Development of a Practical Highly Sensitive Diagnostic Method for African Swine Disease in Vietnam and Elucidation of Its Environmental Dynamics

• Project/Area Number: 22KK0097
• Research Category: Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 42:Veterinary medical science, animal science, and related fields
• Research Institution: Kyoto University
• Principal Investigator: 山崎 渉 京都大学, 東南アジア地域研究研究所, 教授 (70393262)
• Co-Investigator(Kenkyū-buntansha): 目堅 博久 宮崎大学, 産業動物防疫リサーチセンター, 准教授 (90633264) ; 一二三 達郎 鹿児島大学, 農水産獣医学域獣医学系, 准教授 (90790949)
• Project Period (FY): 2022-10-07 – 2027-03-31
• Project Status: Granted (Fiscal Year 2024)
• Budget Amount: ¥20,150,000 (Direct Cost: ¥15,500,000、Indirect Cost: ¥4,650,000); Fiscal Year 2026: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2025: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2024: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2023: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2022: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: アフリカ豚熱 / 環境動態 / 豚 / 口腔液 / 高感度 / LAMP / 簡易DNA抽出 / 全血 / ポイントオブケア / 診断法

Outline of Research at the Start

グローバル化に伴うアフリカ豚熱の世界流行は世界の脅威である。アフリカ豚熱はアフリカ豚熱ウイルスによる豚とイノシシの熱性伝染病で、強い伝染性と90％を超える高い致死率を特徴とする。中国や朝鮮半島、東南アジアでも感染爆発が続いており、日本にも侵入の危機が迫っている。ベトナムでは、脆弱な水際防疫の強化が急務となっている。本研究では研究代表者らが新開発する高感度・経済的かつ簡易・迅速的なアフリカ豚熱診断法を用いて、イノシシや軟ダニサンプルからの高精度なフィールド調査を行う。

Outline of Annual Research Achievements

日本国内でウイルスの高感度検出法の開発に成功し、論文公表をした。ベトナム国立農業大学との共同研究により、この成果を豚の口腔液に応用したところ、従来法よりも高感度にアフリカ豚熱ウイルス（ASFV）を検出することができた。豚の口腔液63サンプルをベトナム国内の農場で収集し、アフリカ豚熱（ASF）未発生国である日本国内で収集した5サンプルを真の陰性参照として、新開発した方法の性能評価を行った。
その結果、リアルタイムPCR法による解析において、新開発した方法の診断的感度は100% (9/9)、診断的特異度は76.3% (45/59)をそれぞれ示した。新開発した方法では14サンプルが新たに陽性を示し、検出感度の明確な改善が認められた。さらに、LAMP法による解析において、新開発した方法の診断的感度は100% (1/1)、診断的特異度は92.5% (62/67)をそれぞれ示した。新開発した方法では5サンプルが新たに陽性を示し、やはり検出感度の明確な改善が認められた。
添加試験による検出限界比較では、前者では1000倍、後者では100倍以上の高感度化が確認された。臨床サンプルにおいては添加試験ほどの高感度化は認められなかったが、検出感度の明確な改善は確認された。添加試験では培養した新鮮なASFVを使用したことがこの差異が生じた一因ではないかと推測している。
なお、イノシシの口腔液5サンプルを収集し、検査を行ったが、いずれもASFVは陰性を示した。これとは別途、ベトナム国内で40サンプルのマダニを収集することができたので、現在、解析を進めている。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

豚口腔液中に含まれる微量なアフリカ豚熱ウイルスの濃縮検出法の開発に成功した（論文投稿中）。マダニのサンプリングも進んでいる。さらに、日本側から研究代表者と研究分担者の計2名が共同研究加速のためにベトナムを訪問するとともに、若手研究者・大学院生・学部生向けにマダニ媒介性感染症に関する特別セミナーを研究分担者が行った。さらにベトナム側から3名の若手研究者を京都大学に招聘し、知見と技術の共有を進めることができた。学術交流と若手を含む研究者の国際経験の涵養が順調に推進している。

Strategy for Future Research Activity

ベトナム側の共同研究者にサンプリングを継続していただき、新開発した方法の性能評価や現地での調査を継続する。日本側研究者も引き続き現地を訪問し、建設的批判に基づく議論、情報共有、技術支援、サンプリング、相互交流などをすすめる。

Research Products

• [Int'l Joint Research] ベトナム国立農業大学(ベトナム)
• [Int'l Joint Research] ベトナム国立農業大学(ベトナム)
• [Int'l Joint Research] ベトナム国立農業大学(ベトナム)
• [Journal Article] Urine of Cats with Severe Fever with Thrombocytopenia Syndrome: A Potential Source of Infection Transmission (2025)
