JP2014060990A - Killing method for gliding bacteria in aquaculture fishing place - Google Patents
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Abstract
Description
本発明は、養殖漁場における滑走細菌の殺滅方法に関する。さらに詳しくは、本発明は、過酸化水素または過酢酸で薬浴処理することにより、養殖魚の滑走細菌症を治療およびその発症を効果的に予防する方法に関する。 The present invention relates to a method for killing gliding bacteria in an aquaculture fishery. More specifically, the present invention relates to a method for treating and effectively preventing the onset of gliding bacteriosis in farmed fish by treatment with hydrogen peroxide or peracetic acid.
滑走細菌症は、ブリ、マダイ、ヒラメ、トラフグなどの海産魚類が発症する魚病であり、その原因菌である滑走細菌は、テナキバクラム マリティマム（Tenacibaculum maritimum）（「Flexibacter maritimus」または「Gliding-bacterial」ともいう）であることが判明している（例えば、非特許文献１参照）。
滑走細菌症は、最初にアメリカでサケ科魚類にその発症が報告され、日本でも１９７０年頃から種々の海産養殖魚に発生して、ときには大きな被害をもたらすようになった（例えば、非特許文献２参照）。
Gliding bacteriosis is a fish disease that occurs in marine fish such as yellowtail, red sea bream, Japanese flounder, and tiger puffer fish. (For example, see Non-Patent Document 1).
Gliding bacteriosis was first reported to salmonids in the United States, and it occurred in various sea-cultured fish in Japan from around 1970, and sometimes caused great damage (for example, Non-Patent Document 2). reference).
その症状は、稚魚では口唇部や尾鰭に糜爛や壊死あるいは崩壊が起こり、幼魚や成魚では頭部、躯幹、鰭、鰓などに発赤や出血、ときには潰瘍が見られ、特に稚魚期における被害が多い（例えば、非特許文献３参照）。
海産養殖場は、閉鎖系であることが多く、海産魚の個体が高密度に存在していることから、一旦養殖魚に滑走細菌症が発症すると、その感染の影響は大きく、海産養殖産業において深刻な問題となっている。
Symptoms include larvae and necrosis or disintegration in the lip and caudal fins of juveniles, and redness and bleeding, and sometimes ulcers in the head, trunk, coral, and pupae of juvenile and adult fish, especially during the fry stage (For example, refer nonpatent literature 3).
Marine aquaculture farms are often closed, and there are a large number of marine fish individuals. Once gliding bacteriosis develops in farmed fish, the effects of infection are significant and serious in the marine aquaculture industry. Has become a serious problem.
滑走細菌は、ニフルスチレン酸ナトリウムやテトラサイクリン系の抗生物質に感受性があり、滑走細菌症の防除対策としては、滑走細菌の不活化菌体またはそれを有効成分とする魚類滑走細菌症ワクチン（特許文献１参照）、ニフルスチレン酸ナトリウム（特許文献２参照）および特定のベンジルアミン誘導体またはその酸付加塩と有効量のオキシテトラサイクリンなどの抗菌活性物質を含む組成物（特許文献３参照）などの薬剤に頼っているのが現状である。例えば、ニフルスチレン酸ナトリウムは、かれい目魚類の薬浴剤で、農林水産省の水産用医薬品として承認されている。 The gliding bacterium is sensitive to sodium niflustyrene and tetracycline antibiotics. As a control measure against gliding bacteriosis, an inactivated cell of the gliding bacterium or a fish gliding bacteriosis vaccine containing it as an active ingredient (Patent Literature) 1), sodium niflustyrate (see Patent Document 2) and a specific benzylamine derivative or an acid addition salt thereof and an effective amount of an antibacterial active substance such as oxytetracycline (see Patent Document 3). The current situation depends on it. For example, sodium niflustyrene is a medicinal bath for fish of the order of fish and is approved as a marine product by the Ministry of Agriculture, Forestry and Fisheries.
しかしながら、これらの抗菌性薬剤などの使用は、多剤耐性菌の増加や薬剤の魚体への残留による食品衛生上の問題および環境中への拡散による公衆衛生上の問題を孕んでいる。また、薬剤の経口投与では、餌を摂取しない重症魚には効果が得られないという問題がある。
さらに、ニフルスチレン酸ナトリウムには使用制限があり、ひらめ、ほしがれい、まこがれい、まつかわなどのかれい目魚類のうち、重量５０ｇ以下の稚魚にしか使用できないという問題がある。
また、ニフルスチレン酸ナトリウムは、発がん性や変異原性をもつ可能性のあるニトロフラン系の合成抗菌剤であり、今後水産用医薬品の承認が取り消しとなる可能性がある。
However, the use of these antibacterial drugs and the like has been associated with food hygiene problems due to an increase in multi-drug resistant bacteria and drug residues in fish and public health problems due to diffusion into the environment. In addition, there is a problem that oral administration of a drug cannot provide an effect on severe fish that do not consume food.
Furthermore, sodium niflustyrate has a limitation in use, and there is a problem that it can be used only for juvenile fish with a weight of 50 g or less among the fishes such as flounder, hosihore, komagurei, matsukure, and the like.
In addition, sodium niflustyrene is a nitrofuran synthetic antibacterial agent that may have carcinogenicity and mutagenicity, and the approval of marine products may be canceled in the future.
