. Ohashi

  INSECT MOLECULAR BIOLOGY 19 (3) 359 - 365 0962-1075 2010/06 [Refereed][Not invited]
   

  Salp15, a 15-kDa tick salivary gland protein, is known for several suppressive activities against host immunity and critical functions for the transmission of Lyme borrelia in Ixodes scapularis and Ixodes ricinus, the major vectors found in North America and Western Europe. Salp15 inhibits the activation of cluster of differentiation (CD)4+T-cells through the repression of T-cell receptor (TCR)-triggered calcium fluxes and interleukin (IL)-2 production. Furthermore, Salp15 adheres to the spirochaeta and specifically interacts with its outer surface protein C. The binding of Salp15 to Borrelia burgdorferi protects it from antibody-mediated killing in vitro. The aim of this study is to identify the Salp15 genes in Ixodes persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan. Two cDNA clones encoding the Salp15-like sequence were obtained from salivary glands of fed female ticks. These genes encode 135- and 132-amino acid proteins, designated Salp15 Iper-1 and Salp15 Iper-2, respectively, both having signal peptide sequences and predicted to be secretory proteins. Salp15 Iper-1 and -2 showed 51.8 and 68.2% similarity to I. scapularis Salp15, respectively. Reverse transcriptase PCR analysis showed that Salp15 Iper genes were expressed specifically in the salivary glands throughout life cycle stages of the ticks and were up-regulated by blood feeding. In the I. persulcatus-derived sequences, the C-terminal part, which is the binding domain to the CD4 molecule of T-cells in I. scapularis Salp15, was well conserved. In the future, it will be necessary to analyse immunosuppressive functions of I. persulcatus Salp15 and their interaction with Borrelia spp. in Japan.
• Molecular cloning and expression analysis of bovine programmed death-1

  Ryoyo Ikebuchi, Satoru Konnai, Yuji Sunden, Misao Onuma, Kazuhiko Ohashi

  MICROBIOLOGY AND IMMUNOLOGY 54 (5) 291 - 298 0385-5600 2010/05 [Refereed][Not invited]
   

  Recent work has shown that PD-1, an immune inhibitory receptor, is involved in mechanisms for down-regulating immune responses during tumor progression or chronic viral infection. However, in the case of bovine diseases, there have been no reports on this molecule due to lack of information about bovine PD-1. In this study, we performed identification and preliminary characterization of the bovine PD-1 gene in two breeds of cattle. We cloned full cDNA sequences encoding for PD-1 from both Holstein-Friesian and Japanese Black breeds, and found that both of the genes encoded a 282-amino acid protein, which had a signal sequence, transmembrane domain and an immunoreceptor tyrosine-based inhibitory motif. This bovine PD-1 showed 72.9% and 65.6% homology to human and mouse PD-1, respectively, both of which have been well characterized and documented. Quantitative real-time PCR analysis showed that bovine PD-1 is expressed predominantly in T-cells (such as CD4+ and CD8+ cells) and among PBMCs, and is strongly upregulated on T-cell stimulation via ConA. A limited number of cattle were tested yet, as expected, the degree of PD-1 mRNA expression in CD4+ and CD8+ T-cells was greater in cattle with bovine leukemia virus-induced lymphoma than in uninfected cattle. Further studies to characterize the functions of bovine PD-1 are therefore warranted, in order to elucidate the mechanism of the immunosuppression associated with progression of several diseases and therapy in cattle.
• Molecular detection of trypanosomes in cattle in South America and genetic diversity of Trypanosoma evansi based on expression-site-associated gene 6

  Hirohisa Mekata, Satoru Konnai, William H. Witola, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi

  INFECTION GENETICS AND EVOLUTION 9 (6) 1301 - 1305 1567-1348 2009/12 [Refereed][Not invited]
   

  In South American countries, trypanosomiasis as a result of Trypanosoma evansi and Trypanosoma vivax infections causes significant economic losses in livestock. The objectives of this study were to characterize the epidemiology of bovine trypanosomiasis in South America and to draw a comparison between South American and Asian T. evansi isolates based on the polymorphisms in their transferrin receptor encoding gene 6. We assessed the prevalence rates of T. evansi and T vivax infections in cattle in different regions of Peru and Bolivia using the polymerase chain reaction (PCR) and found that, in Lima and Pucallpa in the Republic of Peru, T. evansi infection rates were 5.8% (6/104) and 2.5% (5/195), respectively, while in Santa Cruz, Republic of Bolivia, the infection rate for T. evansi was 11.5% (59/510). The prevalence rates of T. vivax in Lima and Santa Cruz were 3.8% (4/104) and 0.9% (5/510), respectively. In T. evansi, uptake of host transferrin is mediated by a receptor derived from the two expression site-associated genes 6 and 7 (ESAG6 and ESAG7). We previously showed that the ESAG6 depicts genetic diversity among different isolates of T. evansi in Asia. In this study, we cloned and sequenced the ESAG6 genes from T evansi isolates from South America, and found, in addition to some of the previously observed variants, 20 novel variants of ESAG6 genes which could be categorized into three new clades among the various isolates. To conclude, the results obtained in this study suggest that T evansi isolates from South America are more diverse than the Asian isolates. (C) 2009 Elsevier B.V. All rights reserved.
• PCR-based detection of blood parasites in cattle and adult Rhipicephalus appendiculatus ticks

  Shinji Yamada, Satoru Konnai, Saiki Imamura, Martin Simuunza, Mwelwa Chembensofu, Amos Chota, Andrew Nambota, Misao Onuma, Kazuhiko Ohashi

  VETERINARY JOURNAL 182 (2) 352 - 355 1090-0233 2009/11 [Refereed][Not invited]
   

  To ascertain the infection rate for tick-borne pathogens in Zambia, an epidemiological survey of Theileria parva, Babesia bigemina and Anaplasma marginale in traditionally managed Sanga cattle was conducted using PCR. Of the 71 native Zambian cattle, 28 (39.4%) were positive for T parva, 16 (22.5%) for B. bigemina and 34 (47.9%) for A. marginale. The mixed infection rate in cattle was 8.5% (6/71), 16.9% (12/71), 7.0% (5/71) and 2.8% (2/71) for T. parval B. bigemina, T. parval A. marginale, B. bigeminal A. marginale and T. parval B. bigeminal A. marginale, respectively. To predict the risk for transmission of tick-borne pathogens from ticks to cattle, a total of 74 Rhipicephalus appendiculatus ticks were collected from a location where cattle had been found positive for T parva. Of the ticks collected, 10 (13.5%) were found to be PCR-positive for T parva. The results suggest that the infection rate for tick-borne pathogens was relatively high in Sanga cattle and that adult R. appendiculatus ticks were highly infected with T parva. (C) 2008 Elsevier Ltd. All rights reserved.
• Quantification of water buffalo (Bubalus bubalis) cytokine expression in response to inactivated foot-and-mouth disease (FMD) vaccine

  Claro N. Mingala, Satoru Konnai, Fe A. Venturina, Misao Onuma, Kazubliko Ohashi

  RESEARCH IN VETERINARY SCIENCE 87 (2) 213 - 217 0034-5288 2009/10 [Refereed][Not invited]
   

  This study describes the quantification of cytokine expression of vaccinated water buffaloes with FMD inactivated vaccine. Using real-time PCR quantification assay, expression of Th1 (IL-2, IL-12p40, IFN gamma); Th2 (IL-4, IL-10) and inflammatory (IL-6, TNF alpha) cytokines were quantified weekly for the entire three-week duration of the experiment. It was noted that IFN gamma, IL-10 and TNF alpha had peaked on week three post-vaccination while the remaining cytokines peaked on the second week and decreased by the third week. The counteraction between IFN gamma and IL-4 was noted as well as the possible suppressive action of IL-10 to that of IL-2 and IL-12, which is a common phenomenon between Th1 and Th2 cytokines. Synergy between TNFa and IL-6 was also observed. These findings suggest that within the immune system of water buffalo there is a dynamic cell-mediated and humoral interaction in response to immunogen. This assessment of the cytokine expressions is vital for the study of water buffalo disease progression and concurring protective immune responses. (C) 2009 Elsevier Ltd. All rights reserved.
• Classification of new BVDV isolates from Philippine water buffalo using the viral E2 region

  Claro N. Mingala, Satoru Konnai, Motoshi Tajima, Misao Onuma, Kazuhiko Ohashi

  JOURNAL OF BASIC MICROBIOLOGY 49 (5) 495 - 500 0233-111X 2009/10 [Refereed][Not invited]
   

  Characterization of bovine viral diarrhea virus (BVDV) isolates has been focused of several studies this last decade. Until now lots of new strains are being unfolded maybe due to the viral fast mutation ability. As we focused our research on water buffalo immunology, we were able to identify a probable new BVDV isolates. RNA was extracted from water buffalo blood in the Philippines. The extracted RNA was reverse-transcribed and synthesized cDNA. Oligonucleotide primers from the viral E2 region were used to amplify the target viral gene and later purified, cloned and sequenced. The E2 region with 420 bp nucleotides long was compared with existing published sequences in the GenBank. Based on the constructed phylogenetic tree, the isolated strain showed to be a BVDV type 1b along with Osloss and CP7 strains. Further classification of the new isolates was done within the BVDV type 1b1 group, which was compared with other strains in the sub-group. The analysis revealed that Lamspringe/738, KE9 and 2543/87 were the closest with 92% homology. Additional study is being done to further qualify and quantify the extent of the existence of this new BVDV isolates in water buffalo in the Philippines. This is the first report of BVDV in the Philippines and first concerning BVDV in Philippine water buffalo.
• Cloning and characterization of Rhipicephalus appendiculatus voraxin alpha and its effect as anti-tick vaccine

  Shinji Yamada, Satoru Konnai, Saiki Imamura, Takuya Ito, Misao Onuma, Kazuhiko Ohashi

  VACCINE 27 (43) 5989 - 5997 0264-410X 2009/10 [Refereed][Not invited]
   

  Male tick-derived voraxin alpha and voraxin beta, a pair of testicular proteins, are transferred to female via copulation to stimulate female blood feeding in the tick Amblyomma hebraeum (A. hebraeum). Immunized animals with recombinant (r-)voraxin alpha and voraxin beta have been shown as highly resistant to the tick infestation. In this study, we describe the cloning and characterization of voraxin alpha homologue from the tick Rhipicephalus appendiculatus (R. appendiculatus), the major vector for East Coast fever in Eastern Africa. The sequence analysis of the R. appendiculatus voraxin alpha indicated that the deduced amino acid sequence had high similarity with voraxin alpha of the tick A. hebraeum and Dermacentor variabilis, suggesting that voraxin alpha is conserved in different tick genera. Quantitative RT-PCR and Western blotting analysis showed that male voraxin alpha was predominantly expressed in testis and its expression was induced by blood feeding. X appendiculatus voraxina was not secreted into the host during tick feeding and was detected in mated female hemolymph as measured by Western blotting. Preliminary vaccination of rabbits with r-voraxin alpha elicited the humoral immunity and conferred protective immunity against female ticks, resulting in the reduced fed weight. These results suggest that r-voraxin alpha could be a good candidate as anti-tick vaccine. (C) 2009 Elsevier Ltd. All rights reserved.
• Effect of vaccination with a recombinant metalloprotease from Haemaphysalis longicornis

  Saiki Imamura, Itabajara da Silva Vaz, Satoru Konnai, Shinji Yamada, Chie Nakajima, Misao Onuma, Kazuhiko Ohashi

  EXPERIMENTAL AND APPLIED ACAROLOGY 48 (4) 345 - 358 0168-8162 2009/08 [Refereed][Not invited]
   

  We report the cloning, expression and characterization of an Haemaphysalis longicornis metalloprotease (named HLMP1). The gene encodes a predicted 550 aminoacid protein with similarity to metalloproteases of the reprolysin family. The protein sequence contains a signal sequence, the zinc-binding motif (HEXXHXXGXXH) common to metalloproteases and a cysteine-rich region. Reverse transcription-PCR expression analysis indicates the presence of mRNA in the salivary gland of larva, nymph and adult ticks. Rabbit repeatedly infested with H. longicornis recognized rHLMP1, suggesting that the immune-response against HLMP1 is naturally induced through the feeding of ticks. Vaccination of rabbit with rHLMP1 produced protective immunity against ticks, resulting in 15.6 and 14.6% mortality in nymph and adult ticks, respectively. This work provides information to understand the tick's defense system, and offers new insights to develop strategies to block this defense system with an anti-tick vaccine based on a metalloprotease.
• Identification and characterization of antimicrobial peptide, defensin, in the taiga tick, Ixodes persulcatus

  Y. Saito, S. Konnai, S. Yamada, S. Imamura, H. Nishikado, T. Ito, M. Onuma, K. Ohashi

  INSECT MOLECULAR BIOLOGY 18 (4) 531 - 539 0962-1075 2009/08 [Refereed][Not invited]
   

  Ixodes persulcatus is the primary vector for human tick-borne diseases in Japan. A cDNA library was constructed from whole body homogenates of fed nymphs of I. persulcatus. From this library, one cDNA encoding defensin-like antimicrobial peptide was identified. The amino-acid sequence showed high similarity to those of the defensins of other ticks and arthropods. I. persulcatus defensin mRNA transcripts were detected at all life cycle stages of fed ticks and found to be predominantly expressed in the midguts of adult female ticks, but not in the salivary glands, a finding corroborated by Western blotting analysis. To investigate the function of I. persulcatus defensin, we examined its antibacterial activity by evaluation of growth of several bacterial strains in the presence of the synthetic peptide. The defensin from I. persulcatus markedly inhibited the growth of Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis and Corynebacterium renale, but not Gram-negative bacteria except Escherichia coli O157. In conclusion, these results suggest that I. persulcatus defensin may be playing a significant role in the defence against microbes from bloodmeals.
• Development and application of a quantitative real-time PCR for the diagnosis of Surra in water buffaloes

  Satoru Konnai, Hirohisa Mekata, Claro N. Mingala, Nancy S. Abes, Charito A. Gutierrez, Jesus Rommel V. Herrera, Alan P. Dargantes, William H. Witola, Libertado C. Cruz, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi

  INFECTION GENETICS AND EVOLUTION 9 (4) 449 - 452 1567-1348 2009/07 [Refereed][Not invited]
   

  Trypanosoma evansi (T. evansi) causes the disease called Surra in domestic animals, which is of great economic importance in South Asian countries. In order to improve the diagnosis of Surra, we endeavored to develop a real-time PCR assay for the detection and quantification of parasites in water buffaloes using specific primers for the T. evansi Rode Trypanozoon antigen type (RoTat) 1.2 Variable Surface Glycoprotein (VSG) gene, which is a known diverse DNA region in trypanosomes. The quantitative detection limit of the assay was 10(2) trypanosomes per mL of blood, and the identity of the amplicon was confirmed in all assays by melting curve analysis. To evaluate the clinical applicability of this procedure, detection and estimation of parasitemia in blood samples obtained from water buffaloes and horses were conducted. T evansi was detected in 17/607 (2.8%) blood samples, with parasitemia levels ranging from >10(1) to 10(7) parasites per mL of blood. Interestingly, out of the 17 PCR positive animals, 3 had previously received trypanocidal treatment and 1 had abortion history. These data indicate that real-time PCR for the estimation of putative parasitemia levels is a quantitatively and objectively applicable technique for clinical diagnosis of Surra, and could help to understand disease stage and risk of transmission of T. evansi. (C) 2009 Elsevier B.V. All rights reserved.
• Expression and activity of glycogen synthase kinase during vitellogenesis and embryogenesis of Rhipicephalus (Boophilus) microplus

  Carlos Logullo, William H. Witola, Caroline Andrade, Leonardo Abreu, Josiana Gomes, Itabajara da Silva Vaz, Saiki Imamura, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma

  VETERINARY PARASITOLOGY 161 (3-4) 261 - 269 0304-4017 2009/05 [Refereed][Not invited]
   

  Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism in mammals. GSK-3 belongs to a highly conserved family of serine/threonine protein kinases, whose members are involved in hormonal regulation, nuclear signaling, and cell fate determination in higher eukaryotes. We have cloned and characterized the RmGSK-3 gene from Rhipicephalus (Boophilus) microplus tick embryos. DNA and protein sequence analysis depicted high similarity to the corresponding enzyme, from both vertebrate and invertebrate animals. In addition, the mRNA transcription profile identified during embryogenesis was analyzed. We observed that the RmGSK-3 mRNA rapidly decreases from the 1st to 3rd day of development, and increases from the 3rd to 15th day. After the 15th day of development, we observed a near 50% reduction in RmGSK-3 mRNA transcription in comparison to the 1st day. We detected the GSK-3 P isoform in egg homogenates throughout embryogenesis using Western blot analysis. RmGSK-3 mRNA was present in fat body, midgut and ovary from partially and fully engorged adult female ticks. The highest mRNA level was observed in ovaries from both developmental stages and in first-day eggs. Furthermore, RmGSK-3 activity correlated with glycogen content variation. Finally, kinase activity in egg homogenates was inhibited by the specific inhibitor, SB-216763. These data suggest that RmGSK-3 beta may be involved in glycogen metabolism regulation during R. microplus embryogenesis. (c) 2009 Elsevier B.V. All rights reserved.
• Comparative moleculo-immunological analysis of swamp- and riverine-type water buffaloes responses

  Claro N. Mingala, Satoru Konnai, Libertado C. Cruz, Misao Onuma, Kazuhiko Ohashi

  CYTOKINE 46 (2) 273 - 282 1043-4666 2009/05 [Refereed][Not invited]
   

  This moleculo-epidemiological and immunological study through cytokine response assessment was done to know the dynamics of cytokines in the initiation, persistence and association to physiological changes of a particular pathogen in water buffaloes. This is important to understand the magnitude and behavior of disease progression. Water buffalo blood samples gathered from different places in the Philippines revealed a 9.4%. 27.6%, 10.3% and 4.4% prevalence of bovine viral diarrhea virus (BVDV), bovine leukemia virus (BLV), Anaplasma marginale and Babesia bigemina infection, respectively. This was the first surveillance study of BVDV and BLV in the country. Furthermore, cytokine expression of these naturally infected animals was also quantified. BVDV-infected animals had up-regulated expressions of TNF alpha, IL-2 and IL-4; and down-regulated expressions of IFN gamma and IL-12p40 while BLV positive animals had an up-regulated IL-4 and IL-6, and highly expressed IL-10 and IL-12p40 with unchanged IFN gamma expression. Meanwhile, animals infected with A. marginale had all interleukins and IFN gamma up-regulated with significant expression of IL-10 and IL-12p40 similar to the BLV positive animals. Since it was also observed that swamp-type buffaloes were more disease tolerant than riverine-type buffaloes based on the gathered infection rate of each examined pathogen, further assessment was done focusing on the two vital cytokines, IFN gamma and TNF alpha. We quantified IFN gamma and TNF alpha expressions in ConA-stimulated PBMC from both swamp and riverine buffaloes by real-time PCR. Cytokine expression from ConA-stimulated PBMC revealed that both IFN gamma and TNF alpha were more highly expressed in swamp than in riverine buffalo. To further examine the probable cause of expression differences, the proximal promoter region of these two cytokines were sequenced for the presence of nucleotide polymorphism followed by luciferase assay to analyze the effect of these polymorphisms in gene transcription. A single nucleotide polymorphism was found in the IFN gamma (-299) while eight polymorphisms in the TNF alpha promoter (-541, -553, -562, -596, -609, -655, -659, -688). Luciferase assay showed that both IFN gamma promoter and TNF alpha promoter in swamp-type water buffalo had higher transcription activity compared to riverine-type water buffalo. These findings confirm that IFN gamma and TNF alpha transcriptions in these animals were highly affected by the disparity in the cytokine promoter region. This suggests that disease tolerance or susceptibility of these buffaloes could be due to the differences in their relative cytokine transcription and may relate to pathogen-host specific pathogenesis. (C) 2009 Elsevier Ltd. All rights reserved.
• Microarray Analysis of Host Immune Responses to Marek's Disease Virus Infection in Vaccinated Chickens

  Rika Kano, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 71 (5) 603 - 610 0916-7250 2009/05 [Refereed][Not invited]
   

  Marek's disease (MD) is a commercially important disease of chickens caused by MD virus (MDV). Although avirulent MDV strains have been used for vaccination to prevent MD outbreaks, the protective mechanism of the vaccine has not been elucidated. In this Study, a comprehensive transcriptional analysis using microarray wits conducted in MDV-infected chickens with and without vaccination at 7 and 21 days post-infection (dpi). The data Suggested that the expression of T cell receptor (TCR) 1-related genes was up-regulated in vaccinated-challenged compared to unvaccinated-challenged chickens during the latent phase of infection. Consistently, this induction was confirmed by quantitative PCR. Flow cytometric analysis revealed that most of TCR1(+) cells expressed CD8 alpha chain brightly. The number of this subpopulation was significantly and specifically increased in vaccinated-challenged chickens at 21 dpi compared to univaccinated-challenged chickens, though it was not the major Population in spleen of chickens. The number of CD8 alpha(high) TCR2(+) cells, the major subpopulation of chicken CD8 alpha(high) cells, was increased in vaccinated chickens with or without challenge compared to univaccinated control chickens. These data suggested that both CD8 alpha(high) TCR1(+) and CD8 alpha(high) TCR2(+) cells could be induced by the vaccination. It is also possible that CD8 alpha(high) TCR1(+) cells might be primed by the vaccination and specifically induced by the challenge with virulent strain of MDV during the latent phase of infection. Thus, CD8 alpha(high) TCR1(+) cell Population is probably one of the key factors involved in the protective mechanism induced by a vaccine strain, CV1988.
• Cytokine profiles in chickens infected with virulent and avirulent Marek's disease viruses: Interferon-gamma is a key factor in the protection of Marek's disease by vaccination

  Rika Kano, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi

  MICROBIOLOGY AND IMMUNOLOGY 53 (4) 224 - 232 0385-5600 2009/04 [Refereed][Not invited]
   

  Marek's disease has been controlled by vaccination with avirulent strains of MDV. However, the protection mechanism following vaccination is not fully understood. In this study immune responses of PBMC and splenocytes derived from vaccinated chickens challenged with virulent MDV were examined using real-time PCR and ELISA. Higher levels of IFN-gamma induction were observed in chickens vaccinated during the latent phase of infection with virulent MDV than in similarly challenged, unvaccinated chickens. Furthermore, the mean expression of IFNGR2 and IFN regulatory factor-3 mRNAs was significantly higher in vaccinated than in unvaccinated chickens. These results show that IFN-gamma could be one of the important factors in prevention of MD by vaccination and is effective during the latent phase of the infection.
• Suppression of cell proliferation and cytokine expression by HL-p36, a tick salivary gland-derived protein of Haemaphysalis longicornis

  Satoru Konnai, Chie Nakajima, Saiki Imamura, Shinji Yamada, Hideto Nishikado, Michi Kodama, Misao Onuma, Kazuhiko Ohashi

  IMMUNOLOGY 126 (2) 209 - 219 0019-2805 2009/02 [Refereed][Not invited]
   

  Previously, a putative immunosuppressant-coding gene was identified from a complementary DNA library derived from the salivary glands of partially-fed Haemaphysalis longicornis. Using real-time polymerase chain reaction, the gene was shown to be predominantly expressed during blood feeding with the site of expression being mainly in the salivary glands; this was confirmed by Western blotting analysis. To investigate the function of this novel protein, in this study, we examined the proliferative responses of bovine mononuclear cells and murine splenic cells as well as the expression of profiles of several cytokines in these cells in the presence of the recombinant protein (H. longicornis-derived 36 000 molecular weight protein: rHL-p36). The addition of rHL-p36 at the beginning of the 72 hr cultivation period clearly inhibited proliferation of several mitogen-stimulated cells in a dose-dependent manner, with concomitantly significant down-regulation of messenger RNA levels for interleukin-2. The inhibitory response could be abrogated by blockage of HL-p36 with antibody, suggesting the direct involvement of rHL-p36 in the cell proliferation. Furthermore, the proliferative response of splenocytes isolated from rHL-p36-inoculated mice was significantly lower than for those from control mice, suggesting that rHL-p36 could also directly suppress immune responses in vivo. Interestingly, microarray analysis of the splenocytes showed that the expression of several immunomodulating genes was down-regulated by rHL-p36 inoculation. In conclusion, these results suggest that HL-p36 is an immunosuppressor that might play an important role in the modulation of host immune responses.
• Quantitative Analysis of Cytokine mRNA Expression and Protozoan DNA Load in Theileria parva-Infected Cattle

