JP2003171291A - Mucosal prophylactic for mastitis - Google Patents

Mucosal prophylactic for mastitis

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Publication number
JP2003171291A
JP2003171291A JP30911699A JP30911699A JP2003171291A JP 2003171291 A JP2003171291 A JP 2003171291A JP 30911699 A JP30911699 A JP 30911699A JP 30911699 A JP30911699 A JP 30911699A JP 2003171291 A JP2003171291 A JP 2003171291A
Authority
JP
Japan
Prior art keywords
cells
cell
preparation
inactivated
mastitis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP30911699A
Other languages
Japanese (ja)
Inventor
Takuya Furumichi
琢也 古路
Toshikatsu Hayashi
俊克 林
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takeda Schering Plough Animal Health KK
Original Assignee
Takeda Schering Plough Animal Health KK
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Schering Plough Animal Health KK filed Critical Takeda Schering Plough Animal Health KK
Priority to JP30911699A priority Critical patent/JP2003171291A/en
Priority to PCT/JP2000/007445 priority patent/WO2001032205A1/en
Priority to AU79564/00A priority patent/AU7956400A/en
Publication of JP2003171291A publication Critical patent/JP2003171291A/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0041—Mammary glands, e.g. breasts, udder; Intramammary administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Reproductive Health (AREA)
  • Oncology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Endocrinology (AREA)
  • Communicable Diseases (AREA)
  • Gynecology & Obstetrics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Fodder In General (AREA)

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mucosal prophylactic for mastitis of a mammal, having excellent practicability. <P>SOLUTION: This mucosal prophylactic comprises an oral or a nasal preparation for preventing the mastitis of the mammal, wherein the preparation contains inactivated cell bodies or a processed product given by eluting the cell bodies and containing outside wall proteins of the cell bodies. The preparation has an excellent phylactic effect on the mastitis of the mammal and is orally or nasally administered as a mucosal vaccine, so that the preparation is extremely practicable. <P>COPYRIGHT: (C)2003,JPO

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【発明の属する技術分野】本発明は、家畜などの哺乳動
物、とりわけ牛を主とする細菌感染による***炎の疾病
被害の予防に有用な経口または経鼻の粘膜予防剤および
これを含有してなる家畜用飼料に関する。TECHNICAL FIELD The present invention relates to an oral or nasal mucosal prophylactic agent useful for preventing the damage of mastitis caused by bacterial infection mainly in mammals such as domestic animals, especially cows, and the same. To livestock feed.

【０００２】[0002]

【従来の技術】牛、馬、羊、山羊、豚などの家畜は分娩
後産仔に授乳することによって育児をする。この家畜の
哺乳期間あるいは人による搾乳期間において、***に細
菌感染を受けて***炎は発症する。なお、牛が分娩する
前の乾乳期にも、***炎に罹患することが認められてい
る。特に牛の***炎の罹患率は高く、日本国およびアメ
リカ合衆国の乳牛の５０％が罹患していると報告されて
いる（安里、獣医界、１３６号、２７−３６頁（１９９
３年）、Cullor、臨床獣医、１２巻（１号）、１７−２
７頁（１９９４年））。牛の***炎には熱感、疼痛、発
赤、腫脹を示す臨床型***炎と、これらの症状は示さな
いものの体細胞数の増加など牛乳に異常を示す潜在性乳
房炎がある。***炎に罹患した場合の損害としては乳量
の減少が著しく、例えば北海道における経済的損失は年
間約３００億円と試算されている。また品質低下による
牛乳販売価格の低下あるいは牛の死亡、淘汰による経済
的損失も甚大である。１９９６年の乳用牛の***・泌尿
生殖器疾患の死廃事故発生率は、外科疾患についで２位
であり、１万頭当たり１２９頭と死廃事故発生率の１９
％を占めた（濱岡ほか、臨床獣医、１７巻（５号）、１
９８−２１７頁、（１９９８年））。牛の***炎の予
防、治療には、ペニシリン、クロキサシリン、セファゾ
リン、ストレプトマイシン、オキシテトラサイクリン、
エリスロマイシンなどの抗生物質軟膏が用いられてい
る。予防には分娩２カ月前の乾乳する当日において抗生
物質軟膏の***内注入が行われている。また、治療には
数日間にわたる抗生物質軟膏の***内注入および抗生物
質の筋肉内投与が行われている。なお、抗生物質投与期
間およびその薬剤の休薬期間中は法律の規制により牛乳
の販売ができないため、経済的損失は著しい。また獣医
師に対する報酬および治療に要する薬剤にも、多大な出
費が労される。2. Description of the Related Art Livestock such as cows, horses, sheep, goats, pigs, etc., raise their offspring by breastfeeding their offspring. During the feeding period of this livestock or the milking period of human beings, mastitis develops due to bacterial infection of the breast. In addition, it has been recognized that mastitis is also affected during the dry period before the calving of calves. In particular, the prevalence of mastitis in cattle is high, and it is reported that 50% of dairy cattle in Japan and the United States are affected (Asato, Veterinary Science, 136, 27-36 (199).
3 years), Cullor, Clinical Veterinary Medicine, Volume 12 (No. 1), 17-2
7 (1994)). Bovine mastitis includes clinical mastitis, which shows heat, pain, redness, and swelling, and latent mastitis, which does not show these symptoms but shows abnormalities in milk such as an increase in the number of somatic cells. As a loss caused by mastitis, the decrease in milk production is remarkable. For example, the economic loss in Hokkaido is estimated to be about 30 billion yen per year. In addition, the milk sales price is reduced due to the deterioration of quality, and the death of cows and the economic loss due to selection are also significant. In 1996, the mortality and urogenital accident rate of dairy cows was the second highest after surgical disease, with 129 per 10,000 cows, which is 19 of the death and accident rates.
% (Hamaoka et al., Clinical Veterinary Medicine, Volume 17 (No. 5), 1
98-217, (1998)). For the prevention and treatment of bovine mastitis, penicillin, cloxacillin, cefazoline, streptomycin, oxytetracycline,
Antibiotic ointments such as erythromycin have been used. For prevention, intramammary injection of antibiotic ointment is performed on the day of dry-drying 2 months before delivery. In addition, treatment involves intramammary injection of antibiotic ointment and intramuscular administration of antibiotics for several days. During the antibiotic administration period and the drug withdrawal period, milk cannot be sold due to legal regulations, so economic loss is significant. A great deal of money is also spent on veterinarian rewards and medications required for treatment.