独立行政法人水産総合研究センター養殖研究所（現 増養殖研究所）では、ニフルスチレン酸ナトリウムに代わる医薬品開発の支援を目的として、候補となる薬剤の各種魚病細菌に対する抗菌活性の測定および魚介類に対する安全性と効果を把握する飼育試験などを実施している。
候補となる薬剤としては、二酸化塩素、ブロノポール（2-ブロモ-2-ニトロプロパン-1,3-ジオール）およびグルタルアルデヒド、さらには特定保健食品やヒトに使用されている薬剤（カテキン、プロピレングリコール、キトサン、アクリノール、アクリフラビン、チモール、ソルビン酸カリウム、グルコン酸クロルヘキシジン、ポリリン酸、ポリリン酸ナトリウム、塩化ヘキサデシルピリジニウム）や観賞魚用の駆虫剤に含有されているメチレンブルーが挙げられている。
これまでの試験結果によれば、ブロノポールや安定化二酸化塩素において、シマアジ、マダイおよびヒラメにおいて生菌数の減少効果が確認されている（例えば、非特許文献４参照）。
The National Institute of Aquaculture, National Fisheries Research Center (currently the Institute for Increased Aquaculture) measures the antibacterial activity of various candidate drugs against fish disease bacteria and fishery products in order to support the development of pharmaceuticals that can replace sodium niflustyrene. A breeding test is conducted to ascertain the safety and effectiveness of this.
Candidate drugs include chlorine dioxide, bronopol (2-bromo-2-nitropropane-1,3-diol) and glutaraldehyde, as well as drugs used in certain health foods and humans (catechin, propylene glycol, And chitosan, acrinol, acriflavine, thymol, potassium sorbate, chlorhexidine gluconate, polyphosphoric acid, sodium polyphosphate, hexadecylpyridinium chloride) and methylene blue contained in an anthelmintic agent for ornamental fish.
According to the test results so far, in bronopol and stabilized chlorine dioxide, the effect of reducing the number of viable bacteria was confirmed in striped mackerel, red sea bream, and Japanese flounder (for example, see Non-Patent Document 4).
そこで、本発明は、養殖魚の滑走細菌症を治療およびその発症を効果的に予防すると共に、食品衛生上の問題や環境中への拡散による公衆衛生上の問題を生じさせない養殖漁場における滑走細菌の殺滅方法を提供することを課題とする。 Accordingly, the present invention treats and effectively prevents gliding bacteriosis in cultured fish, and also prevents gliding bacteria in aquaculture fisheries that do not cause food hygiene problems or public health problems due to diffusion into the environment. It is an object to provide a killing method.
本発明者は、上記課題を解決するために鋭意研究を行った結果、養殖魚の寄生虫駆除剤として用いられる過酸化水素や過酢酸などの過酸化水素を発生させる物質が優位に滑走細菌を殺滅する効果を有すること、特に比較的低濃度の過酸化水素を含む閉鎖水系内で養殖魚を長時間、薬浴処理することにより、より効果的に養殖魚の滑走細菌症を治療およびその発症を予防できることを見出し、本発明を完成するに至った。 As a result of diligent research to solve the above problems, the present inventor has predominately killed gliding bacteria by substances that generate hydrogen peroxide such as hydrogen peroxide and peracetic acid used as parasite control agents for cultured fish. In particular, by treating the farmed fish for a long time in a closed water system containing a relatively low concentration of hydrogen peroxide, it is possible to treat and develop gliding bacteriosis in the farmed fish more effectively. The present inventors have found that it can be prevented and have completed the present invention.
かくして、本発明によれば、養殖漁場の閉鎖水系内に養殖魚を収容し、過酸化水素または過酢酸を添加し、前記閉鎖水系内で前記養殖魚を薬浴処理して、前記閉鎖水系内および／または前記養殖魚に生存する滑走細菌を殺滅することを特徴とする養殖漁場における滑走細菌の殺滅方法が提供される。 Thus, according to the present invention, the cultured fish is accommodated in the closed water system of the aquaculture ground, hydrogen peroxide or peracetic acid is added, and the cultured fish is subjected to a chemical bath treatment in the closed water system. There is provided a method for killing gliding bacteria in an aquaculture fishery characterized by killing gliding bacteria that live in the cultured fish.
本発明によれば、養殖魚の滑走細菌症を治療およびその発症を効果的に予防すると共に、食品衛生上の問題や環境中への拡散による公衆衛生上の問題を生じさせない養殖漁場における滑走細菌の殺滅方法を提供することができる。
すなわち、本発明の方法は、既に滑走細菌症を発症した養殖魚の治療と、養殖魚が滑走細菌と共存しており発症する可能性のある滑走細菌症の予防とを合せて実施することができる。しかも、過酸化水素は、水中で水と酸素に自然分解するので、食品衛生や公衆衛生における問題も生じない。
According to the present invention, it is possible to treat and effectively prevent gliding bacteriosis in cultured fish, and to prevent the occurrence of gliding bacteria in aquaculture fisheries that do not cause food hygiene problems or public health problems due to diffusion into the environment. A killing method can be provided.
That is, the method of the present invention can be carried out in combination with treatment of cultured fish that has already developed gliding bacteriosis and prevention of gliding bacteriosis that may occur because the cultured fish coexists with gliding bacteria. . Moreover, since hydrogen peroxide is naturally decomposed into water and oxygen in water, there is no problem in food hygiene and public health.
