JP5240811B2 - Japanese red sea bream sliding bacteriosis vaccine, Japanese red sea bream sliding bacteriosis vaccine composition and method for preventing red sea bream sliding bacteriosis - Google Patents
Japanese red sea bream sliding bacteriosis vaccine, Japanese red sea bream sliding bacteriosis vaccine composition and method for preventing red sea bream sliding bacteriosis Download PDFInfo
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Description
本発明はマダイ滑走細菌症ワクチンに関し、より詳細には、テナキバクラム マリティマム（Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ）不活化菌体を有効成分とする、主に浸漬用のマダイ滑走細菌症ワクチンに関する。 The present invention relates to a red sea bream gliding bacteriosis vaccine, and more particularly, to a red sea bream gliding bacteriosis vaccine mainly for immersion, which contains tenacibacumum maritimum inactivated cells as an active ingredient.
滑走細菌症は、マダイ、カンパチ、ヒラメ、フグ等の海産魚類に発病する病気で、その原因菌はテナキバクラム マリティマム〔Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ（以前はＦｌｅｘｉｂａｃｔｅｒ ｍａｒｉｔｉｍｕｓ）〕であることが判明している（非特許文献１）。テナキバクラム マリティマムによる滑走細菌症は、最初にアメリカでサケ科魚類に発生が報告された魚病で、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（非特許文献２）。
特に稚魚期に於いて、滑走細菌症による被害が多く、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、鰓等に発赤や出血ときには潰瘍が見られる（非特許文献３）。閉鎖系である養殖領域に於ける滑走細菌症は、個体が高密度に存在していることから、それら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。
Gliding bacteriosis is a disease that occurs in marine fish such as red sea bream, amberjack, Japanese flounder, and puffer fish, and the causative fungus is known to be Tenachibacrum maritimum (formerly Flexibacterium maritimum) (non-patent literature) 1). Gliding bacteriosis due to Tenakibacrum maritimum was the first fish disease reported to occur in salmonids in the United States, and has occurred in various sea farmed fish in Japan since around 1970, and has sometimes caused serious damage ( Non-patent document 2).
Especially during the fry stage, there are many damages caused by gliding bacteriosis, and the symptoms are that the larvae and necrosis or collapse occur in the larvae and caudal fins, and redness and An ulcer is seen at the time of bleeding (nonpatent literature 3). Gliding bacteriosis in a closed culturing area has a high density of individuals, so that the effects of these infections are significant and a serious problem in the marine aquaculture industry.
滑走細菌は、ニフルスチレン酸（特許文献１）や、テトラサイクリン系の抗生物質に感受性があるので、滑走細菌症の防除対策としては、その大部分をこれら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に関しては、水産用医薬品としても承認されているし、オキシテトラサイクリン等の抗生物質を経口投与することが有効である（特許文献２）ことも報告されている。
しかしながら、これら抗菌性薬剤等の使用は、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。さらに経口投与は餌を喰わない重症魚には効果が無いことや、ニフルスチレン酸ナトリウムは、現在ヒラメの稚魚にしか使用できないこと等の問題もある。
以上より、マダイ、カンパチ等の海産魚類の滑走細菌症予防に有効であって、且つ環境汚染等の問題が生じない新規製剤の開発が望まれている。
Since gliding bacteria are sensitive to niflustyrene acid (Patent Document 1) and tetracycline antibiotics, most of them depend on these drugs as countermeasures against gliding bacteriosis. For example, regarding sodium niflustyrene, it has been approved as a pharmaceutical for marine products, and it has been reported that oral administration of antibiotics such as oxytetracycline is effective (Patent Document 2).
However, the use of these antibacterial drugs and the like has been associated with food hygiene problems due to an increase in multidrug-resistant bacteria, drug residues in fish bodies, and public health problems due to diffusion into the environment. Furthermore, oral administration is not effective for severe fish that do not eat food, and sodium niflustyrate is currently available only for flounder larvae.
From the above, it is desired to develop a new preparation that is effective for preventing gliding bacteriosis in marine fish such as red sea bream and amberjack and that does not cause problems such as environmental pollution.
本発明の目的は、上記従来技術の問題点に鑑み、マダイが、テナキバクラム マリティマムに感染して、滑走細菌症を発病するのを効果的に防止することができるとともに、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新規製剤を提供することにある。 SUMMARY OF THE INVENTION In view of the problems of the related art, Mada Lee is infected with Tenakibakuramu Maritimamu, it is possible to effectively prevent the onset of gliding bacteria disease, food hygiene problems Ya It is to provide a new preparation that does not cause public health problems due to its diffusion into the environment.