【０００３】さて、細菌など病原微生物の感染症に対す
る抗生物質投与以外の対策となれば、ワクチン投与によ
る予防療法が一般的である。しかしながら、日本国にお
いては未だ一品目も***炎ワクチンは販売されていな
い。アメリカ合衆国においてはエシェリヒア・コリ （E
scherichia coli）Ｊ−５株を用いたワクチン１品目が
アップジョン社から販売されているものの（Morinほ
か、J. Am. Vet. Med. Assoc. ２１２巻、１４２３−１
４３１頁（１９９８年））、他のワクチンは販売されて
おらず、皮下投与あるいは筋肉内投与のいわゆる注射に
よる投与法を採用しているスタフィロコッカス・アウレ
ウス（Staphylococcus aureus）、ストレプトコッカス
・ウベリス（Streptococcus uberis）のワクチンが試
験的に検討されているにすぎない（Giraudoほか、J. Da
iry Sci.８０巻、８４５−８５３頁（１９９７年）、Fi
nchほか、Vaccine １５巻、１１３８−１１４３（１９
９７年））。As a countermeasure other than the administration of antibiotics against infectious diseases of pathogenic microorganisms such as bacteria, prophylactic therapy by vaccination is generally used. However, none of the mastitis vaccines are sold in Japan. Escherichia coli (E
Although one vaccine using the S. scherichia coli J-5 strain is sold by Upjohn (Morin et al., J. Am. Vet. Med. Assoc. 212, 1423-1).
431 (1998)), other vaccines are not sold, and Staphylococcus aureus, Streptococcus aureus, which employs a so-called injection method of subcutaneous administration or intramuscular administration, is used. uberis) vaccine is only being tested experimentally (Giraudo et al., J. Da.
iry Sci. 80, 845-853 (1997), Fi
nch et al., Vaccine 15 volumes, 1138-1143 (19
1997)).

【０００４】[0004]

【発明が解決しようとする課題】本発明は、経口または
経鼻投与方法によるより実用性のある哺乳動物の***炎
用粘膜ワクチン、すなわち従来知見よりはるかに有効性
の高いワクチンを提供するものである。DISCLOSURE OF THE INVENTION The present invention provides a more practical mucosal vaccine for mammalian mastitis by the oral or nasal administration method, that is, a vaccine which is far more effective than the conventional findings. is there.

【０００５】[0005]

【課題を解決するための手段】本発明者らはかかる背景
のもとに、鋭意研究を重ねたところ、哺乳動物の***炎
の起因菌であるスタフィロコッカス・アウレウス（Stap
hylococcus aureus）ほかの培養菌体をホルマリンなど
を用いて不活化処理した菌体、または菌体を溶菌化した
ものに助成剤（アジュバント）を加え、経口または経鼻
投与することが、そのスタフィロコッカス・アウレウス
（Staphylococcus aureus）等に起因する***炎に対し
て予防効果のあることを見出し、これらの知見に基づい
てさらに研究した結果、本発明を完成した。すなわち、
本発明は〔１〕不活化された菌体もしくは菌体の外壁蛋
白を含む菌体溶出処理物を含有する、哺乳動物の***炎
を予防するための経口または経鼻投与用製剤、〔２〕不
活化された菌体もしくは菌体の外壁蛋白を含む菌体溶出
処理物が、菌体もしくは菌体の外壁蛋白を含む菌体溶出
処理物のホルマリン処理により得られる上記〔１〕項記
載の製剤、〔３〕不活化された菌体の外壁蛋白を含む菌
体溶出処理物が、菌体の加熱処理により得られる上記
〔１〕項記載の製剤、〔４〕不活化された菌体の外壁蛋
白を含む菌体溶出処理物が、不活化された菌体の溶菌処
理により得られる上記〔１〕項記載の製剤、〔５〕さら
に、アジュバントを含有してなる上記〔１〕項記載の製
剤、〔６〕(1)脂溶性物質および腸溶性高分子化合物か
ら選ばれる被覆剤で被覆された製剤、(2)油中水型乳化
液剤または(3)リポソーム製剤である上記〔１〕項記載
の製剤、および〔７〕不活化された菌体もしくは菌体の
外壁蛋白を含む菌体溶出処理物を含有してなる家畜用飼
料に関する。[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies based on such a background and found that Staphylococcus aureus (Stapococcus aureus), which is a causative bacterium of mastitis in mammals,
hylococcus aureus) other cultured bacterial cells are inactivated with formalin or the like, or lysed bacterial cells are supplemented with an auxiliary agent and administered orally or nasally. The present invention has been completed as a result of further discovery based on these findings that it was found to have a preventive effect on mastitis caused by Staphylococcus aureus and the like. That is,
The present invention [1] a preparation for oral or nasal administration for preventing mastitis in mammals, which comprises a microbial cell elution-treated product containing inactivated microbial cells or outer wall proteins of the microbial cells, [2] The preparation according to the above item [1], wherein the microbial cell-eluted product containing the inactivated microbial cell or outer cell protein of the microbial cell is obtained by formalin treatment of the microbial cell or cell-eluted product containing the outer cell protein of the microbial cell. [3] The preparation of the above-mentioned [1], wherein the microbial cell elution-treated product containing inactivated microbial cell outer wall protein is obtained by heat treatment of the microbial cell, [4] outer wall of the inactivated microbial cell The preparation as described in the above item [1], wherein the microbial cell elution-treated product containing the protein is obtained by lysing the inactivated cells, and the preparation according to the above item [1], which further comprises an adjuvant. [6] (1) Coating with a coating material selected from fat-soluble substances and enteric polymer compounds Preparation, (2) water-in-oil emulsion or (3) preparation of the above-mentioned [1], which is a liposome preparation, and [7] inactivated cells or cells containing outer wall proteins of the cells. The present invention relates to a livestock feed containing an eluate-treated product.