従来から過酸化水素は、養殖魚の寄生虫を駆除するための薬浴剤として用いられているが、その薬浴処理は、寄生虫を刺激し、異常な屈曲・収縮運動と吸盤の開閉運動を促し、吸盤の付着能力を消失させ、虫体を付着面から脱落せしめることにより駆除するものであり、本発明の作用機構と異なっている。また、処理の薬剤濃度や時間についても、最適条件は異なっている（株式会社片山化学工業研究所、製品名「マリンサワーSP」の農林水産省に提出された動物用医薬品製造承認申請書 概要書5.2参照）。
例えば、海水系養殖魚の外部寄生虫の最適な駆除方法としては、過酸化水素濃度が２００〜３０００ｍｇ／Ｌで薬浴時間が１〜２０分であり、トラフグのヘテロボツリウム症の予防方法では、過酸化水素濃度が４００〜２０００ｍｇ／Ｌで薬浴時間が２０〜１２０分である。
Hydrogen peroxide has traditionally been used as a chemical bath to combat parasites in farmed fish, but the chemical bath treatment stimulates the parasite, causing abnormal bending / contraction movements and suction cup opening and closing movements. It promotes and eliminates the adhering ability of the sucker, and removes the insect body from the adhering surface, which is different from the action mechanism of the present invention. In addition, the optimal conditions for treatment chemical concentration and time are also different (Katayama Chemical Industry Research Institute Co., Ltd., veterinary drug manufacturing approval application submitted to the Ministry of Agriculture, Forestry and Fisheries of the product name “Marine Sour SP”) See 5.2).
For example, as an optimal method for controlling ectoparasites in seawater-cultured fish, the hydrogen peroxide concentration is 200 to 3000 mg / L and the bath time is 1 to 20 minutes. In the method for preventing trough pufferfish heterobotulosis, The hydrogen peroxide concentration is 400 to 2000 mg / L and the chemical bath time is 20 to 120 minutes.
一方、本発明の方法の中でとりわけ有効な処理方法は、
養殖漁場の閉鎖水系内に養殖魚を収容し、過酸化水素濃度が０．１〜１００ｍｇ／Ｌになるように過酸化水素または過酢酸を添加し、その濃度が維持された状態で、閉鎖水系内で養殖魚を０．５〜６時間薬浴処理して、閉鎖水系内および／または養殖魚に生存する滑走細菌を殺滅する方法であり、
養殖魚がブリ、マダイ、カンパチ、ヒラメおよびトラフグから選択される海産魚類である、および
滑走細菌がTenacibaculum maritimumである
のいずれか１つの条件を満たす場合に上記の効果がさらに発揮される。
On the other hand, a particularly effective processing method among the methods of the present invention is:
In the state where the cultured fish is accommodated in the closed water system of the aquaculture ground, hydrogen peroxide or peracetic acid is added so that the hydrogen peroxide concentration is 0.1 to 100 mg / L, and the concentration is maintained, the closed water system In which the cultured fish is treated with a chemical bath for 0.5 to 6 hours to kill gliding bacteria living in the closed water system and / or in the cultured fish,
The above-described effect is further exhibited when the cultured fish is a marine fish selected from yellowtail, red sea bream, amberjack, flounder and trough puffer and the gliding bacterium is Tenacibaculum maritimum.
本発明の養殖漁場における滑走細菌の殺滅方法は、養殖漁場の閉鎖水系内に養殖魚を収容し、過酸化水素または過酢酸を添加し、前記閉鎖水系内で前記養殖魚を薬浴処理して、前記閉鎖水系内および／または前記養殖魚に生存する滑走細菌を殺滅することを特徴とする。 The method for killing gliding bacteria in the aquaculture fishery of the present invention is to contain the aquaculture fish in a closed water system of the aquaculture fishery, add hydrogen peroxide or peracetic acid, and bathe the cultured fish in the closed water system. And killing gliding bacteria that live in the closed water system and / or in the cultured fish.
本発明で使用する薬剤は、過酸化水素または過酢酸である。
過酸化水素としては、通常、工業用として市販されている濃度３〜６０％の過酸化水素水溶液が挙げられ、安全性や作業性の点で濃度３０〜４５％程度が好ましい。
また、過酢酸としては、通常、工業用として市販されているものを用いることができ、例えば、過酢酸６％、酢酸３２％、過酸化水素８％および水５４％の組成のもの、過酢酸３２％、酢酸４％および水６４％の組成のもの、過酢酸２６％、酢酸５４％および水２０％の組成のもの、ならびに過酢酸１４〜１７％、酢酸２０〜３０％、過酸化水素１０〜１５％および水３８〜５６％の組成のものなどが挙げられる。
過酢酸は、水中で過酸化水素と酢酸に分解し、分解により生成された過酸化水素が本発明の効能に寄与する。
過酸化水素または過酢酸を養殖漁場の閉鎖水系に添加するにあたっては、所望の濃度になるようにこれらを海水で適宜希釈または溶解して用いてもよい。
The drug used in the present invention is hydrogen peroxide or peracetic acid.
Examples of hydrogen peroxide include a commercially available aqueous hydrogen peroxide solution having a concentration of 3 to 60%, and a concentration of about 30 to 45% is preferable in terms of safety and workability.
