{"created":"2023-12-20T07:17:56.790985+00:00","id":2000037,"links":{},"metadata":{"_buckets":{"deposit":"85989801-5650-4ef8-8a96-c70035e755be"},"_deposit":{"created_by":17,"id":"2000037","owner":"17","owners":[17],"pid":{"revision_id":0,"type":"depid","value":"2000037"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:02000037","sets":["370:15:392"]},"author_link":[],"control_number":"2000037","item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2023-08-31"}]},"item_10006_degree_grantor_9":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"麻布大学"}],"subitem_degreegrantor_identifier":[{"subitem_degreegrantor_identifier_name":"32701","subitem_degreegrantor_identifier_scheme":"kakenhi"}]}]},"item_10006_degree_name_8":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士(獣医学）"}]},"item_10006_description_22":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"Erysipelothrix rhusiopathiae causes swine erysipelas (SE), which results in considerable economic loss on pig farms and recognized as one of the most important diseases in pig farm industry. In the 1970s, the spread of live vaccines in Japan decreased the occurrence of SE but it still occurs in around 2000 pigs annually, which cannot be ignored in the pig farms. Especially during SE outbreaks that occurred sporadically from 2008 to 2011 in Japan, new E.rhusiopathiae strains were isolated with a specific surface protective antigen (Spa)A protein characterized by methionine at position 203 and isoleucine at position 257 (M203/I257-SpaA type).\n\tBased on these backgrounds, we investigated the subsequent trend of the E.rhusiopathiae M203/I257-SpaA type, which was first discovered in the survey from 2008 to 2012, with the aim of clarifying the factors of the epidemic. This research paper consists of 3 chapters: investigation of field samples affected by E.rhusiopathiae, bacteriological characterization by serotypes of E.rhusiopathiae, pathogenicity investigations in mice and pigs, the efficacy of inactivated vaccines against E.rhusiopathiae in pigs and obtained the following results.\n\tIn the first chapter, we isolated 79 strains of E.rhusiopathiae from pigs in Japan between 2012 and 2019 and identified their serovars and SpaA types. Genetic lineages of some serovar 1a strains were also identified. The pathogenicity of representative E.rhusiopathiae isolates in mice was also evaluated in comparison with the Fujisawa reference strain. Our epizootic investigation showed that serovar 1a was the most common serovar among the isolated strains (59/79) during the study period. None of the 59 serovar 1a strains had the SpaA type of the Fujisawa reference strain, while three strains were the I257-SpaA type and 56 strains were the M203/I257-SpaA type. This indicated that the M203/I257-SpaA type of E.rhusiopathiae was the prevalent strain in Japan, and that the outbreak of the M203/I257-SpaA type from 2008 to 2011 has continued until the time of this writing. Furthermore, the lineage Ⅳb-1 and Ⅳb-2 strains were isolated in the Kyushu and Honshu regions of Japan respectively, but the region-specific distribution of the E.rhusiopathiae lineages Ⅳb-1 and Ⅳb-2 were not clear in 2013, and both strains eventually co-circulated in Japan because they were isolated in the same prefecture in the same year. The disappearance thus seemed to occur within these few years from 2013 to 2019. In addition, the serovar 1a M203/I257 SpaA type of E.rhusiopathiae were more pathogenic than other serovars in the virulence confirmation test of using mice, and it was suggested that there is a high tendency