【０００６】ワクチンの経口または経鼻投与による免疫
は、分泌液中の分泌型IgA（secretory IgA； sIgA）の
誘導による。すなわち乳腺、消化管、呼吸器などの全身
の粘膜は、外界と直接接しているが、ここでの防御機構
には、（１）リゾチーム、ラクトフェリンなどの非特異
的な防御機構と、（２）特定の外来抗原に対し特異的に
対応する免疫反応による防御機構があげられる。後者の
免疫反応による防御機構もさらに二つに大別され、局
所免疫機構（local immune system）と全身免疫機構
（systemic immune system）がある。局所免疫機構とは
粘膜に広く分布しているリンパ装置より産生分泌される
IgAクラスの免疫グロブリンによって担われる免疫機構
である。これは全身の粘膜系での免疫反応の主体をなし
ているところから、粘膜免疫システム（mucosal immune
system）と呼ばれることが多い。全身免疫機構とは、
こうした粘膜面での特異的、非特異的防御機構を破り、
生体内へ侵入した外来抗原に対して応答する主としてIg
Gと、それに関連したリンパ装置によって担われている
免疫系である。***炎の場合、新たな感染を予防するの
に必要なものは上記の非特異的免疫機構とsIgAによる粘
膜免疫機構である。細菌が粘膜を通過し、乳腺組織内に
侵入して感染が成立するとIgGによる全身免疫機構が作
動する。IgAは粘膜面を介する抗原刺激により誘導され
るが、その過程は哺乳類の腸管について解析されてい
る。すなわち哺乳類の腸管には良く発達したパイエル氏
板（P.P.）が散在し、この部位がIgAの誘導組織である
と考えられている。例えば、経口投与されて腸管に達し
た抗原はP.P.を覆う特殊な上皮細胞であるM細胞より取
り込まれる。M細胞には抗原提示能はなく、その下部の
ドーム領域に存在するマクロファージや樹状細胞が抗原
提示細胞として抗原を処理し、Tリンパ球を活性化す
る。抗原刺激ならびに活性化したTリンパ球の影響をう
けたIgA産生前駆B細胞は腸間膜リンパ節、胸管を経て循
環系に乗り、腸管のみならず呼吸器系、乳腺を含む粘膜
固有層に分布するようになる。以上のことからsIgAによ
る粘膜免疫機構を発動させるためには、経口あるいは経
鼻による経路を介して粘膜に直接ワクチンを感作させる
ことが好ましい。Immunization by oral or nasal administration of a vaccine is based on the induction of secretory IgA (secretory IgA; sIgA) in the secretory fluid. That is, the mucous membranes of the whole body such as the mammary gland, the digestive tract, and the respiratory tract are in direct contact with the outside world. The defense mechanism here is (1) nonspecific defense mechanisms such as lysozyme and lactoferrin, and (2) There is a defense mechanism by an immune reaction that specifically corresponds to a specific foreign antigen. The defense mechanism by the latter immune reaction is roughly divided into two, and there are a local immune system and a systemic immune system. The local immune system is produced and secreted by lymphoid devices widely distributed in the mucosa.
It is an immune mechanism carried by IgA class immunoglobulin. This is because the main body of the immune response is the mucosal system of the whole body,
system) is often called. What is the systemic immune system?
Breaking the specific and non-specific defense mechanism on the mucosal surface,
Primarily Ig that responds to foreign antigens invading the body
It is the immune system that is carried by G and its associated lymphoid apparatus. In the case of mastitis, what is necessary to prevent new infection is the above-mentioned non-specific immune mechanism and mucosal immune mechanism by sIgA. When bacteria pass through the mucous membrane and enter the mammary gland tissue to establish an infection, the systemic immune mechanism by IgG operates. IgA is induced by antigen stimulation via the mucosal surface, and its process has been analyzed in the mammalian intestinal tract. That is, the well-developed Peyer's patches (PP) are scattered in the intestinal tract of mammals, and this site is considered to be the IgA-inducing tissue. For example, the orally administered antigen that reaches the intestinal tract is taken up by M cells, which are special epithelial cells that cover PP. M cells have no antigen presenting ability, and macrophages and dendritic cells existing in the lower dome region process antigens as antigen presenting cells to activate T lymphocytes. IgA-producing precursor B cells affected by antigen stimulation and activated T lymphocytes enter the circulatory system via mesenteric lymph nodes and thoracic duct, and enter not only the intestinal tract but also the respiratory system and lamina propria including the mammary gland. It will be distributed. From the above, in order to activate the mucosal immune mechanism by sIgA, it is preferable to directly sensitize the mucosa with the vaccine via the oral or nasal route.