As the peracetic acid, those commercially available for industrial use can be used. For example, peracetic acid having a composition of 6% peracetic acid, 32% acetic acid, 8% hydrogen peroxide and 54% water, peracetic acid 32%, acetic acid 4% and water 64% composition, peracetic acid 26%, acetic acid 54% and water 20% composition, and peracetic acid 14-17%, acetic acid 20-30%, hydrogen peroxide 10 And those having a composition of -15% and water 38-56%.
Peracetic acid is decomposed into hydrogen peroxide and acetic acid in water, and the hydrogen peroxide produced by the decomposition contributes to the efficacy of the present invention.
When hydrogen peroxide or peracetic acid is added to the closed water system of the aquaculture fishery, these may be appropriately diluted or dissolved in seawater so as to have a desired concentration.
本発明の方法では、過酸化水素および過酢酸の代わりに、水中で過酸化水素を発生し得る過酸化水素発生化合物（または過酸化水素供給化合物）を用いることもできる。
このような化合物としては、過炭酸、過ホウ酸、ペルオキシ硫酸などの無機過酸、過酢酸以外の有機過酸およびこれらの塩類が挙げられ、塩類としては、過炭酸ナトリウム、過ホウ酸ナトリウムなどが挙げられる。
In the method of the present invention, a hydrogen peroxide generating compound (or a hydrogen peroxide supplying compound) capable of generating hydrogen peroxide in water can be used instead of hydrogen peroxide and peracetic acid.
Examples of such compounds include inorganic peracids such as percarbonate, perborate, and peroxysulfuric acid, organic peracids other than peracetic acid, and salts thereof. Examples of salts include sodium percarbonate, sodium perborate, and the like. Is mentioned.
本発明の方法における過酸化水素濃度は、滑走細菌の殺滅効果を考慮して、養殖魚の種類や状態、周辺環境などの状況により適宜設定すればよい。
養殖漁場の閉鎖水系内での過酸化水素濃度は、好ましくは０．１〜１００ｍｇ／Ｌであり、より好ましくは０．１２５〜８０ｍｇ／Ｌであり、さらに好ましくは０．１２５〜６４ｍｇ／Ｌである。
また、その処理時間は、好ましくは０．５〜６時間であり、より好ましくは０．５〜２時間であり、さらに好ましくは０．５〜１時間である。
過酸化水素濃度が０．１ｍｇ／Ｌ未満では、本発明の効果が充分に得られないことがある。また、過酸化水素濃度が１００ｍｇ／Ｌを超えると、魚に対して悪影響を及ぼすことがある。
また、処理時間が０．５時間未満では、本発明の効果が充分に得られないことがある。また、処理時間が６時間を超えると、魚に対して悪影響を及ぼし作業性が悪くなることがある。
The hydrogen peroxide concentration in the method of the present invention may be appropriately set according to the conditions such as the type and state of the cultured fish and the surrounding environment, taking into account the killing effect of the gliding bacteria.
The hydrogen peroxide concentration in the closed water system of the aquaculture fishery is preferably 0.1 to 100 mg / L, more preferably 0.125 to 80 mg / L, and still more preferably 0.125 to 64 mg / L. is there.
Moreover, the processing time becomes like this. Preferably it is 0.5 to 6 hours, More preferably, it is 0.5 to 2 hours, More preferably, it is 0.5 to 1 hour.
When the hydrogen peroxide concentration is less than 0.1 mg / L, the effects of the present invention may not be sufficiently obtained. In addition, if the hydrogen peroxide concentration exceeds 100 mg / L, fish may be adversely affected.
In addition, if the treatment time is less than 0.5 hours, the effects of the present invention may not be sufficiently obtained. Moreover, when processing time exceeds 6 hours, it may have a bad influence with respect to a fish and workability | operativity may worsen.
したがって、より好ましい本発明の方法としては、養殖漁場の閉鎖水系内に養殖魚を収容し、過酸化水素濃度が０．１〜１００ｍｇ／Ｌになるように過酸化水素または過酢酸を添加し、その濃度が維持された状態で、前記閉鎖水系内で前記養殖魚を０．５〜６時間薬浴処理して、前記閉鎖水系内および／または前記養殖魚に生存する滑走細菌を殺滅するのがよい。 Therefore, as a more preferable method of the present invention, the cultured fish is accommodated in the closed water system of the aquaculture fishing ground, hydrogen peroxide or peracetic acid is added so that the hydrogen peroxide concentration becomes 0.1 to 100 mg / L, In the state where the concentration is maintained, the cultured fish is treated with the medicine in the closed water system for 0.5 to 6 hours to kill the sliding bacteria that live in the closed water system and / or in the cultured fish. Is good.
本発明の方法では、養殖漁場の閉鎖水系内に所定の過酸化水素濃度になるように過酸化水素または過酢酸を添加し、その濃度が維持された状態で、閉鎖水系内に養殖魚を所定時間保持、すなわち養殖魚を薬浴処理して、閉鎖水系内および／または養殖魚に生存する滑走細菌を殺滅する。
過酸化水素は水中で水と酸素に自然分解するので、「過酸化水素濃度が維持された状態」とは、閉鎖水系内に過酸化水素または過酢酸が添加された直後、過酸化水素の自然分解が進行しても所定の濃度にある状態、または過酸化水素が所定の濃度以下に低下した後、過酸化水素または過酢酸がさらに添加されて所定の濃度にある状態を意味する。
In the method of the present invention, hydrogen peroxide or peracetic acid is added to the closed water system of the aquaculture fishing ground so as to have a predetermined hydrogen peroxide concentration, and the cultured fish is predetermined in the closed water system in a state where the concentration is maintained. Time retention, i.e., bathing the cultured fish to kill the gliding bacteria that live in the closed water system and / or survive in the cultured fish.