本発明者らは、鋭意研究の結果、テナキバクラム マリティマム（Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ）不活化菌体を有効成分とするワクチンが、マダイ等の海産魚類に発病する滑走細菌症を効果的に防止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, the present inventors have found that a vaccine containing tenacibacumum maritimum inactivated cells as an active ingredient can effectively prevent gliding bacteriomyopathy caused by marine fish such as red sea bream. The invention has been completed.
即ち、請求項１に係る発明は、テナキバクラム マリティマムの不活化菌体を有効成分とするマダイ滑走細菌症ワクチンであって、浸漬ワクチンであるマダイ滑走細菌症ワクチンに関する。
請求項２に係る発明は、テナキバクラム マリティマムの不活化菌体を含有するマダイ滑走細菌症ワクチン組成物であって、浸漬用のワクチン組成物であるマダイ滑走細菌症ワクチン組成物に関する。
請求項３に係る発明は、テナキバクラム マリティマムの不活化菌体の有効量を浸漬法によって投与することを特徴とするマダイ滑走細菌症の予防法に関する。
請求項４に係る発明は、沖出しの１〜２週間前に、テナキバクラム マリティマムの不活化菌体を懸濁した溶液に２０分以上浸漬する請求項３記載のマダイ滑走細菌症の予防法に関する。
That is, the invention according to
The invention according to 請 Motomeko 2 is a red sea gliding bacteria disease vaccine composition containing inactivated cells of Tenakibakuramu Maritimamu relates red sea gliding bacteria disease vaccine composition is a vaccine composition for immersion.
The invention according to 請 Motomeko 3 relates prophylaxis of red sea gliding bacteria disease which comprises administering by immersion method an effective amount of inactivated cells of Tenakibakuramu Maritimamu.
The invention according to
本発明のワクチン又はワクチン組成物を用いれば、マダイ等の魚類滑走細菌症を効率的に予防することができるとともに、元来自然物であるので、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ることもない。 With the vaccine or vaccine composition of this invention, it is possible to effectively prevent the fishes gliding bacteria disease such as red sea bream, because it is inherently natural object, the public due to the diffusion of food hygiene to issues or environment It does not cause hygiene problems.
本発明者らは、鋭意研究の結果、テナキバクラム マリティマム（Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ）（以下「本菌」と称す場合がある）の不活化菌体、又はその成分を有効成分とするワクチンが、滑走細菌症の予防に有効であることを見出し、本発明を完成するに至った。以下、本発明の実施形態について説明する。 As a result of diligent research, the present inventors have found that inactivated cells of Tenacibacumum maritimum (hereinafter sometimes referred to as “the present bacterium”), or a vaccine containing the component as an active ingredient, are The inventors have found that it is effective for prevention, and have completed the present invention. Hereinafter, embodiments of the present invention will be described.
本発明のワクチンは、テナキバクラム マリティマム（Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ）の不活化菌体またはその成分を用いる。詳細には、テナキバクラム マリティマム（Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ）のＲ２株、ＳＭ３３２２ｃ株、ＯＭ３５１０株、ＭＣ９２１０株の不活化菌体またはその成分を用いるのが、より強力な滑走細菌症の予防効果を得る観点から望ましい。 The vaccine according to the present invention uses inactivated cells of Tenacibacumum maritimum or components thereof. Specifically, it is desirable to use inactivated cells or components thereof of Tenacibacumum maritimum R2 strain, SM3322c strain, OM3510 strain, MC9210 strain, or its components, from the viewpoint of obtaining a more powerful preventive effect against gliding bacteriomyopathy.
テナキバクラム マリティマムは、グラム陰性、好気性で、屈曲運動をする黄色色素（カロテノイド）をもつ長桿菌（０．５×２〜３μm）である。その発育は３０℃、ｐＨ７が最適で、海水（３０％）を必要とする他、性状がカラムナリス病菌や、その他の細菌と違っているので、日本で新種として命名された。この細菌はタンパク質を分解するが、デンプンその他の多糖を分解せず、細菌を溶解（溶菌）する作用をもっている。
通常、細菌を培養した場合、誘導期、対数増殖期、定常期、死滅期及び生残期に分けられるが、本発明のワクチンでは、対数増殖期のものを用いるのが望ましい。
Tenakibacrum maritimum is a gram-negative, aerobic, gonococcus (0.5 × 2-3 μm) with a yellow pigment (carotenoid) that bends. Its growth is optimal at 30 ° C,
Usually, when a bacterium is cultured, it is divided into an induction phase, a logarithmic growth phase, a stationary phase, a killing phase, and a survival phase, but it is desirable to use a vaccine in the logarithmic growth phase.
