JP2021510379A - 魚類の体表の寄生虫感染症の処置 - Google Patents
魚類の体表の寄生虫感染症の処置 Download PDFInfo
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Abstract
Description
本特許出願に至るプロジェクトは、欧州連合のホライズン2020研究及び革新的開発プログラムから、合意書第634429号の下で財政的支援を受けている。
[実施例]
寄生虫:
ウオノカイセンチュウのデンマーク株を、元来、無疾患の再循環システム中で養殖したニジマスの実験室集団の中に確立した(Xueqin et al. (2012): Xueqin J, Kania PW and Buchmann K. “Comparative effects of four feed types on white spot disease susceptibility and skin immune parameters in rainbow trout, Oncorhynchus mykiss (Walbaum)” J Fish Dis. 2012 Feb;35(2):127-35)。
シュードモナス・フルオレッセンスH6株のリポペプチドバイオサーファクタントは、(Liu et al. (2015): Yiying Liu, Elzbieta Rzeszutek, Menno van der Voort, Cheng-Hsuan Wu, Even Thoen, Ida Skaar, Vincent Bulone, Pieter C. Dorrestein, Jos M. Raaijmakers, Irene de Bruijn “Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia” PLOSOne; DOI:10.1371/journal.pone.0136241; published 28. August 2016)に記載されている方法に従って抽出した。
それぞれ、30個の凹型ウェル(直径25mm、深さ3mm、最大水容量2000μL)を有するガラスプレート(厚さ6mm)を、各寄生虫生活ステージ(セロント、トモント及びトモシスト)のインキュベーションに使用した。
Leica MZ 95解剖顕微鏡(拡大率6〜40×)を、トモント、トモシスト及びセロントの運動性をモニターするために使用した。
1Lのリポペプチドバイオサーファクタント溶液及びニジマス3匹をそれぞれ含有する魚類用プラスチック水槽(総容量3L)の中で、ニジマス(2×3匹)を、試験した全ての寄生虫生活ステージに対して90分以内に有効性が見出された濃度のリポペプチドバイオサーファクタント(10μg/mL及び13μg/mL)に曝露した。対照魚3匹を、リポペプチドバイオサーファクタントを欠くこと以外は同一の条件下に維持した。
寄生虫の生存率(異なるリポペプチドバイオサーファクタント濃度での3つの異なる寄生虫生活ステージ)に関して、3連のウェル間に有意差が観察されなかったため、これらからのデータをプールした。
トモント
ウオノカイセンチュウのトモントは、最も高い2種の濃度(1000μg/mL及び100μg/mLのPS)にのみ感受性があり、この2種の濃度は、全ての寄生虫を15分以内に死滅させた(図1A)。
ウオノカイセンチュウの、トミーテを囲い込んだトモシスト(図2B)は、リポペプチドバイオサーファクタントに対して、トモントと比較した際に異なる感受性を示した(図1B)。
ウオノカイセンチュウのセロントは、PSに対して高い感受性を示し、1000μg/mL及び100μg/mLのPSに曝露された場合に、セロントは、5分以内に致死率が100%であった(図1C)。20μg/mLのPSでは、この時点で20%未満の生存率が見られ、残存しているセロントは30分後に死滅した。
ニジマスは、10μg/mL及び13μg/mLの濃度のPSに3時間曝露された場合に、即時型の有害反応も遅延型の有害反応も示さなかった。魚類に対する毒性作用は検出できなかった。
ウオノカイセンチュウの採取は上記のように実施した。バイオサーファクタントマセトリドAの抽出物は、(De Bruijn et al (2008): De Bruijn I, De Kock MJD, De Waard P, Van Beek TA, Raaijmakers JM. “Massetolide A Biosynthesis in Pseudomonas fluorescens.” J Bacteriol 190, 2777-2789 (2008))に記載されているようにシュードモナス・フルオレッセンスSS101から得、プチソルビン様バイオサーファクタントは、(Kruijt et al (2008): Kruijt M, Tran H, Raaijmakers JM. “Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267.” Journal of applied microbiology 107, 546-556 (2009))に記載されているようにシュードモナス・プチダ267から得、シュードモナス属H6のビスコシン様バイオサーファクタントは、上記のように得た。各サーファクタントについて、生成物を滅菌蒸留水に溶解させることによって15mg/mLの原液を調製し、その後、寄生虫の曝露用に希釈系列を調製し、曝露を上記のように実施した。全3種のバイオサーファクタントについて0.15mg/ml及び0.015mg/mlの濃度をトモント(1反復)及びセロント(2連で)に対して試験し、致死率を、曝露の1時間後まで15分毎に記録した。セロントの致死率データについて、分散分析により、続いてダネットの事後分析により、有意差を計算した(p<0.05)。
シュードモナス属種H6、シュードモナス・フルオレッセンスSS101及びシュードモナス・プチダ267それぞれからのバイオサーファクタント抽出物である、ビスコシン、マセトリド及びプチソルビンは、濃度0.15mg/mLでの最初の5分間の曝露中にセロントの致死率100%を導き出した。0.015mg/mlの濃度では、マセトリド及びビスコシン様バイオサーファクタントは、15分以内にセロントの致死率90%及び50%を導き出したが、プチソルビンはこの濃度では無効であった(図3)。
−シュードモナス属H6のビスコシン様バイオサーファクタントは、ウオノカイセンチュウの自由生活性のトモント及びセロントの生活ステージに対して、それぞれ、濃度100ug/ml及び10〜20ug/mlで明白な阻害効果を示した。
−構造的に関連するバイオサーファクタントマセトリド(シュードモナス・フルオレッセンスSS101によって産生される)は、セロント及びトモントに対して、シュードモナス属H6に由来するビスコシン様バイオサーファクタントと同様の活性を示した。プチソルビンは、H6のビスコシン様バイオサーファクタントから構造的にそれより遠く、セロントに対して、より低い濃度では活性が少なく、トモントに対しては不活性である。
de Bruijn et al. (2007): de Bruijn I, de Kock MJD, Yang M, de Waard P, van Beek TA, Raaijmakers JM. “Genome-based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species.” Molecular Microbiology. 2007; 63(2):417-28.