Since hydrogen peroxide decomposes spontaneously into water and oxygen in water, the `` state in which the hydrogen peroxide concentration is maintained '' means that hydrogen peroxide or peracetic acid is added immediately after hydrogen peroxide or peracetic acid is added to the closed water system. It means a state in which a predetermined concentration is present even when decomposition proceeds, or a state in which hydrogen peroxide or peracetic acid is further added after hydrogen peroxide has fallen to a predetermined concentration or less and is at a predetermined concentration.
本発明者は、養殖漁場の閉鎖水系内の過酸化水素濃度は、水系内の分解要因の多少により変化するものの、一般に過酸化水素の分解により添加時の初期濃度から３０分から１時間程度までは徐々に低下し、その後、殆ど平衡状態で緩やかに低下すること、平衡状態の過酸化水素濃度が０．１ｍｇ／Ｌ以上であれば、少なくとも本発明の方法における処理時間の範囲であれば、その濃度を維持できることを確認している。また、本発明者は、過酢酸は、過酸化水素よりも初期濃度の低下が急激であることを確認している。 The present inventor found that the hydrogen peroxide concentration in the closed water system of the aquaculture fishery varies depending on the degradation factors in the water system, but generally from 30 minutes to about 1 hour from the initial concentration at the time of addition due to the decomposition of hydrogen peroxide. If the hydrogen peroxide concentration in the equilibrium state is 0.1 mg / L or more, at least within the processing time in the method of the present invention, It has been confirmed that the concentration can be maintained. In addition, the present inventor has confirmed that peracetic acid has a more rapid decrease in the initial concentration than hydrogen peroxide.
本発明の好ましい方法としては、上記の処理時間中に過酸化水素濃度が上記の範囲内にあればよく、処理終了時の過酸化水素濃度が少なくとも０．１ｍｇ／Ｌになるように、すなわち処理時間中の過酸化水素の分解を見越して過酸化水素または過酢酸を添加するのがよい。
例えば、処理終了時における見込みの過酸化水素濃度の１０〜１００倍程度になるように、過酸化水素または過酢酸を添加するのが好ましいが、本発明の方法としては、それらに限定されるものではない。
また、処理途中で水系内の過酸化水素濃度を公知の方法により測定し、過酸化水素濃度の低下度合により、適宜過酸化水素または過酢酸を添加してもよい。
一方、過酸化水素濃度が高い場合には、その分解のためにカタラーゼのような過酸化水素分解能を有する酵素ならびに亜硫酸ナトリウムなどの還元物質を併用してもよい。
As a preferred method of the present invention, it is sufficient that the hydrogen peroxide concentration is within the above-mentioned range during the treatment time, and the hydrogen peroxide concentration at the end of the treatment is at least 0.1 mg / L, that is, the treatment. Hydrogen peroxide or peracetic acid should be added in anticipation of hydrogen peroxide decomposition over time.
For example, it is preferable to add hydrogen peroxide or peracetic acid so that the expected concentration of hydrogen peroxide at the end of the treatment is about 10 to 100 times, but the method of the present invention is not limited thereto. is not.
Further, during the treatment, the hydrogen peroxide concentration in the aqueous system may be measured by a known method, and hydrogen peroxide or peracetic acid may be added as appropriate depending on the degree of decrease in the hydrogen peroxide concentration.
On the other hand, when the hydrogen peroxide concentration is high, an enzyme having the ability to decompose hydrogen peroxide such as catalase and a reducing substance such as sodium sulfite may be used in combination for the decomposition.
本発明の方法は、滑走細菌症を発症した、または発症し得る魚類であれば、海水魚、淡水魚のいずれであってもその効果が期待できる。
それらの中でも、養殖産業の盛んな、ブリ、マダイ、カンパチ、ヒラメおよびトラフグから選択される海産魚類は、その効果が大いに期待できる。
また、滑走細菌症の発症は、海産魚類の稚魚期に多く見られるため、本発明の方法は、海産魚類の稚魚期や魚の移送時に実施するのが特に好ましい。
The method of the present invention can be expected to be effective for any saltwater fish or freshwater fish, as long as it is a fish that has developed or can develop gliding bacteriosis.
Among them, marine fish selected from yellowtail, red sea bream, amberjack, Japanese flounder and trough puffer fish, which are prosperous in the aquaculture industry, can be expected to be highly effective.
Moreover, since the onset of gliding bacteriosis is often observed during the juvenile period of marine fish, the method of the present invention is particularly preferably carried out during the juvenile period of marine fish or when transferring fish.