  Shinji Yamada, Satoru Konnai, Saiki Imamura, Martin Simuunza, Mwelwa Chembensofu, Amos Chota, Andrew Nambota, Misao Onuma, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 71 (1) 49 - 54 0916-7250 2009/01 [Refereed][Not invited]
   

  Theileria parva (T. parva) causes a highly serious bovine disease called Fast Coast fever (ECF), which is characterized by pyrexia, dyspnea and cachexia and is of great economic importance in African Countries. We hypothesize that the clinical symptoms of ECF could he explained by a cytokine dysregulation. In this study, we investigated the relationship between T parva DNA load and expression levels of cytokine mRNAs in leukocytes from experimentally infected calves by quantitative PCR. The p104 gene, which encodes the T parva 104 kDa microneme-rhoptry protein, was detected in cattle blood from day 10 after T. parvo-infected tick infestation, and the protozoan DNA load was increased together with severity of disease. The mRNA expressions of pro-inflammatory cytokines, such as interleukin (IL)-1 beta and IL-6, were up-regulated with protozoan DNA load increasing. In addition, the level of a type-2 cytokine (IL-10) transcript was also increased during the acute phase. In contrast, the down-regulation or no detectable levels of the expression of type-I cytokines, such as IL-2 and interferon (IFN)-gamma were observed in T. parva-infected animals. Thus. Our observations indicated that high protozoan load and resulting intense inflammatory responses might be involved in the severity of clinical signs observed in T. parva-infection.
• Rhipicephalus appendiculatus: Characterization of a testis-associated protein

  Shinji Yamada, Yuko Ito, Saiki Imamura, Satoru Konnai, Takuya Ito, Misao Onuma, Kazuhiko Ohashi

  EXPERIMENTAL PARASITOLOGY 120 (4) 337 - 342 0014-4894 2008/12 [Refereed][Not invited]
   

  A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks. (C) 2008 Elsevier Inc. All rights reserved.
• Prevalence and Source of Trypanosome Infections in Field-Captured Vector Flies (Glossina pallidipes) in Southeastern Zambia

  Hirohisa Mekata, Satoru Konnai, Martin Simuunza, Mwelwa Chembensofu, Rika Kano, William H. Witola, Mwase E. Tembo, Harrison Chitambo, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 70 (9) 923 - 928 0916-7250 2008/09 [Not refereed][Not invited]
   

  The prevalence of trypanosome infections in tsetse flies, Glossina pallidipes, collected front Chiawa and Chakwenga in Zambia with endemic trypanosomosis was assessed by polymerase chain reaction (PCR). Out of the 550 G. pallidipes, 58 (10.5%) flies were found to harbor trypanosome DNA. Infection rates of tsetse with Trypanosoma vivax universal, Trypanosoma congolense savannah, T congolense forest and T. congolense kilifi were 4.2% (23/550), 4.7% (26/550), 1.1% (6/550) and 1.6% (9/550), respectively. To determine the mammalian hosts of T. congolense and T. vivax infections from the tsetse flies, mammalian mitochondrion DNA of blood meal in these flies were analyzed by PCR and subsequent gene sequence analysis of the amplicons. Sequence analysis showed the presence of cytochrome b gene (cyt b) of 7 different mammalian species such as human, elephant, buffalo, goat, warthog, greater kudu and cattle. Goats which were main livestock in these areas were further examined to know the extent of its contribution ill spreading the infection. We examined the prevalence of trypanosome infections in the domestic goat Population in 6 settlements in Chiawa alone. Of the 86 goats sampled, 4 (4.6%), 5 (5.8%), 4 (4.6%) and 4 (4.6%) were positive for T. vivax universal, T. congolense savannah, forest and kilifi, respectively. These findings showed that the host-source of trypanosome infections in vector fly give a vital information about spread of infection. The result of this study will certainly contribute in elucidating more the epidemiology of trypanosomosis.
• Prevalence and Source of Trypanosome Infections in Field-Captured Vector Flies (Glossina pallidipes) in Southeastern Zambia

  Hirohisa Mekata, Satoru Konnai, Martin Simuunza, Mwelwa Chembensofu, Rika Kano, William H. Witola, Mwase E. Tembo, Harrison Chitambo, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 70 (9) 923 - 928 0916-7250 2008/09 [Refereed][Not invited]
   

  The prevalence of trypanosome infections in tsetse flies, Glossina pallidipes, collected front Chiawa and Chakwenga in Zambia with endemic trypanosomosis was assessed by polymerase chain reaction (PCR). Out of the 550 G. pallidipes, 58 (10.5%) flies were found to harbor trypanosome DNA. Infection rates of tsetse with Trypanosoma vivax universal, Trypanosoma congolense savannah, T congolense forest and T. congolense kilifi were 4.2% (23/550), 4.7% (26/550), 1.1% (6/550) and 1.6% (9/550), respectively. To determine the mammalian hosts of T. congolense and T. vivax infections from the tsetse flies, mammalian mitochondrion DNA of blood meal in these flies were analyzed by PCR and subsequent gene sequence analysis of the amplicons. Sequence analysis showed the presence of cytochrome b gene (cyt b) of 7 different mammalian species such as human, elephant, buffalo, goat, warthog, greater kudu and cattle. Goats which were main livestock in these areas were further examined to know the extent of its contribution ill spreading the infection. We examined the prevalence of trypanosome infections in the domestic goat Population in 6 settlements in Chiawa alone. Of the 86 goats sampled, 4 (4.6%), 5 (5.8%), 4 (4.6%) and 4 (4.6%) were positive for T. vivax universal, T. congolense savannah, forest and kilifi, respectively. These findings showed that the host-source of trypanosome infections in vector fly give a vital information about spread of infection. The result of this study will certainly contribute in elucidating more the epidemiology of trypanosomosis.
• Detection of Trypanosoma brucei in field-captured tsetse flies and identification of host species fed on by the infected flies

  Satoru Konnai, Hirohisa Mekata, Raadan Odbileg, Martin Simuunza, Mwelwa Chembensof, William Harold Witola, Mwase Enala Tembo, Harrison Chitambo, Noboru Inoue, Misao Onuma, Kazuhiko Ohashi

  VECTOR-BORNE AND ZOONOTIC DISEASES 8 (4) 565 - 573 1530-3667 2008/08 [Refereed][Not invited]
   

  The prevalence of trypanosome infections in tsetse flies in the Chiawa area of Lower Zambezi in Zambia, with endemic trypanosomosis, was determined by a polymerase chain reaction (PCR) method that allowed the detection of trypanosome DNA and determination of the type of animal host fed on by the tsetse fly Glossina pallidipes, using tsetse-derived DNA extracts as templates. Ninety G. pallidipes (82 females and 8 males; 18.3%) of the 492 flies captured by baited biconical traps tested positive for the presence of Trypanosoma brucei species genomic DNA. Of the 90 T. brucei-positive flies, 47 (52.2%) also tested positive for vertebrate mitochondrial DNA. Sequence analysis of the vertebrate mitochondrial DNA amplicons established that they originated from 8 different vertebrate species, namely, human (Hoino sapiens), African elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), waterbuck (Kobus ellipsipymnus), roan antelope (Hippotragus equinus), greater kudu (Tragelaphus strepsiceros), warthog (Phacochoerus africanus), and goat (Capra hircus). Furthermore, to investigate the prevalence of trypanosome infections in domestic goats in the same area where trypanosomes had been detected in tsetse files, a total of 86 goats were randomly selected from 6 different herds. Among the selected goats, 36 (41.9%) were found to be positive for T. brucei species. This combined detection method would be an ideal approach not only for mass screening for infection prevalence in tsetse populations, but also for the prediction of natural reservoirs in areas endemic for trypanosomosis.
• Serological survey of Toxoplasma gondii in wild waterfowl in Chukotka, Kamchatka, Russia and Hokkaido, Japan

  Takehiro Murao, Yoshitaka Omata, Rika Kano, Shiro Murata, Tsukasa Okada, Satoru Konnai, Mitsuhiko Asakawa, Kazuhiko Ohashi, Misao Onuma

  JOURNAL OF PARASITOLOGY 94 (4) 830 - 833 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.
• Effects of anti-tick cocktail vaccine against Rhipicephalus appendiculatus

  Saiki Imamura, Satoru Konnai, Itabajara da Silva Vaz Junior, Shinji Yamada, Chie Nakajima, Yuko Ito, Tomoko Tajima, Jun Yasuda, Martin Simuunza, Misao Onuma, Kazuhiko Ohashi

  JAPANESE JOURNAL OF VETERINARY RESEARCH 56 (2) 85 - 98 0047-1917 2008/08 [Refereed][Not invited]
   

  Rhipicephalus appendiculatus serpin-3 (RAS-3), R. appendiculatus serpin-4 (RAS-4) and a 36-kDa immuno-dominant protein of R. appendiculatus (RIM36) were reported as candidate antigens for the anti-tick vaccine to control ixodid ticks. In the present study, we generated recombinant proteins of RAS-3 (rRAS-3), RAS-4 (rRAS-4) and RIM36 (rRIM36), and assessed their potency as an anti-tick cocktail vaccine in cattle model. RT-PCR analysis showed that RAS-3, RAS-4 and RIM36 transcripts were detected in both adult male and female ticks during feeding. Immunization of cattle with the combination of rRAS-3, rRAS-4 and rRIM36 had raised antibodies against all recombinants and anti-sera had reacted with the molecules from the tick salivary gland extract. Tick infestation challenge demonstrated protective immunity against female ticks, resulting in mortality rates of 39.5 and 12.8 % for the vaccinated and control groups, respectively. Moreover, the mortality rate of Theileria parva-infected female ticks was 48.5 and 10.8 % in the vaccinated and control group, respectively. In order to evaluate the levels of pathogen transmission capacity by T parva-infected ticks fed on immunized cattle, the occurrence of T parva in the bovine parotid lymph node and peripheral blood was also determined and quantified by real-time PCR. Although the infection with T parva could not be protected by the vaccine, the occurrence of pathogen in peripheral blood was delayed 1 to 2 days after the infestation challenge in vaccinated group. These results suggest that this cocktail vaccine plays a role in the prevention of tick infestation.
• A survey of abortifacient infectious agents in livestock in Luzon, the Philippines, with emphasis on the situation in a cattle herd with abortion problems

  Satoru Konnai, Claro N. Mingala, Misako Sato, Nancy S. Abes, Fe A. Venturina, Charito A. Gutierrez, Takafumi Sano, Yoshitaka Omata, Libertado C. Cruz, Misao Onuma, Kazuhiko Ohashi

  ACTA TROPICA 105 (3) 269 - 273 0001-706X 2008/03 [Refereed][Not invited]
   

  In the Philippines, insufficient consideration has been given to the implementation of systematic control measures against major abortifacient infectious agents in livestock. To elucidate the epidemiology of abortifacient infectious agents in livestock, the prevalence of four abortifacient agents was assessed. Initially, a total of 96 cattle including 17 cows with history of abortion were examined in a herd in Luzon at the request of the farm owner. Six (35.3%) of the 17 aborting cows were found to be serologically positive for Neospora caninum (N. caninum), whereas the seroprevalence in non-aborting cows was 15.9% (10/63). Four of the 6 serologically positive aborting cows were also RT-PCR-positive for bovine viral diarrhea virus (BVDV). Two (12.5%) of the 16 bulls examined were also found to be infected with BVDV, suggesting a putative risk factor of transmission via semen. Based on sequence analysis, the isolates detected belong to BVDV type 1b group. Furthermore, an epidemiological survey of abortifacient infectious agents was conducted with various species of livestock from herds located in Luzon. Out of the 105 water buffalo samples collected, 4 (3.8%) were indicated positive to N. caninum, 2 (1.9%) to Toxoplasma gondii (T gondii) and 2 (1.9%) to Trypanosoma evansi (T evansi). The overall seroprevalence of N. caninum in goat and sheep were 23.6% (21/89) and 26.3% (10/38), respectively. BVDV was not detected in these herds. The findings of this exploratory study indicate a relationship between infection and bovine abortion and that a lager study is required to statistically confirm this relationship. (C) 2007 Elsevier B.V. All rights reserved.
• Cytokine responses in camels (Camelus bactrianus) vaccinated with Brucella abortus strain 19 vaccine

  Raadan Odbileg, Byambaa Purevtseren, Dorj Gantsetseg, Bazartseren Boldbaatar, Tumurjav Buyannemekh, Zagd Galmandakh, Janchivdorj Erdenebaatar, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 70 (2) 197 - 201 0916-7250 2008/02 [Refereed][Not invited]
   

  In the present study, we determined the levels of cytokines produced by camel (Camelus bactrianus) peripheral blood mononuclear cells (PBMCs) in response to live attenuated Brucella abortus (B. abortus) S19 vaccine. Seven camels were vaccinated with commercial B. abortus S 19 vaccine, and their cytokine responses were determined using a real-time PCR assay. Cytokine responses to B. abortus S 19 were examined at 6 hr, 48 hr and 1, 2 and 3 weeks post-vaccination. Serological tests were performed to further confirm these immune responses. The results revealed that IFN-gamma and IL-6 were upregulated during the first week post-vaccination. Low level expressions of IL-1 alpha, IL-1 beta, TNF-alpha and IL-10 and no expression of IL-2 and IL-4 were observed compared with the control camels. The findings showed that B. abortus stimulates cell-mediated immunity by directly activating camel Thl cells to secrete IFN-gamma. This quantification of cytokine expression in camels is essential for understanding of Camelidae disease development and protective immune responses. This is the first report of in vivo camel cytokine quantification after vaccination.
• Establishment of a laboratory colony of taiga tick Ixodes persulcatus for tick-borne pathogen transmission studies

  Satoru Konnai, Yoichi Saito, Hideto Nishikado, Shinji Yamada, Saiki Imamura, Akina Mori, Takuya Ito, Misao Onuma, Kazuhiko Ohashi

  JAPANESE JOURNAL OF VETERINARY RESEARCH 55 (2-3) 85 - 92 0047-1917 2008/01 [Refereed][Not invited]
   

  Ixodes persulcatus Schulze (I. persulcatus) is distributed in Russia and Far East Asia including Japan, and has been implicated as the vector of several human pathogens. In particular, I. persulcatus acts as the only tick vector for human lyme borreliosis in Japan. In order to elucidate the mechanism of transmission of I. persulcatus -borne pathogens, we developed a laboratory colony of I. persulcatus. Ticks were fed on Syrian hamster and engorged ticks that had dropped off the animals were collected and maintained to allow them to molt. Tick rearing was performed in incubator at 20 degrees C with 95% relative humidity and 12-hour light/dark photo-period regimen. We found out that adult females fed for 8 +/- 2 days and had a pre-oviposition period lasting for 7 +/- 2 days. The minimum egg incubation period was 1 month with the hatched larvae feeding for 3 +/- 1 days and molting to nymphs 3-4 months thereafter. Meanwhile, the nymphs fed for 4 +/- 1 days and molted to adult 2-3 months thereafter. For future analysis of gene expression profiles in I. persulcatus, we cloned and sequenced the actin gene (a housekeeping gene), and found that it is 92.7% to 98.6% homologous to the published sequences of related ixodid ticks. This laboratory colony of I. persulcatus will facilitate investigations on the role of tick-derived molecules on the transmission of I. persulcatus -borne pathogens and will be important for identification of potential anti-tick vaccine and acaricide target molecules.
• Development of a nested polymerase chain reaction method to detect oncogenic Marek's disease virus from feather tips

  Shiro Murata, Kyung-Soo Chang, Sung-Il Lee, Satoru Konnai, Misao Onuma, Kazuhiko Ohashi

  JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION 19 (5) 471 - 478 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.
• Detection of the virulent Marek's disease virus genome from feather tips of wild geese in Japan and the Far East region of Russia

  S. Murata, K.-S. Chang, Y. Yamamoto, T. Okada, S.-I. Lee, S. Konnai, M. Onuma, Y. Osa, M. Asakawa, K. Ohashi

  ARCHIVES OF VIROLOGY 152 (8) 1523 - 1526 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.
• Attachment duration required for Rhipicephalus appendiculatus to transmit Theileria parva to the host

  Satoru Konnai, Shinji Yamada, Saiki Imamura, Martin Simuunza, Mwelwa Chembensof, Amos Chota, Andrew Nambota, Kazuhiko Ohashi, Misao Onuma

  Vector-Borne and Zoonotic Diseases 7 (2) 241 - 248 1530-3667 2007/06 [Refereed][Not invited]
   

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

  Claro N. Mingala, Raadan Odbileg, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma

  COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 30 (2) 119 - 131 0147-9571 2007/03 [Refereed][Not invited]
   

  The current research concerned in the cloning, sequencing and phylogenctic analysis of inflammatory cytokine (IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha) genes from swamp buffalo and two bubaline breeds, CB (cross between swamp and riverine type buffalo) and the Bulgarian Murrah buffalo. Multiple sequence comparison showed a high homology between the bubaline breeds, which ranged from 99.3% to 100.0% similarity, whereas from 98.6% to 99.0% compared to cattle. The phylogenetic analysis had confirmed and justified the degree of relationship between these bubaline species and their distinctness to each other by the bootstrap value (%) generated. These findings were discussed with particular attention to the diversity of the inflammatory cytokine proteins within closely related species. The result of this study concluded that a small difference in the cytokine structures might be the reason behind or has a contributory factor on the previous reports about the existence of disease resistance. However, in-depth study is necessary to further qualify these findings. (c) 2007 Elsevier Ltd. All rights reserved.
• Interferon-gamma expression associated with suppression of bovine leukemia virus at the early phase of infection in sheep

  Tatsufumi Usui, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 115 (1-2) 17 - 23 0165-2427 2007/01 [Refereed][Not invited]
   

  Immunological control of bovine leukemia virus (BLV)-infection has been reported as dependent on the expression balance of types 1 and 2 cytokines. In this report, mRNA expression of interferon (IFN)-gamma and interleukin (IL)-2 (type 1 cytokines), and of IL-4 and IL-10 (type 2 cytokines) were evaluated in concanavalin A-stimulated peripheral blood mononuclear cells (PBMC) from BLV-infected sheep. Despite the same dose of BLV-infection, the extent of viral propagation was markedly different between eight individual sheep by 12 weeks post infection. The virus did not propagate well in three sheep, which showed augmented mRNA expression of IFN-gamma, a strong indicator of cell-mediated immunity, immediately after BLV-infection. Among the other five sheep having more than 2% of BLV-infected cells among PBMC at 12 weeks post infection, four sheep developed B-cell leukemia or lymphoma within 2 years after infection. These observations indicate IFN-gamma expression may play an important role in the protective mechanism against BLV propagation at the early phase of the infection. (c) 2006 Elsevier B.V. All rights reserved.
• Comparative assessment of Th1 and Th2 cytokines of swamp type buffalo and other bubaline breeds by molecular cloning, sequencing and phylogenetics

  Claro N. Mingala, Raadan Odbileg, Satoru Konnal, Kazuhiko Ohashi, Misao Onuma

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 113 (3-4) 348 - 356 0165-2427 2006/10 [Refereed][Not invited]
   

  Comparative assessment of Th1 and Th2 cytokines of three bubaline breeds namely swamp buffalo, its crossbreed with riverine buffalo (CB), and the improved breed of Bulgarian Murrah buffalo (BMB), was done by molecular cloning, sequencing and phylogenetic analysis. The Th I cytokines analyzed included IL-2, IL-12p35, IL-12p4O, and IFN-gamma while Th2 cytokines included IL-4 and IL-10. Both groups showed strict conservation in the putative secondary structures and amino acid residues within the tribe Bovini, which indicated functional cross-reactivity. Nucleotide sequence homology ranged from 98.6 to 100.0% and was lowest for IL-12p35. With regard to amino acid sequence, the lowest homology was observed in IL-4 with 97.8%. This substitution was mainly due to differences in mRNA splicing. The phylogenetic relationship of the buffalo breeds was analyzed and showed them as a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle and pigs. A deeper knowledge of these cytokine structures will favor understanding of water buffalo immunology and how much it differs from its closest subspecies and other animals. (c) 2006 Elsevier B.V. All rights reserved.
• Complete cDNA sequences and phylogenetic analyses of the Th1 and Th2 cytokines of the bactrian camel (Camelus bactrianus)

  Raadan Odbileg, Byambaa Purevtseren, Zayat Batsukh, Satoru Konnaii, Kazuhiko Ohashi, Misao Onuma

  JOURNAL OF VETERINARY MEDICAL SCIENCE 68 (9) 941 - 946 0916-7250 2006/09 [Refereed][Not invited]
   

  The complementary DNAs of the Th1 (IL-2, 12-12p35, and IFN-gamma) and Th2 (IL-4, IL-10 and IL-13) cytokine genes of the bactrian camel (Camelus bactrianus) were cloned, sequenced, and analyzed. IL-2, IL-4, IL-10, IL-12p35, IL-13, and IFN-gamma were found to have 465, 402, 537, 669, 411, and 501 bp length open reading frames with 154, 133, 178, 222, 136, and 166 amino acid encodings, respectively. The homology ranged from 58.8% to 100% between the nucleotide sequences of the camel cytokine genes and the published sequences of other mammalian genes, including the llama, pig, cow, horse, human, and mouse. The cDNA had highest homology with orders Artiodactyla (pigs and cattle) and Perissodactyla (horses), especially to the recently cloned llama sequences.
• Expression of tumor necrosis factor-alpha in IgM(+) B-cells from bovine leukemia virus-infected lymphocytotic sheep

  Tatsufumi Usui, Satoru Konnai, Kazuhiko Ohashi, Misao Onuma

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 112 (3-4) 296 - 301 0165-2427 2006/08 [Refereed][Not invited]
   

  Tumor necrosis factor (TNF)-cL is thought to be one of the cytokines that account for bovine leukemia virus (BLV)-induced B-cell lymphoproliferative disorder, however, information on TNF-alpha expression in B-cells is limited. In this study, the expression of TNF-alpha in IgM(+) B-cells from BLV-infected sheep with or without lymphocytosis was determined. Freshly isolated IgM(+) B-cells from three sheep with lymphocytosis constitutively transcribed TNF-alpha mRNA. Although TNF-alpha mRNA expression in IgM(+) B-cells was transiently up-regulated after cell culture, TNF-alpha mRNA expression was markedly higher in lymphocytotic sheep when compared to that of non-lymphocytotic sheep or uninfected sheep. Expression of membranebound TNF-alpha on IgM(+) B-cells was also augmented in lymphocytotic sheep. TNF-alpha expression in lymphocytotic sheep may support the proliferation of B-cells. (c) 2006 Elsevier B.V. All rights reserved.
• Acquisition and transmission of Theileria parva by vector tick, Rhipicephalus appendiculatus

  Satoru Konnai, Saiki Imamura, Chie Nakajima, William Harold Witola, Shinji Yamada, Martin Simuunza, Andrew Nambota, Jun Yasuda, Kazuhiko Ohashi, Misao Onuma

  ACTA TROPICA 99 (1) 34 - 41 0001-706X 2006/08 [Refereed][Not invited]
   

