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Studies on modulation of immune responses by poultry red mite, Dermanyssus gallinae, and development of the novel control strategies

藤澤, 宗太郎 北海道大学

2022.03.24

概要

ワクモ (Dermanyssus gallinae) はニワトリを含む鳥類に寄生して吸血を行うダニの一種であり、日本を含む世界中に分布している。ワクモに重度に吸血されたニワトリは貧血や産卵率の低下を呈し、生産性の著しい低下を招く。ワクモと同じく吸血性の外部寄生虫であるマダニや蚊においては、吸血時に様々な生理活性物質を唾液中に分泌することで、宿主の生体応答を抑制的に調節していることが知られている。このような免疫抑制は病原体の媒介や宿主の易感染化に関与していることが報告されており、外部寄生虫は貧血等の直接的な被害に加えて感染症リスクの増大等、間接的な被害をも引き起こす可能性がある。したがってワクモによる寄生が宿主免疫応答に与える影響を解明することは、養鶏場における吸血被害ならびに感染症の制御を検討する上で重要である。しかしながら、ワクモの寄生に対するニワトリの免疫応答に関する詳細な知見は乏しい。

現在、養鶏場におけるワクモの防除には主に殺ダニ剤が用いられる。しかし、薬剤による生産物や環境の汚染や薬剤耐性ワクモの出現が社会的な問題となっており、新たな防除法の開発が強く望まれている。同じ吸血性の節足動物であるマダニにおいては、ワクチンを用いた防除法が有効性を示し、家畜生産現場での実用例が知られている。近年、ワクモに対する新規防除法として、抗マダニワクチンを応用した抗ワクモワクチンが注目されており、組換えワクモ由来タンパク質を抗原として用いたワクチン試験において一定の抗ワクモ効果が報告されている。しかしながら野外試験において充分な有効性を示すワクチン抗原は報告されておらず、抗ワクモワクチンは未だ実用化に至っていない。そのため、抗ワクモワクチンによる制御法を樹立するためにはより有効なワクチン抗原の探索が不可欠である。抗ワクモワクチンは、免疫鶏により産生されたワクチン抗原に対する抗体が吸血時にワクモに取り込まれることで抗ワクモ効果を発揮するため、ワクチン抗原としては吸血時に高い発現を示す分子が望ましい。そのような分子を効率的、且つ網羅的に探索するためには、遺伝子発現を含むワクモの生物学的特徴を吸血状態別に精査することが必要である。しかしながら、吸血及び飢餓状態における遺伝子発現の変動については未だ解析されていない。

本稿ではワクモの吸血が宿主に及ぼす影響をより詳細に解明すること、及び効果的な抗ワクモワクチンの候補抗原を探索することを目的として、まず吸血状態別のワクモのトランスクリプトーム解析を行った。次に、ワクモ汚染農場及び非汚染農場由来のニワトリから血液を採材し、ワクモの寄生が宿主の免疫応答に与える影響を解析した。さらに、トランスクリプトーム解析の結果をもとに新規ワクチン抗原候補分子を同定し、in vitro における性状解析とワクチン抗原としての有用性の検討を行った。

第 1 章:吸血及び飢餓ワクモを用いた RNA シーケンスにより各吸血状態のワクモのトランスクリプトームを比較解析した結果、吸血によりワクモの遺伝子発現パターンが大きく変動することが明らかとなった。Gene ontology 解析の結果、吸血状態のワクモでは金属イオンの結合に関する遺伝子が高頻度で発現しており、ニワトリ血液中に含まれる金属イオンの運搬、貯蔵、または解毒作用が亢進することが示唆された。また吸血ワクモでは ATP合成や代謝に関する遺伝子の発現頻度が高い一方、飢餓状態のワクモでは代謝抑制やオートファジー、糖新生に関する遺伝子群の発現が高頻度に認められた。

第 2 章:ワクモの吸血に対する宿主免疫応答の解析を行った。第 1 章で行った吸血ワクモを用いた RNA シーケンスで得られたニワトリ由来遺伝子の解析、さらにワクモ汚染農場及び非汚染農場由来のニワトリから得た血液サンプルを用いた解析結果から、ワクモに吸血されたニワトリの末梢血では炎症性ケモカイン C-C chemokine ligand 4 (CCL4) が非常に高く発現していることが明らかとなった。一方、Th1 サイトカインや炎症性サイトカインの発現には変化は認められなかった。それに対して抗炎症性サイトカインや、免疫チェックポイント分子などの免疫抑制因子については汚染農場由来のニワトリにおいて非常に高い発現が認められた。また、CCL4 の受容体である C-C chemokine receptor 5 は汚染農場由来のニワトリで低い発現を示した。そのため、ワクモに慢性的に曝露されたニワトリは免疫抑制状態にあることが示唆された。次に、ニワトリの免疫細胞をワクモ由来可溶性抽出物 (SME) 存在下で培養したところ、特にマクロファージ様細胞における炎症性サイトカイン遺伝子の発現が低下し、さらに M2 様マクロファージのマーカー遺伝子の発現が上昇することが示された。以上より、ワクモ由来分子が直接的にニワトリの免疫応答を抑制的に調節することが示唆された。さらに、SME で刺激した免疫細胞において、自然免疫応答に中心的な役割を担うインターフェロンの発現が低下したことから、ワクモの吸血が宿主の易感染化に寄与している可能性が示された。

第 3 章:第 1 章で行った RNA シーケンスの結果を基にシスタチン様分子 (D.gallinae-cystatin: Dg-Cys)、銅 イ オ ン ト ラ ン ス ポー ター 様分 子 (D. gallinae-coppertransporter 1: Dg-Ctr1)、及び adipocyte plasma membrane-associated protein 様分子 (D.gallinae-adipocyte plasma membrane-associated protein: Dg-APMAP) の 3 種類を同定した。遺伝子発現解析の結果、いずれの候補分子もワクモの全ての発育ステージで吸血状態を問わず発現しており、また吸血された血液が貯蔵される中腸に発現していることが示された。次に各候補分子の組換えタンパク質を免疫したニワトリから得た免疫血漿をワクモに吸血させることで、抗ワクモ効果の検討を行った。その結果、いずれの免疫血漿を吸血したワクモでも陰性対照群と比べて生存率の低下が認められた。さらに、Dg-Cys の免疫血漿を吸血したワクモでは、繫殖能力が低下した。これらの結果より、本研究で同定した3種類の分子のワクチン抗原としての有用性が示唆された。さらに、Dg-Cysの免疫血漿と Dg-Ctr1 または Dg-APMAP の免疫血漿と組み合わせた混合血漿による抗ワクモ効果を検証したところ、単独で免疫血漿を用いた場合と比較して殺ダニ効果の増強が認められた。以上より、Dg-Cys を含む複数の抗原を組み合わせたカクテルワクチンが、ワクモ被害に対する新規防除法として有用である可能性が示された。

本研究では吸血状態別のワクモの遺伝子情報を明らかにし、またワクモによる吸血が宿主の免疫応答を抑制することが示唆された。さらに Dg-Cys、Dg-Ctr1、及びDg-APMAP を標的とした抗ワクモワクチン、さらにそれらを組み合わせたカクテルワクチンが、有効な新規ワクモ制御法となり得ることを示した。今後は実験的寄生モデルを用いることでワクモが病原体の媒介に与える影響やその分子基盤を検討することに加え、免疫鶏を用いたワクチンの臨床試験を行い、本研究を基にした新規制御法の効果をより詳細に評価する必要がある。

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