滑走細菌症の原因細菌には、後述する試験例において用いたTenacibaculum maritimum（テナキバクラム マリティマム）以外にも、種々の滑走細菌およびそれらの亜種が考えられるが、本発明の方法は、これまで研究により原因細菌として最も有力とされているTenacibaculum maritimumである場合に最も効果が期待できる。 In addition to Tenacibaculum maritimum used in the test examples described below, various gliding bacteria and their subspecies can be considered as causative bacteria for gliding bacteriosis. It is expected to be most effective when it is Tenacibaculum maritimum, which is considered to be the most effective causative bacterium.
本発明を試験例により具体的に説明するが、本発明はこれらの試験例により限定されるものではない。 The present invention will be specifically described with reference to test examples, but the present invention is not limited to these test examples.
試験例１（滑走細菌に対する殺菌効果確認試験）
〔薬剤〕
試験例では次の薬剤を使用した。
（ａ）過酸化水素（約３０％水溶液、和光純薬工業株式会社製、特級試薬）
（ｂ）過酢酸（１５％の１４％過酸化水素水溶液、Evonik社製、製品名：Peraclean（登録商標）Ocean）
（ｃ）ブロノポール（ノバルティスアニマルヘルス株式会社製、製品名：パイセス）
（ｄ）二酸化塩素（三栄製薬株式会社製、製品名：グリーンＦクリーン）
（ｅ）ニフルスチレン酸ナトリウム（上野製薬株式会社製、製品名：エルバージュ）
Test Example 1 (Bactericidal effect confirmation test against gliding bacteria)
[Drug]
In the test examples, the following drugs were used.
(A) Hydrogen peroxide (approximately 30% aqueous solution, manufactured by Wako Pure Chemical Industries, Ltd., special grade reagent)
(B) Peracetic acid (15% 14% hydrogen peroxide aqueous solution, manufactured by Evonik, product name: Peraclean (registered trademark) Ocean)
(C) Bronopol (manufactured by Novartis Animal Health Co., Ltd., product name: Piesse)
(D) Chlorine dioxide (product name: Green F Clean, manufactured by Sanei Pharmaceutical Co., Ltd.)
(E) Sodium niflustyrate (manufactured by Ueno Pharmaceutical Co., Ltd., product name: Herbage)
〔液体培地および寒天培地の調製〕
３００ミリリットルの蒸留水を入れた１リットル三角フラスコに、トリプトン（日本製薬株式会社製、製品名：Tryptone）０．５ｇ、酵母エキス（日本製薬株式会社製、製品名：BactoTM Yeast Extract）０．５ｇ、肉エキス（Becton, Dickinson and Company製、製品名：Difco BactoTM Beef Extract）０．２ｇおよび酢酸ナトリウム３水和物（キシダ化学株式会社製、特級試薬）０．２ｇを入れて溶解させ、温度１２１℃で１５分間高圧滅菌を行った。また、別途７００ミリリットルのろ過海水（５Ｃ）を滅菌瓶に入れ、温度１２１℃で１５分間高圧滅菌した。
三角フラスコおよび滅菌瓶の内容物が温度５０〜５５℃に冷却された後、それらをクリーンベンチ内で混合して、液体培地（海水Cytophaga培地）を得た。
寒天培地は、上記液体培地を作成する際、寒天（ナカライテスク株式会社製、製品名：精製寒天末）を１０ｇ添加することにより得た。
[Preparation of liquid medium and agar medium]
In a 1 liter Erlenmeyer flask containing 300 ml of distilled water, 0.5 g of tryptone (manufactured by Nippon Pharmaceutical Co., Ltd., product name: Tryptone), yeast extract (manufactured by Nippon Pharmaceutical Co., Ltd., product name: Bacto ™ Yeast Extract) 0. 5 g, 0.2 g of meat extract (Becton, Dickinson and Company, product name: Difco Bacto ™ Beef Extract) and 0.2 g of sodium acetate trihydrate (Kishida Chemical Co., Ltd., special grade reagent) were added and dissolved. High-pressure sterilization was performed at 121 ° C. for 15 minutes. Separately, 700 ml of filtered seawater (5C) was put into a sterilization bottle and autoclaved at a temperature of 121 ° C. for 15 minutes.
After the contents of the Erlenmeyer flask and the sterilization bottle were cooled to a temperature of 50 to 55 ° C., they were mixed in a clean bench to obtain a liquid medium (seawater Cytophaga medium).
The agar medium was obtained by adding 10 g of agar (manufactured by Nacalai Tesque, product name: purified agar powder) when preparing the liquid medium.
〔滑走細菌株の調製〕
独立行政法人水産総合研究センター増養殖研究所において温度−８５℃で冷凍保存された滑走細菌症原因菌（Tenacibaculum maritimum）３株（TC133、TC134およびFPC453、それぞれＡ株、Ｂ株、Ｃ株）の分譲を受け、これらを滑走細菌株として使用した。
改変Cytophaga寒天（１．５％）培地を用いて温度２５℃で２日間培養し、複数のコロニーの先端を白金耳で触れ、改変Cytophaga液体培地に接種し、温度２５℃で２日間振とう培養して菌液を作製した。なお、菌液は、Cytophaga寒天培地に塗抹後、温度２５℃で５日間培養して生菌数を測定した。
[Preparation of gliding bacterial strain]
Of 3 strains of Tenacibaculum maritimum (TC133, TC134 and FPC453, A strain, B strain, and C strain, respectively) frozen and stored at -85 ° C in the National Institute of Aquaculture, Japan They were sold and used as gliding bacterial strains.
