JP4115090B2 - How to deactivate protozoa - Google Patents
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- JP4115090B2 JP4115090B2 JP2001002622A JP2001002622A JP4115090B2 JP 4115090 B2 JP4115090 B2 JP 4115090B2 JP 2001002622 A JP2001002622 A JP 2001002622A JP 2001002622 A JP2001002622 A JP 2001002622A JP 4115090 B2 JP4115090 B2 JP 4115090B2
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Description
【０００１】
【発明の属する技術分野】
本発明は、クリプトスポリジウム（Ｃｒｙｐｔｏｓｐｏｒｉｄｉｕｍ）やジアルジア（Ｇｉａｒｄｉａ）等の原虫類の汚染が懸念される浄水、飲用水、下水、排水、河川水、湖沼水、修景水、プール用水および食品または食品製造設備等を処理対象物とした消毒方法に関する。
【０００２】
本発明は、感染力のある原虫類を短時間でしかも簡便に不活性化して当該微生物の病原性を低減もしくは消失せしめる消毒方法にも関する。
【０００３】
【従来の技術】
最近、クリプトスポリジウムやジアルジア等の原虫による水系感染症の発生が上・下水道分野をはじめとして世界的に大きな問題となっている（総説として例えば、保坂三継（１９９８）「水系原虫感染症−原因生物と流行発生−」，用水と廃水，第４０巻，第２号，１１頁；金子光美（１９９８）「原虫類やその他の病原性微生物の検出とその除去技術」，用水と廃水，第１０巻，第４号，３２頁など）。当該原虫は極めて少数個の飲用によって疾病を起こしうる危険な病原体であり、飲用水や食品の汚染に由来した甚大な流行感染をも懸念されている。甚大な流行感染の発生を未然に防ぐために原虫類を確実に駆除できる消毒技術の開発が強く求められている。
【０００４】
上・下水道分野では、水の消毒方法として塩素処理法が汎用されている。しかしながら、クリプトスポリジウムやジアルジア等の原虫類は、塩素に対する抵抗性が高く、塩素処理法では原虫類の殺滅が困難とされている（例えば、I. S. Campbell et al. (1982) Effect of disinfectants on survival of Cryptosporidium oocysts, Weterinary Record, Vol.111, p414など）。
【０００５】
塩素消毒に代わる対策技術として、下記のような処理技術が検討されている。ろ過法は、砂ろ過や膜ろ過によって原虫類のシストまたはオーシストを被処理水中から物理的に除去する処理技術である。
加熱処理は、被処理水を加熱することによって原虫類のシストまたはオーシストを殺滅する処理技術である。
【０００６】
オゾン処理は、被処理水にオゾンガスを吹き込み、オゾンの酸化力によって原虫類のシストまたはオーシストを不活性化または殺滅する処理技術である。紫外線照射と併用することによって処理効率がさらに高まるといわれている。
また、細菌やウイルスを殺滅する処理技術として紫外線処理があるが、ジアルジアやクリプトスポリジウム等の原虫類への適用は困難とされている。
【０００７】
【発明が解決しようとする課題】
原虫類は、環境中においてクリプトスポリジウムではオーシスト、ジアルジアではシストと呼ばれる胞子状の殻に包まれた形態で存在しているため、化学薬剤に対する耐性があり、従来の水処理法で汎用される塩素消毒では防除できない。
膜ろ過法は、原虫類の確実な除去が期待できるが、透過水量に限界があり、大容量の水処理においては設備の巨大化や膜の更新等に多大なコスト負担が必要である。
【０００８】
砂ろ過法では、ろ床内での短絡水流や破過によって原虫類が処理水中に漏出する恐れがある。
原虫類は、加熱や乾燥に極めて弱いため加熱処理によって不活性化できるが、大容量の水処理においては大型の加温設備の設置や加温に必要なエネルギーの大量投入等に多大なコスト負担が必要である。
【０００９】
紫外線処理によってジアルジアやクリプトスポリジウム等の原虫類を殺滅するためには出力１５Ｗの紫外線を１５０分間照射し続ける必要があり、概ね７００〜１０００ｍＷ・ｓ／ｃｍ2 程度の紫外線を照射する必要があるとされている。この紫外線照射量は通常の紫外線消毒装置の設計値の概ね１０倍から２０倍に相当するため、原虫類の紫外線処理は実用上の適用が困難である（例えば、E. L.Jaroll (1988) Effect of disinfectants on Giardia cysts, CRC Critical Reviews in Environmental Control, Vol.18, p1；M. J. Lorenzo-Lorenzo et al.(1993) Effect of ultraviolet disinfection of drinking water on the viability Cryptosporidium parvum oocysts, J. Parasitol., Vol.79, No.1, p67）。
【００１０】
上述の如く、処理対象物中の原虫類を除去もしくは殺滅することを目的とした従来の防除技術は、処理効果の安定性やコストの点で課題がある。
一方、原虫類の生育活性あるいは感染能力を評価する方法としてはバイタル染色法、脱嚢試験法、動物実験法が提案されている。しかしながら、バイタル染色法と脱嚢試験法は顕微鏡観察によらねば定量評価ができないため、操作の煩雑さ、効率の悪さ、データ精度の悪さ等の欠点があり、不活性化条件の決定には不適である。また、動物実験法はマウス等の小動物へ被検試料を経口的に接種し、１週間から１ヶ月程度の間にわたって継続的に飼育し、排泄された糞便から原虫類のオーシストもしくはシストを精製して顕微鏡観察等でオーシストもしくはシストの数を計測し、この結果に基づいて被検試料中の原虫類濃度を推定する。もしくは該小動物の消化器官を摘出し、病理学的な観察によって発症の有無を判定し、この結果に基づいて被検試料中の原虫類濃度を推定する。このように動物実験法については、判定に長時間を要することや小動物の個体差が結果に大きく影響することに加え、動物保護の観点からも好ましい方法とはいえない。
【００１１】
動物実験法に代わる原虫類の検査方法として培養細胞法が提案されている。例えば、公開特許公報、特開２０００−２１４１６８において、培養細胞を用いれば感染能力のある原虫類を簡便にかつ感度良く検出できると記載されている。
本発明者は、処理対象物中の個々の原虫類もしくはそのシストまたはオーシストをたとえ完全に殺滅できなくとも、原虫類の生育活性または感染能力を消失せしめること（すなわち“不活性化”）ができれば、人畜に対する病原性の有無を問題とする衛生学の面では対策技術になりえることを見い出し、原虫類の不活性化の程度の正確な判定結果に基づいて処理条件を最適化することによって、従来技術の問題点を克服した効率的かつ効果的な原虫類の不活性方法を提供することを課題とする。
【００１２】
【課題を解決するための手段】
原虫類のシストまたはオーシストの感染能力の程度を指標とすれば不活性化に必要な処理条件を絞り込むことができ、処理コストを最小限に抑えた原虫類の不活性化方法を提供することができる。すなわち、前記した課題は下記の（１）〜（４）によって解決できる。