  In order to investigate the transmission dynamics of Theileria parva (T parva) by the brown ear tick, Rhipicephalus appendiculatus (R. appendiculatus), under experimental conditions, detection of T parva in ticks and cattle was performed by a quantitative real-time PCR assay. A calf inoculated with a T parva mixture became PCR-positive for T parva infection on day 8 post-inoculation, and subsequently, nymphal ticks were introduced and maintained to feed on the infected calf for 6 days. Engorged nymphs were collected daily and allowed to molt into adults, and overall, 70.8% (121/171) of the adult ticks acquired the T parva infection. Furthermore, the T parva infection rate in ticks under field conditions was monitored by real-time PCR in R. appendiculatus ticks collected from a traditionally managed pastoral land of Zambia, on which Sanga breed cattle are traditionally reared and the area has endemic East Coast fever (ECF). A total of 70 cattle were randomly selected in the same area and 67 (95.7%) were found to be serologically positive for R. appendiculatus tick antigen (RIM36). Twenty-nine (43.3%) of the 67 serologically positive cattle were real-time PCR-positive for T parva, although no piroplasms could be detected in the blood smears. Unexpectedly, out of 614 R. appendiculatus nymphal and adult ticks collected by flagging vegetation, 4.1% were positive for T parva DNA. However, since the rate of transmission of T parva from infected cattle to ticks and vice versa and the serological evidence of exposure to R. appendiculatus ticks in naturally exposed cattle were relatively high, it would be wise in such a case to consider vector control as well as vaccination against ECF as control measures. (c) 2006 Elsevier B.V. All rights reserved.
• Tumor necrosis factor-alpha genetic polymorphism may contribute to progression of bovine leukemia virus-infection

  Satoru Konnai, Tatsufumi Usui, Manabu Ikeda, Junko Kohara, Toh-ichi Hirata, Kosuke Okada, Kazuhiko Ohashi, Misao Onuma

  MICROBES AND INFECTION 8 (8) 2163 - 2171 1286-4579 2006/07 [Refereed][Not invited]
   

  In a previous report, we had indicated that in a sheep model, the expression of tumor necrosis factor (TNF)-alpha was closely associated with disease progression in sheep experimentally infected with bovine leukemia virus (BLV). However, individual variabilities are observed in these responses in BLV-infected animals. To attempt to identify genetic factors promoting the progression to BLV-induced lymphoma, we endeavored to determine whether there are any polymorphisms in the TNF-alpha gene among 291 individuals and whether this would affect the level of TNF-alpha expression and concomitant progression of BLV-induced disease or increase in the provirus load in the carriers. We found that the frequency of the TNF-alpha -824G allele, which has been associated with low transcription activity of the promoter/predicted enhancer region of the bovine TNF-alpha gene, was higher in individuals with BLV-induced lymphoma than in asymptomatic carrier individuals. In addition, we observed a tendency for increased BLV-provirus load in cattle with TNF-alpha -824G/G homozygote compared to TNF-alpha -824A/A homozygote or TNF-alpha -824A/G. These data suggest that the observed polymorphism in the promoter region of TNF-alpha gene could at least in part contribute to the progression of lymphoma in BLV-infection. (c) 2006 Elsevier SAS. All rights reserved.
• The effect of TAO expression on PCD-like phenomenon development and drug resistance in Trypanosoma brucei

  A Tsuda, WH Witola, S Konnai, K Ohashi, M Onuma

  PARASITOLOGY INTERNATIONAL 55 (2) 135 - 142 1383-5769 2006/06 [Refereed][Not invited]
   

  Drug resistance in Trypanosoma brucei causes severe problems for people and domestic animals, but molecular mechanisms of the resistance are not well known. Programmed cell death (PCD) is a fundamental process in both multicellular and unicellular organisms, and it is speculated to be one of the important factors contributing to the emergence of drug resistance. We have previously reported that the expression of TAO appears to play a role in the inhibition of the PCD-like phenomenon development in T brucei. In this study, to ascertain the correlation between the development of the PCD-like phenomenon and the expression of TAO in T. brucei, we genetically engineered T. brucei for conditional overexpression of the TAO gene. TAO over-expressing transgenic T. brucei was refractory to the development of the PCD-like phenomenon compared to the wild-type, indicating that expression of TAO might have a regulatory role on PCD development. Furthermore, the transgenic cells showed resistance to suramin and antrycide. We postulated that intracellular reactive oxygen species (ROS) may be involved in the mechanism of resistance to antrycide because augmentation of ROS in transgenic cells was lower than that in the wild-type cells following treatment with antrycide. These results suggest a possible correlation of PCD to drug resistance in T. brucei. (c) 2006 Elsevier Ireland Ltd. All rights reserved.
• A novel gene encoding a thrombin inhibitory protein in a cDNA library from Haemaphysalis longicornis salivary gland

  C Nakajima, S Imamura, S Konnai, S Yamada, H Nishikado, K Ohashi, M Onuma

  JOURNAL OF VETERINARY MEDICAL SCIENCE 68 (5) 447 - 452 0916-7250 2006/05 [Refereed][Not invited]
   

  A novel thrombin inhibitory protein coding gene was identified from a cDNA library derived from salivary gland of partially-fed Haemaphysalis longicornis (hard tick). The gene encoded a 93-amino acid protein. designated chimadanin. Which had a signal peptide sequence and was predicted to be a secretory protein. It showed no similarity to any other previously identified proteins or conserved domain sequences. The gene was expressed during blood feeding and suggested to be expressed mainly in the salivary gland. The predicted mature region of chimadanin was expressed in Escherichia coli and characteristics of the recombinant chimadanin were determined. The activated partial thromboplastin time and the prothrombin time in sheep plasma were significantly prolonged by chimadanin in a dose dependent manner. Amidolytic activity of thrombin was also inhibited by chimadanin in a dose dependent manner and it suggested that chimadanin was an anticoagulant with thrombin inhibitory activity. This newly identified thrombin inhibitor may play an important role in tick blood feeding.
• Experimental transmission of Bovine leukemia virus in cattle via rectal palpation

  Junko Kohara, Satoru Konnai, Misao Onuma

  JAPANESE JOURNAL OF VETERINARY RESEARCH 54 (1) 25 - 30 0047-1917 2006/05 [Refereed][Not invited]
   

  We examined whether Bovine leukemia virus (BLV) was transmitted by rectal palpation using a common sleeve between a BLV-infected cow and BLV-negative steers. Three of four steers developed antibodies against BLV as determined by agar-gel immunodiffusion (AGID) test between 7 to 10 weeks after the first rectal palpation using common sleeves from BLV-infected cow. In the steers, BLV proviral DNA were detected by PCR I to 5 weeks earlier than detection of the antibodies by the AGID test. Our experiments demonstrated that rectal palpation is a potential cause of BLV spread in herds and that detection of BLV proviral DNA in cattle by PCR is useful screening test for early diagnosis of BLV infection.
• Tumor necrosis factor-alpha up-regulation in spontaneously proliferating cells derived from bovine leukemia virus-infected cattle

  S Konnai, T Usui, M Ikeda, J Kohara, T Hirata, K Okada, K Ohashi, M Onuma

  ARCHIVES OF VIROLOGY 151 (2) 347 - 360 0304-8608 2006/02 [Refereed][Not invited]
   

  We previously reported that tumor necrosis factor alpha (TNF-alpha) was one of the cytokines that contributed to the leukemogenesis caused by bovine leukemia virus (BLV). To determine if the spontaneous cell proliferation observed in the late disease stages, such as persistent lymphocytosis and lymphosarcoma, correlated with the expression level of TNF-alpha, we analyzed the mRNA expression levels for TNF-alpha in spontaneously proliferating PBMCs derived from BLV-infected cattle. The mean mRNA expression level for TNF-alpha was higher in the spontaneously proliferating PBMCs derived from BLV-infected cattle than in non-spontaneously proliferating PBMCs from normal cattle. The TNF-alpha protein level in the PBMCs was determined by flow cytometric analysis, and it was noted that most of the cells expressing membrane-bound TNF-alpha in the spontaneously proliferating cells were CD5 or sIgM(+) -cells. Additionally, in order to determine if this spontaneous proliferation can be blocked by anti-bovine TNF-alpha MAb, the spontaneously proliferating PBMCs from a BLV-infected cattle were cultured in the presence of the MAb. The addition of this MAb at the beginning of the 72 h-cultivation clearly inhibited spontaneous proliferation of cells in a dose-dependent manner, indicating the direct involvement of TNF-alpha in the spontaneous proliferation of PBMCs during the late disease stage. These data suggest that an aberrant expression of TNF-alpha might contribute to the progression of bovine leukosis in animals which develop persistent lymphocytosis of B-cells or B-cell lymphosarcoma.
• Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis

  HM Pham, S Konnai, T Usui, KS Chang, S Murata, M Mase, K Ohashi, M Onuma

  ARCHIVES OF VIROLOGY 150 (12) 2429 - 2438 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.
• Bovine leukemia virus high tax molecular clone experimentally induces leukemia/lymphoma in sheep

  K Okada, N Nakae, K Kuramochi, S Yin, M Ikeda, S Takami, T Hirata, M Goryo, S Numakunai, S Takeshima, M Takahashi, S Tajima, S Konnai, M Onuma, Y Aida

  JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (12) 1231 - 1235 0916-7250 2005/12 [Refereed][Not invited]
   

  Sheep were inoculated with high tax coded pBLV-IF (H group, Nos.1-5) of bovine leukemia virus (BLV), wild tax coded pBLV-IF (W group, Nos. 6-11), or control plasmid (C group, Nos. 12-14). During the observation period (4 to 46 months), 5 of 5 cases in H group and 3 of 6 cases (Nos. 6, 7, 9) in W group became positive tor gp51. Only 1 case in H group became leukemic, and one case each of H and W groups developed lymphoma. In No. 3, lesions Were found in multiple organs including the lymph nodes, gastrointestinal tract following abomasum, and heart. In No. 6, lesions of lymphoma were found only in the jejunum, and heart. Morphologically, small to middle-sized lymphocytic neoplastic (NP) cells were found in both cases, but lymphoblastic NP cells were found only in No. 3. By immunohistochemical examination, the phenotypes of NP dwells were determined as CD1(-), CD4(-), CD5(-)-, CD8 alpha(-), sIgM(+), lambda light chain(+), B-B4(+), MHC class II+ in both case. The results of this study indicate that inoculation of pBLV-IF can induce lymphocytic and lymphoblastic leukemia/lymphoma in sheep. Additionally, it is suggested that the expression rate of tax gene is not associated with the development of leukemia/lymphoma in sheep experimentally inoculated with pBLV-IF.
• Random sequencing of cDNA library derived from partially-fed adult female Haemaphysalis longicornis salivary gland

  C Nakajima, ID Vaz, S Imamura, S Konnai, K Ohashi, M Onuma

  JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (11) 1127 - 1131 0916-7250 2005/11 [Refereed][Not invited]
   

  A cDNA library was constructed from salivary glands of partially-fed adult female Haemaphysalis longicornis (hard tick). Randomly selected clones were sequenced and a total of 633 sequences were analyzed by bioinformatic programs. The sequences were grouped into 213 clusters, with each cluster being considered to be composed of mRNAs derived from the same gene or closely related genes. About 36% of the mRNA sequences showed significant similarity to known proteins in the non-redundant protein database by the NCBI blastx program and appeared to be coding for functional predicted proteins, whereas the remaining 64% had no similar sequences. Two thirds of the predicted proteins were annotated as basic cellular proteins (housekeeping proteins). Among the functional predicted protein sequences, other than the housekeeping proteins, several protease inhibitors including anticoagulants, two metalloproteases and a potential immunosuppressive protein could be identified. These proteins may play important roles during tick feeding and could be novel anti-tick vaccine candidates.
• Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

  S Konnai, T Usui, M Ikeda, J Kohara, T Hirata, K Okada, K Ohashi, M Onuma

  VIROLOGY 339 (2) 239 - 248 0042-6822 2005/09 [Refereed][Not invited]
   

  Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-alpha and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-alpha-induced responses, in this study we examined the TNF-alpha-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-alpha (rTNF-alpha) was significantly higher than those from AL cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5(+) or sIgM(+) cells and these cells showed resistance to TNF-a-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-alpha-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection. (c) 2005 Elsevier Inc. All rights reserved.
• Molecular cloning and phylogenetic analysis of inflammatory cytokines of Camelidae (llama and camel)

  R Odbileg, S Konnai, K Ohashi, M Onuma

  JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (9) 921 - 925 0916-7250 2005/09 [Refereed][Not invited]
   

  We cloned, sequenced and analyzed the cDNAs encoding Camelidae inflammatory cytokines, including llama (lama glama) interleukin (IL)-1 alpha, IL-1 beta, IL-6, tumor necrosis factor (TNF)-alpha and camel (Camelus bactrianus) IL-6 and TNF-alpha. The similarity levels of the deduced amino acid sequences of IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha from llama (camel) to those from other mammalian species, ranged from 60.7% to 87.7%, 52.8% to 75.3%, 41.4% to 98.6%, and 72.9% to 99.6%, respectively. Phylogenetic analyses based on nucleic acid sequences showed that llama IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha were more closely related to those of camel, pig, cattle, sheep and horse than to those of human, dog, cat, mouse and rat.
• Immmohistochemical analysis of expression patterns of tumor necrosis factor receptors on lymphoma cells in enzootic bovine leukosis

  M Ikeda, S Konnai, M Onuma, N Ishiguro, M Goryo, K Okada

  JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (4) 425 - 432 0916-7250 2005/04 [Refereed][Not invited]
   

  Tumor necrosis factor-alpha (TNF-α) has been reported to be associated with the progression of lymphoproliferative neoplastic diseases and retroviral infections. Hence we examined immunohistochemically the expression patterns of TNF-receptors (TNF-RI and RII) on lymphoma cells derived from the 29 cases of enzootic bovine leukosis (EBL). Lymphomas obtained in 29 animals with EBL were histopathologically classified into three types: diffuse mixed type (10 cases), diffuse large type (9 cases), and diffuse large cleaved type (10 cases). Immunohistochemically using a monoclonal antibody to a bovine lymphocyte surface antigen, the lymphomas were classified into three phenotypes: B-1a (CD5(+)/CD11b(+)), B-1b (CD5(-)/CD11b(+)) and B-2 (conventional 13) (CD5(-)/CDl lb(-)). Interestingly, the lymphoma cells in all animals expressed TNF-RII, but not TNF-RI. Although, in EBL, lymphoma cells of which the histopathological and immunological property differs has been formed, the expression patterns of TNF-Rs had the universality in all lymphoma cells. TNF-RII which induces cell proliferation, was expressed but TNF-RI, which induces cell apoptosis was not expressed on all lymphoma cells, suggesting that TNF-Rs play an important role in the malignant proliferation of B cells and formation of lymphomas in EBL.
• Quantification of llama inflammatory cytokine mRNAs by real-time RT-PCR

  R Odbileg, S Konnai, T Usui, K Ohashi, M Onuma

  JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (2) 195 - 198 0916-7250 2005/02 [Refereed][Not invited]
   

  We have developed a method by which llama cytokine mRNAs can be quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of llama, reverse transcribed to cDNA, and cytokine profiles for interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF) alpha were quantified by real-time PCR. The expressions of mRNAs of inflammatory cytokines IL-1alpha, IL-1beta, IL-6 and TNFalpha were upregulated upon stimulation with LPS in a dose- and time-dependent manner. Incubation of PBMCs with 100 and 1,000 pg/ml of LPS for 3 to 6 hr resulted in the acceleration of the mRNA levels of inflammatory cytokines. Here, we describe a highly sensitive and reproducible method to quantify the transcription of llama cytokine mRNAs by real-time RT-PCR with the double-stranded DNA-binding dye SYBR Green I.
• Ex vivo survival of peripheral blood mononuclear cells in sheep induced by bovine leukemia virus (BLV) mainly occurs in CD5- B cells that express BLV

  Masahiko Takahashi, Shigeru Tajima, Shin-Nosuke Takeshima, Satoru Konnai, Shan Ai Yin, Kosuke Okada, William C. Davis, Yoko Aida

  Microbes and Infection 6 (6) 584 - 595 1286-4579 2004/05 [Refereed][Not invited]
   

  Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis (EBL). In a previous report, we found that in a sheep model, only CD5- B cells proliferated clonally, while CD5+ B cells rapidly decreased when the disease progressed to the lymphoma stage. We demonstrate here that, although both CD5+ and CD5- B cells, but not CD4+ T, CD8+ T and γδTCR +T cells, are protected from spontaneous ex vivo apoptosis in sheep infected with wild-type and a mutant BLV that encodes a mutant Tax D247G protein with elevated trans-activation activity, only CD5- B cells become the main target for ex vivo survival when the disease proceeds to the persistent lymphocytotic stage, which showed an increased expansion of the CD5- B cells. In addition, we identified, by four-color flow cytometric analysis, that in CD5- B cells, the apoptotic rates of cells that expressed wild-type and mutant BLV were greatly decreased compared with those of BLV-negative cells. There was only a slight reduction in the apoptotic rates in BLV-positive cells from CD5+ B cells. In addition, supernatants from peripheral blood mononuclear cell (PBMC) cultures from wild-type- and mutant BLV-infected sheep mainly protected CD5- B cells from spontaneous apoptosis. Our results suggest that, although BLV can protect both CD5 + and CD5- B cells from ex vivo apoptosis, the mechanisms accounting for the ex vivo survival between these two B-cell subsets differ. Therefore, it appears that the phenotypic changes in cells that express CD5 at the lymphoma stage could result from a difference in susceptibility to apoptosis in CD5+ and CD5- B cells in BLV-infected sheep. © 2004 Elsevier SAS. All rights reserved.
• Protective effects of vaccination with bovine leukemia virus (BLV) tax DNA against BLV infection in sheep

  T Usui, S Konnai, S Tajima, S Watarai, Y Aida, K Ohashi, M Onuma

  JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (11) 1201 - 1205 0916-7250 2003/11 [Refereed][Not invited]
   

  A DNA vaccination trial was performed on sheep to determine whether vaccination with bovine leukemia virus (BLV) transactivator Tax DNA is effective against BLV infection. Immunization was carried out with cationic liposomes containing the Tax-expressing plasmid DNA and subsequently all sheep were challenged with BLV. BLV titers in peripheral blood mononuclear cell (PBMC) determined by syncytium formation assay and BLV provirus load detected by genomic PCR analysis showed higher levels of virus titers in control sheep than those in Tax-vaccinated sheep. Higher levels of IFN-gamma mRNA expression have been demonstrated in vaccinated sheep after the challenge. These results suggested that Th1 type immune response induced by Tax DNA vaccine inhibited BLV propagation in vaccinated sheep at the early phase of infection.
• Technical note: DNA typing for ovine MHC DRB1 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

  S Konnai, Y Nagaoka, S Takesima, M Onuma, Y Aida

  JOURNAL OF DAIRY SCIENCE 86 (10) 3362 - 3365 0022-0302 2003/10 [Refereed][Not invited]
   

  The ovine major histocompatibilty complex (Ovar) class II DRB1 second exon was amplified by polymerase chain reaction (PCR) from DNA samples of 52 Suffolk sheep. Polymerase chain reaction products were characterized by the restriction fragment length polymorphism (RFLP) technique using nine restriction enzymes, RsaI, HaeIII, SacI, SacII, DdeI, NciI, Hin1I, EcoRI, and BstNI, yielding 13 types. Sequencing of cloned PCR products identified 16 Ovar-DRB1 alleles. Collectively, all PCR-RFLP patterns exactly matched those predicted from DNA sequences. These findings strongly indicate that the PCR-RFLP method using a combination of nine restriction endonucleases is a very powerful tool in Ovar typing.
• Sequences and diversity of 17 new Ovar-DRB1 alleles from three breeds of sheep

  S Konnai, Y Nagaoka, S Takeshima, M Onuma, Y Aida

  EUROPEAN JOURNAL OF IMMUNOGENETICS 30 (4) 275 - 282 0960-7420 2003/08 [Refereed][Not invited]
   

  To investigate the genetic diversity of the sheep MHC (Ovar) class II DRB1 locus, we amplified exon 2 of Ovar-DRB1 alleles by polymerase chain reaction (PCR) and determined the nucleotide sequences of both resultant strands after cloning. In our study of a total of 97 sheep of three breeds, namely, Suffolk, Cheviot and Corriedale, we identified 18 previously published alleles and 17 new alleles. These alleles were 83.4 to 94.1% identical at the nucleotide level and 71.4 to 90.9% identical at the amino acid level to Ovar-DRB1 *0101 . We identified six new alleles in Cheviot sheep and 11 new alleles in Suffolk sheep. Furthermore, we identified 15, 6 and 1 allele in Suffolk, Cheviot and Corriedale sheep, respectively, that have only been found in these breeds to date. Analysis of the frequencies of the various Ovar-DRB1 alleles in each breed indicated that Ovar-DRB1 *0702 was the most frequent allele in Suffolk sheep (23.9%), Ovar-DRB1 *0203 was the most frequent allele in Cheviot sheep (27.5%) and Ovar-DRB1 *0201 was the most frequent allele in Corriedale sheep (25.0%). A comparative analysis of the positions of polymorphic residues in the first extracellular domain of the DRB genes of sheep, humans and mice revealed an extraordinary similarity amongst the positions of polymorphic residues that are associated with the antigen recognition site (ARS). Moreover, the extent of polymorphism seems to be similar in sheep, humans and mice.
• The rapid quantitative analysis of bovine cytokine genes by real-time RT-PCR

  S Konnai, T Usui, K Ohashi, M Onuma

  VETERINARY MICROBIOLOGY 94 (4) 283 - 294 0378-1135 2003/07 [Refereed][Not invited]
   

  For a practical need, fast and efficient methods to quantify mRNA expression are expecting. By using real-time reverse transcription polymerase chain reaction (RT-PCR) with the double-stranded DNA-binding dye SYBR Green I as a novel method, cytokine profiles (IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12p40 and IFN-gamma) were analyzed in peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected animals. In aleukemic cattle, IFN-gamma and IL-12p40 mRNA expression was significantly increased compared to those in cattle with persistent lymphocytosis. The similar results were obtained in the case of sheep experimentally infected with BLV. Real-time quantitative PCR technique is an applicable technique for analysis of cytokine profiles in field. (C) 2003 Elsevier Science B.V. All rights reserved.
• 牛白血病ウイルス(BLV)Envに対する抗体反応性とウシMHCクラスII DR抗原との相関性

  竹嶋 伸之輔, 嘉村 浩美, 若本 裕晶, 田島 茂, 今内 覚, 森田 裕, 小沼 操, 岡田 幸助, 間 陽子

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 135回 123 - 123 1347-8621 2003/03
• A mutant form of the Tax protein of bovine leukemia virus (BLV), with enhanced transactivation activity, increases expression and propagation of BLV in vitro but not in vivo

  S Tajima, M Takahashi, S Takeshima, S Konnai, SA Yin, S Watarai, Tana, Y Tanaka, M Onuma, K Okada, Y Aida

  JOURNAL OF VIROLOGY 77 (3) 1894 - 1903 0022-538X 2003/02 [Refereed][Not invited]
   

  In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.
• Seroprevalence of bovine immunodeficiency virus and bovine leukemia virus in dairy and beef cattle in Hokkaido

  T Usui, S Meas, S Konnai, K Ohashi, M Onuma

  JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (2) 287 - 289 0916-7250 2003/02 [Refereed][Not invited]
   

  Serological survey of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) infection was conducted in dairy cattle from 10 different regions of Hokkaido, Japan. Among 390 cattle, 11.0% of cattle were BIV-seropositive and 3.3% were BLV-seropositive. Moreover, in two dairy farms, where bovine leukosis has been reported, prevalence of BIV infections were 6.4 and 9.1%, respectively. In contrast, among 150 beef cattle, 16.6% were BIV-seropositive while none was BLV-seropositive. Dual infections with BLV and BIV in dairy cattle were tested by using 107 BLV-seropositive sera, and 20 sera were found BIV-positive (18.7%). These results indicate that BIN infection was widespread in Hokkaido.
• The influence of ovine MHC class II DRB1 alleles on immune response in bovine leukemia virus infection

  S Konnai, NS Takeshima, S Tajima, SA Yin, K Okada, M Onuma, Y Aida

  MICROBIOLOGY AND IMMUNOLOGY 47 (3) 223 - 232 0385-5600 2003 [Refereed][Not invited]
   