Incubate with modified Cytophaga agar (1.5%) medium at 25 ° C for 2 days, touch the tips of multiple colonies with platinum ears, inoculate into modified Cytophaga liquid medium, and shake culture at 25 ° C for 2 days Thus, a bacterial solution was prepared. The bacterial solution was smeared on a Cytophaga agar medium and then cultured at a temperature of 25 ° C. for 5 days to measure the viable cell count.
〔試験〕
各薬剤（ａ）〜（ｅ）は、有効成分の設定の１０倍濃度になるように滅菌蒸留水を用いて希釈したものを使用した。なお過酢酸については、過酢酸濃度の１０倍濃度になるように希釈したものを使用した。
また、菌液は、７０％滅菌ろ過海水で希釈して、約１０5ｃｆｕ／ミリリットルに調整したものを使用した。
菌液を９ミリリットルに希釈した供試薬剤１ミリリットルを混合して試験液とし、０．５時間、１時間、２時間および６時間経過後に、Cytophaga寒天培地に試験液０．１ミリリットルを塗抹して温度２５℃で５日間培養し、殺菌効果（最小殺菌濃度ｍｇ／Ｌ：ＭＢＣ）を確認した。得られた結果を表１に示す。
ＭＢＣ（最小殺菌濃度）は、１００％の細菌を殺滅することができる最も低い薬剤濃度である。
〔test〕
Each drug (a) to (e) was diluted with sterilized distilled water to a concentration 10 times that of the active ingredient. In addition, about the peracetic acid, what was diluted so that it might become 10 times the concentration of peracetic acid concentration was used.
The bacterial solution was diluted with 70% sterilized filtered seawater and adjusted to about 10 5 cfu / ml.
Mix 1 ml of reagent solution diluted with 9 ml of bacterial solution to make a test solution. After 0.5 hours, 1 hour, 2 hours and 6 hours, smear 0.1 ml of test solution on Cytophaga agar medium. The sterilization effect (minimum sterilization concentration mg / L: MBC) was confirmed by culturing at a temperature of 25 ° C. for 5 days. The obtained results are shown in Table 1.
MBC (Minimum Bactericidal Concentration) is the lowest drug concentration that can kill 100% of bacteria.
表１の測定結果から、薬剤（ｂ）の過酢酸が最も高い殺菌能力を有することがわかる。
また、薬剤（ａ）の過酸化水素は、薬剤（ｂ）の過酢酸の殺菌能力には及ばず、薬剤（ｅ）の現行のニフルスチレン酸ナトリウムと比較するとやや殺菌能力に欠けるものの、代替薬として有望視されている薬剤（ｃ）のブロノポールや薬剤（ｄ）の二酸化塩素よりも高い殺菌能力を有することがわかる。特に薬剤（ｃ）のブロノポールと比較すると１時間未満の短時間の接触での効力の差が顕著である。
From the measurement results in Table 1, it can be seen that peracetic acid of the drug (b) has the highest bactericidal ability.
Further, the hydrogen peroxide in the drug (a) does not reach the sterilizing ability of the peracetic acid in the drug (b), and is slightly inferior to the current sodium niflustyrate of the drug (e). It can be seen that it has a higher bactericidal ability than bronopol, which is a promising drug, and chlorine dioxide, which is drug (d). In particular, compared with the bronopol of the drug (c), the difference in efficacy in a short contact of less than 1 hour is remarkable.
上記の試験例１は、滑走細菌症を発症した魚類を使用せず、滑走細菌およびそれに対する薬剤のみを用いた試験であるが、当業者であれば、それらの結果、具体的にはＭＢＣ（最小殺菌濃度）および従来の他の薬剤による薬浴処理の実績から、本発明の方法を、滑走細菌症を発症した魚類の治療、または滑走細菌と共存する魚類の滑走細菌症の予防に適用できることは充分に予測し得る。すなわち、特定のＭＢＣが得られた薬剤の濃度およびそれによる処理時間で、滑走細菌症を発症したまたは発症することが予想される魚類を薬浴処理することにより、本発明の効果が発揮されることが充分に予測し得る。 The above Test Example 1 is a test using only gliding bacteria and a drug for the same without using the fish that developed gliding bacteriosis. The method of the present invention can be applied to the treatment of fish that develops gliding bacteriosis or the prevention of gliding bacteriosis of fish that coexists with gliding bacteria, based on the results of bath treatment with other conventional agents. Is fully predictable. That is, the effect of the present invention is exerted by treating the fish that has developed or is expected to develop gliding bacteriolysis with the concentration of the drug from which the specific MBC was obtained and the treatment time accordingly. Can be fully predicted.
試験例２（マダイの滑走細菌症に対する予防効果確認試験）
〔薬剤〕
試験例１の薬剤（ａ）過酸化水素（約３０％水溶液、和光純薬工業株式会社製、特級試薬）のみを使用した。
〔液体培地および寒天培地の調製〕
試験例１と同様にして、液体培地および寒天培地を調製した。
〔滑走細菌株の調製〕
滑走細菌症原因菌としてＡ株のみを用いたこと、振とう培養の条件「温度２５℃で２日間」を「２５℃で４５時間」に変更したこと以外は試験例１と同様にして、滑走細菌株を調製した。
Test Example 2 (Test for confirming the prevention effect of red sea bream on gliding bacteriosis)
[Drug]
Only the chemical (a) hydrogen peroxide (about 30% aqueous solution, manufactured by Wako Pure Chemical Industries, Ltd., special grade reagent) of Test Example 1 was used.