【００１３】
（１）処理対象物中に存在するまたは存在が疑われる原虫類の、紫外線照射による感染能力の不活性化の程度を指標として紫外線照射条件を設定して、前記処理対象物に紫外線を照射する原虫類の不活性化方法であって、前記紫外線照射条件の設定が、紫外線未照射の処理対象物の試料、および処理対象物に波長２５４ｎｍ付近の紫外線の照射量が１〜１００ｍＷ・ｓ／ｃｍ2 となるように紫外線を照射した後の試料の、それぞれの試料を培養細胞に接触させ、前記試料中の感染能力のある原虫類が培養細胞に寄生して培養細胞内で増殖した原虫類を、酸素抗体法又は免疫染色法を用いて定量検出して、前記それぞれの試料における前記感染能力のある原虫類の濃度を算出し、酸素抗体法で吸光度を測定して定量検出し、下式（１）で定義される不活性化率を算出し、前記紫外線照射量と前記不活性化率との、紫外線照射量の特定の一定増加量毎に感染能力のある原虫類の９０％が不活性化するという連関から、不活性化率の目標値に応じて処理対象物への紫外線照射条件を設定することであることを特徴とする原虫類の不活性化方法。
【００１４】
不活性化率＝１ − （処理対象物に紫外線を照射した後の試料における原虫類の濃度 ÷ 紫外線未照射の処理対象物の試料における原虫類の濃度）（式１）
【００１５】
（２）処理対象物の試料と接触し、かつ、紫外線を受光可能な位置に、光触媒機能を有する薄膜を配置することによって、紫外線による直接作用とともに光触媒が紫外線を受光することで生じる酸化還元反応を利用する前記（１）に記載の原虫類の不活性化方法。
（３）光触媒機能を有する薄膜が酸化チタンを主成分とする薄膜である前記（２）に記載の原虫類の不活性化方法。
【００１６】
（４）処理対象物が水である前記（１）〜（３）のいずれかに記載の原虫類の不活性化方法。
【００１７】
【発明の実施の形態】
本発明は、以下に述べるクリプトスポリジウムやジアルジア等の原虫類が動物の体内に寄生して疾病を引き起こす過程において、原虫類が細胞に寄生して増殖する能力が消失することを“不活性化”と定義し、完全に殺滅せずとも疾病発現のリスクを回避できる効果的な原虫類の不活性化方法を提供することができる。なお、クリプトスポリジウムやジアルジア等の原虫類が動物の体内に寄生して疾病を引き起こす過程を該述すると、以下のようになる。
【００１８】
１）シストまたはオーシストが経口的に体内へ摂取される。
２）消化管内での脱嚢して感染性虫体が発露する。
３）感染性虫体が腸管の表皮細胞へ付着する。
４）感染性虫体が腸管の表皮細胞内へ侵入する。
５）感染性虫体が腸管の表皮細胞内で増殖する。
【００１９】
６）増殖した感染性虫体がシストまたはオーシストを形成する。
７）細胞内で形成されたシストまたはオーシストが細胞外へ放出される。
８）細胞外へ放出されたシストまたはオーシストが腸管内で脱嚢し、再び別の細胞へ侵入し、増殖する。
９）前記２から８を繰り返して、消化管の機能障害を引き起こし、疾病に至る。
【００２０】
上記の疾病に至る過程を鑑み、経口的に摂取される以前に混入が懸念される原虫類を完全に殺滅しなけれればならないという観点で、消毒技術の開発が検討され、結果的に多大なエネルギーやコストを投じた消毒技術に頼らねばならなかった。しかしながら、発明者らが鋭意検討したところ、殺滅しなくても遺伝子の本体であるＤＮＡを損傷させれば、寄生した細胞内での増殖を阻止でき、前記した５以降の疾病に至る過程を抑制できることを見い出した。例えば、紫外線を照射した原虫類は培養細胞へ感染できず、培養細胞内で増殖できないことを知見した。これは紫外線照射によって原虫のＤＮＡが損傷した結果、ＤＮＡ複製および細胞分裂が不能になり、増殖できないためである。なお、かかる知見は培養細胞を用いた評価法を利用することで詳細に解明できる。
【００２１】
本発明は、原虫類の不活性化に必要な紫外線照射強度や照射時間等の処理条件を最適化でき、最適化した処理条件を満たした処理装置の設計もしくは既存設備の運転条件変更によって原虫類の不活性化技術を提供することができる。
まず、原虫類の不活性化方法について詳述する。原虫類（もしくはそのオーシストまたはシスト）は１〜１００ｍＷ・ｓ／ｃｍ2 （波長２５４ｎｍ）程度の低照射量の紫外線照射処理によって容易に不活性化できる。紫外線光源としては、紫外線を発光するランプであれば特に限定はないが、不活性化処理として紫外線照射単独法を採用する場合と光触媒併用法とで相違がある。前者の場合、波長２５４ｎｍ付近の紫外線を発光するランプとして、例えば、低圧水銀ランプ、中・高圧水銀ランプ、キセノンランプを用いることができ、後者の場合は、これらに加えてブラックランプのような波長３６５ｎｍ付近の紫外線を発光するランプも用いることができるが、波長２５４ｎｍ付近と波長３６５ｎｍ付近の紫外線を発光する中・高圧水銀ランプが好ましい。原虫類の不活性化のために最適化された装置として製造することもできるが、既設の紫外線消毒装置について、紫外線照射強度や照射時間等の運転条件を変更することによっても対応することができる。
【００２２】
ところで、多くの生物は紫外線照射によって損傷したＤＮＡを自己修復するための機構を備えているといわれ、この修復機構は近紫外光や可視光によって誘導されることから光回復と呼ばれており、紫外線消毒が抱える克服すべき課題のひとつといわれている（例えば、鹿島田ら（１９９５）『紫外線照射水処理における光回復の評価』水環境学会誌、第１８巻、第１号、４４頁）。光触媒併用法を用いれば、前記した光回復を抑制することができる（例えば、公開特許公報、特開平１１−１５６３５２）。光触媒併用法においては、処理対象物と接触し、かつ、紫外線を受光可能な位置に、光触媒機能を有する薄膜を配置すればよい。光触媒機能を有する薄膜としてはコスト、触媒活性、耐久性などの点で酸化チタンを主成分とする薄膜が好ましい。また、酸化チタン単独組成の薄膜のみならず、光触媒活性を高めるためにＡｇ、Ａｕ、Ｃｕ、Ｆｅ、Ｍｂ、Ｎｉ、Ｐｄ、Ｐｔ、Ｒｄ、Ｒｈ、Ｒｕ、Ｓｎ、Ｖ、Ｚｎ等の金属またはその酸化物を添加した酸化チタン薄膜を用いることもできる。
【００２３】
次いで、原虫類の不活性化の程度を評価する方法について詳述する。前記したように従来の評価技術では操作性や信頼性の点で問題があったことから、これら原虫類の不活性化の程度を評価すべき従来法が抱える課題を克服した方法を採用することが好ましい。本発明においては原虫類の不活性化の程度を評価する方法として、培養細胞を用いた検査方法を用いることができる。例えば、培養細胞に被検試料を３０分から３時間接触させ、被検試料を新鮮な培養培地に置換し、１日から１週間、好ましくは１〜３日間培養した後に、培養細胞内で増殖した原虫類もしくは原虫類のオーシストまたはシストを酵素抗体法もしくは免疫染色法によって定量検出する方法を用いることができる。検査に供する培養細胞は検出しようとする原虫類が感染しうる細胞株であればよく、例えば、マウス由来のＢＡＬＢ／３Ｔ３、ヒト由来のＢＴ−５４９、Ｃａｃｏ−２、ＨＣＴ−８、ＨＴ−２９、Ｈｓ−７００Ｔ、ＨＴ−１０８０、ＬＳ−１７４Ｔ、ＲＬ５９−２、ウシ由来のＭＤＢＫ、イヌ由来のＭＤＣＫなどを用いることができるが、ヒトに対して感染性があるクリプトスポリジウム（パルバム（ｐａｒｖｕｍ）種）を検出しようとした場合には、例えばヒト腸管内皮細胞由来のＨＣＴ−８を用いればヒトへの感染性を正しく評価できる。細胞を培養する培地組成、培養温度、湿度、炭酸ガス濃度、培養時間等の培養条件は、用いようとする細胞株に応じて選定することができる。
