JP2015038113A - Inactivated vaccine formulation, and method for preventing infectious diseases - Google Patents
Inactivated vaccine formulation, and method for preventing infectious diseases Download PDFInfo
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- JP2015038113A JP2015038113A JP2014204553A JP2014204553A JP2015038113A JP 2015038113 A JP2015038113 A JP 2015038113A JP 2014204553 A JP2014204553 A JP 2014204553A JP 2014204553 A JP2014204553 A JP 2014204553A JP 2015038113 A JP2015038113 A JP 2015038113A
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- Prior art keywords
- inactivated
- lactococcus
- inactivated vaccine
- strain
- garvieae
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Abstract
Description
本発明は、ラクトコッカス・ガルビエ(学名「Lactococcus garvieae」、以下同じ)を起因菌とする魚類レンサ球菌症に対する不活化ワクチン製剤、魚類レンサ球菌症の予防方法、不活化ワクチン製剤製造方法などに関連する。より詳細には、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエの不活化菌体を含有する不活化ワクチン製剤、魚類レンサ球菌症の予防方法、不活化ワクチン製剤製造方法などに関連する。 The present invention relates to an inactivated vaccine preparation against fish streptococcal disease caused by Lactococcus garvieae (scientific name “Lactococcus garvieae”, hereinafter the same), a method for preventing fish streptococci, a method for producing an inactivated vaccine preparation, etc. To do. In more detail, for inactivated vaccine preparations containing inactivated cells of Lactococcus garvier whose serotype is non-KG- and non-KG +, methods for preventing fish streptococcal disease, methods for producing inactivated vaccine preparations, etc. Related.
魚類のレンサ球菌症は、特に養殖魚などにおいて、発生頻度が高く、経済的損失も大きい疾病の一つである。魚類のレンサ球菌症には、主に、ラクトコッカス・ガルビエを起因菌とするα溶血性レンサ球菌症と、ストレプトコッカス・イニエ(学名「Streptococcus iniae」)を起因菌とするβ溶血性レンサ球菌症などがある。 Fish streptococcal disease is one of the diseases with a high occurrence frequency and great economic loss, particularly in cultured fish. The streptococcal disease of fish mainly includes α-hemolytic streptococci caused by Lactococcus garvier and β-hemolytic streptococci caused by Streptococcus iniee (scientific name "Streptococcus iniae") There is.
このうち、ラクトコッカス・ガルビエを起因菌とする魚類レンサ球菌症は、ブリ・カンパチ・ヒラマサなどのブリ属魚類などに多く発生しており、マダイ・チダイなどの海水魚、ウナギ、ニジマスなどでも発症する。ブリ属魚類などにおいては、眼球白濁・突出、躯幹の変形、鰓蓋内側の発赤、心外膜炎、狂奔遊泳などの症状を示し、水温の高い季節の前後に被害が大きい。 Of these, fish streptococci caused by Lactococcus garbiae are common in the species of the genus Buri, such as yellowtail, amberjack, and kingfish, and also occur in saltwater fish such as red sea bream, red sea bream, eel, rainbow trout, etc. To do. In the species of yellowtail, the symptoms such as cloudiness / protrusion of the eyeball, deformation of the trunk, redness of the inside of the lid, epicarditis, crazy swimming, etc. are seriously damaged before and after the high water temperature season.
従来、ラクトコッカス・ガルビエを起因菌とするレンサ球菌症が養殖現場などで発生した際には、一週間以上の絶食による流行の鎮静化や、エリスロマイシンなどの抗生物質による治療などが行われてきた。しかし、特に抗生物質の使用については、薬剤耐性菌の出現や食品などへの残留への懸念などの問題が残る。そこで、近年、同疾患の予防対策として、ワクチンの開発が進められ、既に上市されている。 Conventionally, when streptococcal disease caused by Lactococcus garvier has occurred at farming sites, etc., sedation of the epidemic by fasting for more than a week and treatment with antibiotics such as erythromycin have been carried out . However, the use of antibiotics, in particular, remains a problem such as the emergence of drug-resistant bacteria and concerns about residues in foods. Therefore, in recent years, vaccines have been developed as a preventive measure for the disease, and are already on the market.
現在、ブリ属魚類を対象としたラクトコッカス・ガルビエに対するワクチン製剤として、例えば、ホルマリンにより不活化した不活化ワクチン、ホルマリンで不活化したものを濃縮した不活化ワクチン、培養菌液を酵素処理した後ホルマリンで不活化した不活化ワクチンなどの単味ワクチン製剤、二種混合ワクチン製剤、三種混合ワクチン製剤などが用いられている。 Currently, as vaccine preparations for Lactococcus garbies targeting fish species, for example, inactivated vaccines that have been inactivated by formalin, inactivated vaccines that have been concentrated by inactivation with formalin, Simple vaccine preparations such as inactivated vaccines inactivated with formalin, two-type mixed vaccine preparations, and three-type mixed vaccine preparations are used.
ラクトコッカス・ガルビエには、KG-型及びKG+型の2つの血清型が知られている。KG-型は、抗KG-抗血清による凝集反応がおこり、抗KG+抗血清による凝集反応がおこらない株であり、夾膜を有し、病原性の高い型である。一方、KG+型は、抗KG-抗血清と抗KG+抗血清の両者による凝集反応がおきる株であり、夾膜がなく、KG-型よりも病原性の低い型である(非特許文献1及び非特許文献2参照)。 In Lactococcus garbier, two serotypes, KG− and KG +, are known. The KG-type is a strain that undergoes an agglutination reaction with anti-KG-antiserum and does not undergo an agglutination reaction with anti-KG + antiserum, has a capsule, and is highly pathogenic. On the other hand, the KG + type is a strain that undergoes an agglutination reaction with both anti-KG-antiserum and anti-KG + antiserum, has no capsule, and is less pathogenic than KG-type (Non-patent Documents 1 and 2). Non-patent document 2).
ラクトコッカス・ガルビエに関する魚類のワクチンとして、例えば、特許文献1には、莢膜が極薄いか若しくは莢膜を有しないことを特徴とするラクトコッカス・ガルビエに属する菌株を不活化させた菌体を含有する魚類の腸球菌症用ワクチンが、特許文献2には、新規株を利用した魚類のレンサ球菌症を予防治療するためのワクチンが、それぞれ開示されている。その他、非特許文献3には、2012年9月に魚病検査に持ち込まれたカンパチから、従来のα溶血性レンサ球菌の診断用抗血清による凝集試験では全く凝集しない、ラクトコッカス・ガルビエと同じグループの菌株を分離したことが記載され、既知のものとは異なるレンサ球菌が養殖現場などにおいて散発している可能性が示唆されている。
本発明は、新規なラクトコッカス・ガルビエを分離・同定するとともに、その菌を起因菌とする疾患に対する有効な予防手段を提供することなどを目的とする。 An object of the present invention is to isolate and identify a novel Lactococcus garvier and to provide an effective preventive measure against a disease caused by the bacterium.
本発明者らは、日本国愛媛県の養殖現場において、ラクトコッカス・ガルビエに対する従来の不活化ワクチンを投与したにもかかわらずαレンサ球菌症の発症が疑われたブリより新規なラクトコッカス・ガルビエの菌株を独自に分離し、その菌株が非KG-型かつ非KG+型の血清型であることを同定することに成功した。そして、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエを起因菌とする魚類のレンサ球菌症では、従来のワクチンが有効でないことを実証するとともに、その新型レンサ球菌症に対する不活化ワクチンの開発に成功し、その有効性を実証した。 The inventors of the present invention have a newer Lactococcus garvier than a yellowtail that has been suspected to develop α-streptococcal disease despite administration of a conventional inactivated vaccine against Lactococcus garvier at a farming site in Ehime, Japan. Were successfully isolated and identified as non-KG- and non-KG + serotypes. And in the streptococcal disease of fish caused by non-KG- and non-KG + serotypes of Lactococcus galbiae, it is demonstrated that the conventional vaccine is not effective and the inactivation against the new streptococcal disease Succeeded in developing a vaccine and demonstrated its effectiveness.
そこで、本発明では、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエの不活化菌体を含有する、ラクトコッカス・ガルビエを起因菌とする魚類レンサ球菌症に対する不活化ワクチン製剤を提供する。 Therefore, in the present invention, there is provided an inactivated vaccine preparation against fish streptococci caused by Lactococcus garbier, which contains inactivated cells of Lactococcus garbier whose serotype is non-KG- and non-KG +. provide.
例えば、この不活化ワクチン製剤を魚類に投与などすることにより、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエを起因菌とする魚類レンサ球菌症、即ち、ラクトコッカス・ガルビエに対する従来の不活化ワクチンでは予防できない新型のレンサ球菌症に対し、その発生・伝播・蔓延を有効に予防できる可能性がある。 For example, by administering this inactivated vaccine preparation to fish, the conventional serococcosis of fish caused by Lactococcus garvier with a serotype of non-KG- and non-KG +, ie, Lactococcus garvier It may be possible to effectively prevent the occurrence, spread, and spread of a new type of streptococcal disease that cannot be prevented with this inactivated vaccine.
また、例えば、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエの不活化菌体を含有する不活化ワクチンと、血清型がKG-型又はKG+型のラクトコッカス・ガルビエの不活化菌体を含有する不活化ワクチンと、を含有する混合不活化ワクチン製剤を魚類に投与することにより、既知のレンサ球菌症及び既知のものとは異なる新型のレンサ球菌症の両者を同時に有効に予防できる可能性がある。 In addition, for example, an inactivated vaccine containing inactivated cells of Lactococcus garvier whose serotype is non-KG- and non-KG +, and inactivation of Lactococcus garvier whose serotype is KG- or KG + Infectious vaccine containing fungus body and mixed inactivated vaccine preparation containing the same are effectively administered to fish to simultaneously prevent both known streptococcal disease and a new type of streptococcal disease different from the known one There is a possibility.