本発明のワクチンに用いる菌体（テナキバクラム マリティマム）は、本菌を常法により培養し、対数増殖期に採取することにより得られる。本菌の培養は、本菌を適当な培地に接種し常法に従って培養すればよい。例えば、テナキバクラム マリティマム（Ｔ．ｍａｒｉｔｉｍｕｍ）を、７０％海水サイトファーガ寒天培地で、２５℃、４８時間培養するのが望ましい。 The microbial cells (tenakibacrum maritimum) used in the vaccine of the present invention can be obtained by culturing the bacterium by a conventional method and collecting it in the logarithmic growth phase. The bacterium can be cultured by inoculating the bacterium into an appropriate medium and culturing according to a conventional method. For example, it is desirable to cultivate Tenakibacrum maritimum (T. maritimum) on a 70% seawater cytophaga agar medium at 25 ° C. for 48 hours.
本菌の成分には、菌体の膜成分及び分泌物が含まれる。これらの成分を採取するには、不活化菌体の超音波破砕等により行うのが好ましい。
得られた不活化菌体又はその成分は、濾過、蒸発脱水法、濃縮、凍結乾燥等により濃縮して用いるのが好ましい。
The components of this bacterium include membrane components and secretions of microbial cells. In order to collect these components, it is preferable to carry out ultrasonic disruption of inactivated cells.
The obtained inactivated cells or components thereof are preferably used after being concentrated by filtration, evaporative dehydration, concentration, lyophilization or the like.
本菌の不活化菌体又はその成分は、そのままワクチンとして使用してもよいが、薬学的に許容される液状、又は固体状の担体とともにワクチン組成物として使用してもよい。
当該ワクチン組成物の形態としては、注射用組成物、経口投与組成物、魚類浸漬用組成物、飼料組成物等が挙げられるが、魚類浸漬用組成物とするのが望ましい。
液状の担体としては、リン酸緩衝液が挙げられる。固体状の担体としては、タルク、シュークロースなどの賦形剤が挙げられる。飼料組成物とするには、通常の魚類の飼料に本菌の不活化菌体又はその成分を混合すればよい。又、これらのワクチン組成物にはアジュバントを添加して抗原性を高めてもよい。
The inactivated cells of this bacterium or its components may be used as a vaccine as it is, but may be used as a vaccine composition together with a pharmaceutically acceptable liquid or solid carrier.
Examples of the vaccine composition include an injectable composition, an oral administration composition, a fish immersion composition, a feed composition, and the like, and it is desirable to use a fish immersion composition.
Examples of the liquid carrier include a phosphate buffer. Examples of the solid carrier include excipients such as talc and sucrose. In order to obtain a feed composition, inactivated cells of the present bacterium or components thereof may be mixed with normal fish feed. In addition, an antigen may be added to these vaccine compositions to enhance antigenicity.
本発明のワクチン又はワクチン組成物の対象魚種としては、マダイの滑走細菌症に対するのが最も効果的である。 The target species of the vaccine or vaccine composition of the present invention, that the relative gliding bacteria disease Ma die is most effective.
〔ワクチン接種方法〕
魚類ワクチンの接種法として代表的なものに、経口法、注射法及び浸漬法が知られているが、経口法は、胃の消化酵素等によりワクチンの免疫原性が低下してしまうことから高い有効性・持続性を得ることは困難である等の欠点が、また注射法は、稚魚への適用が困難であること、多大な労力が必要であること、魚に与えるストレスが大きいこと、作業者への誤注射の危険性があること等の欠点がある。特に注射法は、稚魚への適用が困難なので、滑走細菌症のような稚魚期に甚大な被害をもたらす疾病に用いることができない。
一方、浸漬法は（経口法に比べ）有効性が高く大量の魚を同時に処理することができ、注射法のように多大な労力が必要である等といった欠点もない。また稚魚への適用も可能である。但し、魚体に取り込まれるワクチンが少量である為、高い有効性が認められるのは、ビブリオ病など一部の疾病に対するワクチンに限られている。
[Vaccination method]
Oral methods, injection methods, and immersion methods are known as typical vaccination methods for fish vaccines, but oral methods are high because the immunogenicity of the vaccine is reduced by gastric digestive enzymes, etc. Disadvantages such as difficulty in obtaining effectiveness and sustainability, and injection method is difficult to apply to fry, requires a lot of labor, stress applied to fish is large, work There are disadvantages such as the risk of misinjection to the person. In particular, since the injection method is difficult to apply to fry, it cannot be used for diseases that cause enormous damage during fry, such as gliding bacteriosis.
On the other hand, the dipping method is highly effective (compared to the oral method) and can treat a large amount of fish at the same time, and does not have the disadvantages that a great deal of labor is required unlike the injection method. It can also be applied to fry. However, since only a small amount of vaccine is taken into the fish body, high efficacy is limited to vaccines against some diseases such as Vibrio disease.