【００２４】
培養細胞内で増殖した原虫類もしくは原虫類のオーシストまたはシストを定量検出するための酵素抗体法としては、例えば、ホルマリン溶液やアルコール溶液で細胞を固定した後に、必要に応じてウシ血清アルブミン添加リン酸緩衝液や培養培地等でブロッキング処理をしてからＥＬＩＳＡ法［Ｅｎｚｙｍｅ−Ｌｉｎｋｅｄ Ｉｍｍｕｎｏ Ｓｏｒｂｅｎｔ Ａｓｓａｙ］（例えば、加藤ら（２０００）培養細胞へのクリプトスポリジウム・パルバムの感染とELISAによる検出，水環境学会誌，第23巻，第7号，p427）によって原虫類（もしくはそのオーシストまたはシスト）の濃度に応じた吸光度の変化として定量検出することができる。
【００２５】
また、培養細胞内で増殖した原虫類もしくは原虫類のオーシストまたはシストを定量検出するための免疫染色法としては、例えば、ホルマリン溶液やアルコール溶液で細胞を固定した後に、必要に応じてウシ血清アルブミン添加リン酸緩衝液や培養培地等でブロッキング処理をしてから、細胞を蛍光標識抗体で染色し、蛍光顕微鏡観察またはレーザー顕微鏡観察によって培養細胞群におけるｆｏｃｉ（病原体の感染部位）を計数し、その結果から被検試料中の感染性を有した原虫類のオーシストまたはシストの濃度を判断することができる。
【００２６】
ところで、原虫類の不活性化の程度を評価するには、感染性を有した原虫類が不活性化処理の前後でどの程度低減したかを数値化する必要がある。前記した定量検出方法のうち免疫染色法においては、原虫類のオーシストまたはシストの数を直接計数するために直ちに不活性化率を判断できる。一方、前記した定量検出方法のうち酵素抗体法においては、原虫類（もしくはそのオーシストまたはシスト）の濃度と吸光度は必ずしも一次関数で表現できない、つまり正比例の関係ではないことから、原虫類（もしくはそのオーシストまたはシスト）の濃度と吸光度に関する検量線が必要である。そこで、既知の濃度で原虫類（もしくはそのオーシストまたはシスト）を含有している標準試料を複数の濃度段階に希釈した試料を被検試料とし、同様の検査手順を実施することで原虫類（もしくはそのオーシストまたはシスト）の濃度と吸光度に関する検量線を得ることができる。標準試料に関する原虫類の濃度と吸光度との相関関係の数式化は、試験結果に応じてふさわしい近似方法を選定すればよいが、例えば、一次線形近似、多項式近似、対数近似、指数近似、累乗近似あるいは両逆数プロット解析などが挙げ上げられる。当該検量線に基づけば、感染性を有した原虫類が不活性化処理の前後でのどの程度低減したかを数値化することができる。被検試料中の原虫類（もしくはそのオーシストまたはシスト）の濃度を正確に判定するためには、前記した検量線の濃度範囲で判断することが望ましく、検査したい試料を必要に応じて希釈または濃縮した試料を被検試料としても良い。
【００２７】
紫外線未照射の試料および種々の照射量で紫外線照射した複数水準の試料を被検試料として、前記した評価方法を用いてそれぞれの被検試料中の原虫類の濃度を測定し、下式（１）で定義される不活性化率を算出し、
不活性化率＝１ − （紫外線を照射した被検試料における原虫類の濃 度 ÷ 紫外線未照射の被検試料における原虫類の濃度） （式１）
さらに、紫外線照射量と前記不活性化率との連関から、所望の不活性化率を得るに必要な紫外線照射量を決定することができる。決定した紫外線照射量を紫外線照射装置の設計条件としてもよいが、かかる紫外線照射量を基礎データとし更に安全率を考慮して算定される紫外線照射量を設計条件として採用してもよい。
【００２８】
【実施例】
実施例１
クリプトスポリジウムのオーシスト懸濁液に紫外線を照射し、生存オーシストの量を感染試験によって定量評価して紫外線照射によるオーシスト不活性化の程度を検査した。
【００２９】
米国Ｗａｔｅｒｂｏｒｎｅ Ｉｎｃ．より購入したクリプトスポリジウム（パルバム種）のオーシスト懸濁液（１×１０6 個／ｍＬ−リン酸緩衝液）を７０μＬずつ９６穴マルチプレートに分注した。１５Ｗ低圧水銀ランプ点灯下で紫外線照射強度が０．１ｍＷ／ｃｍ2 の位置に当該マルチプレートを置き、３０秒から１０分の範囲で紫外線を照射した。紫外線を照射した後のウェルにオーシスト希釈液（０．２％重炭酸ナトリウム、１０％ウマ血清、１ｍＭピルビン酸ナトリウム、０．１ｇ／Ｌカナマイシン、１ｍｇ／Ｌ葉酸、４ｍｇ／Ｌｐ−アミノ安息香酸、２ｍｇ／Ｌパントテン酸、３５ｍｇ／Ｌアスコルビン酸を添加したＲＰＭＩ１６４０培地からなる溶液）をそれぞれ６３０μＬずつ注入して、オーシスト濃度を１０倍に希釈した（オーシスト濃度は１×１０5 個／ｍＬ）。希釈後のオーシスト懸濁液中に生存しているオーシストの濃度は、ヒト由来培養細胞ＨＣＴ−８株（ＡＴＣＣ ＃ＣＣＬ ２４４）を宿主細胞とした感染試験によって定量評価した。
【００３０】
すなわち、０．２％重炭酸ナトリウム、１０％ウマ血清、１ｍＭピルビン酸ナトリウム、０．１ｇ／Ｌカナマイシンを添加したＲＰＭＩ１６４０培地からなる維持培地にて培養したＨＣＴ−８細胞をトリプシン処理で培養容器から回収し、細胞濃度が２．５×１０5 個／ｍＬとなるように新鮮な増殖培地に懸濁して、予めゼラチンでコートした９６穴マルチプレートに２００μＬずつ分注し、炭酸ガス培養器内で２４時間培養した。培養後のマルチプレートの培地を除去し、検水を各々１００μｌずつ分注し、炭酸ガス培養器内で９０分間培養して、感染させた。感染後の各ウェルより培地を除去し、リン酸緩衝液で２回洗浄した後、オーシストを含まない増殖培地を１００μｌずつ分注し、炭酸ガス培養器内で２日間培養した。培養後の各ウェルより培地を除去し、４％ホルマリンを添加したリン酸緩衝液からなる固定液を１００μｌずつ分注して、室温で２時間反応させて固定した。固定液を除去し、リン酸緩衝液で３回洗浄した後、１％ウシ血清アルブミンと０．００２％Ｔｗｅｅｎ２０を添加したリン酸緩衝液からなるブロッキング液を１００μｌずつ分注して、室温で１時間反応させてブロッキングした。ブロッキング液を除去し、一次処理溶液を３３μｌずつ分注して、室温で１時間反応させた。一次処理溶液は、米国Ｗａｔｅｒｂｏｒｎｅ Ｉｎｃ．より購入したビオチン化抗体（商品名Ａｑｕａ−Ｇｌｏ Ｇ／Ｃ Ｉｎｄｉｒｅｃｔ）を前記したブロッキング液で２０倍に希釈した溶液を用いた。反応後の各ウェルから一次処理溶液を除去し、リン酸緩衝液で３回洗浄した後、二次処理溶液を３３μｌずつ分注して、室温で１時間反応させた。二次処理溶液は、アマシャム株式会社より購入したストレプトアビジン−ペルオキシダーゼ複合体（商品名Ｓｔｒｅｐｔｏａｖｉｄｉｎ−ｈｏｒｓｅｒａｄｉｓｈ ｐｅｒｏｘｉｄａｓｅ ｃｏｍｐｌｅｘ）を前記したブロッキング液で４００倍に希釈した溶液を用いた。反応後の各ウェルから二次処理溶液を除去し、リン酸緩衝液で３回洗浄し、過ホウ酸ナトリウム添加リン酸クエン酸緩衝液（Ｓｉｇｍａ）で１回洗浄した後、発色溶液を１００μｌずつ分注して、室温で１時間反応させて発色させた。発色溶液は、ＯＰＤ［o-phenylenediamine dihydrochloride］（Ｓｉｇｍａ）を発色試薬として０．４ｍｇ／ｍｌとなるように過ホウ酸ナトリウム添加リン酸クエン酸緩衝液（Ｓｉｇｍａ）に溶解したものを用いた。発色反応後のマルチプレートは、マイクロプレートリーダー（バイオラド社製 モデル５５０）を用いて、波長５９５ｎｍをリファレンスとして波長４５０ｎｍの吸光度を測定した。測定した吸光度の大小は、検水中の生存オーシスト濃度の高低に依存していることが知られている（加藤ら（２０００）培養細胞へのクリプトスポリジウム・パルバムの感染とELISAによる検出，水環境学会誌，第23巻，第7号，p427）。