  We have reported previously that the alleles of the ovine leukocyte antigen (OLA)-DRB1 gene that encode the Arg-Lys (RK) motif and the Ser-Arg (SR) motif at positions beta(70/71) of the OLA-DRbeta1 domain are associated with resistance and susceptibility, respectively, to development of bovine leukemia virus (BLV)-induced ovine lymphoma. Here, to investigate the different immune response in sheep that carried alleles associated with resistance and susceptible for 30 weeks after infection with BLV, we selected sheep that had the RK/RK or SR/SR genotype among the 52 sheep analyzed by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing of PCR product for the OLA-DRB1 exon 2 and infected them with BLV. Although the number of BLV-infected cells and virus titer had been maintaining low levels throughout the experimental period, the sheep with the RK/RK genotype could induce expansion of CD5-B-cells and rapid production of neutralizing antibody in the early phase of infection. The level of incorporation of [H-3]thymidine by peripheral blood mononuclear cells from the sheep with RK/RK genotype gave a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-helper epitope of the BLV envelope glycoprotein gp51. In contrast, the sheep with SR/SR genotype showed a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-cytotoxic and B-cell epitopes. In such cases, the animals with the RK/RK strongly expressed IFN-gamma, the animals with SR/SR genotype strongly expressed IL-2. To determine the proliferating cells, we tried a blocking assay with monoclonal antibodies such as anti-CD4, -CD8 and -DR molecule. We found that these proliferating cells were MHC-restricted CD4(+) T-cells.
• Effects of chlorpromazine, pentoxifylline and dexamethasone on mRNA expression of lipopolysaccharide-induced inflammatory cytokines in bovine peripheral blood mononuclear cells

  Y Kiku, H Matsuzawa, H Ohtsuka, N Terasaki, S Fukuda, S Kon-Nai, M Koiwa, Y Yokomizo, H Sato, TJ Rosol, H Okada, TO Yoshino

  JOURNAL OF VETERINARY MEDICAL SCIENCE 64 (8) 723 - 726 0916-7250 2002/08 [Refereed][Not invited]
   

  The effects of chlorpromazine (CPZ), pentoxifylline (PTX) and dexamethasone (DEX) on mRNA expression of lipopolysaccharide (LPS)-induced proinflammatory cytokines were examined in bovine peripheral blood mononuclear cells (PBMCs) in vitro. The expression of inflammatory cytokine mRNAs was analyzed by RT-PCR and Southern blot hybridization in bovine PBMCs. CPZ and DEX decreased the expression of cytokine mRNA (such as interleukin-1beta and tumor necrosis factor-a) after stimulation with LPS in a dose-dependent manner. However, pretreatment with PTX had no inhibitory effect on the mRNA expression of proinflammatory cytokines. These results indicated that pretreatment with CPZ and DEX might be effective to reduce the production of LPS-induced inflammatory cytokines in bovine PBMCs in vitro.
• BLV-Tax発現DNAワクチンのウイルス増殖抑制効果

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

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 133回 95 - 95 1347-8621 2002/03
• ヒツジMHC(OLA)-DRB1の塩基配列の決定と多型解析

  今内 覚, 長岡 淑子, 小沼 操, 間 陽子

  MHC: Major Histocompatibility Complex (一社)日本組織適合性学会 8 (2) 122 - 122 2186-9995 2001/09
• 異なるMHCを有するBLV感染羊の免疫応答

  今内 覚, 竹嶋 伸之輔, 田島 茂, 岡田 幸助, 小沼 操, 間 陽子

  MHC: Major Histocompatibility Complex (一社)日本組織適合性学会 8 (2) 136 - 136 2186-9995 2001/09
• Inflammatory Cytokines and Antigen-Responsive Mononuclear Cells in Peripheral Blood of Cattle Infected with Salmonella Takoradi

  KONNAI Satoru, OHTSUKA Hiromichi, SAKAMOTO Tetsuaki, NAKAOKA Yuji, KIKU Yukio, FUKUDA Sigeo, KOIWA Masateru, TAKAHASHI Junkichi, TANIYAMA Hiroyuki, YOKOMIZO Yuichi, OKADA Hiroyuki, YOSHINO Tomoo

  Journal of Veterinary Medical Science 社団法人日本獣医学会 63 (8) 859 - 865 0916-7250 2001/08/25 [Refereed][Not invited]
   

  To determine the immunological response in lactating dairy cows infected with Salmonella (S.) Takoradi, the relationships among distributions of peripheral blood mononuclear cell (PBMC) subpopulations, endotoxin concentrations and dynamics of inflammatory cytokines in blood were investigated. The ratio of CD4⁺ T cells to CD8⁺ T cells was significantly lower in the affected cattle than in the control cattle (p<0.05) to decrease in the number of CD4⁺ T cells in the infected cattle. In contrast, the numbers of γδ-T cells, MHC class II-positive cells were significantly higher in the affected cattle than in the control cattle (p<0.01 respectively). Endotoxemia was found in all but one of the affected cattle. Serum IL-1 and IL-6 bioactivities were significantly higher in the affected cattle than in the control cattle (IL-1, p<0.05; IL-6, p<0.01). Serum TNF-α activities and levels were not detected in the control and affected cattle. The activities of proinflammatory cytokines determined by the bioassay are important to the relationships between concentration of endotoxin, cytokines and clinical signs, such as leukocytosis, leukopenia, fever or bacterial shedding. Serum IL-2 levels were lower in the affected cattle than in the control cattle. Serum IFN-γ was not detected in the affected cattle except one. These results by the ELISA seemed to reflect the condition of subpopulation in the PBMCs from the shedding cattle. The present results suggest that cellular immunity is suppressed while the humoral immunity is activated in acute bovine salmonellosis.
• 牛白血病ウイルス(BLV)Tax変異体をコードする感染性分子クローン接種ヒツジにおけるウイルス動態の解析

  田島 茂, 今内 覚, 竹嶋 伸之輔, 高橋 雅彦, 渡来 仁, 小沼 操, 岡田 幸助, 間 陽子

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 131回 84 - 84 1347-8621 2001/03
• ウシ白血病ウイルス(BLV)感染初期羊におけるCD4+T細胞のBLV抗原に対する反応性

  尹 善愛, 池田 学, 御領 政信, 沼宮内 茂, 平田 統一, 今内 覚, 小沼 操, 間 陽子, 岡田 幸助

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 131回 92 - 92 1347-8621 2001/03
• 異なるMHCを有するBLV感染羊の免疫応答

  今内 覚, 竹嶋 伸之輔, 田島 茂, 岡田 幸助, 小沼 操, 間 陽子

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 131回 92 - 92 1347-8621 2001/03
• Effects of Lipopolysaccharide on Production of Interleukin-1 and Interleukin-6 by Bovine Mammary Epithelial Cells in vitro

  Hiroyuki Okada, Hiromichi Ohtsuka, Satoru Kon-Nai, Rikio Kirisawa, Yuichi Yokomizo, Tomo-O. Yoshino, Thomas J. Rosol

  Journal of Veterinary Medical Science 61 (1) 33 - 35 1782-155X 1999/01 [Refereed][Not invited]
   

  This investigation was performed to determine the effect of lipopolysaccharide (LPS) on production of interleukin (IL)-1 and IL-6 by bovine mammary epithelial cells in vitro. After confluence, the cells were stimulated with LPS (0.1, 1.0 or 10 μg/m/) for 4, 8, 24, and 48 hr. LPS increased production of both IL-1 and IL-6 production from mammary cells in a dose dependent manner. The expression of mRNA for IL-1 receptor antagonist (IL-1ra) was demonstrated by reverse transcription-polymerase chain reaction in bovine mammary epithelial cells.
• Effects of lipopolysaccharide on production of interleukin-1 and interleukin-6 by bovine mammary epithelial cells in vitro

  H Okada, H Ohtsuka, S Kon-Nai, R Kirisawa, Y Yokomizo, T Yoshino, TJ Rosol

  JOURNAL OF VETERINARY MEDICAL SCIENCE 61 (1) 33 - 35 0916-7250 1999/01 [Refereed][Not invited]
   

  This investigation was performed to determine the effect of lipopolysaccharide (LPS) on production of interleukin (IL)-1 and IL-6 by bovine mammary epithelial cells in vitro. After confluence, the cells were stimulated with LPS (0.1, 1.0 or 10 mu g/ml) for 4, 8, 24, and 48 hr. LPS increased production of both IL-1 and IL-6 production from mammary cells in a dose dependent manner. The expression of mRNA for IL-1 receptor antagonist (IL-1ra) was demonstrated by reverse transcription-polymerase chain reaction in bovine mammary epithelial cells.

Books etc

• 動物衛生学 第2版

  今内 覚 (Joint work分担執筆, 範囲:コラム 牛伝染性リンパ腫 (旧名：牛白血病))
  文永堂出版 2024/03
• 周産期疾病から牛を守る

  今内 覚 他 (Contributor第Ⅰ章 重要疾病の基礎知識と周産期免疫の新知見: ５ 周産期に免疫が低下するメカニズムの一端を解明)
  デーリィマン編集部 2022/11
• 獣医内科学第3版 産業動物編

  今内 覚 (Contributor第11章 血液・造血臓器疾患 5. 血液の感染症 1) 牛伝染性リンパ腫)
  一般社団法人 日本獣医内科アカデミー 編・文永堂出版 2022/03
• 主要症状を基礎にした牛の臨床３

  今内 覚 他 (Contributorトリパノソーマ病、悪性カタル熱)
  デーリィマン社 2020/05
• Academic Fantasista 免疫療法で動物を救う

  今内 覚 (Contributor免疫療法で動物を救う https://www.hokudai.ac.jp/pr/hokudai_af2018.pdf)
  北海道大学 2019/04
• 動物の感染症＜第4版＞

  今内 覚 (Contributor総論:IV感染症の予防と治療: 1. 感染症の予防, 各論 : 11 アデノウイルス病, 19 牛免疫不全ウイルス感染症.)
  近代出版 2019/03
• 北大など、動物用の免疫チェックポイント阻害薬を開発 ー 目指すは病気の違いを越えた汎用免疫療法 ー

  今内 覚 (Others)
  日経バイオテクONLINE 2017/10
• テレビ・ドクター４ よく分かる乳牛の病気100選

  今内 覚 (Contributor近年の重大疾病と予防策 地方病型(流行型)牛白血病、皮膚に異常を示す病気 散発性牛白血病の項)
  デーリィマン社 2017/10
• 動物衛生学

  今内 覚 (Contributorコラム 牛白血病)
  文永堂出版 2017/09
• 動物用ワクチンとバイオ医薬品 -新たな潮流-

  Satoru Konnal (Contributor第1章 総論 4. 抗体医薬: 免疫チェックポイント阻害薬等)
  文永堂出版 2017/07
• 牛白血病防除の観点から

  今内 覚 (Others検定検査乳S)
  検定検査乳S 34:7 2016/01
• 現代農業・牛白血病を出さない！尾鈴のBL対策

  宮崎県JA尾鈴 (Others牛白血病を出さない！尾鈴のBL対策)
  農山漁村文化協会 2015/05
• 獣医免疫学

  今内 覚 ほか (Contributor第13章 ワクチン)
  緑書房 2015
• 新製品の登場で注目集める動物用医薬品

  今内 覚 (Others)
  日経バイオテク 2014/09
• 牛病学〈第三版〉

  今内 覚 (ContributorV. 感染症の制御 3) 免疫疲弊化と免疫賦活化)
  近代出版 2013

Conference Activities & Talks

• Immune Checkpoint Inhibitors 〜Breakthrough Cancer Therapy and its Application to Veterinary Medicine 〜  [Invited]

  Satoru KONNAI

  Seminario Especial in Universidade Federal do Rio Grande do Sul  2024/03
• 牛伝染性リンパ腫の現状から最後の砦：発症予測法について  [Invited]

  今内 覚

  新潟県獣医師会産業動物部会特別講習会  2024/03
• 動物も新薬で病気から救える時代に  [Invited]

  今内 覚

  国民との科学・技術対話事業  2024/02
• 獣医学から生まれる医学への貢献 汎動物学(ズービキティ）とは？

  今内 覚

  One Health リサーチセンター 開所記念 市民公開講座  2024/02
• 牛伝染性リンパ腫ってどういう病気？ 感染・発症・診断の基本と最前線  [Invited]

  今内 覚

  細胞診クラブ主催 牛伝染性リンパ腫フォーラム【基本と最前線×東洋医学的解析×鍼灸治療】  2024/01
• 牛伝染性リンパ腫の現状から最後の砦：発症予測法について  [Invited]

  今内 覚

  第41回日本獣医師会獣医学術学会年次大会（令和5年度）特別企画  2023/12
• 牛伝染性リンパ腫とは？ 最後の砦！発症予測診断法RAISINGとは？  [Not invited]

  今内 覚

  2023アグリビジネス創出フェア in Hokkaido －北海道の食と農の明日へ－  2023/11
• 早期発症診断によって経済損失低減を実現する 牛のがん検診  [Not invited]

  今内 覚

  知の集積と活用の場 産学官連携協議会2023  2023/11
• Novel livestock disease prevention method using probiotics instead of antibiotics  [Invited]

  Satoru KONNAI

  7th International Livestock Biotechnology Symposium  2023/10
• 牛伝染性リンパ腫対策の最新知見  [Invited]

  今内 覚

  令和5年岩手県獣医師会研修会  2023/10
• イヌの鼻腔内腺癌や骨肉腫に免疫チェックポイント阻害剤が有効であることを初めて報告 ～イヌ用抗PD-L1抗体による免疫療法の適用拡大に期待～

  今内 覚ら

  プレスリリース(https://www.hokudai.ac.jp/news/pdf/231005_pr.pdf)  2023/10
• 酪農教育ファームにおける安全・衛生  [Invited]

  今内 覚

  中央酪農会議 令和5年度酪農教育ファームスキルアップ研修会  2023/09
• 牛伝染性リンパ腫における最近の知見、新たな発症予測診断について  [Invited]

  今内 覚

  北海道獣医師会留萌支部獣医新技術講習会  2023/09
• 牛伝染性リンパ腫への対策に向けて  [Invited]

  今内 覚

  松前町肉牛改良センター特別講演会  2023/06
• Combination therapy effective against canine melanoma

  Konnai S

  Research Press Release (https://www.global.hokudai.ac.jp/blog/combination-therapy-effective-against-canine-melanoma/)  2023/06
• イヌ悪性黒色腫に対して放射線治療との併用で 抗PD-L1抗体の効果が高まることをはじめて報告 ～イヌ用免疫チェックポイント阻害薬のより良い使用法の実現に期待～

  今内 覚ら

  プレスリリース(https://www.hokudai.ac.jp/news/pdf/230602_pr.pdf)  2023/06
• 動物難治性疾病に対する新規予防・治療法の開発  [Invited]

  今内 覚

  第15回大動物臨床研究会特別セミナー  2023/05
• 動物難治性疾病に対する新規予防・治療法の開発  [Invited]

  今内 覚

  北里大学第486回獣医学科セミナー＜特別講演＞  2023/05
• 牛のリンパ腫発症を予測するがん検診技術  [Invited]

  今内 覚

  岩手県中央家畜保健衛生所セミナー  2023/04
• PD-L1の阻害により既存のワクチン効果を増強 ～子牛のワクチンプログラムへの応用に期待～

  今内 覚ら

  プレスリリース(https://www.hokudai.ac.jp/news/pdf/230316_pr2.pdf)  2023/03
• 動物も新薬で病気から救える時代に  [Invited]

  今内 覚

  国民との科学・技術対話事業  2023/03
• 牛伝染性リンパ腫 (旧名：牛白血病)の現状と早期発症診断による経済損失低減の実現  [Invited]

  今内 覚

  山口県畜産振興協会令和4年度家畜生産農場衛生対策事業  2023/02
• Development of effective preventive and therapeutic methods for animal diseases  [Invited]

  Satoru KONNAI

  Special seminar in Mongolian University of Life Sciences  2023/02
• 動物も新薬で病気から救える時代に  [Invited]

  今内 覚

  国民との科学・技術対話事業  2022/11
• 牛のリンパ腫発症を予測するがん検診技術を開発～発症予測法の実用化による畜産被害の軽減に期待～

  今内 覚ら

  プレスリリース(https://www.hokudai.ac.jp/news/2022/10/post-1113.html)  2022/10
• Breakthrough in cancer therapy  [Invited]

  Satoru KONNAI

  Capability building of ASEAN biotechnology researchers for livestock resiliency and sustainability  2022/07
• Importance of building global network for livestock resiliency and sustainability  [Invited]

  Satoru KONNAI

  Capability building of ASEAN biotechnology researchers for livestock resiliency and sustainability  2022/07
• Novel livestock disease prevention method using probiotics instead of antibiotics  [Invited]

  Satoru KONNAI

  Capability building of ASEAN biotechnology researchers for livestock resiliency and sustainability  2022/07
• 家畜伝染病ヨーネ病の現状と病態発生機序について  [Invited]

  今内 覚

  第97回日本結核・非結核性抗酸菌症学会学術講演会エキスパートセミナー  2022/07
• 動物難治性疾病に対する治療と実際、未来  [Invited]

  今内 覚

  北里大学第471回獣医学科セミナー＜Cutting edge＞  2022/05
• 母牛はわが身を削って子牛を出産する～牛伝染性リンパ腫と分娩との関係，周産期に疾病が多発するメカニズムの一端を証明～  [Not invited]

  今内 覚ら

  プレスリリース(https://www.hokudai.ac.jp/news/pdf/220310_pr.pdf)(https://www.hokudai.ac.jp/news/2022/03/post-1006.html)  2022/03
• 牛伝染性リンパ腫の防除戦略  [Invited]

  今内 覚

  全国農業共済協会 令和3年度 家畜診療等技術全国研究集会  2022/02
• 動物用バイオ医薬品の開発と応用研究  [Invited]

  今内 覚

  令和3年度日本薬剤師会農林水産薬事薬剤師部会動物薬事研修会  2022/02
• 動物難治性疾病に対する治療と実際、未来  [Invited]

  今内 覚

  国民との科学・技術対話事業  2022/02
• 牛伝染性リンパ腫における免疫学的解析を基盤とした新制御法の試み  [Invited]

  今内 覚

  日本獣医学会市民公開講座  2022/02
• 動物難治性疾病に対する治療と実際、未来  [Invited]

  今内 覚

  国民との科学・技術対話事業  2022/02
• 牛伝染性リンパ腫の病態発生機序解析を基盤とする新制御法の試み  [Invited]

  今内 覚

  日本家畜衛生学会 第94回大会・家畜衛生フォーラム2021  2021/12
• 牛伝性性リンパ腫の最新知見について  [Invited]

  今内 覚

  令和3年度家畜保健衛生所病性鑑定技術検討会  2021/11
• 牛伝染性リンパ腫の現状と対策  [Invited]

  今内 覚

  ささえや製薬学術セミナー  2021/10
• 動物の慢性感染症と免疫チェックポイント分子  [Invited]

  今内 覚

  十勝家畜保健衛生所講演会  2021/09
• 牛伝染性リンパ腫の新たな知見  [Invited]

  今内 覚

  北海道獣医師会十勝支部産業講習会  2021/09
• 牛伝染性リンパ腫の最新知見  [Invited]

  今内 覚

  オホーツク獣医師会獣医新技術講習会  2021/07
• 免疫チェックポイント阻害薬による腫瘍免疫療法について 〜北海道大学動物医療センターにおける臨床研究成果から〜  [Invited]

  今内 覚

  One Healthリサーチセンター キックオフシンポジウム  2021/05
• Research Press Release: New therapy target for malignant melanomas in dogs

  Konnai S

  Research Press Release (https://www.global.hokudai.ac.jp/blog/new-therapy-target-for-malignant-melanomas-in-dogs/)  2021/04
• Probiotics keep calves healthy, too!  [Not invited]

  Probiotics keep, calves healthy, too

  Research Press Release (https://www.global.hokudai.ac.jp/blog/probiotics-keep-calves-healthy-too/)  2021/04
• 続報・肺転移のあるイヌ悪性黒色腫に抗PD-L1抗体が有効であることをはじめて実証 ～イヌ用免疫チェックポイント阻害薬の実現に大きく前進～  [Not invited]

  今内 覚, 前川直也ら

  プレスリリース(https://www.hokudai.ac.jp/news/pdf/210215_pr.pdf) (https://www.hokudai.ac.jp/news/2021/02/pd-l1.html)  2021/02
• Deeper insight into how tick spit suppresses cattle immunity  [Not invited]

  Konnai S

  Research Press Release (https://www.global.hokudai.ac.jp/blog/deeper-insight-into-how-tick-spit-suppresses-cattle-immunity/) (https://conexao.ufrj.br/2021/01/28/parceria-nipo-brasileira-estuda-como-carrapatos-afetam-bovinos/) (https://www.correiodopovo.com.br/especial/chance-de-al%C3%ADvio-para-bovinos-1.573426)  2021/01
• プロバイオティクスで子牛を下痢から守る～発酵哺乳飼料による子牛の腸炎防御効果を証明～  [Not invited]

  今内 覚, 茅先 史, 岡川朋弘ら

  プレスリリース (https://www.hokudai.ac.jp/news/pdf/210118_pr.pdf) (https://www.hokudai.ac.jp/news/2021/01/post-783.html)  2021/01
• マダニ唾液が免疫チェックポイント因子の発現を誘導～マダニ媒介性病原体の伝播機序の解明に期待～  [Not invited]

  今内 覚, 佐治木大和ら

  プレスリリース (https://www.hokudai.ac.jp/news/pdf/210114_pr3.pdf)(https://www.hokudai.ac.jp/news/2021/01/post-779.html)  2021/01
• プロスタグランジンE2を介した免疫チェックポイント阻害薬の新たな耐性獲得機構の解明～新たな免疫療法への応用に期待～  [Not invited]

  今内 覚, 佐治木大和ら

  プレスリリース(https://www.hokudai.ac.jp/news/pdf/201222_pr.pdf)(https://www.hokudai.ac.jp/news/2020/12/e2.html)  2020/12
• 子牛の下痢症に対する発酵代用乳の腸炎抑制効果の検証  [Invited]

  今内 覚

  令和2年度獣医療提供体制整備推進総合対策事業：新規獣医師臨床研修促進事業 実践実習  2020/10
• 家畜の慢性感染症制御の難しさ 〜免疫から逃れる病原体たち〜  [Invited]

  今内 覚

  令和2年度獣医療提供体制整備推進総合対策事業：新規獣医師臨床研修促進事業  2020/10
• 牛伝染性リンパ腫(旧名：牛白血病) の最新知見 北海道大学による臨床検査診断結果に基づく実践的対策の紹介  [Invited]

  今内 覚

  令和2年家畜診療等技術北海道地区研修会  2020/10
• 動物用バイオ医薬品実用化を可能とする大量生成技術の構築  [Invited]

  今内 覚

  イノベーション創出強化研究推進事業研究紹介2020 (http://www.naro.affrc.go.jp/laboratory/brain/innovation/results/2019/26058BC.html)  2020/10
• 免疫療法 〜免疫チェックポイント阻害剤について〜  [Invited]

  今内 覚

  北海道大学One Healthフロンティア卓越大学院プログラムOne Health Relay Report https://onehealth.vetmed.hokudai.ac.jp/news/220  2020/03
• 動物の難病(がん・感染症)に対する創薬研究  [Invited]

  今内 覚

  日本学術振興会 科研費 研究成果トピックス https://www.jsps.go.jp/j-grantsinaid/37_topics/data/10101-40396304.pdf  2020/03
• 動物用バイオ医薬品の開発と応用研究  [Invited]

  今内 覚

  農林水産省動物医薬品検査所 令和元年度第１回特別講演会  2020/01
• 牛白血病の最新知見  [Invited]

  今内 覚

  北海道獣医師会宗谷支部令和元年度新技術講習会  2019/11
• 牛白血病の最新知見〜北海道大学臨床検査診断結果に基づく実践的対策の紹介〜  [Invited]