本発明により、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエを起因菌とする魚類レンサ球菌症に対し、その発生・伝播・蔓延を予防できる。 According to the present invention, it is possible to prevent the occurrence, spread, and spread of a fish streptococcal disease caused by Lactococcus galbiae whose serotype is non-KG− and non-KG +.
<ラクトコッカス・ガルビエLC1301株について>
本発明者らは、日本国愛媛県の養殖現場において、ラクトコッカス・ガルビエに対する従来の不活化ワクチンを投与したにもかかわらずαレンサ球菌症の発症が疑われたブリより、新規なラクトコッカス・ガルビエの菌株を独自に分離し、その菌株が非KG-型かつ非KG+型の血清型であることを同定することに成功した。この分離・同定した菌株をラクトコッカス・ガルビエLC1301株(Lactococcus garvieae LC1301)と命名した。
<About Lactococcus garbier LC1301 strain>
The inventors of the present invention at the aquaculture site in Ehime, Japan, developed a novel lactococcus ・ from a yellowtail that was suspected to develop α-streptococcal disease despite administration of a conventional inactivated vaccine against Lactococcus garbier. We successfully isolated a strain of Garbier and identified it as a non-KG- and non-KG + serotype. This isolated and identified strain was named Lactococcus garvieae LC1301 strain (Lactococcus garvieae LC1301).
LC1301株の形態的性状としては、通常のラクトコッカス・ガルビエの形態と一致し、通性嫌気性のグラム陽性レンサ球菌の形状を示す。運動性、芽胞形成はない。培養的性質としては、一般的に用いられる肉エキス寒天平板培地、カゼイン・ダイズ混合ペプトン寒天平板培地などで白色のコロニーを形成する。また、一般的に用いられる肉エキス液状培地、カゼイン・ダイズ混合ペプトン液状培地などで振とう培養することにより増殖する。培養温度は20〜30℃が好適である。 The morphological properties of the LC1301 strain are consistent with those of the normal Lactococcus garvieae and show the shape of facultative anaerobic gram-positive streptococci. There is no motility or spore formation. As a culture property, a white colony is formed by using a meat extract agar plate medium generally used, a casein / soybean mixed peptone agar plate medium, or the like. In addition, it grows by shaking culture in a commonly used meat extract liquid medium, casein / soybean mixed peptone liquid medium, or the like. The culture temperature is preferably 20-30 ° C.
LC1301株の生化学的性状を以下に示す。
(1)グラム染色性:グラム陽性
(2)硝酸塩の還元:−
(3)脱窒反応:−
(4)VPテスト:+
(5)インドールの生成:−
(6)硫化水素の生成:−
(7)クエン酸の利用:−
(8)色素産生:−
(9)ウレアーゼ:−
(10)オキシダーゼ:−
(11)アラニン-フェニルアラニル-プロリンアリルアミダーゼ活性:+
(12)ピログルタミン酸アリルアミダーゼ活性:−
(13)N-アセチル-β-グルコサミニダーゼ活性:−
(14)グリシル-トリプトファン-アリルアミダーゼ活性:−
(15)生育の範囲:pH4.5〜9.5、温度10〜45℃
(16)酸素に対する態度:通性嫌気性
(17)炭素源の利用性; D-リボース:+、D-マンニトール:+、D-ソルビトール:−、ラクトース:−、D-トレハロース:+、D -ラフィノース:−、サッカロース:−、L-アラビノース:−、D -アラビトール:−、シクロデキストリン:+、グリコーゲン:−、プルラン:−、マルトース:+、D-メリビオース:−、メレチトース:−、タガトース:+
(18)糖類の分解:β-グルコシダーゼ活性:+、β-グルクロニダーゼ活性:+、β-ガラクトシダーゼ活性:−、α-ガラクトシダーゼ活性:−、βマンノシダーゼ活性:−
(19)馬尿酸の分解:−
(20)アルギニンの分解:+
(21)溶血性:α溶血型
(22)抗KG-抗血清:−
(23)抗KG+抗血清:−
The biochemical properties of LC1301 strain are shown below.
(1) Gram staining: Gram positive
(2) Reduction of nitrate:
(3) Denitrification reaction: −
(4) VP test: +
(5) Production of indole: −
(6) Production of hydrogen sulfide: −
(7) Use of citric acid:-
(8) Pigment production:-
(9) Urease:-
(10) Oxidase: −
(11) Alanine-phenylalanyl-proline allylamidase activity: +
(12) pyroglutamate allylamidase activity: −
(13) N-acetyl-β-glucosaminidase activity: −
(14) Glycyl-tryptophan-allylamidase activity:
(15) Range of growth: pH 4.5-9.5, temperature 10-45 ° C
(16) Attitude toward oxygen: facultative anaerobic
(17) Availability of carbon source; D-ribose: +, D-mannitol: +, D-sorbitol:-, lactose:-, D-trehalose: +, D-raffinose:-, saccharose:-, L-arabinose :-, D-arabitol:-, cyclodextrin: +, glycogen:-, pullulan:-, maltose: +, D-melibiose:-, meretitose:-, tagatose: +
(18) Decomposition of saccharide: β-glucosidase activity: +, β-glucuronidase activity: +, β-galactosidase activity: −, α-galactosidase activity: −, β mannosidase activity: −
(19) Decomposition of hippuric acid:-
(20) Decomposition of arginine: +
(21) Hemolytic: alpha hemolytic type
(22) Anti-KG-antiserum:-
(23) Anti-KG + antiserum: −
ラクトコッカス・ガルビエLC1301株の特許微生物寄託を行った(寄託機関:独立行政法人製品評価技術基盤機構特許微生物寄託センター、所在地:日本国千葉県木更津市かずさ鎌足2-5-8、受託番号:NITE P-01653、受領日:2013年7月9日、日本において採取された菌株)。 Deposited a patent microorganism for Lactococcus garbier LC1301 strain (Deposit organization: National Institute for Product Evaluation Technology Patent Microorganism Deposit Center, Location: 2-5-8 Kazusa Kama feet, Kisarazu-shi, Chiba, Japan, accession number: NITE P-01653, date of receipt: strain collected in Japan on July 9, 2013).
なお、本発明は、不活化することにより、血清型が非KG-型かつ非KG+型であるラクトコッカス・ガルビエを起因菌とする新型のレンサ球菌感染症を有効に予防できるものであればよく、このLC1301株を用いる場合のみに狭く限定されない。 The present invention is not limited as long as it can effectively prevent a new type of streptococcal infection caused by Lactococcus garvier whose serotype is non-KG- and non-KG +. However, the present invention is not limited to a narrow case only when this LC1301 strain is used.
<本発明に係る不活化ワクチン製剤について>
本発明は、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエの不活化菌体を含有する、ラクトコッカス・ガルビエを起因菌とする魚類レンサ球菌症に対する不活化ワクチン製剤をすべて包含する。また、本発明は、不活化菌体を有効成分として含有するもののみに狭く限定されず、例えば、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエの培養菌液を不活化処理することにより得られた菌体及び菌液を有効成分として含有する不活化ワクチン製剤、即ち、例えば、不活化菌体と培養液を分離せずに用いることにより、若しくは不活化菌体と培養液を分離しないまま濃縮することにより、不活化菌体と、該菌体以外の菌体由来成分とを含有した場合も広く包含する。
<About an inactivated vaccine preparation according to the present invention>
The present invention includes all inactivated vaccine preparations against fish streptococcal disease caused by Lactococcus garbies, which contain inactivated cells of Lactococcus garbies whose serotypes are non-KG- and non-KG +. To do. Further, the present invention is not narrowly limited to those containing inactivated cells as an active ingredient. For example, a culture solution of Lactococcus galvier whose serotype is non-KG-type and non-KG + type is inactivated. An inactivated vaccine preparation containing the bacterial cells and bacterial liquid obtained as an active ingredient, that is, for example, by using the inactivated bacterial cells and the culture solution without separation, or the inactivated bacterial cells and the culture solution It is widely encompassed when the inactivated cells and components derived from the cells other than the cells are contained by concentrating the cells without separation.
不活化に供する菌体は、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエであればよい。例えば、ラクトコッカス・ガルビエに対する従来の不活化ワクチンを投与したにもかかわらずαレンサ球菌症を発症した魚類から分離して用いてもよい。分離菌は、公知の固形培地・液体培地、例えば、肉エキス寒天平板培地、カゼイン・ダイズ混合ペプトン寒天平板培地、肉エキス液状培地、カゼイン・ダイズ混合ペプトン液状培地などで培養し、増殖させることができる。 The cells to be used for inactivation may be any Lactococcus garbier with a serotype of non-KG− and non-KG +. For example, you may isolate | separate and use from the fish which developed alpha streptococcus despite having administered the conventional inactivation vaccine with respect to Lactococcus garvier. The isolated bacteria can be cultured and propagated in a known solid medium / liquid medium, for example, meat extract agar plate medium, casein / soybean mixed peptone agar plate medium, meat extract liquid medium, casein / soybean mixed peptone liquid medium, etc. it can.
LC1301株は、不活化することによりワクチンとして用いることができる点、特に濃縮や酵素処理などを行わなくてもワクチンとしての効力が高い点、及び、アジュバントとの相性がよく、不活化したものとアジュバントとを混合して用いることにより相乗的なワクチン効力を奏する点などから、本発明に用いるクトコッカス・ガルビエの菌体として最も好適である。 The LC1301 strain can be used as a vaccine by being inactivated, particularly has high efficacy as a vaccine without performing concentration or enzyme treatment, etc., and has good compatibility with adjuvants and has been inactivated. From the standpoint of synergistic vaccine efficacy by using a mixture with an adjuvant, etc., it is most suitable as a cell of C. galvia used in the present invention.
不活化菌体は、例えば、培養菌液に対し、物理的処理(紫外線照射、X線照射、熱処理、超音波処理など)、化学的処理(ホルマリン・クロロホルムなどによる有機溶媒処理、酢酸などの弱酸による酸処理、アルコール・塩素・水銀などによる処理)などを行うことにより作製できる。 Inactivated cell bodies are, for example, physical treatment (ultraviolet irradiation, X-ray irradiation, heat treatment, ultrasonic treatment, etc.), chemical treatment (organic solvent treatment with formalin / chloroform, etc., weak acid such as acetic acid, etc. Acid treatment with alcohol, treatment with alcohol, chlorine, mercury, etc.).