【０００７】本発明の経口または経鼻投与用製剤作製の
ための供試菌としては、スタフィロコッカス・アウレウ
ス（Staphylococcus aureus）、コアグラーゼ陰性スタ
フィロコッカス（Coagulase negative Staphylococc
i）、ストレプトコッカス・アガラクチア（Streptococc
us agalactiae）、ストレプトコッカス・ディスガラク
チア（Streptococcus dysgalactiae）、ストレプトコッ
カス・ウベリス（Streptococcus uberis）、ストレプト
コッカス・ボビス（Streptococcus bovis）、エンテロ
コッカス・フェーカリス（Enterococcus faecalis）、
コリネバクテリウム・ボビス（Corynebacterium bovi
s）、アクチノマイセス・ピオゲネス（Actinomyces pyo
genes）、エシェリヒア・コリ（Escherichia coli）、
およびクレブシェラ・ニューモニエ（Klebsiella pneum
oniae）が挙げられる。ワクチン作製のための菌体は培
養により得ることができる。菌体が効率的に得られるよ
うに培地および培養条件を適宜選択すればよい。培地と
しては、公知の液体培地または固形培地のいずれを用い
てもよく、ミルクホエーなどを加えて菌体の収量を上げ
たり、起病性を高めるための改良をしてもよい。培養は
前記の培地をその使用法に従って滅菌したものに上記の
菌を殖菌し、３７℃で8時間〜１週間程度、好ましくは2
0〜48時間振盪あるいは静置して行えばよい。菌体は培
養液に懸濁された状態のものでもよいが、後の処理をよ
り効率的に行うためには、培養後、培養物から遠心沈
殿、濾過または凝集沈降などの公知の方法により沈殿物
または濃縮物として回収することが好ましい。本発明に
おける「不活化」処理とは、本菌の有する病原性即ち感
染力を失わせしめる処理のことを言う。具体的には、増
殖能さえ失わせしめればよく、殺菌または後述の「少な
くとも菌体外壁蛋白を溶出せしめる処理」と同一処理に
よってもこの目的が達せられる場合が多い。菌体の不活
化は、ホルマリン、酢酸などの弱酸、クロロホルムなど
で菌体を処理することにより達成できる。特にホルマリ
ン処理により菌体を不活化するのが好ましく、この場
合、ホルマリンは通常0.2〜1.0％の濃度において作用さ
せる。本発明における「不活化された菌体の外壁蛋白を
含む菌体溶出処理物」を得る工程には上記培養菌体に
「少なくとも菌体外壁蛋白を溶出せしめる処理」 および
「不活化処理」 を施すが、どちらの処理を先に行っても
よく、両処理が同時に行われる操作を選択することもで
きる。本発明における菌体溶出処理物は少なくとも菌体
外壁蛋白を含むように調製されるが、これ以外に菌体内
成分などの他の菌体成分が含まれていてもよい。このよ
うな菌体溶出処理物の調製において、「少なくとも菌体
外壁蛋白を溶出せしめる処理」 (以下、単に「溶出処理」
と称することがある) とは、少なくとも菌体外壁蛋白
を部分的にでも菌体から遊離または溶出せしめる処理の
ことを言う。この処理の代表例として、加熱処理または
溶菌処理が挙げられる。加熱処理は、培養菌体またはそ
の懸濁液を、６０〜１００℃好ましくは７０〜８０℃
で、１０〜６０分間、好ましくは２０〜３０分間処理す
ることにより行われる。該処理条件は、菌体量および菌
体を入れ加熱を行う処理容器の材質および形状などに応
じて適宜選択される。溶菌処理とは、菌体細胞壁または
菌体外膜のムコ多糖類または蛋白質の、少なくとも一部
を溶解または遊離せしめる処理であり、例えば、溶菌酵
素処理、物理的破砕法（例、超音波処理破砕法）または
凍結と解凍を繰り返す方法 (凍結融解法)などの物理的
処理、またはこれらを組み合わせて行う処理が挙げられ
る。さらに、培養条件によって起こりうる菌の自己消化
(autolysis) も含まれる。[0007] As a test bacterium for preparing the preparation for oral or nasal administration of the present invention, Staphylococcus aureus, Coagulase negative Staphylococc
i), Streptococcus agalactiae (Streptococc
us agalactiae), Streptococcus dysgalactiae (Streptococcus dysgalactiae), Streptococcus uberis, Streptococcus bovis, Enterococcus faecalis,
Corynebacterium bovi
s), Actinomyces pyogenes
genes), Escherichia coli,
And Klebsiella pneum
oniae). Bacteria for vaccine production can be obtained by culturing. The medium and culture conditions may be appropriately selected so that the bacterial cells can be efficiently obtained. As the medium, any known liquid medium or solid medium may be used, and milk whey or the like may be added to improve the yield of bacterial cells or improve the pathogenicity. Culture is carried out by sterilizing the above-mentioned medium according to its usage method, and culturing the above-mentioned bacteria at 37 ° C. for about 8 hours to 1 week, preferably 2
It may be shaken or left standing for 0 to 48 hours. The cells may be in a state of being suspended in the culture solution, but in order to perform the subsequent treatment more efficiently, the cells may be precipitated from the culture by a known method such as centrifugal precipitation, filtration or aggregation precipitation after the culture. It is preferable to collect it as a product or a concentrate. The “inactivation” treatment in the present invention refers to a treatment for losing the pathogenicity of the bacterium, that is, the infectivity. Specifically, it suffices that the proliferative ability is lost, and this purpose can often be achieved by sterilization or the same treatment as "treatment for eluting at least bacterial outer wall protein" described later. The inactivation of the bacterial cells can be achieved by treating the bacterial cells with a weak acid such as formalin and acetic acid, chloroform and the like. In particular, it is preferable to inactivate the cells by treatment with formalin, and in this case, formalin is usually allowed to act at a concentration of 0.2 to 1.0%. In the step of obtaining the “bacterial cell elution-treated product containing inactivated bacterial outer wall protein” in the present invention,
Although "at least a process for eluting bacterial outer wall protein" and "inactivation process" are performed, either process may be performed first, or an operation in which both processes are performed simultaneously may be selected. The microbial cell elution-treated product in the present invention is prepared so as to contain at least the microbial cell outer wall protein, but other microbial cell components such as microbial cell components may also be contained. In the preparation of such a bacterial cell elution-treated product, "at least a process for eluting bacterial outer wall proteins" (hereinafter, simply "elution treatment")
May be referred to as) means a treatment of at least partially releasing or eluting bacterial outer wall proteins from bacterial cells. Typical examples of this treatment include heat treatment or bacteriolysis treatment. The heat treatment is carried out by culturing cultured cells or a suspension thereof at 60 to 100 ° C, preferably 70 to 80 ° C
For 10 to 60 minutes, preferably 20 to 30 minutes. The treatment conditions are appropriately selected depending on the amount of cells and the material and shape of the treatment container in which the cells are placed and heated. The lysing treatment is a treatment for dissolving or releasing at least a part of the mucopolysaccharide or protein in the cell wall of the microbial cell or the outer membrane of the microbial cell, for example, lytic enzyme treatment, physical disruption method (eg, ultrasonic treatment disruption). Method) or a method of repeating freezing and thawing (freezing and thawing method), or a combination thereof. In addition, autolysis of bacteria that can occur depending on culture conditions
(autolysis) is also included.