to be isolated from acute symptoms pigs. On the other hand, no significant difference in virulence was found in a mice infection experiment with the Fujisawa reference strain which is the same serovar 1a. Based on these results, the epidemics of SE that have been observed in Japan in recent years are not due to strains in which a new mutation has been inserted into the base sequence of the SpaA gene of E.rhusiopathiae, and since in 2008, M203/I257 SpaA type of E.rhusiopathiae continues to infiltrate, and although it tends to be isolated from pigs with acute symptoms, its pathogenicity is not significantly different from that of the serovar 1a Fujisawa reference strain, at least in mice.\n\tIn the second chapter, to find the reason why the M203/I257-SpaA type of E.rhusiopathiae is currently the major causative agent in outbreaks, four recent E.rhusiopathiae isolates comprising of two serovar 1a with M203/I257-SpaA type strains, one serovar 1b strain, and one serovar 2a strain were compared with each other and with the serovar 1a Fujisawa reference strain regarding in vitro phenotypes and in vivo virulence in mice and pigs. The serovar 1b and 2a strains, which are the less prevalent strains in the field in Japan, showed lower growth in liquid culture and lower virulence in animals than the serovar 1a variants. Adhesion of the serovar 2a strain to porcine endothelial cells was weaker than that of the serovar 1a and 1b strains. Several advantages of serovar 1a strains were found, but no plausible cause of the M203/I257-SpaA type variants to be selected for the most prevalent strains among serovar 1a strains was identified in this study. Other feasible phenotypic assay such as the antimicrobial susceptibility test, or the studies on phagocytosis resistance and intracellular persistence may resolve this issue. We think these approaches as the further study and will continue this research from the viewpoints of antigenicity and vaccine-induced immunity as the next step.\nIn the third chapter, we used SER-ME containing E.rhusiopathiae Tama-96 (serovar 2a) as a representative of current inactivated vaccines to investigate whether it was effective for protecting pigs against infection with the Fujisawa reference strain or the M203/I257-SpaA type variant. Sixteen pigs were used in this study. Half of pigs (8/16) were vaccinated, and the rest were unvaccinated and used as control. Challenge tests were performed using the Fujisawa reference strain or the M203/I257-SpaA type 2012 Miyazaki variant, both of which belong to serovar 1a of E.rhusiopathiae. Vaccinated pigs in groups 1 and 3 showed no apparent clinical signs regardless of the E.rhusiopathiae strain used during the challenge, indicating that vaccination protected pigs not only from the Fujisawa reference strain but also from the newly emerged M203/I257-SpaA type 2012 Miyazaki variant. However, all the unvaccinated pigs in group 2 and 4 developed SE. In group 2, two of the four pigs died (including the humane endpoint, the same shall apply hereafter) on the third day after Fujisawa strain challenge, one pig died on the fourth day, and the remaining pig died on the ninth day. In group 4, all four pigs died on the third day after challenge. The sum of the clinical scores were not significantly different (p<0.01) between unvaccinated pigs challenged with the Fujisawa strain (group 2) versus the Miyazaki variant (group 4). Therefore, it was clarified that the cause of the domestic epidemic of M203/I257-SpaA type of E.rhusiopathiae was not caused by the vaccine failure.