例えば、培養菌液にホルマリンを0.001〜2.0%、より好適には0.01〜1.0%の容量濃度で添加し、培養菌液を4〜30℃で、1〜3日間感作することにより、ホルマリンによる不活化を行うことができる。例えば、緩衝液などで不活化処理菌体を洗浄してホルマリンなどの不活化剤を除去したり、不活化処理菌体に中和剤を添加して中和したりしてもよい。また、膜ろ過や遠心分離などにより不活化処理菌体を回収したり、菌体と培養液を分離しないまま培養菌液を濃縮したりしてもよい。 For example, formalin is added to the cultured bacterial solution at a volume concentration of 0.001 to 2.0%, more preferably 0.01 to 1.0%, and the cultured bacterial solution is sensitized at 4 to 30 ° C. for 1 to 3 days. Inactivation can be performed. For example, the inactivated cells may be washed with a buffer solution to remove inactivating agents such as formalin, or neutralized by adding a neutralizing agent to the inactivated cells. Alternatively, the inactivated cells may be collected by membrane filtration or centrifugation, or the culture solution may be concentrated without separating the cells and the culture solution.
不活化ワクチン製剤に含まれる不活化菌体の量は、特に制限はないが、例えば、不活化前の菌体の量が103〜1011CFU/mLの範囲が好適で、107〜1011CFU/mLの範囲がより好適である。 The amount of the inactivated cells contained in the inactivated vaccine preparation is not particularly limited, but for example, the amount of the cells before inactivation is preferably in the range of 10 3 to 10 11 CFU / mL, 10 7 to 10 A range of 11 CFU / mL is more preferred.
本発明に係る不活化ワクチン製剤は、アジュバントを含有するもの、即ち、上述の不活化菌体とアジュバントとを有効成分として少なくとも含有するものであってもよい。 The inactivated vaccine preparation according to the present invention may contain an adjuvant, that is, one containing at least the above-described inactivated cells and an adjuvant as active ingredients.
例えば、LC1301株のように、アジュバントとの相性のよい菌の不活化菌体とアジュバントとを混合して用いることにより相乗的なワクチン効力を発揮させることができる。 For example, synergistic vaccine efficacy can be exerted by using a mixture of an inactivated bacterial cell having good affinity with an adjuvant and an adjuvant, such as the LC1301 strain.
アジュバントには、公知のものを広く用いることができる。例えば、動物油(スクアレンなど)又はそれらの硬化油、植物油(パーム油、ヒマシ油など)又はそれらの硬化油、無水マンニトール・オレイン酸エステル、流動パラフィン、ポリブテン、カプリル酸、オレイン酸、高級脂肪酸エステルなどを含む油性アジュバント、PCPP、サポニン、グルコン酸マンガン、グルコン酸カルシウム、グリセロリン酸マンガン、可溶性酢酸アルミウム、サリチル酸アルミニウム、アクリル酸コポリマー、メタクリル酸コポリマー、無水マレイン酸コポリマー、アルケニル誘導体ポリマー、水中油型エマルジョン、第四級アンモニウム塩を含有するカチオン脂質などの水溶性アジュバント、水酸化アルミニウム(ミョウバン)、水酸化ナトリウムなどの沈降性アジュバント、コレラ毒素、大腸菌易熱性毒素などの微生物由来毒素成分、その他、ベントナイト、ムラミルジペプチド誘導体、インターロイキンなどが挙げられる。また、これらを混合したものでもよい。 A wide variety of known adjuvants can be used. For example, animal oil (such as squalene) or hydrogenated oil thereof, vegetable oil (such as palm oil, castor oil) or hydrogenated oil thereof, mannitol / oleic anhydride, liquid paraffin, polybutene, caprylic acid, oleic acid, higher fatty acid ester, etc. Oil-based adjuvants, including PCPP, saponin, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymer, methacrylic acid copolymer, maleic anhydride copolymer, alkenyl derivative polymer, oil-in-water emulsion, Derived from microorganisms such as water-soluble adjuvants such as cationic lipids containing quaternary ammonium salts, precipitation adjuvants such as aluminum hydroxide (alum), sodium hydroxide, cholera toxin, and E. coli heat-labile toxin Examples include toxin components, bentonite, muramyl dipeptide derivatives, and interleukins. Moreover, what mixed these may be used.
本発明は、上記の不活化ワクチン、例えば、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエの不活化菌体を含有する不活化ワクチンと、血清型がKG-型又はKG+型のラクトコッカス・ガルビエの不活化菌体を含有する不活化ワクチンと、を含有する混合不活化ワクチン製剤であってもよい。この混合不活化ワクチン製剤を魚類に投与することにより、既知のレンサ球菌症及び既知のものとは異なる新型のレンサ球菌症の両者を同時に有効に予防できる可能性がある。 The present invention provides the inactivated vaccine described above, for example, an inactivated vaccine containing an inactivated cell of Lactococcus garvier having a serotype of non-KG-type and non-KG + type, and a serotype of KG-type or KG + type. A mixed inactivated vaccine preparation containing an inactivated vaccine containing inactivated cells of Lactococcus garbier. By administering this mixed inactivated vaccine preparation to fish, both known streptococcal disease and a new type of streptococcal disease different from known ones may be effectively prevented at the same time.
また、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエの不活化菌体を含有する不活化ワクチン、又は、その不活化ワクチンと血清型がKG-型又はKG+型のラクトコッカス・ガルビエの不活化菌体を含有する不活化ワクチンと、を含有する混合不活化ワクチンに加え、他の疾患に対するワクチン、例えば、β溶血性レンサ球菌症不活化ワクチン、ビブリオ病不活化ワクチン、イリドウイルス病不活化ワクチン、類結節症不活化ワクチン、ストレプトコッカス・ジスガラクチエ感染症不活化ワクチンなどのいずれか又は複数との混合ワクチン製剤であってもよい。 In addition, an inactivated vaccine containing inactivated serotypes of Lactococcus galbiae whose serotype is non-KG-type and non-KG +, or the inactivated vaccine and serotype of KG-type or KG + type Lactococcus In addition to a mixed inactivated vaccine containing an inactivated bacterium of Garbier, and a vaccine against other diseases, such as a β-hemolytic streptococcal inactivated vaccine, a vibrio disease inactivated vaccine, an iridovirus It may be a mixed vaccine preparation with any or a plurality of disease inactivated vaccines, nodular disease inactivated vaccines, Streptococcus dysgalactie infectious disease inactivated vaccines, and the like.
その他、目的・用途などに応じて、緩衝剤、等張化剤、無痛化剤、防腐剤、抗菌剤、抗酸化剤などを適宜添加してもよい。 In addition, a buffer, an isotonic agent, a soothing agent, a preservative, an antibacterial agent, an antioxidant, and the like may be added as appropriate according to the purpose and use.
緩衝剤の好適な例として、例えば、リン酸塩、酢酸塩、炭酸塩、クエン酸塩等の緩衝液などを用いることができる。 As a suitable example of a buffering agent, buffer solutions, such as a phosphate, acetate, carbonate, citrate, etc. can be used, for example.
等張化剤の好適な例として、例えば、塩化ナトリウム、グリセリン、D-マンニトールなどを用いることができる。 As a suitable example of an isotonizing agent, sodium chloride, glycerin, D-mannitol etc. can be used, for example.
無痛化剤の好適な例として、例えば、ベンジルアルコールなどを用いることができる。 As a suitable example of the soothing agent, for example, benzyl alcohol or the like can be used.
防腐を目的とした薬剤の好適な例として、例えば、チメロサール、パラオキシ安息香酸エステル類、フェノキシエタノール、クロロブタノール、ベンジルアルコール、フェネチルアルコール、デヒドロ酢酸、ソルビン酸、その他、各種防腐剤、抗生物質、合成抗菌剤などを用いることができる。 Suitable examples of antiseptic agents include, for example, thimerosal, paraoxybenzoates, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, other antiseptics, antibiotics, and synthetic antibacterials. An agent or the like can be used.
抗酸化剤の好適な例として、例えば、亜硫酸塩、アスコルビン酸などを用いることができる。 As a suitable example of an antioxidant, a sulfite, ascorbic acid, etc. can be used, for example.
その他、この薬剤には、補助成分、例えば、保存・効能の助剤となる光吸収色素(リボフラビン、アデニン、アデノシンなど)、安定化のためのキレート剤・還元剤(ビタミンC、クエン酸など)、炭水化物(ソルビトール、ラクトース、マンニトール、デンプン、シュークロース、グルコース、デキストランなど)、カゼイン消化物、各種ビタミンなどを含有させてもよい。 In addition, this drug includes auxiliary ingredients such as light-absorbing dyes (riboflavin, adenine, adenosine, etc.) that serve as storage and efficacy aids, and chelating / reducing agents (vitamin C, citric acid, etc.) for stabilization. , Carbohydrates (sorbitol, lactose, mannitol, starch, sucrose, glucose, dextran, etc.), casein digests, various vitamins, and the like may be included.
ワクチン製剤の剤型などについては、公知のものを採用でき、特に限定されない。例えば、液体製剤として用いてもよいし、凍結乾燥などの処置の後、餌などに混入させてもよい。 About the dosage form etc. of a vaccine formulation, a well-known thing can be employ | adopted and it does not specifically limit. For example, it may be used as a liquid preparation, or may be mixed in food after treatment such as lyophilization.
<本発明に係る不活化ワクチン製剤製造方法について>
本発明は、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエの菌体を不活化する工程を含む、ラクトコッカス・ガルビエを起因菌とする魚類レンサ球菌症に対する不活化ワクチン製剤製造方法をすべて包含する。
<About the inactivated vaccine preparation manufacturing method according to the present invention>
The present invention provides an inactivated vaccine preparation against fish streptococcal disease caused by Lactococcus garbier, comprising a step of inactivating cells of Lactococcus garbier whose serotype is non-KG- and non-KG + Includes all methods.
