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毒素原性大腸菌による離乳後下痢症における子豚実験感染モデルの確立および対策混合飼料の開発について

松本 弘輝 大阪府立大学 DOI:info:doi/10.24729/00017529

2021.11.19

概要

子豚の離乳後下痢(Post-weaning diarrhea: PWD)は、離乳後 3~10 日で主に腸管毒素原性大腸菌(Enterotoxigenic Escherichia coli: ETEC)によって引き起こされる。ETEC に感染した豚は通常、1~5 日間続く泥状から水様性の下痢を呈し、脱水による斃死もしくは発育低下・遅延が生じ、世界中の養豚産業に深刻な経済的損失をもたらしている(Dubreuil et al., 2016)。我が国においても、下痢を呈した離乳豚を検査したところ、大腸菌単独感染およびロタウイルス、コクシジウムなどとの混合感染を合わせると、半数以上(52.6%)の豚から大腸菌が検出されており、本疾病に対する ETEC の重要性が伺える(Katsuda et al., 2006)。PWDの原因となる ETEC は、易熱性エンテロトキシン(LT)や耐熱性エンテロトキシン(STa および STb)を産生し、腸管上皮細胞へ付着するための線毛を保有している。豚では主に、F4および F18 線毛を発現する ETEC が検出され、F5、F6、F41 線毛を発現する ETEC の検出も報告されているが、検出頻度は低い(Fairbrother et al., 2005)。

一般に、ETEC のような細菌による PWD をコントロール(予防・治療)するために抗菌剤が使用されることが多く、中でも Paenibacillus polymyxa によって産生されるポリミキシン系抗生物質であるコリスチンが豚で多く用いられていた(Tambadou et al., 2015)。一方、ヒトでは、コリスチンは現在、Pseudomonas aeruginosa、Acinetobacter baumannii、Klebsiella pneumoniae、Enterobacter spp.などの多剤耐性グラム陰性菌による感染症治療のための最後サルモネラなどの特定のグラム陰性菌において、コリスチンに対する耐性を付与する安定なプラスミド媒介遺伝子 mcr-1 が同定された(Rebelo et al., 2018)。これまでコリスチン耐性機構は、非移行性のゲノム媒介変異しか確認されていなかったが、mcr-1 の発見により水平伝播および種間移行の可能性もでてきたことから、ヒト医療においてコリスチンの有効性が低 下することが懸念されている。実際に、国内の豚から分離された主要 O 群血清型(O139、O149、O116、OSB9)の病原性大腸菌 684 株について、コリスチン耐性および mcr-1 の保有状況を調査した報告では、mcr-1 保有株は 2007 年分離株で初めて確認され、調査した 684 株における mcr-1 の保有率は 2011 年以降に急増しており、mcr-1 保有株はすべてがコリスチン耐性株であった(Kusumoto et al., 2016)。これらの事態を受け、我が国では内閣府食品安全委員会により家畜に対して硫酸コリスチンを使用することは中程度のリスク(ヒト用抗菌性物質による治療効果が減弱又は喪失する可能性は否定できない)であるとされ、2018 年 7 月 1 日をもって硫酸コリスチンの飼料添加物としての使用が禁止された。

国内では、飼料添加物として抗菌剤および抗生物質の添加がいくつか承認されているが、 EU 加盟国では、耐性菌増加を抑制するために成長促進目的での抗菌剤および抗生物質の添加が 2006 年より禁止されており、世界中で同様の動きがみられる(Casewell et al., 2003,Dębski., 2016, Nhung et al., 2016)。耐性菌出現を管理し、同時に家畜の生産性を維持するためには、抗菌剤、特に豚用のコリスチンに代わるものを見つけることが強く求められている有機酸、プロバイオティクス、プレバイオティクスなどの飼料添加が広く研究されている(Badia et al., 2012, Suiryanrayna et al., 2015, Yang et al., 2016, Zhu et al., 2017)。PWDを予防し得る飼料を探索する最も効果的な評価方法は、豚を用いた感染試験である。しかしながら、これらの有効性評価において、発症に個体差が大きい不適切な感染モデルを使用した場合、新たな製品を開発するためにより多くの豚を用いた試験が必要となり、動物福祉の理念に相反する事態となり得る(Luise et al., 2019a)。近年、豚における 13 番染色体上に位置するムチン 4 遺伝子(MUC4)は F4 線毛受容体の発現と関連があるとされている(Jensenet al., 2006, Roubos-van et al., 2017)。豚の MUC4 を標的とした DNA ベースのテストによ り、F4 ETEC に対する抵抗性および感受性を調査することが可能となり、ETEC 実験感染に よる下痢の発症率は大きく改善したものの 100%には達しなかった(Roubos-van et al., 2017)。

本研究では、第一章で、豚由来腸管上皮細胞株を用い ETEC の細胞付着能を評価し、遺伝子上では同一の F4 線毛遺伝子を有する ETEC であっても、株毎に細胞付着能が異なることを見出した。さらに、細胞に高い付着性を有する ETEC 株を感受性(ホモ)もしくは感受性/抵抗性(ヘテロ)の豚に攻撃することにより、100%下痢が発症する PWD 感染モデルを確立した。

第二章では、PWD 感染モデルを用いて、有機酸(フマル酸、乳酸)、乳酸菌死菌体末、さとうきび抽出物の PWD 予防効果についてスクリーニングした。予防効果の高かった二つの飼料を組み合わせて、低濃度(安価)でも効果が発揮される混合飼料を開発した。

第三章では、新規に開発した混合飼料を豚に給与し、次世代シーケンサー(Miseq)による腸内細菌叢メタゲノム解析や、CE-TOF MS による血漿メタボローム解析、リアルタイム PCRによる腸管のサイトカイン等発現量を確認することにより、PWD 予防メカニズムを検証した。

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参考文献

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