本発明のワクチン又はワクチン組成物を接種する方法としては「浸漬法」によるのが好ましい。即ち、本発明のワクチン又はワクチン組成物は「浸漬法」によっても十分に高い有効性が認められ、浸漬法の長所である多大な労力が必要ないこと、稚魚への適用も可能であること等から、主に浸漬用として、本発明のワクチン又はワクチン組成物が、特に稚魚期に甚大な被害をもたらす滑走細菌症に対するワクチンとして極めて有用である。 As a method for inoculating the vaccine or vaccine composition of the present invention, the “immersion method” is preferred. That is, the vaccine or vaccine composition of the present invention has a sufficiently high effectiveness even by the “immersion method”, does not require a great amount of labor, which is an advantage of the immersion method, and can be applied to fry, etc. Therefore, mainly for immersion, the vaccine or vaccine composition of the present invention is extremely useful as a vaccine against gliding bacteriomyopathy that causes enormous damage especially in the fry stage.
本発明のワクチン又はワクチン組成物の投与は成魚でもよいが、滑走細菌症に罹患する前、例えば稚魚の段階が好ましい。
投与方法は、上記したとおり「浸漬法」によるのが好ましいが、浸漬に用いるワクチン溶液としては、本発明のワクチン又はワクチン組成物を１μｇ／ｍＬ〜１００ｍｇ／ｍＬの濃度で、海水に懸濁したもの等が望ましい。
Administration of the vaccine or vaccine composition of the present invention may be adult fish, but is preferably performed before, for example, the stage of juvenile fish.
The administration method is preferably the “immersion method” as described above, but as the vaccine solution used for immersion, the vaccine or vaccine composition of the present invention is suspended in seawater at a concentration of 1 μg / mL to 100 mg / mL. Things are desirable.
〔ワクチンの浸漬時間〕
ワクチンの浸漬（投与）時間としては、１０分以上行うのが好ましいが、２０分以上行うのがより好ましい。この理由は、１０分を下回る時間、浸漬したとしても十分なワクチン効果を発揮することができないからである。
尚、後述する実施例に於いて、１５分間浸漬した試験区よりも３０分間浸漬した試験区の方が、高いワクチン効果が得られることが実証されている（図３参照）。
[Vaccine immersion time]
The immersion (administration) time of the vaccine is preferably 10 minutes or more, more preferably 20 minutes or more. This is because a sufficient vaccine effect cannot be exerted even if immersed for less than 10 minutes.
In the examples described later, it has been demonstrated that the test group immersed for 30 minutes has a higher vaccine effect than the test group immersed for 15 minutes (see FIG. 3).
〔ワクチンの浸漬濃度〕
投与方法は、上記したとおり「浸漬法」によるのが好ましく、海水に本発明のワクチン又はワクチン組成物を１μｇ／ｍＬ〜１００ｍｇ／ｍＬの濃度で懸濁したものが好ましく使用される。
[Vaccine immersion concentration]
As described above, the administration method is preferably the “immersion method”, and a suspension of the vaccine or vaccine composition of the present invention in seawater at a concentration of 1 μg / mL to 100 mg / mL is preferably used.
〔ワクチンの浸漬回数〕
浸漬（投与）回数は１回でもよいが、２回以上行うのがワクチン効果をより強固に発揮するためには好ましい。後述する実施例に於いても、１回投与の試験区よりも２回投与の試験区で、より高いワクチン効果（有効率が高い傾向）が認められている（図６参照）。
[Number of times of immersion of vaccine]
The immersion (administration) may be performed once, but it is preferable to perform the immersion twice or more in order to exert the vaccine effect more firmly. Also in the examples described later, a higher vaccine effect (a tendency of higher efficacy rate) was observed in the test group administered twice than in the test group administered once (see FIG. 6).
〔ワクチン浸漬後、沖出しする迄の経過日数〕
より高いワクチン効果（有効率が高い効果）を得る為には、ワクチンの浸漬時間は２０分以上行うのがより好ましいが、この場合、ワクチンの浸漬（投与）は沖出しする少なくとも１週間前（好ましくは１〜２週間）に行うのが、好ましい（図１及び２参照）。
[Elapsed days after immersing the vaccine until it goes offshore]
In order to obtain a higher vaccine effect (effect with a high effective rate), it is more preferable to carry out the immersion time of the vaccine for 20 minutes or more. In this case, the immersion (administration) of the vaccine is at least one week before the offing ( Preferably, it is performed for 1 to 2 weeks (see FIGS. 1 and 2).
以下に実施例を示すが、本発明はこれらによって何ら限定されるものではない。 Examples are shown below, but the present invention is not limited to these examples.