【００３１】
また、オーシスト濃度算定のための検量線は、米国Ｗａｔｅｒｂｏｒｎｅ Ｉｎｃ．より購入したクリプトスポリジウム（パルバム種）のオーシストを前記したオーシスト希釈液で０〜１０万個／ｍｌの範囲で種々の濃度に希釈したオーシスト懸濁液（以下、標準液という）を検水として前記した感染試験に基づいて定量評価し、作成した。
【００３２】
まず、オーシスト濃度算定のための検量線を図１に示した。図１では、ウェルに添加したクリプトスポリジウムのオーシスト濃度の対数を横軸として、各ウェルについての発色強度を示す吸光度を縦軸にプロットした。回帰式を計算した結果、相関係数０．９８で下式（２）が得られた。
ｙ ＝ ０．０２３５Ｌｎ（ｘ） ＋ ０．０２１９ （式２）
式（２）を変形した下式（３）によれば吸光度（ｙ）から生存オーシスト濃度（ｘ）を算出することができる。
【００３３】
ｘ ＝ Ｅｘｐ｛（ｙ−０．０２１９）÷０．０２３５｝ （式３）
ついで、種々の時間で紫外線照射した実施例の検水に関する感染試験の結果を図２に示した。図２では、紫外線照射強度（本実施例においては０.１ｍＷ／ｃｍ2 ）と照射時間［秒］の積から紫外線照射量［ｍＷ・ｓ／ｃｍ2 ］を算定し、その数値を横軸上に標記した。それぞれの照射量の紫外線を照射して得た検水に関する感染試験の結果を吸光度の値として縦軸にプロットした。なお、紫外線未照射のオーシスト懸濁液に関して同様の感染試験で評価した結果を最左列に併記した。本図より明らかなように、紫外線照射量を高めると吸光度が顕著に低下しており、紫外線照射によって生存オーシスト濃度が減少、すなわち不活性化が可能であることが容易に推定される。
【００３４】
さらに、図１に示した感染試験の検量線に基づいて、図２に示した実施例の感染試験における吸光度測定値から検水中の生存オーシスト濃度を算出した。具体的には、式（２）中のｙに図２の吸光度を代入することによって生存オーシスト濃度を算出した。紫外線照射後の検水の生存オーシスト濃度を紫外線未照射時のそれと比較することによって、紫外線照射によるクリプトスポリジウムのオーシストの不活性化率を算出した。すなわち、不活性化率は下式（４）で算出した。
【００３５】
不活性化率［％］ ＝ ｛１−（紫外線照射後の生存オーシスト濃度÷ 紫外線未照射時の生存オーシスト濃度）｝ × １００ （式４）
結果を図３に示した。図３では、紫外線照射量を横軸とし、それぞれの照射量における不活性化率を縦軸にプロットした。本図より明らかなように紫外線照射量が高まると指数関数的に不活性化が可能であることは明らかである。本実施例の結果では、紫外線照射量にして２０ｍＷ・ｓ／ｃｍ2 毎に９０％のクリプトスポリジウムのオーシストが不活性化することが可能であった。つまり、２０、４０、６０ｍＷ・ｓ／ｃｍ2 の紫外線を照射した場合、それぞれ、９０、９９、９９．９％が不活性化することができる。
【００３６】
実施例２
内側を二酸化チタン光触媒被膜したガラスシャーレにクリプトスポリジウムのオーシスト懸濁液を入れ、液面上部から紫外線を照射し、生存オーシストの量を感染試験によって定量評価して紫外線照射によるオーシスト不活性化の程度を検査した。
【００３７】
内側を二酸化チタン光触媒被膜した外径６０ｍｍのガラスシャーレに、米国Ｗａｔｅｒｂｏｒｎｅ Ｉｎｃ．より購入したクリプトスポリジウム（パルバム種）のオーシスト懸濁液（１×１０6 個／ｍＬ−リン酸緩衝液）を５ｍＬを注ぎ、４００Ｗ高圧水銀ランプ点灯下で紫外線照射強度が０．１ｍＷ／ｃｍ2 の位置に当該マルチプレートを置き、液面上部から紫外線を照射した。３０秒から１０分の範囲で経時的にオーシスト懸濁液を１００μＬずつ採取し、実施例１に記したオーシスト希釈液を用いて１０倍に希釈した後の試料を検水とした。当該検水実施例１に記した手順に従って、培養細胞に感染させ、酵素抗体法で吸光度を測定し、さらに図１に示した検量線に基づいて検水中の生存オーシスト濃度を算出した。
【００３８】
結果を図４に示した。図４では、紫外線照射量を横軸とし、それぞれの照射量における不活性化率を縦軸にプロットした。本図より明らかなように紫外線照射量が高まると指数関数的に不活性化が可能であることは明らかである。本実施例の結果では、紫外線照射量にして１５ｍＷ・ｓ／ｃｍ2 毎に９０％のクリプトスポリジウムのオーシストが不活性化することが可能であった。つまり、１５、３０、４５ｍＷ・ｓ／ｃｍ2 の紫外線を照射した場合、それぞれ、９０、９９、９９．９％が不活性化することができる。本実施例で得られた光触媒併用法を用いれば、実施例１に記した紫外線照射単独法に比べて２割程度少ない紫外線照射量で不活性化が可能であった。
【００３９】
以上の２例の実施例の結果より、紫外線照射することでクリプトスポリジウムを効果的に不活性化できることは明らかである。また、光触媒併用法によれば紫外線単独法に比べてより少ない紫外線照射量で原虫類を不活性化できる。さらに、実際の不活性化処理へ適用に際しては所望の不活性化目標値に応じて紫外線照射量の最適値を決定することができる。
【００４０】
【発明の効果】
本発明によれば、従来、紫外線による不活性化が困難とされた原虫類のシストやオーシストを容易に不活性化できる。さらに、所望の不活性化目標値に応じて紫外線照射量の最適値を決定することができる。
実際の処理への適用に際しては、濁度、色度、紫外線吸収率等による不活性化効果の阻害因子が存在するが、その場合にはそれぞれの処理対象物の水質を考慮して不活性条件を見極めて、最適化すればよい。また、前記した紫外線照射量の最適値に安全率を乗じて照射条件を算定してもよい。
【００４１】
また、一般に微生物を紫外線照射した後に可視光や近紫外光を受光すると紫外線照射によって損傷したＤＮＡが修復され、再び活性化するいわゆる光回復現象が存在することが知られているが、紫外線とはことなる機構で不活性化する光触媒法を併用することによって光回復現象を抑制することができる。
【図面の簡単な説明】
【図１】実施例１および２において検水中の生存オーシスト濃度の算定根拠とした、感染試験における検量線例を示す図である。
【図２】クリプトスポリジウム原虫に関して本願の不活性化方法の実施例１における感染試験結果例を示す図である。
【図３】クリプトスポリジウム・パルバムに関して本願の不活性化方法の実施例１における紫外線照射量と不活性率との相関性を示す例図である。
【図４】クリプトスポリジウム・パルバムに関して本願の不活性化方法の実施例２における紫外線照射量と不活性率との相関性を示す例図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to purified water, potable water, sewage, drainage, river water, lake water, landscape water, pool water, food for food and food production in which contamination with protozoa such as Cryptosporidium and Giardia is concerned. The present invention relates to a disinfection method using equipment as a processing target.