  今内 覚

  みなみ北海道農業共済組合南部家畜診療センター講習会  2019/11
• 抗生剤に頼らないプロバイオティクスを応用した新規家畜疾病予防法の開発 〜発酵哺乳飼料による子牛の下痢抑制効果〜  [Invited]

  今内 覚

  みなみ北海道農業共済組合南部家畜診療センター講習会  2019/11
• 動物の難病(がん・感染症)に対する 創薬研究(新薬の開発)  [Invited]

  今内 覚

  国民との科学・技術対話  2019/10
• 動物用抗体医薬品の開発および臨床研究  [Invited]

  今内 覚

  公益社団法人日本動物用医薬品協会第51回学術講習会  2019/10
• 動物難治性疾病に対する創薬研究  [Invited]

  今内 覚

  WISE特別セミナー2019  2019/10
• Effects of bovine tumor necrosis factor alpha decoy receptors on cell death and inflammatory cytokine kinetics: potential for bovine inflammation therapy  [Not invited]

  Fujisawa S, Konnai S, Okagawa T, Maekawa N, Goto S, Murata S, Ohashi K

  International Veterinary Immunology Symposium 2019  2019/08
• Functional analysis of bovine CTLA-4 in bovine leukemia virus infection  [Not invited]

  Watari K, Konnai S, Okagawa T, Maekawa N, Goto S, Murata S, Ohashi K

  International Veterinary Immunology Symposium 2019  2019/08
• Development of a sensitive PD-L1 immunohistochemistry of canine cancers and clinical efficacy of an anti-PD-L1 antibody in canine oral malignant melanoma with pulmonary metastasis  [Not invited]

  Maekawa N, Konnai S, Okagawa T, Murata S, Ohashi K

  International Veterinary Immunology Symposium 2019  2019/08
• Immunomodulatory effects of sialostatin L and sialostatin L2 from Ixodes persulcatus Schulze, Taiga tick  [Not invited]

  Satoru Konnai, Yamato Sajiki, Tomohiro Okagawa, Naoya Maekawa, Masayoshi Isezaki, Shiro Murata, Kazuhiko Ohashi

  International Veterinary Immunology Symposium 2019  2019/08
• The induction of immunosuppression via prostaglandin E2 and the enhancement of anti-bacterial effects by anti-PD-L1 antibody combined with COX-2 inhibitor in Mycoplasma bovis infection  [Not invited]

  Goto S, Konnai S, Okagawa T, Maekawa N, Murata S, Ohashi K

  International Veterinary Immunology Symposium 2019  2019/08
• Contribution of prostaglandin E2 to disease progression and enhancement of antiviral effects by anti-PD-L1 antibody combined with COX-2 inhibitor in bovine leukemia virus infection  [Not invited]

  Sajiki Y, Konnai S, Okagawa T, Maekawa N, Murata S, Ohashi K

  International Veterinary Immunology Symposium 2019  2019/08
• Establishment of anti-bovine PD-1 chimeric antibody and a pilot clinical study  [Not invited]

  Satoru Konnal, Tomohiro Okagawa, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi

  International Veterinary Immunology Symposium 2019  2019/08
• ウシマイコプラズマ感染症における免疫疲弊化  [Invited]

  今内 覚

  日本マイコプラズマ学会第46会学術集会  2019/05
• 牛白血病臨床検査診断結果に基づく実践的対策  [Invited]

  今内 覚

  上川青年獣医師会特別講演  2019/05
• 獣医学領域における感染症の制御  [Invited]

  今内 覚

  微生物化学研究所特別講演  2019/05
• 免疫チェックポイントを標的としたイヌ免疫療法の試み  [Invited]

  今内 覚

  麻布大学獣医学部臨床特別セミナー  2019/04
• Developing immunotherapy to save animals  [Invited]

  Satoru Konnai

  Research Highlight: https://www.global.hokudai.ac.jp/blog/developing-immunotherapy-to-save-animals/  2019/04
• 動物難治性疾病に対する創薬研究  [Invited]

  今内 覚

  東北大学大学院医学系研究院抗体創薬研究分野 第6回抗体創薬研究セミナー  2019/03
• Six scientists whose transformative work reaches across borders.  [Invited]

  Satoru Konnai

  Nature index: https://www.nature.com/articles/d41586-019-00831-5  2019/03
• 牛白血病の最新知見とウイルス量を基盤とした対策について  [Invited]

  今内 覚

  大分県畜産協会家畜生産農場清浄化支援事業講習会  2019/03
• 静かなる脅威・牛白血病 〜最新知見から〜  [Invited]

  今内 覚

  日本中央競馬会(JRA)畜産振興事業シンポジウム 地方病性牛白血病・清浄化へのアプローチ ~牛白血病清浄化に向けて今しなければならないこと~  2019/03
• 家畜の慢性感染症制御の難しさ 〜免疫から逃れる病原体たち〜  [Invited]

  今内 覚

  第9回大動物臨床研究会東京シンポジウム  2019/03
• 動物難治性疾病に対する創薬研究 〜免疫チェックポイントを標的としたイヌ免疫療法の試み〜  [Invited]

  今内 覚

  第15回日本獣医内科学アカデミー学術大会  2019/02
• Immune checkpoint inhibitors-related diseases in animals: Application of immunotherapy targeting the immune checkpoints for veterinary medicine  [Invited]

  Satoru KONNAI

  Seminario Especial in Universidade Federal do Rio Grande do Sul  2019/02
• 動物の難病(がん・感染症)に対する 創薬研究(新薬の開発)  [Invited]

  今内 覚

  国民との科学・技術対話  2019/01
• 牛白血病臨床検査診断結果に基づく実践的対策  [Invited]

  Satoru Konnal

  平成30年度小平町和牛生産改良組合牛白血病研修会  2019/01
• 動物難治性疾病に対する創薬研究 〜免疫チェックポイントを標的とした新規免疫療法の試み〜  [Invited]

  Satoru Konnal

  大阪市立大学大学院工学研究科 医工・生命工学教育研究センター第１回セミナー  2018/12
• イヌ難治性腫瘍疾患に対する免疫チェックポイントを 標的とした新規免疫療法の試み  [Not invited]

  今内 覚, 前川直也, 細谷謙次, 金 尚昊, 高木 哲, 賀川由美子, 岡川朋弘, 村田史郎, 大橋和彦

  北海道小動物獣医師会年次大会2018  2018/11
• 牛白血病の最新知見、現状と対策  [Invited]

  今内 覚

  兵庫県畜産協会平成30年度食の安全・消費者の信頼確保対策事業平成30年度牛白血病講習会  2018/10
• 動物難治性疾病に対する創薬研究 〜免疫学的解析を基盤とした新規制御法の開発〜  [Invited]

  今内 覚

  第161回日本獣医学会学術集会微生物学分科会シンポジウム「感染症対策の新たな突破口を探る」  2018/09
• 牛白血病の最新知見 〜臨床データーからの更新知見〜  [Invited]

  今内 覚

  平成30年度みなみ北海道農業共済組合第1回診療等体験発表会 特別講演  2018/07
• 免疫チェックポイントを標的とした動物用抗体医薬の開発  [Invited]

  今内 覚

  第2回ヒトと伴侶動物の比較医学研究会 ヒト医療と獣医療の先端技術を駆使した次世代のがん治療〜がん撲滅への新たな挑戦〜  2018/06
• 牛難治性疾病における免疫疲弊化機序の 解析と制御法への応用  [Invited]

  今内 覚

  第74回九州・山口病性鑑定協議会  2018/06
• 動物難治性疾病に対する創薬研究  [Invited]

  今内 覚

  北里大学獣医学部特定講義  2018/06
• 牛白血病の最新知見 現状と対策  [Invited]

  今内 覚

  国立大学法人岐阜大学応用生物科学部附属家畜衛生地域連携教育研究センター家畜衛生講演会  2018/06
• 牛白血病の最新知見  [Invited]

  今内 覚

  鹿児島県獣医師会 産業動物部会曽於・肝属地区獣医師講習会  2018/03
• 牛白血病の最新知見 帯広食肉衛生検査所との共同研究成果から  [Invited]

  今内 覚

  北海道獣医師会十勝支部公衆衛生講習会  2018/03
• 牛白血病の疫学と公衆衛生分野での留意点  [Invited]

  今内 覚

  北海道獣医師会平成29年度獣医公衆衛生講習会  2018/03
• 地方病性牛白血病対策 について  [Invited]

  今内 覚

  平成29年度岩手県牛白血病防疫推進講習会  2018/01
• 牛の免疫応答を利用した難治性疾病の新規制御法開発 〜免疫チェックポイントを標的とした動物用抗体創薬〜  [Invited]

  今内 覚

  家畜衛生フォーラム2017 「抗菌剤に頼らない新しい家畜疾病の制御法 －モデルとしての難治性・慢性疾病克服のための研究－」  2017/12
• Immune exhaustion during chronic diseases in animals  [Invited]

  Satoru Konnal

  The 20th Joint Symposium Between Seoul National University and Hokkaido University  2017/11
• 牛難治性疾病に対する多機能型バイオ医薬(抗体医薬)の創出と発展的応用  [Invited]

  今内 覚

  農林水産業・食品産業科学技術研究推進事業 「知」の集積と活用の場ポスターセッション  2017/11
• 動物用バイオ医薬品の開発  [Invited]

  今内 覚

  東北大学大学院医学系研究科抗体創薬研究分野 第二回抗体創薬研究セミナー  2017/11
• 増加している牛白血病 - 北海道での現状と対策について -  [Invited]

  今内 覚

  第22回北海道肉牛研究会大会  2017/10
• Epidemiological and immunological study for intractable infectious diseases in Mongolian livestock  [Invited]

  Satoru KONNAI

  JICA Project: Infectious Diseases Seminar 2017 in Mongolia  2017/10
• 動物の難病に対する新規治療法の開発  [Invited]

  今内 覚

  国民との科学・技術対話  2017/10
• Theileria parva感染症における免疫チェックポイント因子の動態解析  [Not invited]

  今内 覚, 岡川朋弘, 山田慎二, Martin Simuunza, Timothy Connelley, Ivan Morrison, 村田史郎, 大橋和彦

  第160回日本獣医学会学術集会  2017/09
• Role of Inhibitory Molecules in Bovine Chronic Infectious Diseases and as Target for Therapy  [Invited]

  Satoru Konnal

  宮崎大学産業動物防疫リサーチセンター(CADIC)特別セミナー  2017/09
• Therapeutic intervention in cancer and chronic infections in animals: Antibody mediated manipulation of PD-1/PD-L1 interaction  [Invited]

  Satoru Konnal

  Hokkaido University & Colorado State University Kick-off Symposium 2017  2017/07
• 牛白血病の最新知見  [Invited]

  Satoru Konnal

  平成29年度第108回上川家畜衛生研究会  2017/07
• 動物における免疫チェックポイント標的抗体による治療戦略  [Invited]

  Satoru Konnal

  化学及血清療法研究所 三風会  2017/06
• The role of tick saliva-derived factors in pathogen transmission  [Invited]

  Satoru Konnal

  第90回日本細菌学会総会シンポジウム わが国におけるダニ媒介性細菌感染症研究の現状  2017/03
• 動物難治性疾病における免疫回避機序の解明 〜免疫チェックポイントを標的とした動物用医薬品開発〜  [Not invited]

  今内 覚

  第2回北海道大学・部局横断シンポジウム 『免疫・癌・感染』  2017/03
• Immune exhaustion during chronic infections in cattle  [Not invited]

  Satoru KONNI

  Seminar in the Loslin institute, University of Edinburgh, UK  2017/02
• 牛白血病撲滅対策  [Invited]

  Satoru Konnal

  公益社団法人千葉県畜産協会牛白血病感染拡大防止研修会 ー牛白血病の感染拡大防止を図るためー  2017/01
• 獣医療における抗体医薬の開発と現状  [Invited]

  Satoru Konnal

  徳島大学先端酵素学研究所免疫制御学セミナー  2016/12
• 牛の感染免疫に関する最新の知見  [Invited]

  Satoru Konnal

  平成28年度岩手県獣医師会職域部会合同研修会  2016/12
• 家畜法定伝染病ウシアナプラズマ病の免疫回避機構の解析  [Not invited]

  Satoru Konnal

  第23回 リケッチア研究会  2016/12
• 牛白血病の最新の知見について  [Invited]

  Satoru Konnal

  神奈川県畜産技術協会生物科学安全研究所支部地方競馬益金補助事業主催講演会  2016/11
• Immunotherapy for canine malignant cancers  [Invited]

  Satoru KONNAI

  The 19th Joint Symposium Between Seoul National University and Hokkaido University 〜Advanced Veterinary Sciences: From Bench to Clinic〜  2016/11
• 牛白血病の現状と対策  [Invited]

  Satoru Konnal

  日高町和牛生産改良組合防疫技術講習会  2016/11
• 免疫チェックポイントを標的とする免疫療法とは  [Invited]

  Satoru Konnal

  平成28年宮崎大学テニュアトラック推進機構主催セミナー  2016/11
• 牛白血病対策  [Invited]

  Satoru Konnal

  八紘牧場衛生対策会議  2016/10
• 牛白血病について  [Invited]

  Satoru Konnal

  平成2８年度第3回標茶町技連学習会  2016/10
• Identification of immunomodulatory factors in Ixodes persulcatus Schulze ‘Taiga tick’, the vector for Lyme disease and relapsing fever in Japan  [Invited]

  Satoru KONNAI

  XXV International Congress of Entomology (ICE-2016)  2016/09
• Development of novel strategy for disease control in animal-engineering animal antibodies for development of novel therapeutics  [Invited]

  Satoru KONNAI

  Texas A&M University, College of Veterinary Medicine, Veterinary Pathology Seminar Series  2016/09
• Engineering antibodies for development of novel therapeutics and vaccine for livestock  [Invited]

  Satoru KONNAI

  1st International Symposium on Livestock Biotechnology  2016/08
• Increases of cells expressing PD-1 and PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via its blockade  [Not invited]

  Satoru KONNAI

  International Veterinary Immunology Symposium 2016  2016/08
• Research on immune checkpoint in veterinary medicine  [Invited]

  Satoru KONNAI

  Joint Workshop on Livestock Disease Research in University of Sydney  2016/08
• むしからうつる感染症  [Invited]

  Satoru Konnal

  札幌幌南ロータリークラブ市民講話  2016/08
• 牛白血病における免疫応答 〜免疫学的解析に基づく新規動物用バイオ医薬品の開発〜  [Invited]

  今内 覚

  第20回大動物臨床教育セミナー  2016/05
• 牛白血病について  [Invited]

  今内 覚

  平成28年度JA新はこだて酪農生産部会長万部支部講習会  2016/04
• 牧野における牛白血病対策  [Invited]

  今内 覚

  後志家畜自衛防疫推進協議会産業動物飼養管理講習会  2016/04
• 牛白血病の免疫学的解析  [Invited]

  今内 覚

  理化学研究所シンポジウム  2016/03
• 牛白血病の制御法について  [Invited]

  Satoru Konnal

  道東NOSAI牛白血病拡大防止事業教育講座  2016/03
• 牛白血病の感染防御対策  [Invited]

  Satoru Konnal

  道東NOSAI牛白血病拡大防止対策事業教育講座  2016/03
• 牛白血病における免疫性状と制御方法の開発  [Invited]

  Satoru Konnal

  平成27年度日本獣医師会獣医学術学会年次大会特別企画シンポジウム「牛白血病の現状と課題 －清浄化に向けて－」  2016/02
• 牛白血病の現状と対策について  [Invited]

  Satoru Konnal

  静岡県東部家畜保健衛生推進協議会畜産講演会  2016/02
• 牛白血病における免疫応答 〜免疫学的解析に基づく新規疾病予防法の開発〜  [Invited]

  Satoru Konnal

  大動物臨床研究会第6回東京シンポジウム  2016/02
• 獣医医療における抗体医薬の開発と現状  [Invited]

  Satoru Konnal

  東北大学大学院医学系研究科特別講義  2016/01
• Development of therapeutic antibodies targeting immunoinhibitory molecules in veterinary medicine  [Invited]

  Satoru KONNAI

  IPR/CRED/PDIS Joint International Seminar -From protein structural science to development of therapeutics-  2016/01
• 動物難治性疾病に対するバイオ医薬品の開発  [Invited]

  Satoru Konnal

  JA全農家畜衛生研究所特別講演  2015/11
• 牛白血病の現状と対策の例  [Invited]

  Satoru Konnal

  青森県上十三地区家畜衛生推進協議会H27年度畜産講習会  2015/11
• 初乳の衛生管理について 〜牛白血病防除の観点から〜  [Invited]

  Satoru Konnal

  第23回乳房炎防疫対策研究会  2015/11
• 牛白血病の最新知見  [Invited]

  Satoru Konnal

  第２回ミニ宮崎しゃくなげ会研修会  2015/11
• 牛白血病における免疫応答  [Invited]

  Satoru Konnal

  第39回大動物臨床研究会シンポジウム  2015/11
• 牛白血病 最近の知見と対策について  [Invited]

  今内 覚

  大分県畜産協会特別講演  2015/09
• 牛白血病の病態発生機序および制御法に関する研究  [Invited]

  Satoru Konnal

  第158回日本獣医学会学術集会特別企画  2015/09
• 牛白血病に認められる細胞性免疫の抑制 - PL牛を優先淘汰する意義 -  [Invited]

  今内 覚

  2015年度 牛臨床寄生虫研究会・九州沖縄産業動物臨床研究会合同研究集会  2015/07
• 地方病型牛白血病の対策と新規制御法研究の現状  [Invited]

  今内 覚

  長野県獣医師会産業動物臨床部会講習会  2015/04
• 牛白血病の対策  [Invited]

  今内 覚

  北海道公共牧場会職員春期研修会  2015/04
• 慢性感染症における免疫応答と免疫回避機構  [Invited]

  今内 覚

  新潟県農業共済組合連合会平成26年度家畜診療等技術講習会  2015/03
• 牛白血病への対策について  [Invited]

  今内 覚

  新潟県農業共済組合連合会平成26年度家畜診療等技術講習会  2015/03
• 牛白血病の現状について  [Invited]

  今内 覚

  平成26年度放牧衛生技術検討会ならびに釧路地区家畜自衛防疫研修会  2015/02
• 牛白血病ウイルス(BLV)感染症の現状と対策  [Invited]

  今内 覚

  北海道獣医師会十勝支部産業動物講習会  2015/02
• 慢性感染症に認められる免疫疲弊  [Invited]

  Satoru Konnal

  平成26年度日本獣医師会獣医学術集会年次大会特別企画 シンポジウム  2015/02
• Increases of cells expressing PD-1 and PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via its blockade  [Invited]

  Satoru Konnal

  Keystone Symposia on Immunity to Veterinary Pathogens: Informing Vaccine Development  2015/01
• 牛白血病 ー最新知見・現状と対策ー  [Invited]

  今内 覚

  平成26年度留萌和牛振興協議会研修会  2015/01
• 感染症と免疫細胞の役割  [Invited]

  今内 覚

  中央畜産会平成26年度海外家畜伝染病等危機管理対策強化講習会 第一次診療臨床診断技術強化講習会  2014/12
• 牛白血病の現状と対策  [Invited]

  今内 覚

  北海道獣医師会道南支部産業動物講習会  2014/11
• 牛白血病の現状と感染免疫  [Invited]

  今内 覚

  平成26年度千葉県農業共済組合連合会紫葉会技術懇談会  2014/11
• 牛白血病の現状と北海道大学の取り組み  [Invited]

  Satoru Konnal

  日本ウイルス学会北海道支部 第 48 回 夏季シンポジウム  2014/07
• 牛の難治性疾病に対する新規ワクチン戦略  [Not invited]

  今内 覚

  農業・食品産業技術総合研究機構 生物系特定産業技術研究支援センター 2013年度研究成果発表会  2014/03
• 牛白血病の新規制御法の開発と清浄化モデルの構築  [Invited]

  今内 覚

  北海道の元気創出フォーラム～知的財産を活かした地域潜在力の発掘に向けて～  2014/02
• 獣医医療における免疫抑制機序を標的とした新規疾病制御法の開発  [Invited]

  今内 覚

  大阪大学微生物病研究所セミナー  2014/01
• 牛白血病の病態発生機序の解明  [Invited]

  今内 覚

  平成25年度動物衛生研究所北海道支所集談会  2013/12
• 牛白血病 ー最新知見・現状と対策ー  [Invited]

  今内 覚

  北海道しりべし獣医師会産業動物飼養管理講習会  2013/11
• 牛白血病ウイルスの感染防御対策  [Invited]

  今内 覚

  平成25年宮崎県畜産技術研修会特別講演  2013/11
• 牛白血病ウイルス(BLV)感染症の現状と対策  [Invited]

  今内 覚

  埼玉県獣医師会北支部・埼玉県八日会・しゃくなげ会埼玉県支部・埼玉県畜産技術振興会合同定例研修会  2013/11
• 家畜臨床免疫学研究: 牛白血病における病態発生機序の解明と新規制御法の確立  [Invited]

  今内 覚

  岐阜大学大学院連合獣医学研究科 平成25年度若手研究者育成プログラム  2013/11
• 牛白血病における病態発生機序の解明と新規制御法の確立に関する研究  [Invited]

  今内 覚

  微生物化学研究所特別講演  2013/10
• 牛白血病の感染防御対策  [Invited]

  今内 覚

  北海道獣医師会留萌支部平成25年度新技術講習会  2013/09
• ウシ難治性感染症の現状と対策および新規制御法の開発  [Invited]

  今内 覚

  ゼノアック 家畜感染症研修会  2013/09
• 気をつけよう 虫さされによる感染症  [Invited]

  今内 覚

  札幌幌南ロータリークラブ市民講話  2013/08
• 牛白血病の現状と対策  [Invited]

  今内 覚

  化学及血清療法研究所第30回家畜衛生講習会並びに研究協議会  2013/08
• 牛白血病の現状と対策について  [Invited]

  今内 覚

  NOSAI山形平成25年度家畜診療所技術研究会  2013/07
• 農場の感染症リスクコントロール  [Invited]

  今内 覚

  NOSAI山形平成25年度家畜診療所職員研修会  2013/07
• 牛白血病 ー最新知見、現状と対策ー  [Invited]

  今内 覚

  第104回上川家畜衛生研究会  2013/07
• 感染症(牛白血病)について 現状と対策  [Invited]

  今内 覚

  第35回鹿児島・宮崎しゃくなげ会合同技術研修会  2013/07
• 牛白血病清浄化対策について  [Invited]

  今内 覚

  宮崎県尾鈴農業協同組合BL対策研修会  2013/06
• 牛白血病の対策について  [Invited]

  今内 覚

  北海道獣医師会牛白血病対策検討委員会  2013/03
• 牛白血病ウイルス(BLV)感染症の 現状と対策  [Invited]

  今内 覚

  第51回愛知県獣医師会学術研究発表会特別講演  2013/03
• Role of Inhibitory Molecules in Bovine Chronic Infectious Diseases and as Target for Therapy  [Invited]

  Satoru Konnal

  Philippines Carabao Center Special Seminar  2013/03
• 牛難治性疾病に対する免疫抑制因子を標的とした新規制御法の開発  [Invited]

  今内 覚

  日本大学生物資源科学部 動物医科学研究センター特別セミナー  2013/01
• 牛白血病ウイルス感染症の伝播阻止トライアルについて  [Invited]

  今内 覚

  本川牧場疾病対策講習会  2012/12
• 牛白血病ウイルス感染症の現状と対策  [Invited]

  今内 覚

  沖縄県獣医師会家畜衛生畜産部会沖縄畜産技術者協会特別講演  2012/12
• 牛の白血病について  [Invited]

  今内 覚

  北海道獣医師会胆振支部特別講演  2012/11
• 牛白血病ウイルス感染症の現状と対策  [Invited]

  今内 覚

  宮崎県獣医師会学術委員会産業動物部門研修会  2012/11
• 牛白血病の現状と対策  [Invited]