\n　　Summarizing these results, we indicated three conclusions. First: the epidemic of M203/I257-SpaA type of E.rhusiopathiae continues to dominate of the isolates in Japan, Second: there was no significant difference of the growth in liquid culture, adherence of porcine endothelial cells, and the virulence in mice and pigs between M203/I257-SpaA type and Fujisawa reference strain, The third: current vaccines in Japan, including SER-ME, suggest that outbreaks in Japan are unlikely caused by vaccine failure. Since it remains unclear why the M203/I257-SpaA type variant of E. rhusiopathiae is currently the major causative agent in outbreaks, we will further analyze the pathogenicity of the variants. In the future, it will be necessary to examine the superiority of the M203/I257-SpaA type of Erysipelas swine, for example, in animals that can infect and spread E. rhusiopathiae, or in sewage and soil where E. rhusiopathiae can exist. We hope that the series of findings obtained in this study will be of help in the future control of Erysipelas swine in the pig farming industry."}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"豚丹毒菌（Erysipelothrix rhusiopathiae）はグラム陽性の桿菌である。豚に主たる病原性を示し、世界中の養豚業界において経済的損失を与える重要な人獣共通感染症であり、わが国では届出伝染病に指定されている。豚における豚丹毒の症状は主に3つに分類され、急性・亜急性・慢性症状がある。急性症状では敗血症による突然死、亜急性症状では発熱や食欲不振に加えて最も特徴的とされる蕁麻疹（ダイヤモンド・スキン）の出現、慢性症状では関節炎、リンパ節炎および心内膜炎などを引き起こし、その症状は多岐に渡る。疾病対策には他の疾病との類症鑑別が重要となる。豚丹毒菌の菌体表層に発現するSpa（Surface protective antigen）Aタンパク質は、感染防御ならびに病原性に関わる抗原として、多くの研究が報告されている。\n　日本における豚丹毒菌の発生は1970年代に生ワクチンが普及してから減少しているものの、未だに年間2000頭前後の発生があり、養豚業界において決して無視できない疾病である。特に2008年から20011年にかけては血清型1aのSpaAタンパク質の203位と257位のアミノ酸がそれぞれメチオニンとイソロイシンに変化したM203/I257-SpaA変異株が新たな変異株として流行した。この他にも、少数ながら同じ血清型1aにおけるSpaAタンパク質の203位と242位のアミノ酸が、それぞれメチオニンとアラニンに置換したM203/A242-SpaA変異株および203位のアミノ酸のみがメチオニンに置換したM203-SpaA変異株も発見された。このように、これまで分離されてこなかった豚丹毒菌血清型1aのSpaA変異株の台頭が近年では見受けられている。\n　これらの背景をふまえ、本研究は2008年から2011年の調査で初めて見つかった豚丹毒菌SpaA変異株のその後の動向および2012年以降、我が国で流行している株の病原性について検討した。本研究は3章から構成される。野外サンプル調査、血清型別、SpaA変異株と血清型別の細菌学的特性調査並びにマウス及び豚での病原性調査、不活化ワクチンの豚丹毒菌SpaA変異株に対する豚での有効性を中心に検討し、以下のような成果を得た。\n　第一章：2012年から2019年に分離した豚丹毒菌株を中心に血清型、SpaA遺伝子の表現型を調査し、SpaA変異株の国内分離状況および、マウスにおける病原性を検討した。2012年から2019年に日本各地から集めた79株の豚丹毒菌の血清型、SpaA遺伝子塩基配列の変異部分の解析およびLineage型別を行った。調査した79株のうち、59株が血清型1aに属しており、その59株のうち56株が2008年から2011年の調査で初めて見つかった株と同じ血清型1aのM203/I257-SpaA変異株であった。2012年以降も血清型1aのM203/I257-SpaA変異株の流行の継続が明らかとなった。さらには、血清型1aのM203/I257-SpaA変異株のLineage型別をすると、これまで主に九州地域（LineageⅣb-1）と本州地域（LineageⅣb-2）で分離した株が、2012年以降では日本全国に分布し、地域性が消失していることが確認できた。また、マウスを用いた病原性確認試験において血清型1aのM203/I257-SpaA変異株は血清型2aの株と比較して病原性が強いことが示唆された。一方、同じ血清型1aで病原性比較対照株である藤沢株とM203/I257-SpaA変異株においては大きな病原性の違いはないことが明らかとなった。これらの結果より、わが国における2012年以降の豚丹毒菌の流行は豚丹毒菌のSpaA遺伝子の塩基配列に新たな変異が挿入された株などによるものではなく、2008年から引き続いて血清型1aのM203/I257-SpaA変異株の地域性が混在し、流行が拡大していることが明らかとなった。また血清型1aのM203/I257-SpaA変異株はマウスを用いた病原性試験では藤沢株と同様な病原性を示す強毒株であることが判明した。\n　第二章：2012年以降の分離株から血清型1aのM203/I257-SpaA変異株を2株、血清型1b、2a株をそれぞれ1株ずつ選択した合計4株を、病原性比較対照株として広く用いられている血清型1aの藤沢株とin vitro の細菌学的特性およびin vivoの病原性の違いを比較検討し、M203/I257-SpaA変異株が国内で流行拡大を続ける原因を検索した。国内での分離数が少ない血清型2a株は血清型1a株と比較してin vitroでの増殖性が低く、豚血管内皮細胞との細胞接着性が低く、マウスや豚におけるin vivo病原性も低いことが明らかとなった。豚を用いた動物試験では、病原性を臨床症状スコアで評価すると、血清型1a株と血清型2a株の間、血清型1a株と血清型1b株の間、さらには血清型1b株と血清型2a株の間においても病原性の臨床症状スコアに有意差が認められた。以上のことより、血清型1a株と1b株、2a株の間に病原性の違いを見出すことが出来た。さらに血清型1b株と血清型2a株は第一章より分離数が少ないことも踏まえると、我が国で分離数が最も多い血清型1aのM203/I257-SpaA変異株は藤沢株同様に、豚へも強い病原性をもち、我が国に広く浸潤していることを裏付ける結果となった。\n　第三章では豚丹毒菌不活化ワクチンの血清型1aのM203/I257-SpaA変異株に対する豚での有効性を検討した。豚を4頭ずつ4つの群に振り分け、群1と群3に豚丹毒菌不活化ワクチンSER-ME（スワインテクトSER-ME、日生研株式会社）を投与し、群2と群4を非接種対照群とした。接種菌として群1、2に病原性比較対照株である血清型1aの藤沢株を、群3、4に血清型1aのM203/I257-SpaA変異株（2012年宮崎株）を用いた。