実験Ｉ 「供試菌株の相違によるワクチン有効性の比較」
マダイ由来１４株、ヒラメ由来１株およびトラフグ由来１株で不活化ワクチンを作製し、その有効性をマダイにおいて検討した。
Experiment I “Comparison of vaccine efficacy due to differences in test strains”
An inactivated vaccine was prepared with 14 red sea bream-derived strains, 1 flounder-derived strain, and 1 tiger puffer-derived strain, and the effectiveness was examined in red sea bream.
〔供試魚〕
近畿大学水産研究所白浜実験場で２００３年に生産されたマダイ（平均全長５．４ｃｍ，平均体重３．１ｇ）を４５０尾使用した。
[Test fish]
450 red sea bream (average length: 5.4 cm, average weight: 3.1 g) produced in 2003 at the Kinki University Fisheries Research Institute Shirahama Experiment Station were used.
〔供試ワクチン〕
マダイ由来１２株（強毒株６株，弱毒株６株），ヒラメ由来１株（弱毒株），トラフグ由来１株（強毒株）を７０％海水改変サイトファーガ寒天培地で２５℃、４８時間培養した後、１．５％ホルマリンＰＢＳで４℃，２４時間不活化した菌体を供試ワクチンとした。
[Test vaccine]
12 strains of red sea bream (severely virulent strain, 6 attenuated strains), 1 strain of flounder (attenuated strain), 1 strain of troughfish (strongly virulent strain) at 25 ° C. and 48% in 70% seawater modified cytophaga agar After culturing for a period of time, bacterial cells inactivated with 1.5% formalin PBS at 4 ° C. for 24 hours were used as test vaccines.
〔試験区設定およびワクチンの投与〕
２００Ｌ容ポリカーボネイト水槽１５基に３０尾ずつ収容した。１４基をワクチン投与区、１基を対照区に設定した。ワクチン投与は浸漬法によって行い、浸漬濃度は湿菌重量で２０μｇ／ｍＬ、浸漬時間は３０分とした。
[Test area setting and vaccine administration]
Thirty fish were housed in 15 200 L polycarbonate tanks. 14 groups were set as the vaccine administration group and 1 group as the control group. Vaccine administration was carried out by an immersion method, the immersion concentration was 20 μg / mL in terms of wet bacterial weight, and the immersion time was 30 minutes.
〔人為感染〕
７０％海水改変サイトファーガ寒天培地で２５℃、約２４時間培養したテナキバクラム マリティマム（Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ）Ｒ２株を、濾過海水で１００μｇ／ｍＬに懸濁し、供試魚を３０分間浸漬した。人為感染の翌日から１０日後まで約１２時間ごとに死亡魚を取り上げ、有効率を〔数１〕における式において算出した。
Ｒ２株を１００ｍｇ／ｍＬの濃度に懸濁した菌液をＢＣＧ接種用管針で供試魚体表に接種し、人為感染の翌日から１４日後まで約１２時間ごとに死亡魚を取り上げた。
結果を下記の〔表１〕に示す。
[Artificial infection]
Tenacibacurum maritimum R2 strain cultured in a 70% seawater-modified cytophaga agar medium at 25 ° C. for about 24 hours was suspended in filtered seawater at 100 μg / mL, and the test fish was immersed for 30 minutes. Dead fish were picked up approximately every 12 hours from the day after the artificial infection until 10 days later, and the effective rate was calculated by the equation in [Equation 1].
A bacterial solution in which the R2 strain was suspended at a concentration of 100 mg / mL was inoculated on the surface of the test fish with a BCG inoculation tube needle, and dead fish were taken up about every 12 hours from the next day to 14 days after the artificial infection.
The results are shown in [Table 1] below.
〔結果〕
・ 強毒株群では生残率２０〜９０％、有効率１７．２〜８９．７％であった。
・ また、弱毒株群では生残率０〜７２．４％，有効率０〜７１．５％であった。
・ 対照区の生残率は３．３％であった。
・ 強毒株群では、ＳＭ２２０２ｓを除き、４０％以上の有効率が認められた。
・ 特に、ＳＭ３３２２ｃ区およびＯＭ３５１０区では有効率７５％以上であった。
・ 弱毒株群ではＭＣ９２１０区で７１．５％の有効率が得られたものの、それ以外の試験区では０〜３４．５％であった。
以上より、テナキバクラム マリティマムの中でも、Ｒ２株、ＳＭ３３２２ｃ株、ＯＭ３５１０株、ＭＣ９２１０株の不活化菌体またはその成分を用いるのが望ましいことが示された。
〔result〕
-The virulent strain group had a survival rate of 20 to 90% and an effective rate of 17.2 to 89.7%.