【０００８】溶菌酵素としては、菌体細胞壁または菌体
外膜のムコ多糖類または蛋白質に抗原性を損なわない程
度に作用する加水分解酵素であればいずれでもよく、リ
ゾチーム、セルラーゼ、アクロモペプチダーゼ、セラチ
オペプチダーゼ(商品名：ダーゼン、武田薬品工業株式
会社製) などが挙げられ、とりわけ、リゾチームが好ま
しく用いられる。リゾチームは市販の容易に入手出来る
卵白製のものが適当である。溶菌酵素処理は使用する加
水分解酵素により公知の方法に従って行えばよいが、菌
体が溶菌し難い場合には特に、溶菌酵素処理後または溶
菌酵素処理と同時にアニオン性界面活性剤を加えて反応
させるとよい。アニオン性界面活性剤としては、その疎
水基が脂肪族炭化水素基または芳香族炭化水素基である
ことが好ましく、親水基がカルボン酸塩 (一般式は、R1
COONa)、親水基が硫酸エステル塩 (一般式は、R2OSO3N
a)または親水基がスルホン酸塩(一般式は、R2SO3Naまた
はR2C6H4SO3Na)である界面活性剤が挙げられる 〔上記
一般式において R1はCH3(CH2) m (mは２以上１１以下
の整数を示す) を、R2はCH3(CH2)n(nは４以上１５以下
の整数、好ましくは７以上１１以下の整数を示す) で表
される基をそれぞれ示す〕。上記アニオン性界面活性剤
の中でも特に、溶菌酵素が作用し得る温度範囲とりわけ
室温付近で水溶性に優れかつ溶菌性が強いものが好まし
く、また容易に入手出来るという点で、具体的には例え
ば、次のようなものが挙げられる。R1COONaで表される
化合物としては、R1＝CH3(CH2)8のデカン酸またはR1＝C
H3(CH2)10 のラウリン酸などが好ましく用いられる。R2
OSO3Naで表される化合物としては、R2＝CH3(CH2)11のラ
ウリル硫酸ナトリウムなどが好ましく用いられる。R2SO
3Naで表される化合物としては、R2＝CH3(CH2)9のデカン
スルホン酸ナトリウムなどが好ましく、またR2C6H4SO3N
aで表される化合物としては、R2＝CH3(CH2)11であるラ
ウリルベンゼンスルホン酸ナトリウムなどが好ましく用
いられる。物理的破砕法における超音波処理破砕法は市
販の超音波発生機を使用して公知の方法で行えばよく、
例えば、菌体懸濁液を発振周波数２０KHZ、出力が２０
０Wで５〜３０分処理するとよい。凍結融解法は公知の
方法で行えばよく、例えば、−２０℃ 程度に菌体を凍
結した後３０〜４０℃で融解する操作を繰り返すことに
より菌体の１部を破壊する方法が挙げられる。アセトン
等の有機溶媒を用いた脱水乾燥、加温真空乾燥、凍結乾
燥などを行い、さらに固体や粉体の破砕に用いる機械処
理を加えてもよい。「不活化処理」は、上記「少なくと
も菌体外壁蛋白を溶出せしめる処理」と同時に達成され
ることも多いが、必要に応じて、ホルマリン、酢酸など
の弱酸、クロロホルムなどで菌体溶出処理物を処理すれ
ばよい。特にホルマリン処理が好ましい。ホルマリン処
理は該菌体溶出処理物をホルマリン0.2〜1.0％の濃度に
おいて作用させればよい。上記のように作製した不活化
された菌体もしくは菌体の外壁蛋白を含む菌体溶出処理
物を含有する溶菌液はそのまま哺乳動物に経口または経
鼻投与できるが、凍結または冷蔵保存することも可能で
あり、必要時に解凍し、必要に応じて水などで適当に希
釈後、飼料（例、マイロ、トウモロコシ、大麦、大豆油
かす、なたね油かす、ふすま等）に添加した後、家畜な
どの哺乳動物に経口投与してもよい。また、さらに凍結
乾燥、アセトン脱水乾燥などで乾燥粉末化し、さらに適
度に澱粉、糖類で希釈、調製してから投与してもよい。
保存は、雑菌の混入、有効成分の安定性等から、菌体を
処理したものを遠心沈降や濾過などの方法、アセトンや
エタノール等の有機溶媒で蛋白物質を沈殿させる方法、
または硫酸ソーダや硫酸アンモニウムなどの飽和水溶液
を用いる塩析法などにより濃縮し、沈殿として回収後、
乾燥粉末化して保存することが好ましい。乾燥粉末化を
行う場合、適当な媒体、例えばジャガ芋澱粉、とうもろ
こし澱粉等の澱粉類、大豆粉、米糖類の穀類 (粕) 粉末
に吸着させて真空、通風、凍結乾燥などにより乾燥粉末
化してもよい。これらの不活化された菌体もしくは菌体
の外壁蛋白を含む菌体溶出処理物を効果的にとりこませ
るためには、通常アジュバントを加える。アジュバント
としては、コレラ毒素(cholera toxin、CT)、大腸菌易
熱性外毒素(heat-lablile enterotoxin、LT)、またはこ
れらの毒力を除いた変異株由来の無毒性のCT及びLT、も
しくはモノホスフォリルリピッドＡ(monophosphoryl li
pid A)、サポニン(Quillaja saponin)などがあげられ
る。The lytic enzyme may be any hydrolyzing enzyme which acts on the mucopolysaccharide or protein in the cell wall or outer membrane of the cell to the extent that the antigenicity is not impaired. Lysozyme, cellulase, achromopeptidase, Ceratiopeptidase (trade name: Dazen, manufactured by Takeda Pharmaceutical Co., Ltd.) and the like can be mentioned, and lysozyme is particularly preferably used. Suitable lysozyme is commercially available egg white lysozyme. The lytic enzyme treatment may be carried out according to a known method depending on the hydrolase used, but particularly when the cells are difficult to lyse, particularly after the lytic enzyme treatment or simultaneously with the lytic enzyme treatment, an anionic surfactant is added and reacted. Good. As the anionic surfactant, the hydrophobic group is preferably an aliphatic hydrocarbon group or an aromatic hydrocarbon group, the hydrophilic group is a carboxylate (general formula R 1
COONa), a hydrophilic group is a sulfate ester salt (general formula is R 2 OSO 3 N
a) or a surfactant whose hydrophilic group is a sulfonate (general formula is R 2 SO 3 Na or R 2 C 6 H 4 SO 3 Na) (in the above general formula, R 1 is CH 3 (CH 2 ) m (m is an integer of 2 or more and 11 or less) and R 2 is CH 3 (CH 2 ) n (n is an integer of 4 or more and 15 or less, preferably 7 or more and 11 or less) The respective groups are shown below]. Among the above-mentioned anionic surfactants, particularly those having excellent water solubility and strong bacteriolysis in a temperature range in which a lytic enzyme can act, particularly around room temperature, are preferable, and specifically, in that they are easily available, specifically, for example, Some examples are as follows. Examples of the compounds represented by R 1 COONa, R 1 = CH 3 (CH 2) 8 of decanoic acid or R 1 = C