  • Author(s): Mekata Hirohisa、Yamamoto Mari、Kaneko Yasuyuki、Yamada Kentaro、Okabayashi Tamaki、Saito Akatsuki
  • Journal Title: Pathogens Volume: 14 Pages: 254-254
  • DOI: 10.1016/j.tvjl.2025.106301
  • Peer Reviewed / Open Access
• [Journal Article] Apoptosis in kidney tissue of senior and geriatric cats with chronic kidney disease (2025)
  • Author(s): KURAHARA Natsume、YUTSUDO Ayami、YAMATO Osamu、MIYOSHI Noriaki、HIFUMI Tatsuro、YABUKI Akira
  • Journal Title: Journal of Veterinary Medical Science Volume: 87 Issue: 3 Pages: 248-256
  • DOI: 10.1292/jvms.24-0296
  • ISSN: 0916-7250, 1347-7439
  • Peer Reviewed / Open Access
• [Journal Article] Apoptosis in kidney tissue of senior and geriatric cats with chronic kidney disease (2025)
  • Author(s): KURAHARA Natsume、YUTSUDO Ayami、YAMATO Osamu、MIYOSHI Noriaki、HIFUMI Tatsuro、YABUKI Akira
  • Journal Title: Journal of Veterinary Medical Science Volume: 87 Issue: 4 Pages: 248-256
  • DOI: 10.1292/jvms.24-0444
  • Peer Reviewed / Open Access
• [Journal Article] Phyllodes tumor in the mammary gland of a northern tree shrew (<i>Tupaia belangeri</i>) (2025)
  • Author(s): HIFUMI Tatsuro、TANAKA Yuki、TSUKIYAMA-KOHARA Kyoko、MIYOSHI Noriaki
  • Journal Title: Journal of Veterinary Medical Science Volume: 87 Pages: 384-388
  • DOI: 10.1016/j.virusres.2025.199550
  • Peer Reviewed / Open Access
• [Journal Article] Development of a concentration method for simple and sensitive detection of SARS-CoV-2 in saliva using a magnetic nanoparticle kit (2025)
  • Author(s): Yamazaki Yasuko、Tanaka Riku、Castillo Gladys、Macalanda Adrian Miki C.、Talactac Melbourne R.、Yamazaki Wataru
  • Journal Title: Journal of Virological Methods Volume: 331 Pages: 115059-115059
  • DOI: 10.1016/j.jviromet.2024.115059
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] MALDI-TOF MS analysis for detection of bovine coronavirus with tryptic peptides from viral proteins (2024)
  • Author(s): HAYASHI KATSUHIKO、OHYA KENJI、YOSHINARI TOMOYA、HIROSE SHOUHEI、SHIMIZU SOUTA、MORITA YUJI、OHNISHI TAKAHIRO、WATANABE MAIKO、TAHARAGUCHI SATOSHI、MEKATA HIROHISA、TANIGUCHI TAKAHIDE、HARA-KUDO YUKIKO
  • Journal Title: Journal of Microorganism Control Volume: 29 Issue: 4 Pages: 143-151
  • DOI: 10.4265/jmc.29.4_143
  • ISSN: 2758-6383, 2758-6391
  • Peer Reviewed / Open Access
• [Journal Article] Nine-year seroepidemiological study of severe fever with thrombocytopenia syndrome virus infection in feral horses in Cape Toi, Japan (2024)
  • Author(s): Mekata Hirohisa、Yamada Kentaro、Umeki Kazumi、Yamamoto Mari、Ochi Akihiro、Umekita Kunihiko、Kobayashi Ikuo、Hirai Takuya、Okabayashi Tamaki
  • Journal Title: BMC Veterinary Research Volume: 20 Issue: 1 Pages: 1-8