In the attenuated strain group, the survival rate was 0 to 72.4% and the effective rate was 0 to 71.5%.
・ Survival rate in the control plot was 3.3%.
In the highly toxic strain group, an effective rate of 40% or more was recognized except for SM2202s.
・ Effective rate was 75% or more especially in SM3322c and OM3510.
In the attenuated strain group, an effective rate of 71.5% was obtained in MC9210, but 0 to 34.5% in other test plots.
From the above, it was shown that it is desirable to use inactivated cells of R2 strain, SM3322c strain, OM3510 strain, MC9210 strain or components thereof among Tenakibacrum maritimum.
〔ホルマリン不活化ワクチンの浸漬投与によるマダイの滑走細菌症の防除〕
実験 II ： ワクチン浸漬時間および浸漬後経過日数の長短による比較
実験 III： ワクチン２回投与の検討
[Control of red sea bream gliding bacteriosis by immersion of formalin-inactivated vaccine]
Experiment II: Comparative experiment with vaccine immersion time and length of days elapsed after immersion III: Examination of two doses of vaccine
実験 II ： ワクチン浸漬時間および浸漬後経過日数の長短による比較
「材料と方法」
（１）供試魚 ： マダイ稚魚４００尾（平均全長８．８ｃｍ，平均体重１４．６ｇ）
（２）供試ワクチン ： Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ Ｒ２株を、７０％海水サイトファーガ寒天培地で、２５℃、４８時間培養し、１．５％ホルマリンＰＢＳで４℃，４８時間の条件で不活化した。このようにして得られたワクチンを、以下「ＦＫＣ」（Formalin killed cells）と称す。尚、ＦＫＣは本発明にかかるワクチンである。
（３）飼育水槽 ： ２００Ｌ容パンライト水槽
（４）収容尾数 ： ５０尾ずつ
（５）ワクチン（ＦＫＣ）投与 ： ２０ｍｇ／ｍＬのＦＫＣ懸濁海水に浸漬させることにより行った。ＦＫＣ投与は、１５分間浸漬、３０分間浸漬の２通りで実施した。
（６）平均水温 ： ２０．７℃
（７）人為感染 ： 人為感染は、Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ Ｒ２を１．１×１０８ＣＦＵ／ｍＬに懸濁した海水に２０分間浸漬することにより行った。
Experiment II: Comparison of vaccine immersion time and length of days elapsed after immersion "Materials and methods"
(1) Test fish: 400 red sea bream (average length 8.8cm, average weight 14.6g)
(2) Test vaccine: Tenacibacumum maritimum R2 strain was cultured in a 70% seawater cytophaga agar medium at 25 ° C. for 48 hours, and inactivated in 1.5% formalin PBS at 4 ° C. for 48 hours. The vaccine thus obtained is hereinafter referred to as “FKC” (Formalin killed cells). FKC is a vaccine according to the present invention.
(3) Breeding tank: 200L panlite tank (4) Accommodating number of fish: 50 fish each (5) Vaccine (FKC) administration: It was performed by immersing in 20 mg / mL FKC suspended seawater. FKC administration was carried out in two ways: immersion for 15 minutes and immersion for 30 minutes.
(6) Average water temperature: 20.7 ° C
(7) Artificial infection: Artificial infection was carried out by immersing Tenacibacumum maritimum R2 in seawater suspended in 1.1 × 10 8 CFU / mL for 20 minutes.
供試魚にＦＫＣを投与（５）した後、人為感染（７）を行い、ＦＫＣの感染防御効果を検討した。ＦＫＣ投与から３日後、１週間後および２週間後に、人為感染させた試験区を夫々設け、ＦＫＣ非投与の対照区と比較することにより、ＦＫＣの有効率を算出した。
尚、有効率は〔数１〕における式において算出した。表２に各試験区を示す。
After FKC was administered to the test fish (5), artificial infection (7) was performed, and the infection protective effect of FKC was examined. Three days after FKC administration, one week later and two weeks later, an artificially infected test group was provided, and the FKC efficacy rate was calculated by comparing with the control group not administered with FKC.
The effective rate was calculated by the equation in [Equation 1]. Table 2 shows each test section.