H 3 (CH 2 ) 10 lauric acid and the like are preferably used. R 2
As the compound represented by OSO 3 Na, sodium lauryl sulfate having R 2 = CH 3 (CH 2 ) 11 and the like are preferably used. R 2 SO
As the compound represented by 3 Na, sodium decanesulfonate having R 2 = CH 3 (CH 2 ) 9 is preferable, and R 2 C 6 H 4 SO 3 N
As the compound represented by a, sodium laurylbenzenesulfonate in which R 2 = CH 3 (CH 2 ) 11 is preferably used. Ultrasonic treatment crushing method in the physical crushing method may be performed by a known method using a commercially available ultrasonic generator,
For example, bacterial cell suspension the oscillation frequency 20 kHz Z, output 20
Treatment with 0 W for 5 to 30 minutes is recommended. The freeze-thawing method may be carried out by a known method, and for example, a method of destroying a part of the cells by repeatedly freezing the cells at about -20 ° C and then thawing at 30-40 ° C can be mentioned. Dehydration drying using an organic solvent such as acetone, vacuum drying under heating, freeze drying, etc. may be performed, and mechanical treatment used for crushing solids or powder may be added. The "inactivation treatment" is often achieved at the same time as the above "treatment for elution of at least the outer cell wall protein", but if necessary, formalin, a weak acid such as acetic acid, a microbial cell elution product with chloroform, etc. Just process it. Formalin treatment is particularly preferable. The formalin treatment may be performed by allowing the cell-eluted product to act at a concentration of 0.2 to 1.0% formalin. The lysate containing the inactivated bacterial cells or bacterial cell elution-treated product containing the outer wall protein of the bacterial cells prepared as described above can be orally or nasally administered to a mammal as it is, but may also be frozen or stored under refrigeration. It is possible and thawed when necessary, appropriately diluted with water, etc. if necessary, added to feed (eg, milo, corn, barley, soybean oil meal, rapeseed meal, bran etc.), and then fed to livestock. It may be administered orally to the animal. Further, it may be further lyophilized, dried by acetone dehydration and the like into a dry powder, further appropriately diluted with starch and saccharide, and then prepared and then administered.
Storage is due to contamination of various bacteria, stability of the active ingredient, etc., a method of treating the bacterial cells by centrifugation or filtration, a method of precipitating a protein substance with an organic solvent such as acetone or ethanol,
Alternatively, after concentration by a salting out method using a saturated aqueous solution of sodium sulfate, ammonium sulfate, etc., and collecting as a precipitate,
It is preferable to dry powder and store. In the case of performing dry powdering, it is adsorbed to an appropriate medium, for example, starches such as potato starch, corn starch, soybean powder, rice sugar grain (meal) powder, and dried and powdered by vacuum, ventilation, lyophilization, etc. Good. In order to effectively incorporate these inactivated cells or cells-eluting product containing the outer wall protein of the cells, an adjuvant is usually added. As an adjuvant, cholera toxin (CT), Escherichia coli heat-labile exotoxin (heat-lablile enterotoxin, LT), or nontoxic CT and LT derived from mutant strains excluding these virulence, or monophosphoriol Lulipid A (monophosphoryl li
pid A) and saponin (Quillaja saponin).