De Bruijn et al (2008): De Bruijn I, De Kock MJD, De Waard P, Van Beek TA, Raaijmakers JM. “Massetolide A Biosynthesis in Pseudomonas fluorescens.” J Bacteriol 190, 2777-2789 (2008).
Liu et al. (2015): Yiying Liu, Elzbieta Rzeszutek, Menno van der Voort, Cheng-Hsuan Wu, Even Thoen, Ida Skaar, Vincent Bulone, Pieter C. Dorrestein, Jos M. Raaijmakers, Irene de Bruijn “Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia” PLOSOne; DOI:10.1371/journal.pone.0136241; published 28. August 2016.
Xueqin et al. (2012): Xueqin J, Kania PW and Buchmann K. “Comparative effects of four feed types on white spot disease susceptibility and skin immune parameters in rainbow trout, Oncorhynchus mykiss (Walbaum)” J Fish Dis. 2012 Feb;35(2):127-35.
Aihua, L., Buchmann, K. (2001). “Temperature- and salinity-dependent development of a Nordic strain of Ichthyophthirius multifiliis from rainbow trout.” J Appl Ichthyol 17, 273-276.
Kruijt et al (2008): Kruijt M, Tran H, Raaijmakers JM. “Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267.” Journal of applied microbiology 107, 546-556 (2009).
Claims (15)
- 淡水魚及び海水魚などの魚類における白点病の処置に使用するための、リポペプチドバイオサーファクタント。
- 魚類における白点病の処置に使用するための、(a)ビスコシンリポペプチド又はその誘導体、(b)マセトリド又はその誘導体、及び(c)プチソルビン又はその誘導体から選択される、請求項1に記載のリポペプチドバイオサーファクタント。
- 魚類におけるウオノカイセンチュウの感染の処置に使用するための、(a)ビスコシン又はその誘導体、(b)マセトリド又はその誘導体、及び(c)プチソルビン又はその誘導体から選択される、請求項1に記載のリポペプチドバイオサーファクタント。
- ビスコシンリポペプチドがシュードモナス・フルオレッセンスH6株から取得可能なビスコシン様リポペプチドである、請求項2又は3に記載のリポペプチドバイオサーファクタント。
- バイオサーファクタントが10〜1000μg/mlの濃度を有する、請求項1〜4のいずれかに記載のリポペプチドバイオサーファクタント。
- 淡水魚及び海水魚などの魚類における白点病の処置に使用するための、少なくとも1種のリポペプチドバイオサーファクタントを含む組成物。
- (a)ビスコシンリポペプチド又はその誘導体、(b)マセトリド又はその誘導体、及び(c)プチソルビン又はその誘導体から選択される少なくとも1種のリポペプチドバイオサーファクタントを含む、請求項6に記載の組成物。
- リポペプチドバイオサーファクタントが、シュードモナス・フルオレッセンスH6株から取得可能なリポペプチドである、請求項6又は7に記載の組成物。
- 淡水魚におけるウオノカイセンチュウ感染の処置に使用するための、少なくとも1種のリポペプチドバイオサーファクタントを含む、請求項5に記載の組成物。
- 魚類におけるウオノカイセンチュウ感染の処置に使用するための、(a)シュードモナス・フルオレッセンスH6株から取得可能なリポペプチド、(b)マセトリド及び(c)プチソルビンから選択される細菌性リポペプチドバイオサーファクタントを含む、請求項6に記載の組成物。
- バイオサーファクタントが10〜1000μg/mlの濃度を有する、請求項6〜10のいずれかに記載の組成物。
- 細菌分離株であって、前記細菌が、淡水魚及び海水魚などの魚類における白点病の処置に使用するためのリポペプチドサーファクタントを産生する、前記細菌分離株。
- 細菌が、魚類における白点病の処置に使用するためのマセトリド又はその誘導体を産生する、請求項12に記載の細菌分離株。
- 魚類における白点病の処置に使用するためのプチソルビン又はその誘導体を産生する、請求項12に記載の細菌分離株。
- 淡水魚におけるウオノカイセンチュウ感染の処置に使用するための、シュードモナス・フルオレッセンスH6株の細菌分離株。
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