上述の通り、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエの菌体を不活化することにより、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエを起因菌とする新型の魚類レンサ球菌症に有効な不活化ワクチン製剤を製造できる。 As described above, by inactivating cells of non-KG- and non-KG + serotypes of Lactococcus garvier, serotypes of non-KG- and non-KG + -type Lactococcus garvier A new type of inactivated vaccine preparation effective against fish streptococcal disease can be produced.
本発明に係る不活化ワクチン製剤は、例えば、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエの菌体を増殖する工程、その菌体を不活化する工程などにより行うことができる。また、不活化工程の後に、適宜、その不活化菌体にアジュバントなどを添加する工程を加えてもよい。 The inactivated vaccine preparation according to the present invention can be performed by, for example, a step of growing a non-KG-type and non-KG + -type Lactococcus galvier cell, a step of inactivating the cell, etc. . Moreover, you may add the process of adding an adjuvant etc. to the inactivated microbial cell suitably after the inactivation process.
用いる菌体、及び、その菌体の不活化方法については、上記の通りである。また、目的・用途に応じて、上述のアジュバント、緩衝剤、等張化剤、無痛化剤、防腐剤、抗酸化剤などを適宜添加してもよい。 The cells used and the method for inactivating the cells are as described above. Further, according to the purpose and use, the above-mentioned adjuvant, buffer, tonicity agent, soothing agent, preservative, antioxidant and the like may be appropriately added.
<不活化ワクチン製剤製造のための菌の使用について>
本発明は、血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエを起因菌とするレンサ球菌症に対する不活化ワクチン製剤製造のための該菌の使用を広く包含する。
<About the use of bacteria for the production of inactivated vaccine preparations>
The present invention broadly encompasses the use of the bacterium for the production of an inactivated vaccine preparation against streptococcal disease caused by Lactococcus galbiae whose serotype is non-KG- and non-KG +.
血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエを起因菌とするレンサ球菌症に対する不活化ワクチン製剤製造のために、上述の血清型が非KG-型かつ非KG+型のラクトコッカス・ガルビエを広く用いることができる。 In order to produce an inactivated vaccine preparation against streptococcal disease caused by non-KG- and non-KG + serotypes of Lactococcus garvier, the above-mentioned serotypes are non-KG- and non-KG + lactococcus・ Garbier can be widely used.
<本発明に係る魚類レンサ球菌症の予防方法>
上述の不活化ワクチン製剤、又は上述の混合不活化ワクチン製剤を投与する、ラクトコッカス・ガルビエを起因菌とする魚類レンサ球菌症の予防方法を広く包含する。
<Method for preventing fish streptococcal disease according to the present invention>
Widely encompassing a method for preventing fish streptococci caused by Lactococcus garvier, which comprises administering the inactivated vaccine preparation described above or the mixed inactivated vaccine preparation described above.
上述の不活化ワクチン製剤を魚類に投与することにより、従来の不活化ワクチンでは予防できない新型の魚類レンサ球菌症の発生・伝播・蔓延を有効に予防できる。また、上述の混合不活化ワクチン製剤を魚類に投与することにより、既知のレンサ球菌症及び既知のものとは異なる新型のレンサ球菌症の両者を同時に有効に予防できる可能性がある。 By administering the above-mentioned inactivated vaccine preparation to fish, it is possible to effectively prevent the occurrence, spread, and spread of a new type of fish streptococcal disease that cannot be prevented by conventional inactivated vaccines. Moreover, by administering the above-mentioned mixed inactivated vaccine preparation to fish, there is a possibility that both known streptococci and a new type of streptococci different from known ones can be effectively prevented at the same time.
適用対象となる魚類として、例えば、ブリ属魚類(ブリ、カンパチ、ヒラマサなど)、マダイ・チダイ・ヒラメ・シマアジ・マアジ・サバ・マグロなどの海水魚、ウナギ、ニジマスなど、ラクトコッカス・ガルビエを起因菌とするレンサ球菌症に罹患する魚類が挙げられる。 Examples of applicable fishes include, for example, yellowtail fish (yellowtail, amberjack, flatfish, etc.), saltwater fish such as red sea bream, sea bream, Japanese flounder, sea horse mackerel, mackerel, mackerel, tuna, eel, rainbow trout, etc. Examples include fish suffering from streptococcal disease.
不活化ワクチン製剤の投与方法として、例えば、注射法、浸漬法、経口法などが挙げられる。 Examples of the administration method of the inactivated vaccine preparation include an injection method, a dipping method, and an oral method.
注射法の場合、例えば、不活化前の菌体の量を103〜1011CFU/mLの範囲に調製した不活化ワクチン製剤を、0.05〜3.0mL筋肉内又は腹腔内に投与する。即ち、不活化前の菌体の量が103〜1011CFU/mLであり、一回当たりの投与量が0.05〜3.0mLである不活化ワクチン製剤、その用量で筋肉内又は腹腔内に投与する不活化ワクチン製剤は、ラクトコッカス・ガルビエに対する従来の不活化ワクチンでは予防できない新型の魚類レンサ球菌症の予防に有効である。 In the case of the injection method, for example, an inactivated vaccine preparation prepared by adjusting the amount of bacterial cells before inactivation in the range of 10 3 to 10 11 CFU / mL is administered intramuscularly or intraperitoneally to 0.05 to 3.0 mL. That is, the inactivated vaccine preparation in which the amount of bacterial cells before inactivation is 10 3 to 10 11 CFU / mL and the dose per dose is 0.05 to 3.0 mL, administered intramuscularly or intraperitoneally at that dose The inactivated vaccine preparation is effective in preventing a new type of fish streptococcal disease that cannot be prevented by the conventional inactivated vaccine against Lactococcus garbier.
浸漬法の場合、例えば、不活化前の菌体の量を103〜109CFU/mLの範囲に調製した不活化ワクチン製剤含有液に、対象魚を0.05〜48時間浸漬する。即ち、不活化前の菌体の量が103〜109CFU/mLであり、一回当たり0.05〜48時間の浸漬を行う不活化ワクチン製剤は、ラクトコッカス・ガルビエに対する従来の不活化ワクチンでは予防できない新型の魚類レンサ球菌症の予防に有効である。 In the case of the immersion method, for example, the target fish is immersed for 0.05 to 48 hours in an inactivated vaccine preparation-containing solution prepared in a range of 10 3 to 10 9 CFU / mL of the bacterial cells before inactivation. That is, the inactivated vaccine preparation in which the amount of bacterial cells before inactivation is 10 3 to 10 9 CFU / mL and soaked for 0.05 to 48 hours per time is a conventional inactivated vaccine against Lactococcus garvier. It is effective in preventing a new type of fish streptococcal disease that cannot be prevented.
経口法の場合、例えば、不活化前の菌体の量を103〜1011CFU/mLの範囲に調製した不活化ワクチン製剤を混合した飼料を自由摂餌させ、1〜20日間の連続投与を行う。即ち、不活化前の菌体の量が103〜1011CFU/mLであり、1〜20日間の経口連続投与を行う不活化ワクチン製剤は、ラクトコッカス・ガルビエに対する従来の不活化ワクチンでは予防できない新型のレンサ球菌症の予防に有効である。 In the case of the oral method, for example, a feed mixed with an inactivated vaccine preparation prepared in a range of 10 3 to 10 11 CFU / mL of the amount of bacterial cells before inactivation is freely fed and continuously administered for 1 to 20 days I do. That is, the inactivated vaccine preparation in which the amount of bacterial cells before inactivation is 10 3 to 10 11 CFU / mL and continuous oral administration for 1 to 20 days is a preventive in the conventional inactivated vaccine against Lactococcus garvier It is effective in preventing a new type of streptococcal disease that cannot be achieved.
このうち、注射法による腹腔内投与が、感染予防効果が高く、免疫持続期間が長いため、最も好適である。 Of these, intraperitoneal administration by the injection method is most preferred because of its high infection prevention effect and long immunity duration.
不活化ワクチン製剤の投与回数は、その作用が持続する限り1回でよいが、対象魚類の大きさ、ワクチン効果の度合いなどに応じて、1〜60日間隔で複数回投与してもよい。その他、複数の投与方法を適宜組み合わせて、対象魚類に不活化ワクチン製剤を投与してもよい。 The inactivated vaccine preparation may be administered once as long as its action continues, but may be administered multiple times at 1-60 day intervals depending on the size of the target fish, the degree of vaccine effect, and the like. In addition, the inactivated vaccine preparation may be administered to the target fish by appropriately combining a plurality of administration methods.
実施例1では、Lactococcus garvieaeに対する従来の不活化ワクチンを投与したにもかかわらずαレンサ球菌症の発症が疑われたブリより、原因菌の分離・同定を試みた。 In Example 1, an attempt was made to isolate and identify the causative bacteria from yellowtail that was suspected to develop α-streptococcal disease despite administration of a conventional inactivated vaccine against Lactococcus garvieae.
愛媛県愛南町のブリ養殖場において、2013年4月、分養殖作業の際、L.garvieaeに対する従来の不活化ワクチンを投与した養殖魚群中より、α溶血性レンサ球菌症が疑われる個体5尾を認めた。 At the yellowtail farm in Ainan-cho, Ehime Prefecture, in April 2013, five individuals suspected of having α-hemolytic streptococcal disease from among a group of farmed fish that were administered a conventional inactivated vaccine against L. garvieae Admitted.
剖検所見では、心外膜炎、肝臓黄白色斑の散在、腎臓及び脾臓の軽度肥大、脳の軽度発赤が認められた。 Autopsy findings showed epicarditis, liver yellow spots, mild enlargement of the kidneys and spleen, and mild redness of the brain.