【０００９】上記に説明した不活化された菌体もしくは
菌体の外壁蛋白を含む菌体溶出処理物は、哺乳動物に対
して低毒性で安全性に優れていることから、例えば、ラ
ット、マウス、モルモット、ヒツジ、ヤギ、ブタ、ウ
シ、ウマ、ネコ、イヌ、サル、ヒトなどの哺乳動物（好
ましくは、ウシ、ウマ、ヒツジ、ヤギ、ブタなどの家
畜）の***炎予防剤として用いることができる。例え
ば、家畜などの哺乳動物に経口投与する場合には、通常
は上記したごとくに不活化された菌体もしくは菌体の外
壁蛋白を含む菌体溶出処理物を含有する溶菌液を飼料に
添加して与えればよい。また経鼻投与する場合には、不
活化された菌体もしくは菌体の外壁蛋白を含む菌体溶出
処理物を含有する溶菌液を手動または電動の噴霧器(ネ
ブライザー)等を使用して、鼻腔内に噴霧すればよい。
また、経口投与の際には、哺乳動物の消化管内酵素や微
生物による分解をできるだけ少なくすることで、腸管か
らの吸収利用性を高め、より少ない量で強い感染防御能
を発揮させるために、乾燥菌体成分をデカン酸などの脂
肪酸あるいは硬化油脂などの生理的に許容できる脂溶性
物質あるいは腸溶出性製剤の製造に用いられる腸溶性高
分子化合物などの被覆剤で被覆された製剤にしたり、油
脂および界面活性剤を用いた溶菌液を水に分散しない油
中水型乳化液剤としたり、あるはリポソーム製剤などの
既知の製剤技術を応用して調製してもよい。もちろんそ
れらの製剤は、経口投与だけでなく経鼻投与に使用して
もよい。被覆化に用いられる脂溶性物質としては、デカ
ン酸 (カプリン酸)、 ステアリン酸、ラウリン酸、パル
ミチン酸などの脂肪酸、硬化牛脂または硬化大豆油など
の油脂が挙げられる。脂肪酸油脂による被覆化は例え
ば、菌体粉末に溶解した脂溶性物質を添加して混合した
後、冷却固化したものを粉砕造粒するなどの公知の被覆
製剤技術により行われる。また、腸溶性高分子化合物に
よる被覆化としては、ヒドロキシプロピルメチルセルロ
ースフタレートやカルボキシメチルエチルセルロースな
どのセルロース誘導体をアルコールなどの溶媒に溶かし
た中に菌体粉末を分散後、溶媒を除去して粉砕する等の
公知方法を利用してもよい。油中水型乳化に用いられる
油脂としては、大豆油、パーム油、コーン油などが挙げ
られ、界面活性剤としては、グリセリン脂肪酸エステ
ル、ソルビタン脂肪酸エステル、プロピレングリコール
脂肪酸エステルなどが挙げられる。油中水型乳化は例え
ば、水溶性とした菌体溶液と界面活性剤を添加した油脂
とを攪拌又は超音波処理にて乳化するなどの公知の方法
で行われる。リポソーム製剤に用いられるリン脂質とし
ては卵黄レシチン、ジパルミトイルフォスファチジルコ
リン、ジステアロイルフォスファチジルコリン、ジオレ
オイルフォスファチジルコリン等のリン酸ジエステルの
アシル基が飽和又は不飽和の脂肪酸残基で、親水基がコ
リン、エタノールアミン、セリン、イノシトール、グリ
セロール等であるリン脂質やリゾレシチン等があげら
れ、もちろん上記複数のリン脂質を組み合わせて使用し
てもよい。製剤化は、逆相蒸発法、超音波処理法などの
自体公知の方法で行われる。上記で説明した調製物（製
剤または飼料）を哺乳動物（例、牛、馬、羊、山羊、豚
などの家畜等）に経口または経鼻投与する際の投与量と
その期間ないし投与間隔については、投与する哺乳動物
の種類などの要因で一概には言えないが、投与量は哺乳
動物の体重１ｋｇ、１日１回当たりの菌体湿重量当たり
0.01〜１０００ｍｇを１〜２１日間隔で１〜１０回経口
または経鼻投与すればよい。The bacterium-eluting product containing the inactivated bacterium or the outer wall protein of the bacterium described above has low toxicity to mammals and is excellent in safety. , A guinea pig, sheep, goat, pig, cow, horse, cat, dog, monkey, human and other mammals (preferably cattle, horses, sheep, goats, pigs and other livestock) can be used as a mastitis preventive agent it can. For example, when orally administered to mammals such as livestock, usually, a lysate containing an inactivated bacterial cell or a bacterial cell elution-treated product containing an outer wall protein of the bacterial cell as described above is added to the feed. And give it. When administered intranasally, the lysate containing the inactivated bacterial cells or the bacterial cell elution-treated product containing the outer wall proteins of the bacterial cells is intranasally administered using a manual or electric sprayer (nebulizer). You can spray it on.
In addition, when administered orally, by minimizing the degradation by enzymes and microorganisms in the digestive tract of mammals, the availability of absorption from the intestinal tract is increased, and in order to exert a strong infection defense ability in a smaller amount, dry The microbial component is a fatty acid such as decanoic acid or a physiologically acceptable fat-soluble substance such as hardened oil or fat, or a preparation coated with a coating agent such as an enteric polymer used in the production of enteric-elutable preparations. Alternatively, the lysate solution containing a surfactant may be used as a water-in-oil emulsion that does not disperse in water, or may be prepared by applying a known formulation technique such as liposome formulation. Of course, those formulations may be used for nasal as well as oral administration. Fat-soluble substances used for coating include fatty acids such as decanoic acid (capric acid), stearic acid, lauric acid and palmitic acid, and fats and oils such as hardened beef tallow or hardened soybean oil. The coating with the fatty acid oil is carried out by a known coating preparation technique such as adding and mixing a fat-soluble substance dissolved in bacterial cell powder, mixing and cooling and solidifying the mixture, and pulverizing and granulating. Further, as the coating with an enteric polymer, as a dispersion of the bacterial cell powder in a cellulose derivative such as hydroxypropylmethylcellulose phthalate or carboxymethylethylcellulose dissolved in a solvent such as alcohol, the solvent is removed and pulverized, etc. You may utilize the well-known method of. Examples of fats and oils used for water-in-oil emulsification include soybean oil, palm oil, corn oil and the like, and examples of surfactants include glycerin fatty acid ester, sorbitan fatty acid ester and propylene glycol fatty acid ester. The water-in-oil emulsification is carried out by a known method such as emulsifying a water-soluble microbial cell solution and an oil and fat added with a surfactant by stirring or ultrasonic treatment. Phospholipids used in liposome preparations include egg yolk lecithin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, etc. Examples of the phospholipids having hydrophilic groups such as choline, ethanolamine, serine, inositol, glycerol, lysolecithin and the like, and of course, a plurality of the above phospholipids may be used in combination. The formulation is performed by a method known per se such as a reverse phase evaporation method and an ultrasonic treatment method. Regarding the dose and the period or administration interval when the preparation (formulation or feed) explained above is orally or nasally administered to mammals (eg, livestock such as cattle, horses, sheep, goats and pigs) Although it cannot be generally stated due to factors such as the type of mammal to be administered, the dose is 1 kg of mammal's body weight, per wet weight of bacterial cells per day.
0.01 to 1000 mg may be orally or nasally administered 1 to 10 times at intervals of 1 to 21 days.