  今内 覚

  埼玉県獣医師会特別講演  2012/10
• 牛慢性疾患の新たなる治療法の可能性 -免疫抑制因子を標的とした新規治療薬の開発-  [Invited]

  今内 覚

  酪農学園大学大動物研究セミナー  2012/09
• 牛の白血病について  [Invited]

  今内 覚

  北海道獣医師会根室支部特別講演  2012/05
• 牛難治性疾病に対する新規戦略(多機能型ワクチンによる牛疾病制御の試み)  [Invited]

  今内 覚

  第153回日本獣医学会学術集会微生物分科会シンポジウム  2012/03
• 牛白血病のコントロールについて〜ある農場における清浄化の取り組み事例から〜  [Invited]

  Satoru Konnal

  平成23年度 北海道家畜保健衛生所病性鑑定技術検討会  2012/01
• 牛白血病の現状と感染予防対策について  [Invited]

  今内 覚

  道央農業振興公社 畜産研修会  2011/12
• 牛の難治性疾病に対する多機能型ワクチン戦略  [Invited]

  今内 覚

  第51回家畜衛生学会シンポジウム  2011/11
• 国内の牛白血病の現状と今後の対策について  [Invited]

  今内 覚

  牛臨床寄生虫研究会・家畜臨床学会ジョイントシンポジウム  2011/10
• 牛白血病の現状とその対策  [Invited]

  今内 覚

  栃木県獣医師会特別講演  2011/10
• 牛の難治性疾病に対する多機能型ワクチン戦略  [Invited]

  今内 覚

  宮崎大学農学部特別シンポジウム(集談会)  2011/09
• 牛白血病の現場でのコントロールについて  [Invited]

  今内 覚

  宮崎大学農学部特別シンポジウム(教育プログラム)  2011/09
• Research on host-pathogen interactions for establishment of novel strategies for disease control  [Invited]

  Satoru Konnal

  Special seminar in Federal University of Rio Grande do Sul  2011/08
• Research on host-pathogen interactions for establishment of novel strategies for disease control  [Invited]

  Satoru Konnal

  Special seminar in Northern Fluminense State University Darcy Ribeiro (Campos campas)  2011/07
• Research on host-pathogen interactions for establishment of novel strategies for disease control  [Invited]

  Satoru Konnal

  Special seminar in Northern Fluminense State University Darcy Ribeiro (Makae campus)  2011/07
• 牛白血病ウイルス感染症の現状と対策  [Invited]

  今内 覚

  福岡県家畜保健衛生所講習会  2011/02
• 牛白血病の感染防御対策  [Invited]

  今内 覚

  第43回北海道しゃくなげ会  2011/02
• 生産現場における白血病対策 —現状と課題—  [Invited]

  今内 覚

  平成22年度日本獣医師会 獣医学術学会年次大会  2011/02
• 増加傾向にある牛白血病の現状と対策  [Invited]

  今内 覚

  日本家畜臨床学会第41回学術集会  2010/11
• 原虫病疾病制御のための新戦略 -ダニワクチン開発-  [Invited]

  今内 覚

  日本家畜衛生学会  2010/11
• 増加傾向にある牛白血病の現状と対策  [Invited]

  今内 覚

  平成22年度北海道地区家畜診療技術研修会  2010/09
• メガファームにおける取り組み紹介 —牛白血病ウイルス感染伝播阻止トライアルの実例—  [Invited]

  今内 覚

  平成22年度獣医学術北海道地区学会シンポジウム  2010/09
• Pro-inflammatory cytokine storm in Theileria parva-infected cattle  [Invited]

  Satoru Konnal

  The 9th International Veterinary Immunology Symposium (Concurrent Session)  2010/08
• 牛白血病ウイルス感染症の現状と対策  [Invited]

  今内 覚

  北海道獣医師会オホーツク支部大動物講習会2  2010/07
• 牛白血病ウイルス感染症の現状と対策  [Invited]

  今内 覚

  北海道獣医師会オホーツク支部大動物講習会1  2010/07
• 新規動物用ワクチンならびに新規治療法の開発  [Invited]

  今内 覚

  日本生物科学研究所シンポジウム  2010/03
• 新規動物用ワクチンならびに新規治療法の開発  [Invited]

  今内 覚

  動物用ワクチンーバイオ医薬研究会改組設立記念シンポジウム  2010/03
• PD-1/PD-L1システム制御による牛白血病ワクチン戦略  [Invited]

  今内 覚

  平成21年度日本獣医師会学会年次大会・特別企画  2010/01
• 牛難治性慢性感染症における免疫疲弊化機序の解明と免疫賦活化の試み  [Invited]

  今内 覚

  第4回日本家畜臨床感染症研究会総会・学術集会  2009/12
• 牛白血病ウイルス感染症の現状と対策  [Invited]

  今内 覚

  北海道獣医師会根室支部大動物臨床部会講演会  2009/11
• 牛白血病ウイルス(BLV)感染症対策  [Invited]

  今内 覚

  第60回北海道獣医師大会産業動物獣医学会シンポジウム  2009/09
• ダニのEST解析とダニワクチンの現状  [Invited]

  今内 覚

  第15回 ダニと疾患のインタフェースに関するセミナー(SADI) 教育講演  2007/05

MISC

• 子牛用ワクチン効果の新規増強法の試み

  今内 覚, 岡川朋弘  臨床獣医  42-  (2)  38  -41  2024/02  [Not refereed][Invited]
  
• 小動物臨床最前線！ 二次診療の現場より

  今内 覚  北小獣通信  1-  (93)  2024/01  [Not refereed][Invited]
  
• One HealthアプローチにもとづくBLV/HTLV-Ⅰ感染症におけるリンパ腫の診断と早期発症予測法の開発

  今内 覚, 岡川朋弘, 斎藤益満  臨床とウイルス  51-  (4)  192  -198  2023/10  [Refereed][Invited]
  
• 牛伝染性リンパ腫診断の新技術・早期発症予測診断法RAISINGとは

  今内 覚, 岡川朋弘  家畜診療  70-  (5)  259  -266  2023/05  [Not refereed][Invited]
  
• 成果紹介：ウシ伝染性リンパ腫の早期発症診断による経済損失低減の実現

  今内 覚, 岡川朋弘  JATAFFジャーナル  11-  (4)  45  -50  2023/04  [Refereed][Invited]
  
• 研究紹介：牛のリンパ腫発症を予測するがん検診技術を開発 ～発症予測法の実用化による畜産被害の軽減に期待～

  今内 覚  北海道獣医師会誌  67-  44  -47  2023/02  [Not refereed][Invited]
  
• 牛伝染性リンパ腫の現状から最後の砦：発症予測法について

  今内 覚, 岡川朋弘  臨床獣医  41-  (2)  46  -31  2023/02  [Not refereed][Invited]
  
• ピックアップ新技術：牛のリンパ腫発症を予測するがん検診技術を開発. リスク高い牛の優先淘汰による発生予防に期待

  今内 覚, 岡川朋弘  DAIRYMAN  73-  (2)  64  -66  2023/02  [Not refereed][Invited]
  
• 牛伝染性リンパ腫の診断と早期発症予測法の開発

  富永みその, 今内覚, 岡川朋弘, 神谷可菜, 齋藤麻矢, 安富一郎, 目堅博久, 前川直也, 村田史郎, 大橋和彦  北海道獣医師会雑誌  67-  (8)  2023
• 免疫チェックポイント分子Programmed death-ligand1(PD-L1)を標的とした抗体薬による犬の鼻腔内腺癌に対する免疫療法の検討

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

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

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

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

  もたい吉之介, 村田史郎, 佐藤純平, 西明仁, 前川直也, 岡川朋弘, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  166th-  2023
• 特集：ワクチン戦略を考える：ワクチンの基本

  今内 覚  臨床獣医  11-  16  -21  2022/11  [Not refereed][Invited]
  
• 牛伝染性リンパ腫ウイルス感染牛における分娩が及ぼす免疫抑制メカニズムの解析

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

  今内 覚, 岡川朋弘, 前川直也, 村田史郎, 大橋和彦  家畜衛生学雑誌  47-  (3)  147  -150  2021/12  [Not refereed][Invited]
  
• 発酵哺乳飼料による子牛の腸炎防御効果の直接証明

  今内 覚, 岡川朋弘  JATAFFジャーナル  9-  (10)  21  -26  2021/10  [Not refereed][Invited]
  
• 京都府の1酪農場における牛白血病ウイルス対先の乳生産性・繁殖性ならびに疾病発生に及ぼす効果

  北野菜奈, 吉田 陽, 今内 覚, 高橋俊彦  家畜感染症学雑誌  10-  (3)  71  -77  2021/10  [Refereed][Not invited]
  
• 子牛下痢症対策のアップデート：子牛下痢症への発酵代用乳の適応

  岡川朋弘, 今内 覚  臨床獣医  9-  34  -38  2021/09  [Not refereed][Invited]
  
• 技術ワイド：特集：哺育牛の飼養管理を見直す① 下痢の重症化を抑止し哺乳量も早期回復 乳酸菌添加した発酵代用乳による下痢対策

  今内 覚  DAIRYMAN  71-  (8)  44  -46  2021/08  [Not refereed][Invited]
  
• プロバイオティクスで子牛を下痢から守る 〜高品質で安全な発酵代用乳〜

  岡川 朋弘, 今内 覚  MPアグロジャーナル  45-  (7)  34  -37  2021/07  [Not refereed][Invited]
  
• 臨床免疫を基盤とする産学協同応用研究の推進

  今内 覚  臨床獣医  6-  82  -84  2021/06  [Not refereed][Invited]
  
• Expression and functional analysis of swine PD-1 /PD-L1 pathway

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

  藤澤宗太郎, 村田史郎, 伊勢崎政美, 佐藤匠, 大石英司, 種子野章, 前川直也, 岡川朋弘, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• 犬の口腔内悪性黒色腫に対する抗Programmed death-Ligand 1抗体療法と放射線治療におけるアブスコパル効果の誘導解析

  大脇稜, 出口辰弥, 細谷謙次, 今内覚, 前川直也, 立花由莉加, 中村基司, 金尚昊, 須永隆文, 奥村正裕  北海道獣医師会雑誌  65-  (8)  2021
• 牛白血病の病態発生機序解析を基盤とする新制御法の試み

  今内 覚, 岡川朋弘, 前川直也, 村田史郎, 大橋和彦  畜産技術  (784)  36  -40  2020/09  [Not refereed][Invited]
  
• Immunotherapy〜Immune checkpoint inhibitor〜

  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  Japan Veterinary Products Association Digest  67-  (2)  1  -10  2020/02  [Not refereed][Invited]
  
• 分子標的薬-将来の展望 免疫チェックポイント阻害薬を用いた腫瘍免疫療法

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

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

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

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

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

  Satoru Konnal  Med. Entomol. Zool.  70-  (4)  189  -197  2019/12  [Refereed][Invited]
   

  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。
• 技術ワイド 徹底防除―ヨーネ病 病態発生のメカニズムと新規制御法

  Satoru Konnal  DAIRYMAN  69-  (10)  40  -41  2019/10  [Not refereed][Invited]
  
• 免疫チェックポイント分子Programmed death‐ligand 1(PD‐L1)を標的とする抗体薬による免疫療法が奏功した肺転移のある口腔内悪性黒色腫の犬の1例

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

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

  Satoru Konnal  臨床獣医  37-  (3)  37  -41  2019/03  [Not refereed][Invited]
  
• 免疫チェックポイント阻害剤による犬の悪性腫瘍に対する新規治療法

  高木哲, 前川直也, 今内覚  日本獣医師会獣医学術学会年次大会講演要旨集  2018-  2019
• 免疫チェックポイントを標的とした免疫療法

  Satoru Konnal  The Japanese Society for Animal Vaccine and Biomedical Research News Letter  (18)  2  -3  2018/12  [Not refereed][Invited]
  
• 動物難治性疾病に対する創薬研究

  今内 覚, 岡川朋弘, 前川直也, 中島千絵, 鈴木定彦, 山本啓一, 戸田幹洋, 村田史郎, 大橋和彦  MPアグロジャーナル  35-  (10)  22  -25  2018/10  [Not refereed][Invited]
  
• ヨーネ病の病態発生機序の解析

  今内 覚, 永田礼子, 川治聡子, 森 康行  畜産技術  761-  2  -6  2018/10  [Not refereed][Invited]
  
• 免疫チェックポイント分子を標的とした腫瘍免疫療法 抗PD-1/PD-L1抗体療法

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

  渡慧, 今内覚, 佐治木大和, 岡川朋弘, 前川直也, 後藤伸也, 村田史郎, 大橋和彦  北海道獣医師会雑誌  62-  (8)  282  -282  2018/08/24  [Not refereed][Not invited]
  
• マレック病ウイルスの病原性進化機構の解明―病原性試験や全ゲノム解析の試み

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

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

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

  田中晶菜, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 戸塚知恵, 千葉由純, 池田昌穂, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  353  -353  2018/08/21  [Not refereed][Not invited]
  
• ヨーネ病におけるプロスタグランジンE₂(PGE₂)動態解析及びCOX‐2阻害剤の免疫活性化効果の検討

  佐治木大和, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 後藤伸也, 永田礼子, 川治聡子, 森康行, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  341  -341  2018/08/21  [Not refereed][Not invited]
  
• ウシCytotoxic T‐lymphocyte antigen‐4(CTLA‐4)の機能解析および慢性感染症における発現解析

  渡慧, 今内覚, 岡川朋弘, 前川直也, 後藤伸也, 佐治木大和, 村田史郎, 鈴木定彦, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  340  -340  2018/08/21  [Not refereed][Not invited]
  
• 肺転移のある悪性黒色腫罹患犬に対するPD‐L1を標的とした抗体薬の治療効果

  前川直也, 今内覚, 高木哲, 細谷謙次, 賀川由美子, 岡川朋弘, 和泉雄介, 出口辰弥, 鈴木定彦, 山本啓一, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  161st-  429  -429  2018/08/21  [Not refereed][Not invited]
  
• イヌCytotoxic T‐lymphocyte antigen‐4(CTLA‐4)を標的としたバイオ医薬品の開発に向けた基礎的検討

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

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

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

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

  村田史郎, 伊勢崎政美, 谷口綾香, 北條巧, 種子野章, 酒井英史, 宇野有紀子, 矢吹卓也, 市居修, 伊東拓也, 今内覚, 大橋和彦  衛生動物  69-  (2)  103  -103  2018/06  [Not refereed][Not invited]
  
• 牛難治性疾病に対するバイオ医薬の創出

  今内 覚  JATAFFジャーナル  6-  (5)  29  -33  2018/05  [Not refereed][Invited]
  
• 獣医療における抗体医薬の現状

  今内 覚, 村田史郎, 大橋和彦  家畜診療  65-  (4)  233  -240  2018/04  [Not refereed][Invited]
  
• 牛白血病の最新知見とウイルス量を基盤とした対策について

  今内 覚  獣医公衆衛生研究  20-  (2)  12  -17  2018/03  [Not refereed][Invited]
  
• 牛白血病の現状と対策について

  今内 覚  北海道肉牛研究会報  22-  1  -11  2018/03  [Not refereed][Invited]
  
• ネコpodoplaninに対するモノクローナル抗体の樹立とネコ扁平上皮がんにおけるpodoplanin発現解析

  山田慎二, 板井俊介, 賀川由美子, 今内覚, 金子美華, 加藤幸成, 加藤幸成  日本獣医学会学術集会講演要旨集  161st-  2018
• 牛白血病発症牛由来細胞株に発現するアンチセンス鎖転写産物

  三苫修也, 原彰浩, 目堅博久, 目堅博久, 今内覚, 岡川朋弘, 関口敏, 乗峰潤三, 乗峰潤三  日本獣医学会学術集会講演要旨集  161st-  2018
• 京都府の1酪農場におけるBLV対策とその効果について

  北野 菜奈, 吉田 陽, 今内 覚, 菊 佳男, 高橋 俊彦  家畜感染症学会誌  6-  (4)  178  -178  2017/12  [Not refereed][Not invited]
  
• 牛の免疫応答を利用した難治性疾病の新規制御法開発

  今内 覚  家畜衛生学雑誌  43-  (3)  101  -103  2017/11  [Not refereed][Invited]
  
• 感染ウイルス定量解析によるリスク評価を基盤とする牛白血病対策

  佐治木大和, 今内 覚, 中島隆二  畜産の研究  71-  (9)  729  -739  2017/09  [Not refereed][Invited]
  
• 日本に分布するマレック病ウイルス野外株の病原性の検討

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

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

  後藤伸也, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 樋口豪紀, 小岩政照, 田島誉士, 小原潤子, 加藤幸成, 鈴木定彦, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  370  -370  2017/08/30  [Not refereed][Not invited]
  
• Theileria parva感染症における免疫チェックポイント因子の動態解析

  今内覚, 岡山朋弘, 山田慎二, SIMUUNZA Martin, CONNELLEY Timothy, MORRISON Ivan, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  338  2017/08/30  [Not refereed][Not invited]
  
• PD‐L1を標的としたイヌ用キメラ抗体医薬の作製と悪性黒色腫および未分化肉腫罹患犬における抗腫瘍効果の検討

  前川直也, 今内覚, 高木哲, 賀川由美子, 岡川朋弘, 西森朝美, 池渕良洋, 和泉雄介, 出口辰弥, 加藤幸成, 鈴木定彦, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  460  -460  2017/08/30  [Not refereed][Not invited]
  
• 牛白血病ウイルス(BLV)感染症においてプロスタグランジンE₂(PGE₂)が及ぼす免疫抑制機序の解明

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

  西森朝美, 今内覚, 岡川朋弘, 前川直也, 池渕良洋, 後藤伸也, 佐治木大和, 鈴木定彦, 小原潤子, 小笠原諭, 加藤幸成, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  370  -370  2017/08/30  [Not refereed][Not invited]
  
• COX‐2阻害薬と抗PD‐L1抗体の併用によるイヌ腫瘍疾患の制御法の検討

  浅野裕美恵, 今内覚, 岡川朋弘, 前川直也, 西森朝美, 後藤伸也, 高木哲, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  160th-  371  -371  2017/08/30  [Not refereed][Not invited]
  
• 牛白血病ウイルス(BLV)感染ハイリスク牛における初めての子宮内感染直接証明

  佐治木 大和, 今内 覚, 西森 朝美, 岡川 朋弘, 永野 昌志, 小原 潤子, 村田 史郎, 大橋 和彦  北海道獣医師会雑誌  61-  (8)  280  -280  2017/08  [Not refereed][Not invited]
  
• わが国におけるダニ媒介性細菌感染症研究の現状 新興ボレリア感染症

  川端 寛樹, 佐藤 梢, 熊谷 由美, 大西 真, 安藤 秀二, 福士 秀悦, 下島 昌幸, 西條 政孝, 阿戸 学, 長谷川 秀樹, 関塚 剛史, 黒田 誠, 高野 愛, 伊藤 幸枝, 古野 希和, 山野 公明, 伊東 拓也, 今内 覚, 李 景利, Taylor Kyle, 中尾 亮, 中尾 稔, 小山 幸次郎, 兼古 稔, 福島 和子, 齊藤 智也, 中嶋 健介, 榊原 佳子, 増沢 俊幸, 藤田 博己  日本細菌学雑誌  72-  (1)  34  -34  2017/02  [Not refereed][Not invited]
  
• マダニ唾液由来因子の病原体伝播における役割

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

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

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

  越智晶絵, 今内覚, 伊東拓也, 川端寛樹, 高野愛, 安藤秀二, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  159th-  339  2016/08/30  [Not refereed][Not invited]
  
• ウシ腫瘍壊死因子(TNF‐α)デコイレセプターによる新規抗炎症剤の開発

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

  村田史郎, 町田柚香, 伊勢崎政美, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  159th-  399  2016/08/30  [Not refereed][Not invited]
  
• 腫瘍血管遮断薬CA4Pの犬の血管内皮細胞への影響の検討

  和泉 雄介, 青島 圭佑, 星野 有希, 今内 覚, 高木 哲  日本獣医学会学術集会講演要旨集  159回-  451  -451  2016/08  [Not refereed][Not invited]
  
• B細胞性リンパ腫罹患犬における免疫チェックポイント分子発現解析

  倉島千尋, 田川道人, 高木哲, 前川直也, 今内覚, 新坊弦也, 富張瑞樹, 松本高太郎, 猪熊壽, 川本恵子, 宮原和郎  日本獣医学会学術集会講演要旨集  159回-  462  -462  2016/08  [Not refereed][Not invited]
  
• 抗ワクモ(Dermanyssus gallinae)ワクチン開発に向けたワクモ由来フェリチン2の性状解析

  伊勢崎政美, 村田史郎, 酒井英史, 矢吹卓也, 種子野章, 市居修, 伊東拓也, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  159回-  399  -399  2016/08  [Not refereed][Not invited]
  
• 牛白血病への対策を練る〜牛白血病について〜

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

  今内 覚, 村田 史郎, 大橋 和彦  農家の友  68-  (2)  108  -110  2016/02  [Not refereed][Invited]
  
• ウシ慢性感染症における免疫応答の解析と新規制御法への応用

  今内 覚  畜産技術  2-  (729)  2  -5  2016/02  [Not refereed][Invited]
  
• 初乳の衛生管理について 〜健康な子牛を育てるために：牛白血病防除の観点から〜

  今内 覚  乳房炎防除対策研究会誌  22-  (22.)  21  -23  2016/02  [Not refereed][Invited]
  
• Programmed death-1 (PD-1)

  今内 覚  Veterinary Oncology  3-  (1)  12  -14  2016/01  [Refereed][Invited]
  
• 牛白血病における免疫応答

  今内 覚  産業動物臨床医学雑誌  6-  (3)  130  -132  2015/11  [Not refereed][Invited]
  
• 牛白血病の現状と対策の例

  今内 覚  宮城県獣医師会会報  68-  (4)  187  -193  2015/10  [Not refereed][Invited]
  
• 牛白血病ウイルス感染症における疲弊化T細胞の動態とその再活性化

  岡川朋弘, 今内覚, 西森朝美, 前川直也, 池渕良洋, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  324  -324  2015/08/30  [Not refereed][Not invited]
  
• イヌ腫瘍浸潤リンパ球におけるPD‐1発現解析と免疫チェックポイント分子を標的とした新規腫瘍免疫療法の検討

  前川直也, 今内覚, 池渕良洋, 岡川朋弘, 足立真実, 高木哲, 賀川由美子, 賀川由美子, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  405  -405  2015/08/30  [Not refereed][Not invited]
  
• 日本に分布するマレック病ウイルス野外株の分離

  町田柚香, 村田史郎, 伊勢崎政美, 酒井英史, 種子野章, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  354  2015/08/30  [Not refereed][Not invited]
  
• シュルツェマダニ(Ixodes persulcatus)唾液腺由来免疫抑制因子sL2の同定および機能解析

  越智晶絵, 今内覚, 岡川朋弘, 西森朝美, 前川直也, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  301  -301  2015/08/30  [Not refereed][Not invited]
  
• 日本国内における牛白血病若齢発症牛の実態調査と表現型解析

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

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

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

  村瀬優介, 今内覚, 伊東拓也, 高野愛, 川端寛樹, 安藤秀二, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  158th-  301  2015/08/30  [Not refereed][Not invited]
  
• 牛の感染症の対策・防除を考える

  今内 覚  臨床獣医  33-  (8)  11  -39  2015/08  [Not refereed][Invited]
  
• 北海道におけるマダニ媒介性新興回帰熱病原体ボレリアの分布調査

  川端寛樹, 川端寛樹, 今内覚, 高野愛, 中尾稔, 伊東拓也, 佐藤梢  衛生動物  66-  (2)  43  -43  2015/06  [Not refereed][Not invited]
  
• 牛白血病を出さない！尾鈴のBL対策

  今内 覚ら 他  現代農業  6-  312  -322  2015/06  [Not refereed][Invited]
  