罹患個体のうちの1尾(体重:1,950g、体長:44.5cm)の脳を採取し、菌分離を試みた。白金耳でSCDb寒天培地(カゼイン・ダイズ混合ペプトン液体培地(SCDb)に寒天を加えた培地、以下同じ)に脳組織を塗布し、25℃で1日間培養した。単集落を釣菌し、新たなSCDb寒天培地に接種し、単集落分離を行った。これを10w/v%グリセリン含むSCD液体培地にMcFarland No.5程度の濁度となるよう懸濁し、保存した。 The brain of one of the affected individuals (weight: 1,950 g, body length: 44.5 cm) was collected and attempted to isolate bacteria. The brain tissue was applied to an SCDb agar medium (a medium obtained by adding agar to casein / soybean mixed peptone liquid medium (SCDb), the same applies hereinafter) with a platinum loop, and cultured at 25 ° C. for 1 day. Single colonies were fished and inoculated on a new SCDb agar medium to separate single colonies. This was suspended in a SCD liquid medium containing 10 w / v% glycerin so as to have a turbidity of about McFarland No. 5, and stored.
分離菌の顕微鏡検査を行った結果、既知のL.garvieaeと同様、グラム陽性レンサ球菌の形状を示した。 As a result of microscopic examination of the isolated bacteria, the shape of Gram-positive streptococci was shown as in known L. garvieae.
L.garvieae、Streptococcus iniae、Streptococcus parauberis、及び、Streptococcus dysgalactiaeに対する特異的プライマーを用いて、PCR法による遺伝子検査を行った結果、L.garvieaeに対する特異的プライマーを用いた場合のみ、増幅が認められた。 As a result of genetic testing by PCR using specific primers for L. garvieae, Streptococcus iniae, Streptococcus parauberis, and Streptococcus dysgalactiae, amplification was observed only when specific primers for L. garvieae were used. .
分離菌の溶血性試験を行った。コロンビア5%ヒツジ血液寒天培地に分離菌を播種し、培養した結果、既知のL.garvieaeと同様の不完全溶血環が形成され、α溶血性を示した。 A hemolytic test of the isolated bacteria was performed. As a result of inoculating and culturing the isolated bacteria on a Columbia 5% sheep blood agar medium, an incomplete hemolytic ring similar to that of known L. garvieae was formed, and α hemolysis was exhibited.
グラム陽性球菌同定キット「Rapid ID32 Strep(日本ビオメリュー)」を用いて、分離菌の生化学的性状試験を行った。結果を表1及び表2に示す。なお、表1及び表2中、「陽性率」はキットの添付文書に記載された既知のL.garvieaeの陽性率を表す。表1及び表2に示す通り、分離菌は、既知のL.garvieaeの陽性率と全項目で一致した。 Biochemical characterization of the isolated bacteria was performed using a Gram-positive cocci identification kit "Rapid ID32 Strep (Nippon Biomelieu)". The results are shown in Tables 1 and 2. In Tables 1 and 2, “positive rate” represents the positive rate of known L. garvieae described in the package insert of the kit. As shown in Tables 1 and 2, the isolated bacteria matched the known positive rate of L. garvieae in all items.
上記の通り、L.garvieaeには、KG-型及びKG+型の2つの血清型が知られている。そこで、抗L.garvieae KS-7M株血清(KG-型)、抗L.garvieae YT-3株血清(KG+型)、抗L.garvieae KG7409株血清(KG+型)の三種類の抗血清を用いて、凝集試験を行った。その結果、いずれの血清においても凝集は確認されなかった。 As described above, two serotypes of KG-type and KG + type are known for L. garvieae. Therefore, anti-L. Garvieae KS-7M strain serum (KG-type), anti-L. Garvieae YT-3 strain serum (KG + type), and anti-L. Garvieae KG7409 strain serum (KG + type) were used. The agglutination test was conducted. As a result, no aggregation was confirmed in any serum.
以上の結果より、愛媛県愛南町においてL.garvieaeに対する従来の不活化ワクチンを投与したにもかかわらずαレンサ球菌症の発症が疑われたブリより分離した菌を、血清型が非KG-型かつ非KG+型の新規のLactococcus garvieaeと同定し、Lactococcus garvieae LC1301と命名した。同菌を独立行政法人製品評価技術基盤機構特許微生物寄託センターに寄託した(受託番号:NITE P-01653)。 Based on the above results, serotypes of non-KG-type serotypes were isolated from yellowtail that was suspected of developing alpha streptococcal disease despite administration of a conventional inactivated vaccine against L. garvieae in Ainan, Ehime Prefecture. Moreover, it was identified as a new non-KG + type Lactococcus garvieae and named Lactococcus garvieae LC1301. The same bacterium was deposited with the Patent Microorganisms Depositary Center, National Institute of Technology and Evaluation (Accession Number: NITE P-01653).
実施例2では、Lactococcus garvieaeに対する従来の不活化ワクチン製剤が、分離菌(L.garvieae LC1301株)に感染した魚類に対して有効かどうかを検証した。 In Example 2, it was verified whether a conventional inactivated vaccine preparation against Lactococcus garvieae was effective against fish infected with an isolated bacterium (L. garvieae LC1301 strain).
水酸化ナトリウム水溶液でpH8に調整したSCD液体培地にLC1301株を播種し、24時間静置培養して、LC1301株の培養菌液を調製した。
The LC1301 strain was inoculated on an SCD liquid medium adjusted to
天然種苗のブリ(投与時平均体重95.0g)10尾及び天然種苗のカンパチ(投与時平均体重113.0g)10尾に、ブリ属魚類用混合不活化ワクチン製剤「ピシバック 注 3混 L-9(共立製薬株式会社製、「ピシバック」は登録商標、以下同じ)」を0.1mLずつ腹腔内注射し、それぞれ120L容水槽で14日間飼育した。対照群として、ブリ及びカンパチ各10尾にPBSを0.1mLずつ腹腔内注射し、それぞれ120L容水槽で14日間飼育した。飼育設定水温を25.0℃とした。 Mixed inactivated vaccine formulation for yellowtail fish “Pishibak * 3 L-9 (Kyoritsu) on 10 natural seedlings (average weight 95.0 g when administered) and 10 natural seedlings amberjack (average weight 113.0 g when administered) “Pishibakku” (registered trademark, the same applies hereinafter) manufactured by Pharmaceutical Co., Ltd. was intraperitoneally injected 0.1 mL at a time and each was reared in a 120 L water tank for 14 days. As a control group, 0.1 ml of PBS was intraperitoneally injected into 10 yellowtails and amberjack each, and each was reared in a 120 L water tank for 14 days. The breeding water temperature was 25.0 ° C.
14日間経過後、調製したLC1301株の培養菌液をPBSで5倍に希釈したものを0.1mLずつ各供試魚に腹腔内注射し、攻撃を行った。攻撃菌量は3.4×107CFU/尾であった。攻撃後、14日間、設定水温25℃の水槽で飼育し、観察した。 After 14 days, 0.1 ml each of the prepared culture solution of LC1301 strain diluted 5 times with PBS was injected intraperitoneally into each test fish for attack. The amount of attacking bacteria was 3.4 × 10 7 CFU / tail. After the attack, the animals were reared in a water tank with a set water temperature of 25 ° C. for 14 days and observed.
結果を図1及び図2に示す。図1は、ブリに対し、L.garvieaeに対する従来の不活化ワクチン製剤で免疫した後、分離菌(LC1301株)で攻撃した場合における生存率を示すグラフ、図2は、カンパチに対し、L.garvieaeに対する従来の不活化ワクチン製剤で免疫した後、分離菌(LC1301株)で攻撃した場合における生存率を示すグラフである。両図のグラフの横軸(攻撃後日数)は、分離菌による攻撃を行ってからの経過日数を、縦軸(生存率、単位:%)は、分離菌による攻撃後の生存率を、それぞれ表す。両グラフ中、「ピシバック」の折れ線はブリ属魚類用混合不活化ワクチン製剤で免疫した場合の生存率を、「対照群」の折れ線は対照としてワクチン製剤の代わりにPBSを投与した場合の生存率を、それぞれ表わす。 The results are shown in FIGS. FIG. 1 is a graph showing the survival rate when a yellowtail is immunized with a conventional inactivated vaccine preparation against L. garvieae and then challenged with an isolated bacterium (LC1301 strain), and FIG. It is a graph which shows the survival rate at the time of immunizing with the conventional inactivated vaccine formulation with respect to garvieae, and attacking with the isolation bacteria (LC1301 strain | stump | stock). In both graphs, the horizontal axis (days after attack) represents the number of days elapsed since the attack with the isolate, and the vertical axis (survival rate, unit:%) represents the survival after the attack with the isolate. Represent. In both graphs, the “Pishibac” line indicates the survival rate when immunized with a mixed inactivated vaccine preparation for the fish species of the genus Buri, and the “Control group” line indicates the survival rate when PBS is administered instead of the vaccine preparation as a control. Respectively.
図1及び図2に示す通り、ブリとカンパチのいずれにおいても、対照群では生存率が0%であったのに対し、ブリ属魚類用不活化ワクチン製剤で免疫した場合でも生存率は10%にとどまった。この結果は、血清型が非KG-型かつ非KG+型の新規に同定されたL.garvieaeによる魚類レンサ球菌症に対し、L.garvieaeに対する従来の不活化ワクチン製剤が有効でないこと、即ち、従来のL.garvieaeに対する従来の不活化ワクチン製剤では、この新型レンサ球菌症を有効に予防できないことを示す。 As shown in FIG. 1 and FIG. 2, in both yellowtail and amberjack, the survival rate was 0% in the control group, whereas the survival rate was 10% even when immunized with an inactivated vaccine preparation for the fish species Stayed in. This result shows that the conventional inactivated vaccine preparation against L. garvieae is not effective against fish streptococci caused by newly identified L. garvieae whose serotype is non-KG- and non-KG +, This shows that the conventional inactivated vaccine preparation against L. garvieae cannot effectively prevent this new streptococcal disease.
実施例3では、血清型が非KG-型かつ非KG+型のLactococcus garvieaeに対する不活化ワクチンを作製し、ワクチンとしての効力を調べた。 In Example 3, an inactivated vaccine against Lactococcus garvieae whose serotype was non-KG− and non-KG + was prepared, and its efficacy as a vaccine was examined.