  • DOI: 10.1186/s12917-024-04042-7
  • Peer Reviewed / Open Access
• [Journal Article] Complete genome sequence of the avian paramyxovirus serotype 9 strain duck/Miyazaki/128/2021 (2024)
  • Author(s): Mekata Hirohisa、Yamamoto Mari、Matsui Yuto、Niazi Ahmad Massoud、Yamada Kentaro、Okabayashi Tamaki、Cha Se-Yeoun、Jang Hyung-Kwan
  • Journal Title: Microbiology Resource Announcements Volume: 13 Issue: 11
  • DOI: 10.1128/mra.00060-24
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Direct TaqMan assay for the detection and genotyping of bovine viral diarrhea virus types 1 and 2 (2024)
  • Author(s): Ullah Shakir、Notsu Kosuke、Saito Akatsuki、Okabayashi Tamaki、Mekata Hirohisa、Isoda Norikazu、Sekiguchi Satoshi
  • Journal Title: Archives of Virology Volume: 170 Issue: 1 Pages: 1-10
  • DOI: 10.1007/s00705-024-06207-z
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Bilateral hibernomas in the femoral regions of a dog (2024)
  • Author(s): Tatsuro Hifumi, Yoshinori Inokuchi, Masashi Tsujio, Shogo Tanaka, Shinji Hirano, Noriaki Miyoshi
  • Journal Title: The Canadian veterinary journal Volume: 65 Pages: 367-370
  • Peer Reviewed / Open Access
• [Journal Article] Molecular detection and diversity of tick-borne rickettsial pathogens in ticks collected from camel (Camelus dromedarius) in Upper Egypt (2024)
  • Author(s): Soliman Ahmed M.、Mahmoud Hassan Y.A.H.、Amer Moaz M.、Hifumi Tatsuro、Tanaka Tetsuya
  • Journal Title: Acta Tropica Volume: 253 Pages: 107172-107172
  • DOI: 10.1016/j.actatropica.2024.107172
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Discovery of Colpodella spp. in ticks (Hyalomma dromedarii) infesting camels in southern Egypt (2024)
  • Author(s): Soliman Ahmed M.、Mahmoud Hassan Y.A.H.、Hifumi Tatsuro、Tanaka Tetsuya
  • Journal Title: Ticks and Tick-borne Diseases Volume: 15 Issue: 5 Pages: 102352-102352
  • DOI: 10.1016/j.ttbdis.2024.102352
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Cabot rings in a cat with myeloproliferative disease (2024)
  • Author(s): Fujimoto Mizuki、Takahashi Masashi、Hifumi Tatsuro、Yabuki Akira、Furusawa Yu、Hatai Hitoshi、Endo Yasuyuki
  • Journal Title: Veterinary Clinical Pathology Volume: 53 Issue: 2 Pages: 196-201
  • DOI: 10.1111/vcp.13340
  • Peer Reviewed / Open Access
• [Journal Article] A case report of feline mast cell tumour with intertumoral heterogeneity: Identification of secondary mutations c.998G＞C and c.2383G＞C in KIT after resistance to toceranib (2024)
  • Author(s): Tani Hiroyuki、Hifumi Tatsuro、Ito Keita、Kuramoto Tomohide、Miyoshi Noriaki、Fujiki Makoto、Nakayama Tomohiro
  • Journal Title: Veterinary Medicine and Science Volume: 10 Issue: 5
  • DOI: 10.1002/vms3.70003
  • Peer Reviewed / Open Access