〔結果〕
１）図１は、本発明にかかる滑走細菌症ワクチンを「１５分間浸漬させた場合」におけるマダイの生存率（％）の推移を示したグラフであるが、ＦＫＣ投与から人為感染までの日数が１週間のもの（前記表２における第２段目）が、３日のもの（同第１段目）及び２週間（同第３段目）のものと比して、格段に高い生存率（％）が示された。
２）図２は、本発明にかかる滑走細菌症ワクチンを「３０分間浸漬させた場合」におけるマダイの生存率（％）の推移を示したグラフであるが、ＦＫＣ投与から人為感染迄の日数が１週間、２週間のものが、３日のものと比して、格段に高い生存率（％）が示された。
３）以上より、本発明のワクチンを３０分浸漬させた場合の方が、１５分間浸漬させた場合と比して、高いワクチン効果（生存率％）を発揮することが示された。
さらに、１５分浸漬の場合および３０分間浸漬の場合の双方において、ワクチン浸漬後の沖出し迄の日数は、１週間程度（厳密には４日以上であることが本発明者らによって確認されている）、若しくはそれ以上（〜２週間）空けることが、より高いワクチン効果（有効率が高い効果）を得る為には好ましいことが示された。
〔result〕
1) FIG. 1 is a graph showing the transition of survival rate (%) of red sea bream in the case where the gliding bacteriosis vaccine according to the present invention is “immersed for 15 minutes”. The number of days from administration of FKC to artificial infection is Survival rate (2nd stage in Table 2) is significantly higher than that of 3 days (1st stage) and 2 weeks (3rd stage). %)It has been shown.
2) FIG. 2 is a graph showing the transition of survival rate (%) of red sea bream when the gliding bacteriosis vaccine according to the present invention is “immersed for 30 minutes”. The number of days from administration of FKC to artificial infection is One week and two weeks showed a significantly higher survival rate (%) than that of three days.
3) From the above, it was shown that the case of immersing the vaccine of the present invention for 30 minutes exhibits a higher vaccine effect (survival rate%) than the case of immersing for 15 minutes.
Furthermore, in both the case of 15-minute immersion and the case of 30-minute immersion, the present inventors have confirmed that the number of days until the offing after the vaccine immersion is about 1 week (strictly 4 days or more). It is shown that it is preferable to obtain a higher vaccine effect (an effect with a high efficacy rate) to leave it longer (~ 2 weeks).
実験 III： ワクチン２回投与の検討
「材料と方法」
（１）供試魚 ： マダイ稚魚２４０尾（平均全長４．９ｃｍ，平均体重１．６ｇ）
（２）供試ワクチン（ＦＫＣ） ：
Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ Ｒ２株を、７０％海水サイトファーガ寒天培地で、２５℃、４８時間培養し、１．５％ホルマリンＰＢＳで４℃，４８時間の条件で不活化した。尚、ＦＫＣは本発明にかかるワクチンである。
（３）飼育水槽 ： ５００Ｌ容パンライト水槽
（４）収容尾数 ： ６０尾ずつ
（５）ワクチン（ＦＫＣ）投与 ：
２０ｍｇ／ｍＬのＦＫＣ懸濁海水に３０分間浸漬させることにより行った。
ＦＫＣ投与（浸漬）は、２回投与（人為感染２週間前および１週間前の２回）、人為感染２週間前の１回投与、人為感染１週間前の１回投与の３通りで実施した（表３参照）。
（６）平均水温 ： ２０．０℃
（７）人為感染 ： Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ Ｒ２株を、懸濁海水に３０尾ずつ２０分間浸漬することにより行った。
（８）浸漬濃度 ：
前記人為感染（７）において、Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ Ｒ２株の浸漬濃度として、（１）１．６×１０７ＣＦＵ／ｍＬの場合と、（２）２．４×１０７ＣＦＵ／ｍＬの場合の２通りで実施した（表４参照）。
Experiment III: Examination of two doses of vaccine “Materials and Methods”
(1) Test fish: 240 red sea bream (average total length 4.9cm, average weight 1.6g)
(2) Test vaccine (FKC):
Tenacibacumum maritimum R2 strain was cultured in a 70% seawater cytophaga agar medium at 25 ° C. for 48 hours, and inactivated in 1.5% formalin PBS at 4 ° C. for 48 hours. FKC is a vaccine according to the present invention.
(3) Breeding tank: 500L panlite tank (4) Capacity: 60 fish each (5) Vaccine (FKC) administration:
It was performed by immersing in 20 mg / mL FKC-suspended seawater for 30 minutes.
FKC administration (immersion) was performed in three ways: 2 administrations (2 weeks before and 2 days before human infection), 1
(6) Average water temperature: 20.0 ° C
(7) Artificial infection: Tenacibabacterium maritimum R2 strain was immersed in suspended seawater for 30 minutes for 20 minutes.
(8) Immersion concentration:
In the artificial infection (7), the immersion concentration of Tenacibacumum maritimum R2 strain is (1) 1.6 × 10 7 CFU / mL and (2) 2.4 × 10 7 CFU / mL. (See Table 4).