不活化菌液の調製を以下の通り行った。200mL容三角フラスコに、SCD液体培地100mLを入れ、凍結保存したL.garvieae LC1301株を1白金耳接種し、25℃で25時間緩やかに振盪培養した。寒天平板希釈法による生菌数は1.4×109CFU/mLであった。この培養菌液をPBSで10倍又は100倍に希釈し、その希釈菌液を元培養菌液とした。500mL容三角フラスコにSCD液体培地300mLを入れ、元培養菌液0.3mLを接種し、25℃で24時間振盪培養した。これに終濃度0.3vol%となるよう日本薬局方ホルマリンを加え、培養時と同じ条件下で2日間感作させ、不活化菌液を調製した。 The inactivated bacteria solution was prepared as follows. A 200 mL Erlenmeyer flask was charged with 100 mL of SCD liquid medium, and 1 platinum loop of the cryopreserved L. garvieae LC1301 strain was inoculated and cultured with gentle shaking at 25 ° C. for 25 hours. The viable cell count by the agar plate dilution method was 1.4 × 10 9 CFU / mL. This cultured bacterial solution was diluted 10-fold or 100-fold with PBS, and the diluted bacterial solution was used as the original cultured bacterial solution. A 500 mL Erlenmeyer flask was charged with 300 mL of SCD liquid medium, 0.3 mL of the original culture was inoculated, and cultured with shaking at 25 ° C. for 24 hours. To this, Japanese Pharmacopoeia formalin was added to a final concentration of 0.3 vol%, and sensitized for 2 days under the same conditions as in culture to prepare an inactivated bacterial solution.
また、アジュバント乳化抗原の調製を以下の通り行った。上述の不活化菌液を10,000×g、4℃、5分間の条件で遠心分離し、上清を除去した後、PBSで懸濁し、2.0×1011CFU/mLの不活化菌体/PSB溶液60gを調整した。実験室用乳化機を用いて、W/O型のアジュバントであるMONTANIDE ISA-763A VG(SEPPIC社製)140gを氷冷条件で撹拌しながら、不活化菌体/PSB溶液60gを添加し、予備乳化した。氷冷状態を維持しながら、撹拌数を6,000rpmに上昇させ、15分間乳化し、アジュバント乳化抗原とした。 In addition, an adjuvant emulsified antigen was prepared as follows. Centrifugation of the above-mentioned inactivated bacteria solution under conditions of 10,000 xg, 4 ° C, 5 minutes, removing the supernatant, suspending in PBS, 2.0 x 10 11 CFU / mL inactivated bacteria / PSB solution 60 g was adjusted. Using a laboratory emulsifier, add 140 g of inactivated cells / PSB solution while stirring 140 g of MOTANIDE ISA-763A VG (manufactured by SEPPIC), a W / O type adjuvant, under ice-cooling conditions. Emulsified. While maintaining the ice-cooled state, the number of stirring was increased to 6,000 rpm and emulsified for 15 minutes to obtain an adjuvant-emulsified antigen.
天然種苗のブリ(投与時平均体重87.1g)45尾を15尾ずつ三群に分け、各群に、それぞれ、不活化菌液(抗原量:1.4×108CFU/0.1mL)、アジュバント乳化抗原(:1.0×108CFU/0.1mL)、PBS(陰性対照物質)を0.1mLずつ腹腔内注射し、それぞれ500L容水槽で14日間飼育した。飼育設定水温を25.0℃とした。 Forty-nine yellowtail yellowtail (average body weight 87.1g at the time of administration) is divided into three groups of 15 each, and each group is divided into inactivated bacterial solution (antigen amount: 1.4 x 10 8 CFU / 0.1mL), adjuvant emulsified antigen (: 1.0 × 10 8 CFU / 0.1 mL) and 0.1 mL of PBS (negative control substance) were intraperitoneally injected, and each was reared in a 500 L water tank for 14 days. The breeding water temperature was 25.0 ° C.
14日間の飼育期間中、生死、摂餌行動、遊泳行動などを観察した。その結果、14日間の免疫期間を通じて、体色、摂餌行動、遊泳行動などに特に異常は認められなかった。これより、各抗原の安全性には問題はないと判断した。 During the 14-day breeding period, we observed life and death, feeding behavior and swimming behavior. As a result, there were no abnormalities in body color, feeding behavior, swimming behavior, etc. throughout the 14-day immunization period. From this, it was judged that there was no problem in the safety of each antigen.
14日間経過後、実施例2とほぼ同様の手順で調製したLC1301株の培養菌液をPBSで5倍に希釈した後、0.1mLずつ各供試魚に腹腔内注射し、攻撃を行った。攻撃菌量は4.2×106CFU/尾であった。攻撃後、14日間、設定水温25℃の水槽で飼育し、観察した。 After 14 days, the culture solution of the LC1301 strain prepared in substantially the same manner as in Example 2 was diluted 5-fold with PBS, and then 0.1 mL each was intraperitoneally injected into each test fish for attack. The amount of attacking bacteria was 4.2 × 10 6 CFU / tail. After the attack, the animals were reared in a water tank with a set water temperature of 25 ° C. for 14 days and observed.
結果を図3に示す。図3は、ブリに対し、L.garvieae LC1301株の不活化菌体を含有する不活化ワクチンで免疫した後、分離菌(LC1301株)で攻撃した場合における生存率を示すグラフである。図3のグラフの横軸(攻撃後日数)は、分離菌による攻撃を行ってからの経過日数を、縦軸(生存率、単位:%)は、分離菌による攻撃後の生存率を、それぞれ表す。同グラフ中、「不活化菌液」の折れ線は不活化菌液で免疫した場合の生存率を、「アジュバント乳化抗原」の折れ線はアジュバント乳化抗原で免疫した場合の生存率を、「対照群」の折れ線は対照としてPBSを投与した場合の生存率を、それぞれ表わす。 The results are shown in FIG. FIG. 3 is a graph showing the survival rate when yellowtail is immunized with an inactivated vaccine containing inactivated cells of the L. garvieae LC1301 strain and then challenged with a isolate (LC1301 strain). In the graph of FIG. 3, the horizontal axis (days after attack) represents the number of days elapsed since the attack with the isolate, and the vertical axis (survival rate, unit:%) represents the survival after the attack with the isolate. Represent. In the graph, the broken line of “inactivated bacterial solution” indicates the survival rate when immunized with the inactivated bacterial solution, and the broken line of “adjuvant emulsion antigen” indicates the survival rate when immunized with the adjuvant emulsion antigen, “control group”. Each of the polygonal lines represents the survival rate when PBS was administered as a control.
図3に示す通り、PBS投与群(陰性対照)では、ブリの生存率が0%であったのに対し、不活化菌液投与群及びアジュバント乳化抗原投与群では、ブリの生存率が100%であった。また、全ての生存魚について、体色、摂餌行動、遊泳行動などに特に異常は認められず、剖検所見からも特に異常は認められなかった。その他、攻撃に用いた菌は生存魚からは分離されなかった。 As shown in FIG. 3, the survival rate of yellowtail was 0% in the PBS administration group (negative control), whereas the survival rate of yellowtail was 100% in the inactivated bacterial solution administration group and the adjuvant emulsified antigen administration group. Met. In addition, for all surviving fish, there were no abnormalities in body color, feeding behavior, swimming behavior, etc., and no abnormalities were also observed from autopsy findings. In addition, the fungus used for the attack was not isolated from the live fish.
これらの結果は、血清型が非KG-型かつ非KG+型のL.garvieaeの不活化菌体を含有する不活化ワクチンでブリ属魚類を免疫することにより、従来のワクチンが有効でない新型レンサ球菌症を有効に予防できることを示唆する。 These results show that a new streptococci that is not effective against conventional vaccines is obtained by immunizing yellowtail fish with an inactivated vaccine containing inactivated cells of L. garvieae whose serotype is non-KG- and non-KG +. This suggests that the disease can be effectively prevented.
また、本実施例では、不活化菌液単独でも高いワクチン効果を示すとともに、アジュバントを添加した場合でも同様の高いワクチン効果を維持できた。このことは、L.garvieae LC1301株が免疫原性の高い株であることを示唆し、また、同株がアジュバントとの親和性の高い株であることを示唆する。 Moreover, in this Example, the inactivated bacterial solution alone showed a high vaccine effect, and the same high vaccine effect could be maintained even when an adjuvant was added. This suggests that the L. garvieae LC1301 strain is a highly immunogenic strain, and that the strain is a strain having a high affinity with an adjuvant.
実施例4では、血清型が非KG-型かつ非KG+型のL.garvieaeを用いて作製した不活化ワクチンが、既知のL.garvieaeによるレンサ球菌症の予防にも有効かどうか、検討した。 In Example 4, it was examined whether an inactivated vaccine produced using L. garvieae whose serotype was non-KG− and non-KG + was also effective in preventing streptococcal disease caused by known L. garvieae.
実施例3と同様の手順で、不活化菌液及びアジュバント乳化抗原を調製した。実施例3と同様、天然種苗のブリ(投与時平均体重87.1g)45尾を15尾ずつ三群に分け、各群に、それぞれ、不活化菌液(抗原量:1.4×108CFU/0.1mL)、アジュバント乳化抗原(:1.0×108CFU/0.1mL)、PBS(陰性対照物質)を0.1mLずつ腹腔内注射し、それぞれ500L容水槽で14日間飼育した。 Inactivated bacterial solution and adjuvant emulsified antigen were prepared in the same procedure as in Example 3. Similarly to Example 3, 45 yellowtails (average body weight 87.1 g at the time of administration) of natural seedlings were divided into three groups of 15 fish, and each group was given an inactivated bacterial solution (antigen amount: 1.4 × 10 8 CFU / 0.1 mL), adjuvant emulsified antigen (: 1.0 × 10 8 CFU / 0.1 mL), and PBS (negative control substance) were injected intraperitoneally at 0.1 mL each, and each was reared in a 500 L water tank for 14 days.