• [Journal Article] Development of a simple and highly sensitive virion concentration method to detect SARS-CoV-2 in saliva (2024)
  • Author(s): Yamazaki Yasuko、Alonso Uxia Alonso、Galay Remil L.、Yamazaki Wataru
  • Journal Title: Heliyon Volume: 10 Issue: 12 Pages: 199550-199550
  • DOI: 10.1016/j.heliyon.2024.e33168
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Molecular phylogenetic analysis of Echinococcus multilocularis from horses raised in Canada or Japan, using mitochondrial cytochrome b gene-targeted PCR (2024)
  • Author(s): Hifumi Tatsuro、Tanaka Tetsuya、Suzu Ichinosuke、Sato Miho、Akioka Kohei、Fujimata Chiaki、Shinkai Ryohei、Maeda Tsutomu、Kusakisako Kodai、Ikadai Hiromi、Miyoshi Noriaki
  • Journal Title: Food and Waterborne Parasitology Volume: 34 Pages: e00219-e00219
  • DOI: 10.1016/j.fawpar.2024.e00219
  • Peer Reviewed / Open Access
• [Journal Article] Identification of human-to-primates viral spillover in Southeast Asia (2024)
  • Author(s): Youdill Ophinni, Chika Yamada, Michiko Fujisawa, Ryota Sakamo, Herman Hidaya, Wataru Yamazaki
  • Journal Title: 京都大学研究連携基盤持続可能社会創造ユニット 令和5年度研究成果報告書 Volume: 1 Pages: 39-42
• [Journal Article] Detection and phylogenetic analysis of Dabieshan tick virus and Okutama tick virus in ticks collected from Cape Toi, Japan (2023)
  • Author(s): Mekata Hirohisa、Kobayashi Ikuo、Okabayashi Tamaki
  • Journal Title: Ticks and Tick-borne Diseases Volume: 14 Issue: 6 Pages: 102237-102237
  • DOI: 10.1016/j.ttbdis.2023.102237
  • Peer Reviewed / Open Access
• [Journal Article] Rapid detection of alveolar echinococcosis in hepatic nodules of horses by recombinase polymerase amplification assay . (2023)
  • Author(s): Hifumi, T., Tanaka, T., Sato, M., Akioka, K., Fujimata, C. and Miyoshi, N.
  • Journal Title: Vet. Anim. Sci. Volume: 20 Pages: 100291-100291
  • DOI: 10.1016/j.vas.2023.100291
  • Peer Reviewed / Open Access
• [Journal Article] Development of a highly sensitive point‐of‐care test for African swine fever that combines EZ‐Fast DNA extraction with LAMP detection: Evaluation using naturally infected swine whole blood samples from Vietnam (2023)
  • Author(s): Ngan Mai Thi、Thi My Le Huynh、Xuan Dang Vu、Thi Bich Ngoc Trinh、Phan Le Van、Thi Hoa Nguyen、Quang Lam Truong、Thi Lan Nguyen、Notsu Kosuke、Sekiguchi Satoshi、Yamazaki Yasuko、Yamazaki Wataru
  • Journal Title: Veterinary Medicine and Science Volume: - Issue: 3 Pages: 1-8
  • DOI: 10.1002/vms3.1124
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Studies on emerging tick-borne viral diseases in companion and wild animals in Japan (2025)
  • Author(s): Hirohisa Mekata
  • Organizer: Faculty of Veterinary Medicine - Seminar for NCM Microbiology - Veterinary Infectious Diseases
  • Int'l Joint Research / Invited