• 牛白血病 最近の知見と対策について

  今内 覚  動薬研究  6-  (71)  1  -11  2015/06  [Not refereed][Invited]
  
• マレック病ウイルス癌遺伝子(Meq)と病原性との関連

  高木道浩, 村田史郎, 今内覚, 大橋和彦  鶏病研究会報  50-  (4)  199  -205  2015/02/25  [Not refereed][Not invited]
   

  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。
• アフリカのゴミ集積場における化学物質の汚染実態と生体への健康影響の評価:ナイロビのダンドラ地域における家畜の重金属汚染の例

  中田北斗, 中山翔太, 水川葉月, 池中良徳, 石井千尋, YARED YB, YABE John, 今内覚, 石塚真由美  日本獣医学会学術集会講演要旨集  157th-  533  2014/08/11  [Not refereed][Not invited]
  
• 牛白血病ウイルス感染牛におけるナチュラル・キラー細胞の動態および機能解析

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

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

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

  西森朝美, 今内覚, 池渕良洋, 岡川朋弘, 大平浩輔, 中原綾子, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  157th-  424  2014/08/11  [Not refereed][Not invited]
  
• ケニアの大規模ゴミ集積場・ダンドラ地域における家畜の化学物質汚染の実態と住民の健康影響評価

  中田北斗, 中山翔太, 水川葉月, 池中良徳, 石井千尋, YOHANNES Yared B, 今内覚, 石塚真由美  J Toxicol Sci  39-  (Supplement)  S336  -187  2014/07  [Not refereed][Not invited]
   

  ケニア共和国の首都ナイロビ市内の大規模ゴミ集積場・ダンドラ地域は、世界第二位の巨大・高密度スラム街であり、地域内の子供の半数はWHO基準（100 μg/kg）以上の血中Pb濃度であることが報告されているが、家畜の汚染や生体への曝露源に関する報告はない。本研究では、ダンドラおよび対照区として同国・ナクル地域に生息するヤギ、ヒツジ、ブタおよびウシの血中金属類（10元素）濃度およびPb安定同位体比をICP-MSにより、PCBsおよび有機塩素系農薬（OCPs）濃度をGC-MSとGC-ECDにより測定した。<br> その結果、全てのサンプルでCr, Mn, Ni, Cu, Zn, As, Mo, Agの高濃度蓄積は認められず、PCBsおよびOCPsは検出限界以下であった。Pb濃度はナクルに比べてダンドラで高い蓄積傾向を示し、ヤギおよびヒツジでは有意差が認められた。ブタの平均Pb濃度はダンドラ（2600 μg/kg, dry wt:dw）がナクル（73 μg/kg, dw）の約35倍の高値を示した。ダンドラのウシからは、ヘム合成に関与するアミノレブリン酸脱水酵素活性が低下するとされる値（100μg/kg）と同程度のPb濃度（91 μg/kg以上）が検出され、血液毒性等の中毒症状の蔓延が示唆された。Cd濃度の地域差および種差は認められなかったが、総じて高値（570±460 ng/kg, dw）を示し、ウシの摂食によるヒトのCd曝露が示唆された。Pb安定同位体比は地域および動物種により異なる傾向を示し、地域内に複数の曝露源があること、動物種により主要な曝露源が異なることが示唆された。<br> 本研究より、両地域に生息する家畜には高濃度のPb, Cdが蓄積し、特にPb汚染はダンドラで深刻なレベルであり、その曝露源が複数存在することが示唆された。家畜と生活環境を共にするヒトへの同様の汚染も強く示唆された。
• アフリカのゴミ集積場における化学物質の汚染実態と生体への健康影響の評価:ナイロビのダンドラ地域における重金属汚染の例

  中田北斗, 中山翔太, 水川葉月, 池中良徳, 石井千尋, YOHANNES Yared B, 今内覚, 石塚真由美  環境化学討論会要旨集(CD-ROM)  23rd-  ROMBUNNO.3D-01  2014/05/14  [Not refereed][Not invited]
  
• 我が国におけるマダニ媒介性新興回帰熱病原体ボレリアの分布調査

  川端 寛樹, 今内 覚, 高野 愛, 中尾 稔, 伊東 拓也, 門馬 直太, 安藤 匡子, 高田 伸弘, 佐藤 梢, 大西 真  衛生動物  65-  (Suppl.)  59  -59  2014/03  [Not refereed][Not invited]
  
• 遡及調査により明らかとなった新興回帰熱の2症例

  佐藤 梢, 高野 愛, 今内 覚, 中尾 稔, 伊東 拓也, 小山 幸次郎, 兼古 稔, 大西 真, 川端 寛樹  衛生動物  65-  (Suppl.)  59  -59  2014/03  [Not refereed][Not invited]
  
• 新興回帰熱Borrelia miyamotoi感染症に関する調査およびin vitro抗菌薬感受性

  川端 寛樹, 高野 愛, 大西 真, 佐藤 梢, 今内 覚, 中尾 稔, 伊東 拓也, 小山 幸次郎, 兼古 稔  日本細菌学雑誌  69-  (1)  148  -148  2014/02  [Not refereed][Not invited]
  
• Changes in peripheral leukocyte population and cytokine expression in dairy cows infected with bovine leukemia virus after oral administration of interferon-alpha

  柿沼 清市, 大塚 浩通, 今内 覚  家畜感染症学会誌 = The journal of farm animal in infectious disease  3-  (3)  77  -82  2014
• シュルツェマダニ(lxodes persulcatus)由来因子の宿主免疫抑制機能の解析

  今内覚, 伊東拓也, 川端寛樹, 高野愛, 安藤秀二, 村田史郎, 大橋和彦  大原綜合病院年報  53-  65  2013/12/15  [Not refereed][Not invited]
  
• 免疫抑制受容体PD-1のリガンドPD-L2の機能的特徴と臨床応用研究

  西森朝美, 今内覚, 池渕良洋, 岡川朋弘, 村田史郎, 大橋和彦  動物ワクチン-バイオ医薬品研究会ニュースレター  8-  18  -19  2013/12  [Not refereed][Invited]
  
• 牛白血病ウイルスの感染予防対策

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

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

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

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

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

  今村彩貴, 中溝万里, 川西路子, 池渕良洋, 目堅博久, 村田史郎, 今内覚, 大橋和彦, 中島奈緒, 山本欣也, 内山万利小, 平野文哉, 永井英貴, 木島まゆみ  日本獣医学会学術集会講演要旨集  156th-  260  2013/08/30  [Not refereed][Not invited]
  
• ウシ免疫抑制因子PD‐L1およびPD‐L2の組換えタンパク質作製と免疫機能解析

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

  今内覚, 豊間根耕地, 小池菜々子, 伊東拓也, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  156th-  231  2013/08/30  [Not refereed][Not invited]
  
• 国内に潜在すると考えられる新興回帰熱に関する疫学調査研究

  高野愛, 川端寛樹, 大久保(佐藤)梢, 中尾稔, 伊東拓也, TAYLOR Kyle, 李景利, 坪田敏男, 今内覚, 吉村英紘, 豊間根耕地, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  156回-  304  -304  2013/08  [Not refereed][Not invited]
  
• シュルツェマダニにおける病原体伝播関連因子の同定.

  今内覚, 川端寛樹, 伊東拓也, 高野愛, 安藤秀二, 村田史郎, 大橋和彦  衛生動物  64-  (2)  112  2013/06/15  [Not refereed][Not invited]
  
• 回帰熱病原体Borrelia miyamotoiの検出法の確立・検証,およびIxodes属ダニからの病原体検出への応用.

  高野愛, 佐藤梢, 中尾稔, 伊東拓也, TAYLOR Kyle, 今内覚, 坪田敏男, 高田伸弘, 川端寛樹  衛生動物  64-  (2)  113  -113  2013/06  [Not refereed][Not invited]
  
• スーラ病の分子疫学調査および野外分離株を用いた比較病原性解析と病態発生機序の解明

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

  松山智慧麿, 遠藤大二, 大橋和彦, 今内覚, 村田史郎, 有川二郎, 吉松組子, 水谷哲也, 林正信  日本獣医学会学術集会講演要旨集  155th-  229  2013/03/04  [Not refereed][Not invited]
  
• Recent Findings in Bovine Infection Immunity

  KONNAI Satoru  Journal of the Japan Veterinary Medical Association  66-  (3)  171  -179  2013/03  [Refereed][Invited]
   

  病原微生物の侵入に対して宿主は免疫によって対抗する。牛においても病原微生物に対する感染防御機構は，人やマウスと同様である。すなわち，食細胞を中心とした自然免疫とリンパ球による獲得免疫の協調作用により病原微生物に対抗する。自然免疫系における食細胞による病原体の認識機構は，微生物間で共通の分子構造を抗原非特異的に認識するのに対し，獲得免疫系におけるリンパ球は，受容体の遺伝子再構成により多彩なレパートリーを形成し，微細な抗原構造を特異的に認識する機構を備えている。このようなリンパ球の一部は，免疫を記憶した亜集団へ分化し，病原体の生体内への再侵入時に強い免疫応答を誘導する。このように宿主が備え持つ感染免疫は，非特異的防御のみならず抗原特異性や免疫記憶を有しており，病原体排除に重要な役割を担う。しかし，種々の感染症の中には，予防や治療が困難な感染症がまだ数多く存在する。これらの原因の一つは，病原微生物が免疫回避や攪乱を引き起こし病態が進行することにある。このような感染症に対し有効な治療薬やワクチンを開発するためには，病原体と宿主の相互作用を解明することが重要である。本総説では，どのような異常な免疫応答によって病態が形成され死に至るのか，またどのような免疫回避機構で持続感染が成立し病態を発揮するのか，国内外の感染免疫に関する研究動向を簡単に解説するとともに，これらに関する最近の牛における知見について紹介したい。
• シュルツェマダニ由来因子の病原体伝播における機能解析

  今内覚, 川端寛樹, 伊東拓也, 高野愛, 安藤秀二, 村田史郎, 大橋和彦  大原綜合病院年報  52-  105  -106  2012/12/15  [Not refereed][Not invited]
  
• カメキララマダニより検出された新種回帰熱ボレリアの経ステージ感染様式~ボレリア属細菌の進化の軌跡を探る~

  高野愛, 杉森千恵子, 藤田博己, 角坂照貴, TAYLOR Kyle, 坪田敏男, 今内覚, 田島朋子, 佐藤梢, 渡邉治雄, 大西真, 川端寛樹  大原綜合病院年報  52-  106  -107  2012/12  [Not refereed][Not invited]
  
• 感染症と免疫の基礎 臨床現場で役立つ免疫の知識

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

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

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

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

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

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

  岡川朋弘, 今内覚, 池渕良洋, 鈴木紗織, 寸田祐嗣, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  154th-  233  2012/08/31  [Not refereed][Not invited]
  
• シュルツェマダニ(Ixodes persulcatus)唾液腺由来免疫抑制因子sL2の同定および発現解析

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

  池渕良洋, 今内覚, 岡川朋弘, 横山和正, 中島千絵, 鈴木定彦, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  154th-  233  2012/08/31  [Not refereed][Not invited]
  
• マレック病感染鶏由来腫瘍病変部における免疫抑制因子PD‐1/PD‐L1発現の形態学的解析

  村田史郎, 松山あゆ美, 伊勢崎政美, 高崎紗蘭, 市居修, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  154回-  234  -234  2012/08  [Not refereed][Not invited]
  
• カメキララマダニより検出された回帰熱群ボレリアの経ステージ感染様式

  高野愛, 杉森千恵子, 藤田博己, 角坂照貴, TAYLOR Kyle, 坪田敏男, 今内覚, 田島朋子, 佐藤梢, 渡邉治雄, 大西真, 川端寛樹  日本獣医学会学術集会講演要旨集  154回-  270  -270  2012/08  [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]
  
• 増加している牛白血病 〜北海道での現状と対策について〜

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

  岡川朋弘, 今内覚, 池渕良洋, 鈴木紗織, 村田史郎, 大橋和彦  動物ワクチン-バイオ医薬品研究会ニュースレター  5-  21  -23  2012/06  [Not refereed][Invited]
  
• 小誌連載「内科実習」各疾患の群管理・牛白血病対策

  今内 覚  臨床獣医  6-  44  -49  2012/06  [Not refereed][Invited]
  
• 牛白血病ウイルスに対する大規模牛群内での伝播防止コントロール

  今内 覚  酪農ジャーナル  4-  15  -18  2012/04  [Not refereed][Invited]
  
• 地方病性牛白血病腫瘍組織における免疫抑制因子PD‐L1の発現とその役割

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  今内覚, 岡川朋弘, NAFTALY Githaka, 目堅博久, 鈴木紗織, EDWARD Kariuki, ROBERT Skilton, 石塚真由美, 村田史郎, 大橋和彦  日本獣医学会学術集会講演要旨集  152nd-  204  2011/08/31  [Not refereed][Not invited]
  
• ボレリア属細菌のマダニ体内動態と進化に関する研究.

  高野愛, 川端寛樹, 藤田博己, 角坂照貴, 今内覚, 田島朋子, 渡邉治雄, 大西真  衛生動物  62-  (2)  133  2011/06/15  [Not refereed][Not invited]
  
• シュルツェマダニ(Ixodes persulcatus)におけるライム病ボレリア伝播関連因子の同定.

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

  今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  Dairyman  61-  (6)  30  -31  2011/06/01  [Not refereed][Invited]
  
• マダニ媒介性のライム病病原体Borrelia gariniiの維持伝播サイクルに関する研究

  川端 寛樹, 高野 愛, 中尾 稔, 増沢 俊幸, 高田 伸弘, 矢野 泰弘, 石畝 史, 藤田 博己, 伊東 拓也, 及川 陽三郎, 川森 文彦, 熊谷 邦彦, 三上 稔之, 花岡 希, 安藤 秀二, 本田 尚子, Taylor Kyle, 坪田 敏男, 今内 覚, 渡邉 治雄, 大西 真  衛生動物  62-  (Suppl.)  69  -69  2011/04  [Not refereed][Not invited]
  
• 牛の難治性疾病に対する多機能型ワクチン戦略

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

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

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

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

  今内 覚, 田島 誉士, 村田 史郎, 大橋 和彦  牛臨床寄生虫研究会誌  2-  2  -3  2011/02  [Not refereed][Invited]
  
• ワンヘルス理念に基づく動物由来感染症制御に関する研究 国内生態系におけるライム病ボレリアの維持伝播経路に関する研究

  川端寛樹, 石ぐろ史, 藤田博己, 高田伸弘, 矢野泰弘, 増澤俊幸, 中尾稔, 伊東拓也, 及川陽三郎, TAYLOR Kyle, 坪田敏男, 今内覚, 川森文彦, 三上稔之, 熊谷邦彦, 高野愛, 安藤秀二, 花岡希, 本田尚子, 渡邉治雄, 大西真  ワンヘルス理念に基づく動物由来感染症制御に関する研究 平成22年度 総括・分担研究報告書  17  -20  2011  [Not refereed][Not invited]
  
• 獣医学における保全医学の展開―生物多様性と野生動物感染症―マガンなどの野生水禽の疾病抵抗性とマレック病ウイルスの分布

  大橋和彦, 松原綾子, 村田史郎, 今内覚  獣医畜産新報  64-  (1074)  21  -26  2011/01/01  [Not refereed][Not invited]
  
• 牛白血病ワクチン開発への展望

  今内 覚  家畜衛生学雑誌  37-  81  -84  2011/01  [Not refereed][Invited]
  
• 酪農現場における牛白血病対策の現状と課題

  今内 覚  臨床獣医  29-  14  -18  2011/01  [Not refereed][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]
  
• 難治性慢性疾病における免疫疲弊化機序の解明と新規治療法への応用―獣医医療への応用にむけて―

  池渕良洋, 今内覚, 寸田祐輔, 小沼操, 村田史郎, 大橋和彦  日生研たより  56-  (6)  77  -81  2010/11/01  [Not refereed][Not invited]
  
• ザンビア共和国をモデルとした微量元素汚染:ウシにおける微量元素濃度とメタロチオネインおよびサイトカインmRNA発現量

  室谷太郎, 池中良徳, 中山翔太, YABE John, MUZANDU Kaampwe, CHOONGO Kennedy, 今内覚, 梅村孝司, 石塚真由美  日本獣医学会学術集会講演要旨集  150th-  323  2010/09/01  [Not refereed][Not invited]
  
• フィリピン共和国で分離したTrypanosoma evansiの病原性比較解析

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

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

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

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

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

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

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

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

  松原綾子, 村田史郎, 加納里佳, 橋口知幸, 伊勢崎政美, 河東あゆ美, 小沼操, 今内覚, 大橋和彦  日本獣医学会学術集会講演要旨集  150th-  215  2010/09/01  [Not refereed][Not invited]
  
• メガファームにおける取り組み紹介―牛白血病ウイルス感染伝播阻止トライアルの実例―

  今内覚, 安富一郎, 田島誉士, 村田史郎, 大橋和彦  北海道獣医師会雑誌  54-  (8)  378  2010/08/10  [Not refereed][Not invited]
  
• ザンビア共和国をモデルとした重金属類汚染:家畜における重金属類濃度とバイオマーカーmRNA発現量

  室谷太郎, 中山翔太, 池中良徳, YABE John, MUZANDU Kaampwe, CHOONGO Kennedy, 今内覚, 梅村孝司, 石塚真由美  環境化学討論会講演要旨集  19th-  816  -817  2010/06/20  [Not refereed][Not invited]
  
• 難治性慢性疾病における免疫疲弊化機序の解明と新規治療法への応用〜獣医医療への応用に向けて〜

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

  今内 覚, 田島 誉士, 小沼 操, 村田 史郎, 大橋 和彦  産業動物臨床医学雑誌  1-  (2)  110  -114  2010/06  [Not refereed][Invited]
  
• 増加傾向にある牛白血病の現状と対策 〜診療・生産現場からの声に対して〜

  今内 覚, 田島 誉士, 小沼 操, 大橋 和彦  家畜診療  57-  (4)  201  -207  2010/04  [Not refereed][Invited]
  
• ダニ媒介性感染症に対する抗ダニワクチンの開発

  今内 覚  家畜衛生学雑誌  36-  73  -75  2010/04  [Not refereed][Invited]
  
• マガンなどの野生水禽の疾病抵抗性とマレック病ウイルスの分布

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

  Ohashi K, Murata S, Okada T, Konnai S  Animal Viruses  16-  117  -132  2010/01  [Refereed][Invited]
  
• 牛白血病ウイルス感染症の現状と対策〜清浄化への道、まずは新たな感染を防ぐことから〜

  今内 覚  北海道獣医師会雑誌.  53-  529  -534  2009/09  [Not refereed][Invited]
  
• シュルツェマダニにおけるライム病ボレリア伝播関連遺伝子の同定

  今内覚, 村瀬優介, 山田慎二, 肥田野新, 伊東拓也, 川端寛樹, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  148th-  2009
• Theileria parva感染牛に認められた炎症性サイトカインストーム

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

  橋口知幸, 村田史郎, 岡田宰, 加納里佳, 今内覚, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  146th-  189  2008/09/05  [Not refereed][Not invited]
  
• 弱毒および強毒株マレック病ウイルス感染鶏における免疫応答の比較

  加納里佳, 橋口知幸, 岡田宰, 村田史郎, 今内覚, 小沼操, 大橋和彦  日本ウイルス学会北海道支部夏季シンポジウム講演抄録  42nd-  14  2008  [Not refereed][Not invited]
  
• 帯広近郊,ロシアならびにカムチャッカにおける野生水禽類のToxoplasma gondii抗体保有状況

  村尾岳洋, 加納里佳, 村田史郎, 今内覚, 大橋和彦, 小沼操, 前田龍一郎, 小俣吉孝  日本野生動物医学会大会・講演要旨集  13th-  88  2007/09/06  [Not refereed][Not invited]
  
• コイタマダニにおけるオスダニ由来因子の同定と発現解析

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

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

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

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

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

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

  岡田宰, 村田史郎, 加納里佳, 高木道浩, 今内覚, 小沼操, 大橋和彦  日本獣医学会学術集会講演要旨集  144th-  83  2007/08/27  [Not refereed][Not invited]
  
• Interferon-gamma expression associated with suppression of bovine leukemia virus at the early phase of infection in sheep

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

  Satoru Konnai, Tatsufumi Usui, Manabu Ikeda, Junko Kohara, Kosuke Okada, Kazuhiko Ohashi, Misao Onuma  AIDS RESEARCH AND HUMAN RETROVIRUSES  23-  (4)  608  -609  2007/04  [Not refereed][Not invited]
  
• 犬腫瘍細胞溶解産物を感作した樹状細胞ワクチンの有用性に関する基礎的研究

  横川 美穂, 今内 覚, 星野 有希, 高木 哲, 奥村 正裕, 藤永 徹  日本獣医学会学術集会講演要旨集  143回-  211  -211  2007/03  [Not refereed][Not invited]
  
• Theileria parva感染Rhipicephalus appendiculatusによる宿主への実験的伝播

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

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

  村田史郎, 岡田宰, 加納里佳, 林裕子, 今内覚, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  142nd-  80  2006/08/31  [Not refereed][Not invited]
  
• コイタマダニ由来セルピンの抗ダニカクテルワクチン効果

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

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

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

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

  村田史郎, 山本裕己, 岡田宰, 浅川満彦, 長雄一, 池内俊雄, 今内覚, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  140th-  108  2005/08/31  [Not refereed][Not invited]
  
• 発現遺伝子解析によるダニ由来有用物質の探索

  中島千絵, 今村彩貴, 今内覚, 大橋和彦, 小沼操  日本獣医学会学術集会講演要旨集  138th-  65  2004/08/01  [Not refereed][Not invited]
  
• BLV感染における遺伝的抵抗性(抵抗性牛)

  今内 覚  臨床獣医  22-  20  -23  2004/03  [Not refereed][Invited]
  
• Influence of polymorphism of MHC class II alleles on immune response in bovine leukemia virus infection.