[0002]
The present invention also relates to a disinfection method that inactivates infectious protozoa in a short time and simply to reduce or eliminate the pathogenicity of the microorganism.
[0003]
[Prior art]
Recently, the occurrence of water-borne infections caused by protozoa such as Cryptosporidium and Giardia has become a major problem worldwide, including in the field of water supply and sewerage (for example, Mitsuyoshi Hosaka (1998) “Water-borne protozoan infections—causes Biology and outbreaks-", water and wastewater, Vol. 40, No. 2, p. 11; Mitsumi Kaneko (1998)" Technology for detection and removal of protozoa and other pathogenic microorganisms ", water and waste water, No. 1 10 volumes, No. 4, 32 pages). The protozoa is a dangerous pathogen that can cause illness due to a very small number of drinks, and there is a concern about a large epidemic infection resulting from contamination of drinking water and food. There is a strong demand for the development of disinfection technology that can reliably eliminate protozoa in order to prevent the occurrence of massive epidemic infections.
[0004]
In the water and sewerage field, the chlorination method is widely used as a method of disinfecting water. However, protozoa such as Cryptosporidium and Giardia are highly resistant to chlorine, and chlorination methods make it difficult to kill protozoa (for example, IS Campbell et al. (1982) Effect of disinfectants on survival of Cryptosporidium oocysts, Weterinary Record, Vol.111, p414, etc.).
[0005]
The following treatment technologies are being examined as countermeasure technologies to replace chlorine disinfection. The filtration method is a treatment technique for physically removing protozoan cysts or oocysts from water to be treated by sand filtration or membrane filtration.
Heat treatment is a treatment technique that kills protozoan cysts or oocysts by heating water to be treated.
[0006]
Ozone treatment is a treatment technique in which ozone gas is blown into water to be treated, and protozoan cysts or oocysts are inactivated or killed by the oxidizing power of ozone. It is said that the treatment efficiency is further increased by using in combination with ultraviolet irradiation.
In addition, there is ultraviolet treatment as a treatment technique for killing bacteria and viruses, but it is difficult to apply to protozoa such as Giardia and Cryptosporidium.
[0007]
[Problems to be solved by the invention]
Protozoa are resistant to chemical agents because they exist in the form of spore-like shells called oocysts in Cryptosporidium and cysts in Giardia in the environment, and are widely used in conventional water treatment methods. It cannot be controlled by disinfection.
Membrane filtration can be expected to remove protozoa reliably, but the amount of permeated water is limited, and large-capacity water treatment requires enormous costs for enlarging equipment and renewing membranes.
[0008]
In the sand filtration method, protozoa may leak into the treated water due to short-circuit water flow or breakthrough in the filter bed.
Protozoa can be inactivated by heat treatment because they are extremely vulnerable to heating and drying. However, in large-capacity water treatment, a large amount of cost is required for installing large-scale heating equipment and supplying a large amount of energy for heating. is required.
[0009]
To kill protozoa such as Giardia and Cryptosporidium by UV treatmentoutputIt is necessary to continue to irradiate 15 W of ultraviolet rays for 150 minutes, and generally 700 to 1000 mW · s / cm2 It is said that it is necessary to irradiate about ultraviolet rays. Since this UV irradiation dose is roughly 10 to 20 times the design value of a normal UV disinfection device, UV treatment of protozoa is difficult to apply in practice (for example, ELJaroll (1988) Effect of disinfectants on Giardia cysts, CRC Critical Reviews in Environmental Control, Vol. 18, p1; MJ Lorenzo-Lorenzo et al. (1993) Effect of ultraviolet disinfection of drinking water on the viability Cryptosporidium parvum oocysts, J. Parasitol., Vol. 79, No.1, p67).
[0010]
As described above, conventional control techniques aimed at removing or killing protozoa in a processing target have problems in terms of stability of processing effect and cost.
On the other hand, as a method for evaluating the growth activity or infectivity of protozoa, a vital staining method, a decapsulation test method, and an animal experiment method have been proposed. However, the vital staining method and the decapsulation test method cannot be quantitatively evaluated by microscopic observation, and thus have disadvantages such as complicated operation, inefficiency, and poor data accuracy, and are not suitable for determining inactivation conditions. It is. In animal experiment methods, small animals such as mice are orally inoculated with test samples, bred continuously for about 1 week to 1 month, and protozoan oocysts or cysts are purified from excreted feces. Then, the number of oocysts or cysts is measured by microscopic observation or the like, and the protozoan concentration in the test sample is estimated based on this result. Alternatively, the digestive organs of the small animal are removed, the presence or absence of onset is determined by pathological observation, and the protozoan concentration in the test sample is estimated based on this result. As described above, the animal experiment method is not a preferable method from the viewpoint of animal protection, in addition to the fact that it takes a long time for the determination and individual differences of small animals greatly affect the results.
[0011]
A cultured cell method has been proposed as a method for examining protozoa as an alternative to animal experimental methods. For example, it is described in Japanese Patent Application Laid-Open Publication No. 2000-214168 that protozoa capable of infecting can be detected easily and with high sensitivity by using cultured cells.
The inventor is able to abolish the protozoan's growth activity or infectivity (ie “inactivation”) even if the individual protozoa or their cysts or oocysts in the treatment object cannot be completely killed. If possible, find out that it can be a countermeasure technology in terms of hygiene, where the presence or absence of pathogenicity against human livestock is a problem, and by optimizing the processing conditions based on the accurate determination result of the degree of inactivation of protozoa An object of the present invention is to provide an efficient and effective method for inactivating protozoa overcoming the problems of the prior art.