• [Presentation] 豚の口腔液からアフリカ豚熱ウイルスを濃縮検出する手法の開発： LAMP 法とリアルタイム PCR 法を用いた性能評価 (2025)
  • Author(s): Wataru Yamazaki, Mai Thi Ngan, Tran Thi Huong Giang, Dong Van Hieu, Le Van Phan, Huynh Thi My Le, Bui Tran Anh Dao, Ryoko Uemura, Yasuko Yamazaki, Nguyen Thi Lan
  • Organizer: 第16回LAMP研究会
• [Presentation] プロウイルス量を使った繁殖農場でのBLV対策と課題 (2024)
  • Author(s): 目堅博久
  • Organizer: 第64回獣医疫学会学術集会(シンポジウム)
  • Invited
• [Presentation] Urine from cats with severe fever with thrombocytopenia syndrome could be an infectious source (2024)
  • Author(s): Hirohisa Mekata, Yasuyuki Kaneko, Kentaro Yamada, Tamaki Okabayashi, Akatsuki Saito
  • Organizer: The 4th Joint Meeting of Veterinary Science in East Asia
  • Int'l Joint Research
• [Presentation] 2021-23年度に宮崎大学で実施した伴侶動物の SFTS 検査 (2024)
  • Author(s): 目堅博久, 金子泰之, 平井卓哉, 梅木一美, 梅北邦彦, 山田健太郎, 岡林環樹
  • Organizer: 第6回SFTS研究会
• [Presentation] 牛伝染性リンパ腫対策について (2024)
  • Author(s): 目堅博久
  • Organizer: 家畜防疫・衛生指導対策事業に関わる地域自衛防疫取組推進会議
  • Invited
• [Presentation] 伴侶動物と野生動物のSFTS (2024)
  • Author(s): 目堅博久
  • Organizer: 第2回RCGLID&CADIC共同セミナー
• [Presentation] 前腕筋膜張筋への動脈分布における新たな解剖学的知見 (2024)
  • Author(s): 安城駿介, 松屋純人, 一二三達郎, 辻尾祐志
  • Organizer: 日本獣医学会学術集会
• [Presentation] 行政機関と連携した獣医学研究の紹介-馬の多包虫症を題材として- (2024)
  • Author(s): 一二三達郎
  • Organizer: 第28回九州・山口・沖縄病理事例研修会
  • Invited
• [Presentation] 東南アジアにおけるヒトから霊長類へのウイルス感染の特定 (2024)
  • Author(s): Youdill Ophinni, Chika Yamada, Michiko Fujisawa, Ryota Sakamo, Herman Hidaya, Wataru Yamazaki
  • Organizer: 京都大学研究連携基盤持続可能社会創造ユニットミーティング「東南アジアにおける持続可能社会構築に向けた防災・環境・地域研究」
• [Presentation] 鹿児島県大隅地域から検出されたRickettsia spp.におけるgltA、ompB、htrA遺伝子の分子疫学的特徴 (2023)
  • Author(s): 鈴一之将、木村仁美、芦原誠一、榮村奈緒子、一二三達郎、田仲哲也
  • Organizer: 令和5年度獣医学術九州地区学会
• [Presentation] 日本の食用馬の多包虫症における多包条虫のミトコンドリア・シトクロームb（cob）遺伝子を標的とした分子系統解析 (2023)
  • Author(s): 佐藤美穂、一二三達郎、田仲哲也、鈴一之将、秋岡幸兵、藤又千晶、新飼竜平、前田勉、草木迫浩大、筏井宏実、三好宣彰
  • Organizer: 令和5年度獣医学術九州地区学会
• [Presentation] Socio-ecological background to the frequency of emerging infectious diseases- the need for a planetary health approach as an effective countermeasure (2023)
  • Author(s): Wataru Yamazaki
  • Organizer: ドイツ日本研究所(DIJ)とSt. Andrews大学による合同ワークショップ
  • Int'l Joint Research / Invited
• [Presentation] Point-of-care rapid testing for disease detection (2023)
  • Author(s): Wataru Yamazaki
  • Organizer: Keeping Up with Contemporary Veterinary Public Health, UPLB
  • Int'l Joint Research / Invited
• [Presentation] Development of a highly sensitive and cost-effective point-of-care test for African swine fever with loop-mediated isothermal amplification detection: evaluation using naturally infected swine whole blood samples from Vietnam (2023)
  • Author(s): Mai Thi Ngan, Huynh Thi My Le, Vu Ngoc Dang, Trinh Thi Bich Ngoc, Le Van Phan, Nguyen Thi Hoa, Truong Quang Lam, Nguyen Thi Lan, Kosuke Notsu, Satoshi Sekiguchi, Yasuko Yamazaki, Wataru