実施例2などと同様の手順で、既知のLactococcus garvieae株であるKS-7C株(1997年に静岡県で分離されたブリ由来の株、KG-型)の培養菌液を調製した。免疫から14日間経過後、実施例2などと同様の手順で、調製したKS-7C株の培養菌液をPBSで5倍に希釈した後、0.1mLずつ各供試魚に腹腔内注射し、攻撃を行った。攻撃菌量は1.5×107CFU/尾であった。攻撃後、14日間、設定水温25℃の水槽で飼育し、観察した。 By the same procedure as in Example 2 and the like, a culture solution of a known Lactococcus garvieae strain KS-7C strain (a yellowtail-derived strain isolated in Shizuoka Prefecture in 1997, KG-type) was prepared. After 14 days from immunization, the prepared culture solution of KS-7C strain was diluted 5-fold with PBS in the same procedure as in Example 2 and then injected intraperitoneally into each test fish in 0.1 mL units. Attacked. The amount of attacking bacteria was 1.5 × 10 7 CFU / tail. After the attack, the animals were reared in a water tank with a set water temperature of 25 ° C. for 14 days and observed.
結果を図4に示す。図4は、ブリに対し、L.garvieae LC1301株の不活化菌体を含有する不活化ワクチンで免疫した後、既知のL.garvieae(KS-7C株)で攻撃した場合における生存率を示すグラフである。図4のグラフの横軸(攻撃後日数)は、分離菌による攻撃を行ってからの経過日数を、縦軸(生存率、単位:%)は、分離菌による攻撃後の生存率を、それぞれ表す。同グラフ中、「不活化菌液」の折れ線は不活化菌液で免疫した場合の生存率を、「アジュバント乳化抗原」の折れ線はアジュバント乳化抗原で免疫した場合の生存率を、「対照群」の折れ線は対照としてPBSを投与した場合の生存率を、それぞれ表わす。 The results are shown in FIG. FIG. 4 is a graph showing the survival rate when yellowtail is immunized with an inactivated vaccine containing inactivated cells of the L. garvieae LC1301 strain and then challenged with a known L. garvieae (KS-7C strain). It is. In the graph of FIG. 4, the horizontal axis (days after attack) represents the number of days elapsed since the attack with the isolate, and the vertical axis (survival rate, unit:%) represents the survival after the attack with the isolate. Represent. In the graph, the broken line of “inactivated bacterial solution” indicates the survival rate when immunized with the inactivated bacterial solution, and the broken line of “adjuvant emulsion antigen” indicates the survival rate when immunized with the adjuvant emulsion antigen, “control group”. Each of the polygonal lines represents the survival rate when PBS was administered as a control.
図4に示す通り、PBS投与群(陰性対照)及び不活化菌液投与群のブリ生存率は0%、アジュバント乳化抗原投与群のブリ生存率は13%であった。アジュバント乳化抗原投与群では、PBS投与群と比較して、死亡に至る日数が遅く、また、生存魚2尾から攻撃に用いた菌は分離されず、異常な剖検所見も認められなかった。このように、L.garvieae LC1301株を用いて作製した不活化ワクチンには、死亡の遅延をもたらすものの、既知のL.garvieaeによるレンサ球菌症に対するワクチンとしての効力は認められなかった。 As shown in FIG. 4, the yellowtail survival rate of the PBS administration group (negative control) and the inactivated bacterial solution administration group was 0%, and the yellowtail survival rate of the adjuvant emulsified antigen administration group was 13%. In the adjuvant emulsified antigen-administered group, the number of days leading to death was delayed compared to the PBS-administered group, and the bacteria used for the attack were not isolated from the two surviving fish, and no abnormal autopsy findings were observed. Thus, although the inactivated vaccine produced using L. garvieae LC1301 strain caused a delay in death, efficacy as a vaccine against known L. garvieae streptococci was not recognized.
実施例5では、二価混合不活化ワクチンを作製し、ワクチンとしての効力を調べた。 In Example 5, a bivalent mixed inactivated vaccine was prepared, and its efficacy as a vaccine was examined.
L.garvieae LC1301株の不活化菌液の調製を以下の通り行った。200mL容三角フラスコに、SCD液体培地(栄研化学株式会社製)100mLを入れ、凍結保存したLC1301株を1白金耳接種し、25℃で約24時間振盪培養した。この培養菌液をPBSで10倍ずつ102倍まで階段希釈し、その希釈菌液を元培養菌液とした。次に、5L容培養装置に、SCD液体培地(自家配合により、前記と同じ成分組成に調製したもの)2.5Lを入れ、元培養菌液4.5mLを接種し、pH7.0以上の状態を維持しながら25℃で約48時間振盪培養した。これに終濃度0.2vol%となるよう日本薬局方ホルマリンを加え、培養時と同じ条件下で2日間感作させ、不活化菌液を調製した。寒天平板希釈法による生菌数測定試験を行った結果、不活化前生菌数は2.7×108CFU/mLであった。この不活化菌液を遠心分離して上清を除去した後、ホルマリン0.1vol%添加PBSで2倍濃縮し、L.garvieae LC1301株の不活化抗原とした。 The inactivated bacterial solution of L. garvieae LC1301 strain was prepared as follows. A 200 mL Erlenmeyer flask was charged with 100 mL of SCD liquid medium (manufactured by Eiken Chemical Co., Ltd.), and one platinum loop of the cryopreserved LC1301 strain was inoculated and cultured at 25 ° C. for about 24 hours with shaking. This cultured bacterial solution was serially diluted 10-fold with PBS to 10 2 times, and the diluted bacterial solution was used as the original cultured bacterial solution. Next, add 2.5 L of SCD liquid medium (prepared to the same component composition as above) in a 5 L culture device, inoculate 4.5 mL of the original culture, and maintain a pH of 7.0 or higher The mixture was cultured with shaking at 25 ° C. for about 48 hours. To this, Japanese Pharmacopoeia formalin was added to a final concentration of 0.2 vol%, and sensitized for 2 days under the same conditions as in culture to prepare an inactivated bacterial solution. As a result of the viable cell count test by the agar plate dilution method, the viable cell count before inactivation was 2.7 × 10 8 CFU / mL. The inactivated bacterial solution was centrifuged to remove the supernatant, and then concentrated twice with PBS supplemented with formalin 0.1 vol% to obtain an inactivated antigen of the L. garvieae LC1301 strain.
従来の血清型(KG-型)であるL.garvieae KS-7M株の不活化菌液の調製を以下の通り行った。200mL容三角フラスコに、SCD液体培地(栄研化学株式会社製)100mLを入れ、凍結保存したKS-7M株を1白金耳接種し、25℃で約24時間振盪培養した。この培養菌液をPBSで10倍ずつ102倍まで階段希釈し、その希釈菌液を元培養菌液とした。次に、5L容培養装置に、SCD液体培地(自家配合により、前記と同じ成分組成に調製したもの)2.5Lを入れ、元培養菌液4.5mLを接種し、攪拌しながら25℃で24時間培養した。これに終濃度0.3vol%となるよう日本薬局方ホルマリンを加え、培養時と同じ条件下で2日間感作させ、不活化菌液を調製した。寒天平板希釈法による生菌数測定試験を行った結果、不活化前生菌数は1.8×109CFU/mLであった。この不活化菌液を遠心分離して上清を除去した後、ホルマリン0.1vol%添加PBSで2倍濃縮し、L.garvieae KS-7M株の不活化抗原とした。 An inactivated bacterial solution of the conventional serotype (KG-type) L. garvieae KS-7M strain was prepared as follows. A 200 mL Erlenmeyer flask was charged with 100 mL of SCD liquid medium (manufactured by Eiken Chemical Co., Ltd.), one platinum loop of the frozen KS-7M strain was inoculated, and cultured with shaking at 25 ° C. for about 24 hours. This cultured bacterial solution was serially diluted 10-fold with PBS to 10 2 times, and the diluted bacterial solution was used as the original cultured bacterial solution. Next, add 2.5 L of SCD liquid medium (prepared to the same component composition as described above by self-mixing) into a 5 L culture device, inoculate 4.5 mL of the original culture, and stir at 25 ° C. for 24 hours with stirring. Cultured. To this, Japanese Pharmacopoeia formalin was added to a final concentration of 0.3 vol%, and sensitized for 2 days under the same conditions as in culture to prepare an inactivated bacterial solution. As a result of the viable cell count test by the agar plate dilution method, the viable cell count before inactivation was 1.8 × 10 9 CFU / mL. This inactivated bacterial solution was centrifuged to remove the supernatant, and then concentrated twice with PBS supplemented with formalin 0.1 vol% to obtain an inactivated antigen of the L. garvieae KS-7M strain.
新血清型であるL.garvieae LC1301株の不活化抗原と、従来血清型であるL.garvieae KS-7M株の不活化抗原を等量混合し、二価ワクチンとした。 An inactivated antigen of the new serotype L. garvieae LC1301 and an inactivated antigen of the conventional serotype L. garvieae KS-7M were mixed to prepare a bivalent vaccine.
天然種苗のブリ(投与時平均体重45.9g)30尾及び天然種苗のカンパチ(投与時平均体重63.0g)27尾に、本実施例で調製した二価ワクチンを0.1mLずつ腹腔内注射し、それぞれ200L容水槽で14日間飼育した。対照群として、ブリ29尾及びカンパチ25尾にPBSを0.1mLずつ腹腔内注射し、それぞれ200L容水槽で14日間飼育した。飼育設定水温を25.0℃とした。二価ワクチンの投与抗原量は、L.garvieae LC1301株が1.4×108CFU/0.1mL/dose、L.garvieae KS-7M株が9.0×108CFU/0.1mL/doseであった。 30 ml of natural seedlings (average body weight 45.9 g when administered) and 27 species of natural seedlings (average body weight 63.0 g when administered) were intraperitoneally injected with 0.1 mL each of the bivalent vaccine prepared in this example, It was raised for 14 days in a 200L water tank. As a control group, 0.1 ml of PBS was intraperitoneally injected into 29 yellowtails and 25 amberjacks, and each was reared in a 200 L water tank for 14 days. The breeding water temperature was 25.0 ° C. The administered antigen amount of the bivalent vaccine was 1.4 × 10 8 CFU / 0.1 mL / dose for the L. garvieae LC1301 strain and 9.0 × 10 8 CFU / 0.1 mL / dose for the L. garvieae KS-7M strain.