非接種対照群である2つの群は菌接種後、臨床観察期間中に全頭が死亡した。一方でワクチンを投与した2つの群は臨床観察を示すことなく試験終了まで生存した。以上より、豚丹毒菌不活化ワクチンは豚で血清型1aのM203/I257-SpaA変異株に対しても従来株同等に有効であることを示した。従って、血清型1aのM203/I257-SpaA変異株の流行拡大の原因はワクチン投与後のブレイクスルー感染によるものではないことが示唆された。\n　以上の結果より、（１）血清型1aのM203/I257-SpaA変異株の流行拡大は現在も続いていること（２）最も流行している血清型1aのM203/I257-SpaA変異株はマウスや豚への病原性が強いこと（３）血清型1aのM203/I257-SpaA変異株の流行拡大の原因は豚での感染・防御試験によりワクチン投与後のブレイクスルー感染によるものではないことが明らかとなった。\n豚丹毒菌は豚のみならず、家禽や多くの動物、自然環境から分離されている。今後は血清型1aのM203/I257-SpaA変異株が野生動物を含めた他の宿主や自然環境中に分布している可能性などを考慮し、広範囲な調査を続けることが、豚の豚丹毒の予防と制御に必要であると思われる。本研究で得られた一連の知見が今後の養豚業界における豚丹毒菌制御の一助になれば幸いである。"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"乙第443号"}]},"item_10006_identifier_registration":{"attribute_name":"ID登録","attribute_value_mlt":[{"subitem_identifier_reg_text":"10.14944/0002000037","subitem_identifier_reg_type":"JaLC"}]},"item_10006_textarea_23":{"attribute_name":"Rights","attribute_value_mlt":[{"subitem_textarea_value":"本論文の一部は以下に公表した(Part of this dissertation has been published as follows. )\n第一章：Morimoto M, Kato A, Kojima H, Akaike Y, Nogami K, Sasakawa C, Nagai S, To H. Serovars and SpaA Types of Erysipelothrix rhusiopathiae Isolated from Pigs in Japan from 2012 to 2019. Curr Microbiol. 2021 Jan;78(1):55-66. doi: 10.1007/s00284-020-02254-2. Epub 2020 Nov 3. PMID: 33145611.\n第二章：Morimoto M, Kato A, Akaike Y, Nogami K, Ono H, Furusawa T, Kojima H, Sasakawa C. Comparative study of the phenotype and virulence of recent serovar 1a, 1b, and 2a isolates of Erysipelothrix rhusiopathiae in Japan. Vet Microbiol. 2022 Jul;270:109458. doi: 10.1016/j.vetmic. 2022.109458. Epub 2022 May 12. PMID: 35623133.\n第三章：Morimoto M, Kato A, Nogami K, Akaike Y, Furusawa T, Kojima H, Sasakawa C. The Swine Erysipelas Vaccine SER-ME Effectively Protects Pigs against Challenge with the Erysipelothrix rhusiopathiae M203/I257 SpaA-Type Variant. Vet Sci. 2022 Jul 26;9(8):382. doi: 10.3390/vetsci9080382. PMID: 35893775; PMCID: PMC9332197."}]},"item_10006_version_type_18":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_access_right":{"attribute_name":"アクセス権","attribute_value_mlt":[{"subitem_access_right":"open access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_abf2"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"森元, 美紗子"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-12-20"}],"filename":"diss_dv_otsu443.pdf","filesize":[{"value":"3.3 MB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"url":"https://az.repo.nii.ac.jp/record/2000037/files/diss_dv_otsu443.pdf"},"version_id":"0400904f-d4d2-442a-a643-18dd3f16eb03"},{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-12-20"}],"filename":"diss_dv_otsu443_jab&rev.pdf","filesize":[{"value":"195 KB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"url":"https://az.repo.nii.ac.jp/record/2000037/files/diss_dv_otsu443_jab&rev.pdf"},"version_id":"c9ed4e42-7600-4d62-bd7b-c2bf35aed74d"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"doctoral thesis"}]},"item_title":"豚丹毒菌変異株の流行と病原性調査に関する研究","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"豚丹毒菌変異株の流行と病原性調査に関する研究","subitem_title_language":"ja"},{"subitem_title":"Research for outbreaks and virulence of Erysipelothrix rhusiopathiae variant in swine","subitem_title_language":"en"}]},"item_type_id":"10006","owner":"17","path":["392"],"pubdate":{"attribute_name":"PubDate","attribute_value":"2023-12-20"},"publish_date":"2023-12-20","publish_status":"0","recid":"2000037","relation_version_is_last":true,"title":["豚丹毒菌変異株の流行と病原性調査に関する研究"],"weko_creator_id":"17","weko_shared_id":-1},"updated":"2023-12-20T09:53:40.755247+00:00"}