  • Organizer: ISWAVLD 2023
  • Int'l Joint Research
• [Presentation] 東南アジアで流行する動物感染症 (2023)
  • Author(s): 山崎 渉
  • Organizer: 第105回東南アジア学会
  • Invited
• [Presentation] Development and clinical evaluation of a highly sensitive POCT (point-of-care testing) for early diagnosis and early containment of African swine fever (2023)
  • Author(s): Wataru Yamazaki
  • Organizer: East & Southeast Asia Regional Webinar (Part of GARD, or the Global Alliance for Rapid Diagnostics, co-sponsored by Michigan State University and De La Salle University)
  • Int'l Joint Research / Invited
• [Presentation] 簡易迅速なDNA抽出とLAMP検出を組み合わせた、アフリカ豚熱の高感度な ポイントオブケア検査の開発：自然感染豚の全血検体を用いたベトナムでの評価 (2023)
  • Author(s): Wataru Yamazaki, Mai Thi Ngan, Huynh Thi My Le、Vu Ngoc Dang, Trinh Thi Bich Ngoc、Le Van Phan, Nguyen Thi Hoa, Truong, Quang Lam, Nguyen Thi Lan, Yasuko Yamazaki, Kosuke Notsu, Satoshi Sekiguchi
  • Organizer: 第14回LAMP研究会
• [Presentation] Principles of the LAMP method and its application to the early diagnosis of African swine fever (2023)
  • Author(s): Wataru Yamazaki
  • Organizer: Special Lectures on Tick Biology and Rapid Diagnostic (held by UPLB, College of Veterinary Medicine)
  • Invited
• [Presentation] Principles of the LAMP method and its application to the early diagnosis of African swine fever (2023)
  • Author(s): Wataru Yamazaki
  • Organizer: Special Lectures on African swine fever (held by Vietnam National University of Agriculture )
  • Invited
• [Remarks] 簡易DNA抽出法とLAMP検出法を組み合わせたアフリカ豚熱の高感度なフィールド検査法の開発
  • URL: https://kyoto.cseas.kyoto-u.ac.jp/news/2023/04/20230406/
• [Remarks] Development of a highly sensitive POC test for ASF
  • URL: https://kyoto.cseas.kyoto-u.ac.jp/en/news/2023/04/20230406/
• [Remarks] Discussing the implementation ...
  • URL: https://eng.vnua.edu.vn/news-and-events/discussing-the-implementation-of-the-collaborative-research-project-on-african-swine-fever-with-japanese-partners-55379
• [Remarks] Discussing the implementation ...(Vietnamese)
  • URL: https://vnua.edu.vn/tin-tuc-su-kien/hop-tac/thao-luan-trien-khai-du-an-hop-tac-nghien-cuu-ve-dich-ta-lon-chau-phi-voi-cac-doi-tac-nhat-ban-55373
• [Remarks] キックオフミーティングおよびセミナー開催（日本語）
  • URL: https://kyoto.cseas.kyoto-u.ac.jp/news/2023/02/20230215/
• [Remarks] キックオフミーティング開催（ベトナム語）
  • URL: https://khoathuy.vnua.edu.vn/truong-du-an-hop-tac-quoc-te-cua-trung-tam-nghien-cuu-dong-nam-a-dai-hoc-kyoto-nhat-ban-den-tham-va-lam-viec-voi-khoa-thu-y-hoc-vien-nong-nghiep-viet-nam/
• [Remarks] セミナー開催（ベトナム語）
  • URL: https://khoathuy.vnua.edu.vn/seminar-khoa-hoc-voi-chu-de-nguyen-ly-cua-phuong-phap-khuech-dai-gen-dang-nhiet-lamp-va-ung-dung-trong-chan-doan-som-benh-dich-ta-lon-chau-phi/