[0012]
[Means for Solving the Problems]
Providing a method for inactivating protozoa that minimizes the processing cost by limiting the processing conditions required for inactivation by using the degree of infectivity of protozoan cysts or oocysts as an index. it can. That is, the above-mentioned problems are the following (1) to (1) to(4)Can be solved by.
[0013]
(1) UV irradiation conditions are set using, as an index, the degree of inactivation of the infectivity of protozoa present in the processing object or suspected of being present by UV irradiation, and the processing object is irradiated with UV light. A method for inactivating protozoa, wherein the ultraviolet irradiation conditions are set such that a sample of a treatment target that has not been irradiated with ultraviolet rays, and the amount of ultraviolet irradiation with a wavelength of about 254 nm applied to the treatment target is 1 to 100 mW · s / cm2 Each sample of the sample after being irradiated with ultraviolet rays so as to be in contact with the cultured cells, the protozoa with the infectious protozoa in the sample parasitized into the cultured cells and proliferated in the cultured cells,Oxygen antibody method or immunostainingQuantitative detection using the method, the concentration of the infectious protozoa in each sample is calculated, the absorbance is measured by the oxygen antibody method, quantitative detection is performed, and the concentration defined by the following formula (1) is detected. Since the activation rate is calculated, 90% of the infectious protozoa are inactivated for each specific increase in the UV irradiation dose and the inactivation rate. A method for inactivating a protozoan, characterized in that an ultraviolet irradiation condition to a processing object is set according to a target value of an activation rate.
[0014]
Inactivation rate = 1-(Concentration of protozoa in the sample after the object to be treated is irradiated with ultraviolet rays ÷ Object to be treated without UV irradiationSample ofConcentrations of protozoa in) (Formula 1)
[0015]
(2) Object to be processedSample of(1) that utilizes a redox reaction that occurs when a photocatalyst receives ultraviolet rays together with a direct action by ultraviolet rays by disposing a thin film having a photocatalytic function at a position where it can contact ultraviolet rays and receive ultraviolet rays. A method for inactivating protozoa as described.
(3) The method for inactivating protozoa according to (2), wherein the thin film having a photocatalytic function is a thin film mainly composed of titanium oxide.
[0016]
(4(1) to (1), wherein the object to be treated is water.3)One ofA method for inactivating protozoa according to
[0017]
DETAILED DESCRIPTION OF THE INVENTION
The present invention “inactivates” the loss of the ability of protozoa to parasitize cells and proliferate in the process of causing illnesses caused by protozoa such as Cryptosporidium and Giardia described below. It is possible to provide an effective protozoan inactivation method that can avoid the risk of disease development without being completely killed. The process by which protozoa such as Cryptosporidium and Giardia parasitize the body of an animal and cause a disease is described as follows.
[0018]
1) Cyst or oocyst is taken into the body orally.
2) The infectious worms are exposed by decapsulation in the digestive tract.
3) Infectious worms adhere to epidermal cells in the intestine.
4) Infectious worms enter the epidermis cells of the intestinal tract.
5) Infectious worms grow in epidermal cells of the intestinal tract.
[0019]
6) The propagated infectious worms form cysts or oocysts.
7) A cyst or oocyst formed in the cell is released out of the cell.
8) The cyst or oocyst released to the outside of the cell unseals in the intestinal tract, invades another cell, and proliferates.
9) Repeat steps 2 to 8 to cause gastrointestinal dysfunction, leading to disease.
[0020]
In view of the process leading to the above diseases, the development of disinfection technology has been studied from the viewpoint that protozoa that are likely to be mixed before being taken orally must be completely killed. I had to rely on disinfecting technology that invested a lot of energy and costs. However, as a result of intensive studies by the inventors, the DNA that is the body of the gene is damaged without being killed.SereFor example, it has been found that the growth in parasitic cells can be prevented and the process leading to the above-mentioned
[0021]
The present invention is capable of optimizing treatment conditions such as ultraviolet irradiation intensity and irradiation time necessary for inactivation of protozoa, and designing protozoa by changing the operation conditions of existing equipment or changing the operating conditions of existing equipment. Inactivation technology can be provided.
First, a method for inactivating protozoa will be described in detail. Protozoa (or their oocysts or cysts) are 1 to 100 mW · s / cm2 It can be easily inactivated by ultraviolet irradiation treatment with a low irradiation amount of about 254 nm (wavelength 254 nm). The ultraviolet light source is not particularly limited as long as it is a lamp that emits ultraviolet light, but there is a difference between the case where the ultraviolet irradiation single method is adopted as the inactivation treatment and the photocatalyst combined method. In the former case, for example, a low-pressure mercury lamp, a medium / high-pressure mercury lamp, or a xenon lamp can be used as a lamp that emits ultraviolet light having a wavelength of around 254 nm. Although a lamp that emits ultraviolet light around 365 nm can be used, a medium / high pressure mercury lamp that emits ultraviolet light around a wavelength of 254 nm and around 365 nm is preferable. Although it can be manufactured as an apparatus optimized for inactivation of protozoa, it can also be dealt with by changing operating conditions such as ultraviolet irradiation intensity and irradiation time for the existing ultraviolet disinfection apparatus. .
[0022]
By the way, it is said that many organisms have a mechanism for self-repairing DNA damaged by ultraviolet irradiation, and this repair mechanism is induced by near-ultraviolet light or visible light and is called light recovery. It is said to be one of the problems to be overcome by ultraviolet disinfection (for example, Kashimada et al. (1995) “Evaluation of light recovery in ultraviolet irradiation water treatment” Journal of Water Environment Society, Vol. 18, No. 1, p. 44). If the photocatalyst combined method is used, the above-mentioned photorecovery can be suppressed (for example, JP-A-11-156352). In the photocatalyst combined method, a thin film having a photocatalytic function may be disposed at a position in contact with the object to be processed and capable of receiving ultraviolet rays. As the thin film having a photocatalytic function, a thin film containing titanium oxide as a main component is preferable in terms of cost, catalytic activity, durability, and the like. Further, not only a thin film of titanium oxide alone composition but also a metal such as Ag, Au, Cu, Fe, Mb, Ni, Pd, Pt, Rd, Rh, Ru, Sn, V, Zn or the like in order to enhance photocatalytic activity A titanium oxide thin film to which an oxide is added can also be used.
[0023]
Next, a method for evaluating the degree of inactivation of protozoa will be described in detail. As described above, the conventional evaluation technology has problems in terms of operability and reliability, and therefore, a method that overcomes the problems of the conventional method that should evaluate the degree of inactivation of these protozoa should be adopted. Is preferred. In the present invention, a test method using cultured cells can be used as a method for evaluating the degree of inactivation of protozoa. For example, the test sample is brought into contact with the cultured cells for 30 minutes to 3 hours, the test sample is replaced with a fresh culture medium, and cultured in the cultured cells for 1 to 1 week, preferably 1 to 3 days. A method of quantitatively detecting protozoa or oocysts of protozoa or cysts by the enzyme antibody method or immunostaining method can be used. The cultured cells subjected to the test may be cell lines that can be infected with the protozoa to be detected. For example, BALB / 3T3 derived from mouse, BT-549 derived from human, Caco-2, HCT-8, HT-29 , Hs-700T, HT-1080, LS-174T, RL59-2, MDBK derived from bovine, MDCK derived from dog, etc., but Cryptosporidium (parvum species that are infectious to humans) ) Can be detected correctly, for example, by using HCT-8 derived from human intestinal endothelial cells, the infectivity to humans can be correctly evaluated. Culture conditions such as culture medium composition, culture temperature, humidity, carbon dioxide concentration, and culture time can be selected according to the cell line to be used.