  Y Aida, S Konnai, S Takeshima, S Tajima, S Yin, M Onuma, K Okada  AIDS RESEARCH AND HUMAN RETROVIRUSES  19-  S18  -S19  2003  [Not refereed][Not invited]
  
• Comparison of Peripheral Leukocytes in Japanese Black and Holstein Calves

  OHTSUKA Hiromichi, KOMATSU Shoh-ichi, KON-NAI Satoru, FUKUDA Shigeo, KIKU Yoshio, YOSHINO Tomo-o, KOIWA Masateru, KAWAMURA Sei-ichi  Journal of the Japan Veterinary Medical Association  55-  (12)  789  -795  2002/12/20  [Not refereed][Not invited]
   

  黒毛和種(JB)子牛の細胞性免疫を評価する目的で、出生後のJB子牛（JB群；n=10）とホルスタイン種（Hol)子牛（Hol群；n=10）の末梢血白血球の変化を比較した。各群の子牛の出生後3日以内(0週齢)および3、6、9、12週齢の末梢血白血球はフローサイトメトリー（FCM)を用いて解析した。末梢血白血球はFCMの側方散乱光〔side light scatter(SSC)〕と前方散乱光〔forward light scatter (FSC)〕によって得られた3つの大きさ、すなわち小型、中型および大型に分類して単球・リンパ球（ML）とした。各群の中型（M)-ML数は0週齢が最低値で、その後増加した。しかしJB群のM-ML数は6から9週後に減少し、6週にはHol群に比べ有意な低値が認められた。JB群の小型（S)-ML数は3週後に最高値となり、0、3、9、12週にはHol群に比べ有意な高値が認められた。JB群のCD3(+)細胞数はHol群に比べ3週齢に有意な高値を示した。またS、M-ML中のWC(+)細胞比率は、観察期間中JB群がHol群に比べ低値を推移する傾向が認められた。これらのことからJB子牛の細胞性免疫応答はTリンパ球を中心にHol子牛とは異なることが明らかとなった。
• OLA-DRB1アリルの塩基配列の決定とPCR-RFLP法の開発

  今内 覚, 長岡 淑子, 岡田 幸助, 小沼 操, 間 陽子  日本獣医学会学術集会講演要旨集  129回-  95  -95  2000/03

Awards & Honors

• 2019/12 農林水産省 農業技術 2019年10大ニュース
   牛白血病の新たな制御方法、抗ウイルス効果の確認に成功 －牛の難治性疾病に対する応用に期待－ 

  受賞者: 今内 覚
• 2019/06 伊藤記念財団 第4回伊藤記念財団賞
   

  受賞者: 今内 覚
• 2017/01 北海道大学 平成28年度北海道大学研究総長賞
   

  受賞者: 今内 覚
• 2015/09 日本獣医学会 日本獣医学会賞
   

  受賞者: Satoru Konnal
• 2014/02 北海道 平成25年度北海道科学技術奨励賞
   

  受賞者: 今内 覚
• 2005/06 森永奉仕会 森永奉仕会賞
   

  受賞者: 今内覚
• 2003/10 日本獣医学会 獣医学奨励賞
   

  受賞者: 今内覚
• 2003/10 日本獣医学会 日本獣医学会会長賞
   

  受賞者: 今内覚

Research Grants & Projects

• Development of cocktail vaccines against poultry red mites using the recombinant turkey herpesvirus

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/04 -2027/03 

  Author : 村田 史郎, 今内 覚, 大橋 和彦
• Elucidation of the mechanism of tumorigenesis in bovine leukemia by comprehensive genomic analysis of proviral integration sites

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2024/04 -2027/03 

  Author : Tomohiro Okagawa, Satoru Konnai, Naoya Maekawa
• The roles of polymorphism in Meq on the pathogenicity of Marek's disease virus and their application for the vaccine development

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/04 -2026/03 

  Author : 大橋 和彦, 村田 史郎, 今内 覚
• Construction of animal foamy virus vector and development of enzootic bovine leukosis vaccine

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/04 -2026/03 

  Author : 目堅 博久, 齊藤 暁, 今内 覚, 岡川 朋弘
• The search for the candidate molecule for the vaccine to control a wide range of ectoparasite species of chickens

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2020/04 -2023/03 

  Author : 大橋 和彦, 村田 史郎, 今内 覚

   

  ワクモやトリサシダニは、鳥類の代表的な外部寄生虫であり、鶏に貧血や削痩、産卵率低下など養鶏産業に大きな経済的被害をもたらしている。現在、その防除 には主に駆虫剤が使用されているが、薬剤耐性個体の出現や駆虫剤残存による衛生環境の悪化などが問題となっており、抗ダニワクチンなど新規防除法の開発が必要となっている。そこで本研究では、ワクモとトリサシダニ及び近縁なミナミトリサシダニを同時に防除できるダニ種横断的な新規“ユニバーサル”ワクチン の開発を目指して、RNA-SEQ解析や全ゲノム解析と相同性検索により、これらのダニ間で共通の非暴露型抗原の探索・同定を行い、 吸血したダニ個体に致死的に作用して鶏舎内の寄生虫個体数を持続的に減少できるワクチンへの応用を検討することを目的とした。 今年度は、昨年度実施したRNA-SEQによる発現する遺伝子群の網羅的解析の結果を元にFER2など数種類の候補遺伝子を選抜して、その組換え抗原の調製や機能解析を行った。in vitro feeding assayによる今後交差防御試験を行いワクチン候補抗原としての有用性を確認する。またいくつかのワクモ由来分子の抗ワクモワクチンとしての有用性を検討し、抗ワクモ効果（持続的な個体数の減少）を示す新規抗原を同定した。さらに複数種の抗原を用いたカクテル抗原が単独抗原よりも有効な抗ワクモ効果を示すことも明らかとなった。今後、これらの抗原についても“ユニバーサル”ワクチンへの応用を検討していく。
• 血液脳関門を透過する新規DDSによる神経向性ウイルス感染の治療法開発

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2020/04 -2023/03 

  Author : 好井 健太朗, 小林 進太郎, 五十嵐 学, 今内 覚

   

  ダニ媒介性脳炎、日本脳炎、狂犬病など神経向性の人獣共通感染症ウイルスは、脳に侵入・増殖することで重篤な神経症状を引き起こし、致死率も高い。しかし血液脳関門：BBBの存在のため、抗ウイルス分子をウイルスの増殖する脳に到達させる手法が無く、治療法が未開発であった。BBBにはトランスサイトーシスと呼ばれる物質を血流から脳内へと輸送する機構があり、近年の研究により、このBBB透過に関わる分子の機構が明らかになってきている。本研究では、このBBB透過機構に着目し、組換え抗体等の抗ウイルス分子技術と融合させることで、BBBを透過する機能を利用した抗ウイルス分子の脳内導入法を構築し、神経向性ウイルス感染に対する治療応用を試みる。 本年度における研究では、昨年度の研究で構築した、ダニ媒介性脳炎ウイルス（TBEV）の幅広いウイルス株に対して中和効果を示すモノクローナル抗体をベースとして、BBBのトランスサイトーシスへの関与が示されている狂犬病ウイルスのG蛋白上のアミノ酸配列を融合させた組換え抗体を用いての研究を進めた。本抗体に誘導させた、狂犬病ウイルスのG蛋白上のアミノ酸配列は、神経細胞やBBBの血管内皮細胞上に発現するアセチルコリンレセプターとの結合により細胞内輸送小胞に取り込まれて、これがトランスサイトーシスに関与する事が示されている。そこで本研究ではアセチルコリンレセプターを細胞膜上に豊富に発現している神経細胞由来の培養細胞等を用いて検証した所、作製した組換え抗体分子は細胞膜表面に結合している事が示され、これはアセチルコリンレセプターを介したものである事が示唆された。さらに抗体をマウスの末梢に投与した所、マウスの脳内から抗体が検出されたことから、抗体は脳内に移行していることが示され、本研究で検証した組換え抗体分子は、脳内で増殖するウイルスに対する治療応用に有効である可能性が示唆された。
• MHC クラスII DRB3*0902保有牛における牛白血病抵抗性機序

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2019/04 -2023/03 

  Author : 乗峰 潤三, 佐藤 克明, 今内 覚

   

  BLV感染症において牛主要組織適合遺伝子複合体（bovine MHC: BoLA）クラスIIDRB3*0902遺伝子を保有する牛は、極めて低いプロウイルス量を維持し、リンパ球増多症への病態進行に対する抵抗性を持つ。本研究目的は、BLVに感染した牛のBLV抗原特異的CD4+T細胞を解析し、BoLA-DRB3*009:02遺伝子保有牛における抵抗性の免疫学的機序を解明する事である。そのためには、まずBoLA-DRB3*009:02拘束性のCD4＋T細胞エピトープを同定する必要がある。これまで、CD4＋T細胞エピトープ同定に使用する組換BLVgag及びenv 蛋白質(rBLVgag/env)を、全て発現ベクター(pET151)に組み込んだ。また、オリゴペプチドとして、全BLVgag/envをカバーする連続20アミノ酸ペプチドを合成した。さらに、多数のBoLA-DRB3テトラマーを作製し、tetramer-guided epitope mapping (TGEM)法によりBLV抗原特異的CD4+T 細胞エピトープの同定を行った。牛において、MHCクラスIIテトラマーを作製しているのは本研究室のみである。従って、牛のTGEM法によるCD4+T 細胞エピトープ同定の報告は世界初である。 TGEM法によるCD4+T 細胞エピトープ同定に使用したのはフィールドBLV感染牛で、DRB3*009:02/DRB3*015:01ヘテロを保有する牛２頭、DRB3*009:02/DRB3*002:01ヘテロを保有する牛２頭、DRB3*015:01ホモを保有する牛２頭である。それぞれから回収した末梢血PBMCを利用してTGEM法により、BLV抗原特異的CD4+T 細胞エピトープを同定した。
• Development of a method to control poultry red mites using recombinant Marek's disease virus that secretes a vaccine antigen

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2021/03 

  Author : Murata Shiro

   

  Poultry red mites (PRMs, Dermanyssus gallinae) are haematophagous ectoparasites which causes serious economic losses to the poultry industry worldwide, and the development of novel methods to control PRMs is required. We have investigated the application of vaccination and have shown that immunization with vaccine antigens could contribute to the reduction in the number of PRMs. In addition, we investigate the application of virus vectors to develop easy-to-use vaccines for farmers. In this study, we designed the recombinant virus that secrets vaccine antigens to improve the efficiency of vaccines using the recombinant virus. To produce an antigen-secreting virus, we inserted the signal peptides of a virus-derived secretory protein to the virus genome. However, the secretion of antigens was not confirmed, although the expression was observed in infected cells. Further manipulation is required to generate effective vaccines using virus vectors for controlling PRMs.
• Analysis of the molecular mechanism for the increase in the virulence of Marek's disease virus

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2017/04 -2020/03 

  Author : Ohashi Kazuhiko

   

  Marek’s disease virus (MDV), a causative agents of Marek’s disease (MD), which is characterized by the formation of malignant lymphoma, appears to increase its virulence in the fields. To elucidate the molecular mechanisms for the increase in its virulence, we isolated MDVs from vaccinated chickens which still developed clinical MD, and analyzed their virological properties. MDV recently isolated in Japan caused clinical MD in inoculated chickens, although less virulent to chickens compared to virulent MDV strains isolated in United States. In addition, it is suggested that virus-derived factors other than polymorphisms in viral oncogene reported in the United States would be involved in the increase in MDV virulence.
• Analysis of genetic polymorphism of poultry red mites in the world and search for candidate molecules of anti-mite vaccines

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2019/03 

  Author : OHASHI Kazuhiko

   

  Poultry red mites (PRMs), Dermanyssus gallinae, are blood-sucking ectoparasites of chickens, which cause huge economic losses in the poultry industries worldwide. To establish a new effective control method for PRMs, we have searched for and identified candidate antigens which are not polymorphic among PRMs from different areas in the world to develop anti-PRM vaccines. Several antigens identified in this study showed anti-PRM effects determined by in vitro feeding assay using sera from immunized chickens.
• Comprehensive analysis of molecules expressed on the midgut of poultry red mites for the development of new anti-mite vaccines

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2018/03 

  Author : OHASHI Kazuhiko

   

  For the development of new vaccines against poultry red mite, Dermanyssus gallinae, a search was made for novel vaccine candidates expressed on the cell membrane of the midgut of the mite. Three kinds of molecules, whose expressions are predicted on the cell membrane, were identified, and their expressions on the midgut were confirmed by laser capture microdissection and RT-PCR. In addition, cathepsin D-like molecule, catD2, which was already identified in the course of the comprehensive analysis of genes expressed in the red mites done in our laboratory, was shown to be expressed in protonymph, deutonymph and adult and expressed regardless engorgement or starvation. The recombinant catD2 protein was not recognized by the sera from chickens fed by the red mites, suggesting that catD2 is a concealed antigen. Further analyses, including the detailed functions, the expression sites in red mites and the evaluation as vaccine antigens, are required to develop effective vaccines.
• Intramammary infusion of food-grade lactic acid bacteria (Bifidobacterium breve) on cellular and immuno-modulatory functions in mammary glands and therapeutic trials on bovine mastitis in dairy cows.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : NAGAHATA Hajime, ENDOU DAIJI

   

  The aim of this study was to evaluate the efficacy of lactic acid bacteria (LAB) by intramammary infusion for control of bovine mastitis. Methods: LAB, Bifidobacterium breve(3x10^9cells/ml) was infused quarters with mastitis of 48 lactating cows after milking once a day for 2 days. Pathogens, somatic cell counts (SCC), lactoferrin (LF) and IgG and A, chemiluminescence (CL), and mRNA expressions of Th1, 2- cytokines on milk cells were evaluated. Results: SCC and CL responses were increased (P<0.05) in quarters by LAB infusion. LF and IgG,A were also induced in milk on day 3 to day 7. Cytokines IL-1β,TNF-α and IL-6 were elevated in milk by LAB infusion. I Environmental mastitis pathogens, coagulase negative staphylococci and streptococci, were eliminated 50-60% by LAB infusion. In mastitis quarters with no pathogens, SCC in 80% quarters returned to the recovered normal range on day 14 by LAB infusion. The use of LAB appears to be a possible approach for controlling bovine mastitis.
• Influence of the immune status on the malignancy of canine histiocytic sarcoma

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : Takagi Satoshi, KONNAI Satoru, TAGAWA Michihito, YOSHIDA Hiromitsu, YOSHIMOTO Sho

   

  Histiocytic sarcoma (HS) is a progressive neoplastic disease in dogs. It is unclear whether costimulatory molecules, including CD28, CTLA-4, PD-1 are expressed on peripheral blood lymphocytes (PBLs) of canine patients with HS. The expression of CTLA-4 on CD8+ lymphocytes was significantly higher and the expression of PD-1 on CD8+ lymphocytes was significantly higher in the HS group than in the control group. On the other hand, we investigated the presence of tumor associated macrophages (TAMs) and fibroblasts (CAFs) in HS and other tumors. CAFs are rarely observed in HS tissues whereas relatively higher expressions were shown in epithelial tumors such as anal sac tumor and mammary gland tumor. CD204 positive TAMs were frequently observed in HS tissues which showed this tumor suppress host CTLs in situ. In conclusion, HS in dogs may have strong immunosuppressive ability and therapeutic agents such as anti-immune check point molecules can effective for the treatment of this tumor.
• Studies on the molecular mechanisms for the increase in MDV virulence in the field

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : Kazuhiko Ohashi

   

  Marek’s disease virus (MDV), a causative agent of Marek’s disease (MD), which is characterized by the formation of malignant lymphomas, tends to increase its virulence in the fields. To clarify the molecular mechanism for the increase in MDV virulence, recent MDV strains isolated from vaccinated chickens which developed clinical MD were virologically and molecular biologically characterized. Several new diversities/mutations were detected in viral genes of these strains, and a plaque-purified MDV strain was shown to be pathogenic to chicks. However, phylogenetic analysis of the whole genome of the MDV strain and reference strains showed that domestic MDV may increase its virulence through unknown mechanisms which are different from those suggested previously in MDV strains isolated in the United States.
• Defence mechanism against environmental chemicals in livestock animals

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2016/03 

  Author : ISHIZUKA MAYUMI, IKENAKA Yoshinori, TSUBOTA Toshio, KONNAI Satoru

   

  In livestock animals, we identified the enzymatic characters of cytochrome P450 as typical phase I enzyme for xenobiotics. We also analyzed glucronyltrasnferase and sulfotransferase dependent metabolites in urine samples from mammalians including livestock animals, to elucidate the ability of xenobiotic conjugating detoxification. In field surveillance at Asia and African countries, heavy metals are major accumulated environmental pollutants in cattle, and the accumulating pattern is unique in ruminants. In case of poultry species, we clarify the expressed cytochrome P450 and phase II enzymes in liver using transcriptome analyses method and in silico analyses, and we compared the structure of xenobiotic metabolizing enzymes in poultry species to those of mammals.
• Studies on the immunoinhibitory mechanisms of Marek's disease virus and its application for adjuvant

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011/04 -2014/03 

  Author : OHASHI KAZUHIKO, KONNAI Satoru, MURATA Shiro

   

  The programmed death 1 (PD-1) receptor and its ligands, programmed death ligand 1 (PD-L1) and 2 (PD-L2) are known to have an immunoinhibitory function which contribute to the pathogenesis of chronic infections and neoplastic diseases via the PD-1/PD-L pathway. In this study, it has been shown that chicken PD-1 and PD-L1/PD-L2 also have an immunoinhibitory function, such as inhibition of cytokine production. The expressions of PD-1 and PD-L2 were significantly higher in the spleens of infected chickens in the latent phase and in tumor lesions caused by Marek's disease virus (MDV). These results suggest that chicken PD-1/PD-L2 pathway is involved in the establishment of latency and tumor formation by MDV.
• Epidemiological and immunological analyzes of spread manner of bovine viral diarrhea virus in the herd

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2010/04 -2014/03 

  Author : TAJIMA Motoshi, KADOHIRA Mutsuyo, KONNAI Satoshi

   

  In order to estimate the spread manner of bovine viral diarrhea virus (BVDV) in and among the dairy herd, epidemiological and immunological analyzes were done for the BVDV detected and free herd discriminated by the bulk tank milk tests. Although the correlation between the geographical place of farm and outbreak of BVDV was recognized, prevalent viruses were not common based on the genetical analysis of the detected virus gene. On the immunological analyzes, BVDV might prefer to localize in ovary fluid in the infected cattle. Moreover, it was strongly suspected that BVDV might bias the immune status of infected cattle for the easy transmission of virus in uterus.
• Identification and characterization of factors involved in the resistance to Marek' s disease in chickens and wild waterfowls

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2011 

  Author : OHASHI Kazuhiko, KONNAI Satoru

   

  To study the molecular mechanism(s) for the natural resistance to Marek' s disease (MD) in wild waterfowls and genetically resistant lines of chickens, the regulatory mechanisms of the expression of interferon y (IFNy), which plays an important role in the cell-mediated immunity against MD virus (MDV), were analyzed by using the conventional luciferase reporter assay system. The promoter activity was more strongly repressed in white-fronted geese than chickens when Meq, an MDV oncoprotein, was co-expressed. This result suggests that, in white-fronted geese, reduced expression of IFNy by Meq at early after MDV infection could result in the lower degree of T cell activation (activated T cells are targets for MDV) to reduce the chances for the birds to develop MD.
• Studies on the molecular mechanisms for Marek's disease virus to increase its virulence in the field

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2008 -2010 

  Author : OHASHI Kazuhiko, OCHIAI Kenji, KONNAI Satoru

   

  To clarify the molecular mechanisms for Marek's disease virus (MDV) to increase its virulence, several viral genes of MDV strains recently isolated from the field were analyzed. Diversities/polymorphisms were identified in one of the MDV genes, meq, that could be potentially related to the increase in MDV virulence. When we analyzed the effects of these diversities/polymorphisms in the meq gene on the functions of the meq gene product (Meq), the amino acid substitutions resulted from the diversities/polymorphisms significantly altered the transactivation and transforming activities of Meq. These findings suggest that the diversities/polymorphisms in the meq gene could contribute to the recent increase in MDV virulence in the field.
• Genetic analysis of drug resistant trypanosomes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2006 -2008 

  Author : ONUMA Misao, OHASHI Kazuhiko, INOUE Noboru, KONNAI Satoru

   

  トリパノソーマ原虫は人、家畜および野生動物に感染し睡眠病、ナガナ病、スーラ病などを引き起こし、多大な被害を与えている。本症は、ワクチンによる制御が困難なうえ、近年、原虫薬剤耐性株の出現によって、その被害は深刻化している。そこで原虫遺伝子多型および薬剤耐性機序解明を目的として、ザンビア共和国およびフィリピン共和国における本症の分子疫学調査を行った。さらに、媒介昆虫であるツェツェバエの吸血宿主の同定、すなわち本症のレゼルボア動物の推定のために、トリパノソーマ感染ツェツェバエ由来のDNAサンプルに含まれる哺乳類動物のミトコンドリア遺伝子を検出し、その遺伝子配列解析から吸血宿主を同定した。その結果、ヒトおよびアフリカゾウ、アフリカバッファローなどの野生動物に加え、調査地区集落の主要な家畜であるヤギの遺伝子も検出されたことから、ヤギにおける本症の分子疫学調査も行い、同地区の高率な感染率を報告した。一方、フィリピン共和国において集団流産が認められた家畜の原因病原体の調査も実施するとともに、抗原虫薬剤治療歴を持つ家畜からトリパノソーマ原虫を分離し薬剤感受性ならびに病原性解析を行った。
• 抗糖脂質モノクローナル抗体のイヌ型抗体化とメラノーマ治療への応用

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2006 -2007 

  Author : 大橋 和彦, 今内 覚, 奥村 正裕

   

  糖脂質を標的分子とした新規の抗腫瘍療法を開発することを目的として研究として研究を行った。今年度は、平成18年度に調製し、腫瘍細胞、特にイヌ由来メラノーマ細胞株との反応性を確認した各種糖脂質に対するマウスモノクローナル抗体(MAb)パネルのうち、細胞株に広範に反応した抗GM3 MAb(クローンNo.2-14)について、さらに解析を行った。その結果、このクローンMAbは、非常に多くのメラノーマ細胞株に対して試験管内で細胞傷害性を有していることを見出した。また種々のメラノーマ可移植性細胞株を皮下に移植したヌードマウスを用いた治療試験でも、MAbの連続投与により、移植腫瘍の生育が抑制されることが示された(ただし、対照群と治療群の間に統計学的有意差は検出されなかった)。以上より、in vivoにおいても抗GM3 MAbが抗腫瘍効果を発揮できることがマウスモデルで証明された。 昨年度に、抗GM3 MAb(クローンNo.2-14)の抗原結合部位である超可変領域(抗原特異性決定部位)の塩基配列を決定し、イヌ型抗体化を目指して遺伝子発現ベクターへ挿入するため、遺伝子カセットをH鎖・L鎖それぞれにおいて作成したが、今年度は、さらに組み換えイヌ型抗糖脂質MAbの臨床応用を目的として、作成した遺伝子カセットをH鎖・L鎖のバキュロウイルス発現系を用いた組み換えイヌ型抗GM3 MAbの大量発現系の樹立を試みた。しかしながら、その発現量は、あまり高くないことが判明した。発現したL鎖とH鎖について、そのGM3との結合活性を確認したが、単独では結合せず、組み換えL鎖およびH鎖の会合した分子を調製する必要性が示唆された。今後、spacerなどでL鎖およびH鎖を結合した遺伝子を調製し共発現させる系を検討する。
• ダニの唾液由来免疫抑制因子を用いるダニ媒介性病原体の伝播阻止

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2006 -2007 

  Author : 小沼 操, 大橋 和彦, 今内 覚

   

  ダニの発現遺伝子の網羅的解析のため吸血5日目のフタトゲチマダニ(Haemaphysalis longicornis)唾液腺を用いてEST解析(発現タグ解析)を行った。得られた653配列を米国NCBIのblastXプログラムを用いて蛋白データベースと比較したところ、232配列(36%)で有意な相関を示す類似遺伝子が見つかった。その大半は細胞の生存に必要な、いわゆるハウスキーピング遺伝子であったが、1/3は宿主の生理活性に何らかの影響を与え得る因子であると推察された。今回得られた遺伝子の一つは抗凝固因子であるmadanin1と塩基配列で88%、アミノ酸配列で66%の相同性を示した。そこで本因子の生物活性を解析したところ抗凝固活性が認められた。一方、免疫抑制作用が示唆される1配列について宿主免疫への影響を解析した。その結果、牛末梢血単核球(PBMC)やマウス脾臓細胞の増殖反応を濃度依存的に抑制し、サイトカイン(IL-2、IL-12p40およびTNF-α)の発現も強く抑制した。また、BALB/cマウスへの接種では、脾臓の萎縮が認められ、これらのマウス由来の脾臓細胞は各種Mitogenに対する増殖反応が著しく低下し、Microarray解析では免疫活性化因子の発現抑制も認められた。
• Tick vaccine development by using tick testis-derived factors

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2005 -2007 

  Author : ONUMA Misao, OHASHI Kazuhiko, TAJIMA Tomoko, KONNAI Satoru

   

  From the cDNA library constructed from testis/vas deferens, we identified a novel gene of Rhipicephalus appendiculatus (Rappendiculatus) tick, male-specific factor, encoding a secretory component exclusively expressed in the testis/vas deferens. This molecule, tentatively named as Rappendiculatus Mating Factor (RAMF), contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMF mRNA was up-regulated in male ticks during blood feeding. RAMF was detected not only in the testis/vas deferens but also in the postcoitum female ticks by Western blotting, suggesting that this protein could be transferred into the female tick through copulation. The attachment duration had a tendency to prolong in the virgin female ticks microinjected with recombinant RAMF, but there was no obvious effect on blood feeding. These results suggest that RAMF is a male-specific molecule in spermatophore, and other factor(s) might be required for stimulation for female engorgement in R. appendiculatus tick.

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