〔結果〕
１）図４は、Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ Ｒ２株を、１．６×１０７ＣＦＵ／ｍＬの濃度で浸漬させた場合におけるマダイの生存率（％）の推移を示したグラフであるが、人為感染２週間前から１週間置きにＦＫＣを２回投与したもの（前記表３における第１段目）について最も高い生存率（％）が認められ（８０，０％）、次いで、人為感染１週間前にＦＫＣを１回投与したもの（同第２段目）についても高い生存率（％）が認められた（６５，０％）。
２）図５は、Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ Ｒ２株を、２．４×１０７ＣＦＵ／ｍＬの濃度で浸漬させた場合におけるマダイの生存率（％）の推移を示したグラフであるが、人為感染２週間前から１週間置きにＦＫＣを２回投与したものについて最も高い生存率（％）が認められ（７４．１％）、次いで、人為感染１週間前および２週間前にＦＫＣを１回投与したもの（前記表３における第２及び３段目）についても高い生存率（％）が認められた（ともに５９．３％）。
３）以上より、本発明のワクチンを２回浸漬した方が、１回のみの浸漬と比して、高いワクチン効果（生存率％）を発揮することが示された（図６参照）。
〔result〕
1) FIG. 4 is a graph showing the change in survival rate (%) of red sea bream when Tenacibacumum maritimum R2 strain is immersed at a concentration of 1.6 × 10 7 CFU / mL. The highest survival rate (%) was observed for FKC administered twice every other week (first stage in Table 3) (80,0%), and then FKC one week before artificial infection A high survival rate (%) was also observed (65,0%) for those administered once (second stage).
2) FIG. 5 is a graph showing the change in survival rate (%) of red sea bream when Tenacibabacterium maritimum R2 strain is immersed at a concentration of 2.4 × 10 7 CFU / mL. The highest survival rate (74.1%) was observed with FKC administered twice every other week from the previous (74.1%), followed by FKC administered once 1 week and 2 weeks before artificial infection A high survival rate (%) was also observed for (second and third stages in Table 3) (both 59.3%).
3) From the above, it was shown that the case of immersing the vaccine of the present invention twice exhibits a higher vaccine effect (survival rate%) than that of immersing only once (see FIG. 6).
〔まとめ〕
・ 実験II、実験IIIともにＦＫＣの効果が認められた。
・ 実験IIでは１５分浸漬区よりも３０分浸漬区で有効率が高い傾向が認められた。
・ ＦＫＣを投与してから３日後に攻撃した区では生残率が低かった。
・ 実験IIIでは１回投与区よりも２回投与区で有効率が高い傾向が認められた。
[Summary]
・ The effects of FKC were observed in both Experiment II and Experiment III.
-In Experiment II, a tendency was found that the effectiveness rate was higher in the 30-minute immersion zone than in the 15-minute immersion zone.
・ Survival rate was low in the group attacked 3 days after administration of FKC.
・ In Experiment III, a tendency was found that the efficacy rate was higher in the 2 dose groups than in the 1 dose group.
〔結論〕
マダイの滑走細菌症の防除に浸漬ワクチンが有効である。
浸漬時間は３０分以上行うのが好ましい。
ワクチンを投与してから少なくとも４日以上経てから沖出しすることが有効である。
ワクチンの充分な防御効果を得るためには沖出し前に水槽内で２回以上免疫することが望ましい。
[Conclusion]
Immersion vaccine is effective for controlling gliding bacteriosis in red sea bream.
The immersion time is preferably 30 minutes or longer.
It is effective to go offshore at least 4 days after administration of the vaccine.
In order to obtain a sufficient protective effect of the vaccine, it is desirable to immunize twice or more in an aquarium before going offshore.
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| JP5283007B1 (en) * | 2012-08-30 | 2013-09-04 | 株式会社片山化学工業研究所 | How to kill gliding bacteria in aquaculture |
| KR101976764B1 (en) * | 2017-12-19 | 2019-05-09 | 제주대학교 산학협력단 | Tenacibaculum maritimum virulence attenuation technique and live attenuated vaccine for preventing fish Tenacibaculosis disease |
| KR101992455B1 (en) * | 2018-09-11 | 2019-06-24 | 제주대학교 산학협력단 | Tenacibaculum maritimum virulence attenuation technique and live attenuated vaccine for preventing fish Tenacibaculosis disease |
| JP7529430B2 (en) * | 2020-04-15 | 2024-08-06 | 広島県 | Method for preventing infectious diseases in aquatic organisms, vaccine preparation for immersion, and method for producing vaccine preparation for immersion |
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| JP3950500B2 (en) * | 1995-09-23 | 2007-08-01 | 独立行政法人水産総合研究センター | Iridovirus infectious disease vaccine and diagnostic agent for fish and production method thereof |
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