[Preparation of liquid medium and agar medium]
In the same manner as in Test Example 1, a liquid medium and an agar medium were prepared.
[Preparation of gliding bacterial strain]
Gliding in the same manner as in Test Example 1 except that only strain A was used as a causative agent for gliding bacteriosis and the conditions of shaking culture “temperature 25 ° C. for 2 days” were changed to “25 ° C. for 45 hours” A bacterial strain was prepared.
〔試験〕
容量約６リットルのポリプロピレン製の容器に天然滅菌海水（水温２０℃）３リットルを入れ、上記Ａ株を用いて調製した菌液を攻撃菌濃度が１０8ｃｆｕ／ミリリットルになるように添加し、そこにマダイ稚魚（平均体長３０ｍｍ、平均体重０．７ｇ）６０尾を収容し、通気下で１時間浸漬し、マダイ稚魚を滑走細菌症に感染させた。
次いで、容量約１リットルの透明ポリプロピレン製の容器に人工海水（水温２０℃）１リットルを入れ、薬剤（ａ）の過酸化水素をそれぞれ表２に示す濃度０．０５ｍｇ／Ｌ、５０ｍｇ／Ｌおよび１２０ｍｇ／Ｌになるように添加し、そこに滑走細菌症に感染させたマダイ稚魚５尾をそれぞれ収容し、処理時間０．２５〜８時間の薬浴を行なった。
所定時間の薬浴後、４リットルの人工海水を入れた水槽でマダイ稚魚を５日間飼育し、死亡状況および死亡個体の症状を観察した。なお、人工海水は１日１回全量を交換した。
また、薬剤（ａ）無添加の人工海水を用いたこと以外は上記と同様の操作で試験し、薬浴未実施のブランク（比較）とした。得られた結果を表２に示す。
〔test〕
Place 3 liters of naturally sterilized seawater (water temperature 20 ° C.) in a polypropylene container with a capacity of about 6 liters, and add the bacterial solution prepared using the above strain A so that the attacking bacteria concentration is 10 8 cfu / ml, There, 60 red sea bream (average body length 30 mm, average body weight 0.7 g) were accommodated and immersed for 1 hour under ventilation to infect the red sea bream fry with gliding bacteriosis.
Next, 1 liter of artificial seawater (water temperature: 20 ° C.) is placed in a transparent polypropylene container having a capacity of about 1 liter, and the hydrogen peroxide of the drug (a) has a concentration of 0.05 mg / L, 50 mg / L and It added so that it might be set to 120 mg / L, and each 5 fish of red sea bream infected with gliding bacteriosis was accommodated there, and the chemical bath for 0.25-8 hours of processing time was performed.
After a predetermined time of bathing, juvenile red sea bream was raised for 5 days in a tank containing 4 liters of artificial seawater, and the death status and symptoms of the dead individuals were observed. The total amount of artificial seawater was changed once a day.
Moreover, except having used chemical | medical agent (a) artificial seawater without addition, it tested by the same operation as the above, and set it as the blank (comparison) which has not implemented the chemical bath. The obtained results are shown in Table 2.
表２の測定結果から、過酸化水素濃度が０．１〜１００ｍｇ／Ｌで、０．５〜６時間の薬浴処理をすることで滑走細菌を効果的に殺滅し、養殖魚の滑走細菌症を効果的に予防できることがわかった。
また、比較例２および４では薬浴処理中にマダイ稚魚の死亡が確認されたことから、長時間あるいは高濃度の薬浴処理は魚に対して悪影響を与えるおそれがあることもわかった。
From the measurement results of Table 2, the hydrogen peroxide concentration is 0.1 to 100 mg / L, and the bacteria are effectively killed by the chemical bath treatment for 0.5 to 6 hours, and the sliding bacteriosis of the cultured fish It was found that can be effectively prevented.
Further, in Comparative Examples 2 and 4, it was confirmed that the red sea bream larvae died during the chemical bath treatment, so that the chemical bath treatment for a long time or at a high concentration may have an adverse effect on the fish.
かくして、本発明によれば、養殖漁場の閉鎖水系内に海水魚の養殖魚を収容し、過酸化水素濃度が０．１〜１００ｍｇ／Ｌになるように過酸化水素または過酢酸を添加し、その濃度が維持された状態で、前記閉鎖水系内で前記養殖魚を０．５〜６時間薬浴処理して、前記閉鎖水系内および／または前記養殖魚に生存する滑走細菌のTenacibaculum maritimumを殺滅することを特徴とする養殖漁場における滑走細菌の殺滅方法が提供される。 Thus, according to the present invention, accommodate farmed fish marine fish in a closed water system of aquaculture, the addition of hydrogen peroxide or peracetic acid as hydrogen peroxide concentration becomes 0.1-100 mg / L, the In a state where the concentration is maintained , the cultured fish in the closed water system is treated with a chemical bath for 0.5 to 6 hours to kill Tenacibaculum maritimum , a gliding bacterium that survives in the closed water system and / or the cultured fish. There is provided a method for killing gliding bacteria in an aquaculture fishery characterized by:
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