[0024]
As an enzyme antibody method for quantitative detection of protozoa or oocysts or cysts of protozoa grown in cultured cells, for example, after fixing the cells with a formalin solution or an alcohol solution, if necessary, a bovine serum albumin-added phosphate ELISA method [Enzyme-Linked Immuno Sorbent Assay] (for example, Kato et al. (2000) Infection of Cryptosporidium parvum to cultured cells and detection by ELISA, Society of Water Environment Journal, Vol. 23, No. 7, p427) can be quantitatively detected as a change in absorbance according to the concentration of protozoa (or its oocysts or cysts).
[0025]
In addition, as an immunostaining method for quantitatively detecting protozoa or protozoan oocysts or cysts grown in cultured cells, for example, after fixing cells with a formalin solution or an alcohol solution, bovine serum albumin may be used as necessary. After blocking with an added phosphate buffer or culture medium, the cells are stained with a fluorescently labeled antibody, and the foci (site of pathogen infection) in the cultured cell group is counted by fluorescence microscope observation or laser microscope observation. From the results, it is possible to determine the concentration of oocysts or cysts of protozoa having infectivity in the test sample.
[0026]
By the way, in order to evaluate the degree of inactivation of protozoa, it is necessary to quantify how much the infectious protozoa has been reduced before and after the inactivation treatment. Among the quantitative detection methods described above, in the immunostaining method, the inactivation rate can be determined immediately in order to directly count the number of protozoan oocysts or cysts. On the other hand, in the enzyme antibody method among the quantitative detection methods described above, the concentration and absorbance of protozoa (or oocysts or cysts thereof) cannot always be expressed by a linear function, that is, they are not directly proportional. A calibration curve for the concentration and absorbance of oocysts or cysts is required. Therefore, a standard sample containing a protozoan (or its oocyst or cyst) at a known concentration is diluted into a plurality of concentration steps, and a test sample is used to carry out a similar test procedure to obtain a protozoan (or A calibration curve for the concentration and absorbance of the oocyst or cyst) can be obtained. To formulate the correlation between the concentration of protozoa and the absorbance of a standard sample, an appropriate approximation method may be selected according to the test results.For example, linear approximation, polynomial approximation, logarithmic approximation, exponential approximation, exponential approximation, etc. Another example is a reciprocal plot analysis. Based on the calibration curve, how much the infectious protozoa have been reduced before and after the inactivation treatment can be quantified. In order to accurately determine the concentration of protozoa (or its oocysts or cysts) in a test sample, it is desirable to make a determination within the concentration range of the calibration curve described above, and dilute or concentrate the sample to be tested as necessary. The obtained sample may be used as a test sample.
[0027]
Using the above-described evaluation method, the concentration of protozoa in each test sample is measured using a sample that has not been irradiated with UV light and a plurality of levels of samples that have been irradiated with UV light at various doses. ) To calculate the inactivation rate defined by
Inactivation rate = 1-(Concentration of protozoa in test sample irradiated with ultraviolet rays ÷ Concentration of protozoa in test sample not irradiated with ultraviolet rays) (Equation 1)
Furthermore, the amount of ultraviolet irradiation necessary to obtain a desired inactivation rate can be determined from the relationship between the amount of ultraviolet irradiation and the inactivation rate. The determined ultraviolet irradiation amount may be used as the design condition of the ultraviolet irradiation apparatus, but the ultraviolet irradiation amount calculated by considering the ultraviolet irradiation amount as basic data and further considering the safety factor may be adopted as the design condition.
[0028]
【Example】
Example 1
The oocyst suspension of Cryptosporidium was irradiated with ultraviolet rays, and the amount of viable oocysts was quantitatively evaluated by an infection test to examine the degree of oocyst inactivation by ultraviolet irradiation.
[0029]
United States Waterbone Inc. Cryptosporidium (Parvam) oocyst suspension (1 × 106 Pieces / mL-phosphate buffer solution) was dispensed into a 96-well multiplate in an amount of 70 μL. UV irradiation intensity is 0.1mW / cm under 15W low pressure mercury lamp lighting2 The multiplate was placed at the position of and irradiated with ultraviolet rays in the range of 30 seconds to 10 minutes. The oocyst dilution (0.2% sodium bicarbonate, 10% horse serum, 1 mM sodium pyruvate, 0.1 g / L kanamycin, 1 mg / L folic acid, 4 mg / Lp-aminobenzoic acid, 630 μL each of 2 mg / L pantothenic acid and 35 mg / L ascorbic acid-added RPMI1640 medium solution was injected to dilute the
[0030]
That is, HCT-8 cells cultured in a maintenance medium consisting of RPMI1640 medium supplemented with 0.2% sodium bicarbonate, 10% horse serum, 1 mM sodium pyruvate, 0.1 g / L kanamycin were removed from the culture vessel by trypsin treatment. Collected and cell concentration is 2.5 × 10Five The suspension was suspended in a fresh growth medium so that the number of cells / mL was reached, 200 μL each was dispensed into a 96-well multiplate previously coated with gelatin, and cultured in a carbon dioxide incubator for 24 hours. After culturing, the medium of the multiplate was removed, 100 μl each of the test water was dispensed, and cultured in a carbon dioxide incubator for 90 minutes to be infected. After removing the medium from each well after infection and washing twice with a phosphate buffer, 100 μl of a growth medium not containing oocysts was dispensed and cultured for 2 days in a carbon dioxide incubator. The culture medium was removed from each well after culturing, and 100 μl of a fixative solution consisting of a phosphate buffer added with 4% formalin was dispensed and fixed at room temperature for 2 hours. After removing the fixative and
[0031]
A calibration curve for calculating the oocyst concentration was obtained from Waterborne Inc., USA. An oocyst suspension (hereinafter referred to as a standard solution) obtained by diluting oocysts of Cryptosporidium (parvum species) purchased from oocysts with the above-described oocyst diluent to various concentrations in the range of 0 to 100,000 / ml is used as the test water. A quantitative evaluation was made based on the infection test.
[0032]
First, a calibration curve for calculating the oocyst concentration is shown in FIG. In FIG. 1, the horizontal axis represents the logarithm of the oocyst concentration of Cryptosporidium added to the well, and the vertical axis represents the absorbance indicating the color intensity for each well. As a result of calculating the regression equation, the following equation (2) was obtained with a correlation coefficient of 0.98.
y = 0.0235Ln (x) +0.0219 (Formula 2)
According to the following formula (3) obtained by modifying the formula (2), the survival oocyst concentration (x) can be calculated from the absorbance (y).
[0033]
x = Exp {(y−0.0219) ÷ 0.0235} (Formula 3)
Next, FIG. 2 shows the results of an infection test on the sample water of the examples irradiated with ultraviolet rays at various times. In FIG. 2, the ultraviolet irradiation intensity (in this example, 0.1 mW / cm2 ) And irradiation time [second], the amount of UV irradiation [mW · s / cm2 ] Was calculated and the numerical value was marked on the horizontal axis. The result of the infection test regarding the test water obtained by irradiating each irradiation dose of ultraviolet rays was plotted on the vertical axis as the absorbance value. In addition, the result evaluated by the same infection test regarding the oocyst suspension not irradiated with ultraviolet rays is also shown in the leftmost column. As is clear from this figure, when the amount of ultraviolet irradiation is increased, the absorbance is remarkably reduced, and it is easily estimated that the survival oocyst concentration can be reduced, that is, inactivated by ultraviolet irradiation.