飼育期間中、生死、摂餌行動、遊泳行動の観察を行った。その結果、免疫投与群では、対照群との比較においても、14日間の免疫期間を通じて、体色、摂餌行動、遊泳行動に異常を認めなかった。本結果より、この二価ワクチンは、ブリ及びカンパチに対する安全性において問題はないと推測した。 During the breeding period, we observed life and death, feeding behavior, and swimming behavior. As a result, in the immunized group, no abnormality was observed in body color, feeding behavior, and swimming behavior throughout the immunization period of 14 days, even in comparison with the control group. From this result, it was speculated that this bivalent vaccine had no problem in terms of safety against yellowtail and amberjack.
続いて、従来の血清型(KG-型)の攻撃株用として、L.garvieae KS-7C株の培養菌液を調製した。そして、免疫から14日間経過後、免疫投与した供試魚のうちのブリ15尾及びカンパチ15尾、並びに対照群の供試魚のうちのブリ15尾及びカンパチ15尾に、それぞれ、そのKS-7C株の培養菌液をPBSで100倍に希釈したものを0.1mLずつ各供試魚に腹腔内注射し、攻撃を行った。攻撃菌量は2.3×106CFU/尾であった。攻撃後、14日間、設定水温25℃の水槽で飼育し、観察した。 Subsequently, a culture solution of L. garvieae KS-7C strain was prepared for a conventional serotype (KG-type) challenge strain. Then, after 14 days from immunization, 15 bream and 15 amberjack of the immunized test fish, and 15 yellow and amberjack of the control group test fish, respectively, the KS-7C strain Each culture was diluted intraperitoneally with 0.1 mL of the culture solution obtained by diluting the cultured bacterial solution of 100 times with PBS for attack. The amount of attacking bacteria was 2.3 × 10 6 CFU / tail. After the attack, the animals were reared in a water tank with a set water temperature of 25 ° C. for 14 days and observed.
また、新血清型(非KG-型かつ非KG+型)の攻撃株用として、L.garvieae LC1311株の培養菌液を調製した。そして、KS-7C株の場合と同様、免疫から14日間経過後、免疫投与した供試魚のうちの残りのブリ15尾及びカンパチ14尾、並びに対照群の供試魚のうちの残りのブリ14尾及びカンパチ10尾に、それぞれ、そのLC1311株の培養菌液をPBSで100倍に希釈したものを0.1mLずつ各供試魚に腹腔内注射し、攻撃を行った。攻撃菌量は2.0×106CFU/尾であった。攻撃後、14日間、設定水温25℃の水槽で飼育し、観察した。 In addition, a culture solution of L. garvieae LC1311 strain was prepared for use with a new serotype (non-KG-type and non-KG + -type) challenge strain. Then, as in the case of the KS-7C strain, 14 days after immunization, the remaining 15 yellowtails and 14 amberjack of the immunized test fish, and the remaining 14 yellowtails of the control group test fish Each of 10 fishes and amberjack was challenged by intraperitoneally injecting 0.1 mL each of the LC1311 strain culture solution diluted 100-fold with PBS into each test fish. The amount of attacking bacteria was 2.0 × 10 6 CFU / tail. After the attack, the animals were reared in a water tank with a set water temperature of 25 ° C. for 14 days and observed.
結果を図5〜図8に示す。図5は、ブリに対し、二価ワクチンで免疫した後、既知のL.garvieae(KS-7C株)で攻撃した場合における生存率を示すグラフ、図6は、カンパチに対し、二価ワクチンで免疫した後、既知のL.garvieae(KS-7C株)で攻撃した場合における生存率を示すグラフ、図7は、ブリに対し、二価ワクチンで免疫した後、新血清型L.garvieae(LC1311C株)で攻撃した場合における生存率を示すグラフ、図8は、カンパチに対し、二価ワクチンで免疫した後、新血清型L.garvieae(LC1311株)で攻撃した場合における生存率を示すグラフである。各グラフの横軸(攻撃後日数)は、攻撃を行ってからの経過日数を、縦軸(生存率、単位:%)は、攻撃後の生残率を、それぞれ表す。各グラフ中、「二価ワクチン」の折れ線は上記の二価ワクチンで免疫した場合の生残率を、「対照群」の折れ線は対照として免疫時にPBSを投与した場合の生残率を、それぞれ表わす。 The results are shown in FIGS. FIG. 5 is a graph showing the survival rate when a yellowtail is immunized with a bivalent vaccine and then challenged with a known L. garvieae (KS-7C strain). FIG. 6 is a bivalent vaccine against amberjack. FIG. 7 is a graph showing the survival rate when challenged with a known L. garvieae (KS-7C strain) after immunization. FIG. 7 shows a new serotype L. garvieae (LC1311C) after immunization against yellowtail with a bivalent vaccine. FIG. 8 is a graph showing the survival rate when a serotype was challenged with a new serotype L. garvieae (LC1311 strain) after immunization with a bivalent vaccine. is there. In each graph, the horizontal axis (days after attack) represents the number of days elapsed since the attack, and the vertical axis (survival rate, unit:%) represents the survival rate after the attack. In each graph, the “bivalent vaccine” line represents the survival rate when immunized with the above bivalent vaccine, and the “control group” line represents the survival rate when PBS was administered during immunization as a control. Represent.
図5に示す通り、ブリに対し、対照群としてPBSを投与した後KS-7C株で攻撃した場合の生存率は6.7%であったのに対し、二価ワクチンで免疫した後KS-7C株で攻撃した場合の生存率は100%であった。また、図6に示す通り、カンパチに対し、対照群としてPBSを投与した後KS-7C株で攻撃した場合の生存率は0%であったのに対し、二価ワクチンで免疫した後KS-7C株で攻撃した場合の生存率は100%であった。 As shown in FIG. 5, the survival rate when the PBS was administered as a control group and then challenged with the KS-7C strain was 6.7%, whereas the KS-7C strain was immunized with the bivalent vaccine. The survival rate when attacking with was 100%. In addition, as shown in FIG. 6, the survival rate was 0% when camp-7 was administered with PBS as a control group and then challenged with the KS-7C strain, whereas KS- The survival rate when attacked with 7C strain was 100%.
加えて、図7に示す通り、ブリに対し、対照群としてPBSを投与した後LC1311株で攻撃した場合の生存率は0%であったのに対し、二価ワクチンで免疫した後LC1311株で攻撃した場合の生存率は100%であった。また、図8に示す通り、カンパチに対し、対照群としてPBSを投与した後LC1311株で攻撃した場合の生存率は0%であったのに対し、二価ワクチンで免疫した後LC1311株で攻撃した場合の生存率は64.3%であった。 In addition, as shown in FIG. 7, the survival rate was 0% when yellowtail was challenged with LC1311 strain after PBS was administered as a control group, whereas the LC1311 strain was immunized with bivalent vaccine. The survival rate when attacked was 100%. In addition, as shown in FIG. 8, the survival rate was 0% against the amberjack when PBS was administered as a control group and then challenged with the LC1311 strain, whereas the challenge was challenged with the LC1311 strain after immunization with the bivalent vaccine. The survival rate was 64.3%.
以上の結果は、ラクトコッカス・ガルビエを起因菌とする魚類レンサ球菌症に対するこの二価不活化ワクチンが、従来の血清型の魚類レンサ球菌症と新血清型(非KG-型かつ非KG+型)の魚類レンサ球菌症のいずれの防除にも有効であることを示す。従って、本結果は、ラクトコッカス・ガルビエを起因菌とするブリ・カンパチなどの魚類のレンサ球菌症に関して、従来血清型と新血清型の二価不活化混合ワクチンが、新血清型を含む広範な魚類レンサ球菌症の野外での防除に有効であることを示唆する。 The above results show that this bivalent inactivated vaccine against fish streptococcal disease caused by Lactococcus garvier is a conventional serotype of fish streptococci and new serotypes (non-KG- and non-KG +) It is effective in controlling any fish streptococcal disease. Therefore, the present results show that the conventional serotype and new serotype bivalent inactivated mixed vaccines include a wide range of new serotypes in relation to streptococcal disease in fish such as yellowtail and amberjack caused by Lactococcus garbier. This suggests that it is effective in the field control of fish streptococci.
実施例6では、新血清株であるL.garvieae LC1301株の薬剤感受性を調べた。 In Example 6, the drug sensitivity of the new serum strain L. garvieae LC1301 was examined.
薬剤ディスクとして、SNディスク・エリスロマイシン薬剤感受性試験用(日水製薬株式会社製)、及び、BDセンシ・ディスク・オキシテトラサイクリン薬剤感受性試験用(ベクトン・デッキンソン製)を用いて、それらの薬剤に対する感受性を調べた。 Use the SN disk erythromycin drug sensitivity test (manufactured by Nissui Pharmaceutical Co., Ltd.) and the BD sensi disk oxytetracycline drug sensitivity test (manufactured by Becton Dickinson) as drug disks. Examined.
その結果、エリスロマイシン薬剤感受性試験では阻止円径が22.9mmであり、薬剤感受性であると判定された。また、オキシテトラサイクリン薬剤感受性試験では、阻止円径が28.9mmであり、薬剤感受性であると判定された。 As a result, in the erythromycin drug sensitivity test, the inhibition circle diameter was 22.9 mm, and it was determined to be drug sensitive. In the oxytetracycline drug sensitivity test, the inhibition circle diameter was 28.9 mm, and it was determined that the drug was sensitive.
以上の通り、新血清株であるL.garvieae LC1301株は、エリスロマイシン及びオキシテトラサイクリンに感受性であることが分かった。 As described above, the new serum strain L. garvieae LC1301 was found to be sensitive to erythromycin and oxytetracycline.
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