[0034]
Furthermore, based on the calibration curve of the infection test shown in FIG. 1, the survival oocyst concentration in the test water was calculated from the absorbance measurement value in the infection test of the example shown in FIG. Specifically, the survival oocyst concentration was calculated by substituting the absorbance in FIG. 2 for y in the formula (2). The inactivation rate of Cryptosporidium oocysts by UV irradiation was calculated by comparing the concentration of living oocysts in the test water after UV irradiation with that in the case without UV irradiation. That is, the inactivation rate was calculated by the following formula (4).
[0035]
Inactivation rate [%] = {1- (survival oocyst concentration after ultraviolet irradiation / survival oocyst concentration without UV irradiation)} × 100 (Formula 4)
The results are shown in FIG. In FIG. 3, the ultraviolet irradiation amount is plotted on the horizontal axis, and the inactivation rate at each irradiation amount is plotted on the vertical axis. As is clear from this figure, it is clear that inactivation can be performed exponentially as the amount of ultraviolet irradiation increases. In the result of this example, the amount of UV irradiation is 20 mW · s / cm.2 It was possible to inactivate 90% Cryptosporidium oocysts every time. That is, 20, 40, 60 mW · s /
[0036]
Example 2
Cryptosporidium oocyst suspension is placed in a glass petri dish coated with titanium dioxide photocatalyst on the inside, irradiated with ultraviolet rays from the top of the liquid surface, and the amount of viable oocysts is quantitatively evaluated by infection test, and the degree of oocyst inactivation by ultraviolet irradiation Inspected.
[0037]
In a glass petri dish having an outer diameter of 60 mm and coated with a titanium dioxide photocatalyst on the inside, Waterborne Inc., USA. Cryptosporidium (Parvam) oocyst suspension (1 × 106 5 mL of a solution / mL-phosphate buffer solution) is poured, and the UV irradiation intensity is 0.1 mW / cm under the operation of a 400 W high-pressure mercury lamp.2 The multiplate was placed at the position of, and irradiated with ultraviolet rays from the top of the liquid surface. 100 μL of oocyst suspension was sampled over time in the range of 30 seconds to 10 minutes, and the sample after diluting 10 times with the oocyst diluent described in Example 1 was used as test water. According to the procedure described in Example 1 of the test water, the cultured cells were infected, the absorbance was measured by the enzyme antibody method, and the viable oocyst concentration in the test water was calculated based on the calibration curve shown in FIG.
[0038]
The results are shown in FIG. In FIG. 4, the ultraviolet irradiation amount is plotted on the horizontal axis, and the inactivation rate at each irradiation amount is plotted on the vertical axis. As is clear from this figure, it is clear that inactivation can be performed exponentially as the amount of ultraviolet irradiation increases. In the result of this example, the ultraviolet ray irradiation amount is 15 mW · s / cm.2 It was possible to inactivate 90% Cryptosporidium oocysts every time. That is, 15, 30, 45 mW · s /
[0039]
From the results of the above two examples, it is clear that Cryptosporidium can be effectively inactivated by ultraviolet irradiation. In addition, according to the photocatalyst combined method, protozoa can be inactivated with a smaller amount of ultraviolet irradiation as compared with the ultraviolet alone method. Further, when applied to actual inactivation processing, the optimum value of the ultraviolet irradiation amount can be determined according to a desired inactivation target value.
[0040]
【The invention's effect】
According to the present invention, it is possible to easily inactivate protozoan cysts and oocysts conventionally difficult to inactivate by ultraviolet rays. Furthermore, the optimum value of the UV irradiation amount can be determined according to the desired deactivation target value.
In application to actual treatment, there are inhibitors of the inactivation effect due to turbidity, chromaticity, UV absorption rate, etc. In that case, inactive conditions considering the water quality of each treatment object Find and optimize. Further, the irradiation condition may be calculated by multiplying the optimum value of the ultraviolet irradiation amount described above by the safety factor.
[0041]
In addition, it is generally known that there is a so-called photorecovery phenomenon in which DNA damaged by UV irradiation is repaired and activated again when visible light or near UV light is received after UV irradiation of microorganisms. The photorecovery phenomenon can be suppressed by using a photocatalytic method inactivated by a different mechanism.
[Brief description of the drawings]
FIG. 1 is a diagram showing an example of a calibration curve in an infection test as a basis for calculating the survival oocyst concentration in test water in Examples 1 and 2.
FIG. 2 is a diagram showing an example of infection test results in Example 1 of the inactivation method of the present application for Cryptosporidium protozoa.
FIG. 3 is an example diagram showing the correlation between the ultraviolet irradiation amount and the inactivation rate in Example 1 of the inactivation method of the present application for Cryptosporidium parvum.
FIG. 4 is an example diagram showing the correlation between the ultraviolet irradiation amount and the inactivation rate in Example 2 of the inactivation method of the present application for Cryptosporidium parvum.
Claims (4)
前記紫外線照射条件の設定が、紫外線未照射の処理対象物の試料、および処理対象物に波長２５４ｎｍ付近の紫外線の照射量が１〜１００ｍＷ・ｓ／ｃｍ2 となるように紫外線を照射した後の試料の、それぞれの試料を培養細胞に接触させ、前記試料中の感染能力のある原虫類が培養細胞に寄生して培養細胞内で増殖した原虫類を、酸素抗体法又は免疫染色法を用いて定量検出して、前記それぞれの試料における前記感染能力のある原虫類の濃度を算出し、下式（１）：
不活性化率＝１−（処理対象物に紫外線を照射した後の試料における原虫類の濃度
÷紫外線未照射の処理対象物の試料における原虫類の濃度）(式１)
で定義される不活性化率を算出し、前記紫外線照射量と前記不活性化率との、紫外線照射量の特定の一定増加量毎に感染能力のある原虫類の９０％が不活性化するという連関から、不活性化率の目標値に応じて処理対象物への紫外線照射条件を設定することであることを特徴とする原虫類の不活性化方法。For the protozoa that irradiates the treatment object with ultraviolet rays by setting the ultraviolet irradiation condition using as an index the degree of inactivation of the infectivity of the protozoa existing or suspected to exist in the treatment object. An inactivation method comprising:
After setting the ultraviolet irradiation conditions, the sample of the object to be processed that has not been irradiated with ultraviolet light, and the object to be processed are irradiated with ultraviolet rays so that the irradiation amount of ultraviolet rays having a wavelength of about 254 nm is 1 to 100 mW · s / cm 2 . Each sample of the sample is brought into contact with the cultured cell, and the infectious protozoa in the sample is infested with the cultured cell and proliferated in the cultured cell using the oxygen antibody method or immunostaining method. Quantitative detection is performed to calculate the concentration of the infectious protozoa in each sample, and the following formula (1):
Inactivation rate = 1-(Protozoa concentration in the sample after irradiating the object to be treated with ultraviolet rays)
÷ Concentration of protozoa in samples of UV-irradiated objects to be treated) (Equation 1)
90% of infectious protozoa are inactivated for each specific increase in the amount of UV irradiation and the amount of UV irradiation inactivated. Therefore, a method for inactivating protozoa, characterized in that conditions for ultraviolet irradiation of the object to be treated are set according to the target value of the inactivation rate.
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