【００１０】[0010]

【発明の実施の形態】以下に試験例により本発明を具体
的に説明するが、これが本発明の範囲を制限するもので
ないことは言うまでもない。BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below with reference to test examples, but it goes without saying that this does not limit the scope of the present invention.

【実施例】試験例１ スタフィロコッカス・アウレウス菌の20時間培養液をホ
ルマリンにより不活化し、死菌体又は死菌体のリゾチー
ム処理した溶菌体をそれぞれアジュバントとしてコレラ
トキシンと共に経口投与したマウスにおいて、本菌の乳
腺内攻撃に対する感染防御能を検討したところ、感染防
御効果が認められた。Test Example 1 In mice in which a 20-hour culture of Staphylococcus aureus was inactivated with formalin, and killed cells or lysate-treated lysates of killed cells were orally administered together with cholera toxin as an adjuvant. When the ability of this bacterium to protect against infection in the mammary gland was examined, a protective effect was found.

【００１１】即ち、20％グリセロール加トリプチケース
ソイブロス(Trypticase Soy Broth、BBL社製、以下TSB
と略す)に懸濁して-80℃で保存したスタフィロコッカス
・アウレウスのNo.46株（Staphylococcus aureus No.4
6）を融解後、1エーゼをマンニット食塩培地(ニッスイ
社製)に塗沫、37℃で24〜36時間培養後の1コロニーをTS
Bに植菌した。37℃で20時間培養後、10,000r.p.m.、4℃
で10分間、遠心分離して菌体を回収し、0.5％ホルマリ
ン（v/v）を添加したリン酸塩緩衝液(ダルベッコCa、Mg
不含、大日本製薬株式会社製、以下PBS)を加え25℃で24
時間、不活化処理後に得られる死菌体を免疫抗原とし
た。この抗原は免疫処理に供試するまで4℃に保存し
た。免疫処理は、上記の免疫抗原を10,000r.p.m.、4℃
で10分間、遠心分離して回収後、その1、10あるいは50m
g(湿重量)を0.5mlのPBSに懸濁した後、アジュバントと
して10μgのコレラトキシン（和光純薬工業（株）製）
を加え、雄と同居中の8〜10週齢で体重約20gのBALB/c雌
マウスに14日間の間隔で2回経口投与した。乳腺内攻撃
に対する感染防御能の検討は、2回目の免疫後21日に1.5
％のスキムミルクブロスで培養したスタフィロコッカス
・アウレウスのNo.46株（Staphylococcus aureus No.4
6）の104CFUを含む菌液0.1mlを分娩後10〜15日経過した
マウスの左右の第4乳腺内に接種することで確認した。
この時、対照群として免疫を行わなかったマウスにも同
様の条件で攻撃を行った。攻撃後48時間に剖検し、乳腺
よりスタフィロコッカス・アウレウスを分離した。攻撃
後48時間までにへい死した個体は可及的速やかに剖検を
行い、乳腺から菌回収を行った。攻撃後48時間のへい死
数及び乳腺より回収された菌数は表1の通りであった。That is, trypticase containing 20% glycerol Soy Broth, BBL, TSB
No.46 strain of Staphylococcus aureus (Staphylococcus aureus No.4)
6) After thawing, 1 ese is spread on Mannit salt medium (manufactured by Nissui), and 1 colony after culturing at 37 ° C for 24 to 36 hours is TS.
B was inoculated. After culturing at 37 ℃ for 20 hours, 10,000 rpm, 4 ℃
The cells were recovered by centrifugation at 10 minutes for 10 minutes, and 0.5% formalin (v / v) added phosphate buffer (Dulbecco Ca, Mg
Not included, manufactured by Dainippon Pharmaceutical Co., Ltd., hereinafter PBS) and added at 25 ° C for 24
Dead bacterial cells obtained after the inactivation treatment for a period of time were used as an immunogen. This antigen was stored at 4 ° C. until it was subjected to immunological treatment. Immunization is performed by immunizing the above antigens at 10,000 rpm at 4 ℃.
Centrifuge at 10 minutes for 10 minutes to recover after collecting.
After suspending g (wet weight) in 0.5 ml of PBS, 10 μg of cholera toxin (Wako Pure Chemical Industries, Ltd.) as an adjuvant
BALB / c female mice of 8 to 10 weeks of age living with males and weighing approximately 20 g were orally administered twice at 14-day intervals. The ability to protect against infection within the mammary gland was evaluated at 1.5 days 21 days after the second immunization.
No. 46 strain of Staphylococcus aureus (Staphylococcus aureus No. 4 cultured in% skim milk broth)
It was confirmed by inoculating 0.1 ml of the bacterial solution containing 10 4 CFU of 6) into the left and right fourth mammary glands of mice 10 to 15 days after delivery.
At this time, mice that were not immunized as a control group were also challenged under the same conditions. Autopsy was performed 48 hours after the attack, and Staphylococcus aureus was isolated from the mammary gland. Individuals who died 48 hours after the attack were necropsied as soon as possible, and the bacteria were collected from the mammary glands. Table 1 shows the number of deaths and the number of bacteria recovered from the mammary gland 48 hours after the challenge.

【表１】 [Table 1]

【００１２】試験例2 試験例1と同様の方法で処理した免疫抗原のPBS懸濁を調
製し、抗原量の湿重量で1/10量(w/w)の卵白リゾチーム
(和光純薬工業株式会社)を加えた後、37℃に30分間静置
して溶菌処理を行った。その後、アジュバントとしてコ
レラトキシン（和光純薬工業（株）製）を加え、試験例
1と同様の方法で免疫及び攻撃を実施した。攻撃後48時
間のへい死数及び乳腺より回収された菌数は表2の通り
であった。Test Example 2 A PBS suspension of an immune antigen treated in the same manner as in Test Example 1 was prepared, and 1/10 amount (w / w) of egg white lysozyme in wet weight of the antigen amount was prepared.
After adding (Wako Pure Chemical Industries, Ltd.), the mixture was allowed to stand at 37 ° C. for 30 minutes for lysis. Then, cholera toxin (manufactured by Wako Pure Chemical Industries, Ltd.) was added as an adjuvant, and the test example
Immunization and challenge were performed in the same manner as in 1. Table 2 shows the number of deaths and the number of bacteria recovered from the mammary glands 48 hours after the challenge.

【表２】 非免疫群におけるへい死数は、試験例1及び2でそれぞれ
1/5例、3/9例であった。免疫群では、試験例2での1mg免
疫群の1/7例を除いて死亡例は認められなかった。乳腺
から回収されるスタフィロコッカス・アウレウスの平均
菌数は、免疫処理によって低下する傾向が認められた。
特に50mg免疫群における乳腺から回収された平均菌数
は、試験例1及び2でそれぞれ4.1及び4.5 logCFU/gで非
免疫群に比較してそれぞれ有意に（試験例1：p<0.05、
試験例2：p<0.01）減少した。以上の結果、スタフィロ
コッカス・アウレウスNo.46株（Staphylococcus aureus
No.46）のホルマリン不活化抗原又はそのリゾチームに
より溶菌処理した抗原をコレラトキシンとともにマウス
に経口免疫することにより、同菌株の乳腺内攻撃に対す
る感染防御効果が得られることが明らかとなった。[Table 2] The number of deaths in the non-immunized group was measured in Test Examples 1 and 2, respectively.
1/5 cases and 3/9 cases. In the immunized group, no death was observed except 1/7 of the 1 mg immunized group in Test Example 2. The average number of Staphylococcus aureus cells recovered from the mammary gland tended to decrease due to immunization.
In particular, the average number of bacteria recovered from the mammary gland in the 50 mg immunization group was 4.1 and 4.5 log CFU / g in Test Examples 1 and 2, respectively, and significantly different from the non-immunized group (Test Example 1: p <0.05,
Test Example 2: p <0.01) decreased. As a result, Staphylococcus aureus No. 46 strain (Staphylococcus aureus
It was revealed that oral immunization of mice with formalin inactivated antigen (No. 46) or an antigen lysed with lysozyme thereof together with cholera toxin can provide a protective effect against the intramammary attack of the strain.

【００１３】前述の試験例１および２において使用され
たStaphylococcus aureus No.46は、臨床型***炎に罹
患したウシの乳汁より分離され高い***炎の起因性を示
した株であり、平成１１年１０月２８日から財団法人発
酵研究所（ＩＦＯ）にＩＦＯ１６３３３として寄託され
ている。Staphylococcus aureus No. 46 used in the above-mentioned Test Examples 1 and 2 is a strain which was isolated from milk of cattle suffering from clinical mastitis and showed a high causative factor of mastitis. Since October 28, it has been deposited as IFO16333 at the Fermentation Research Institute (IFO).

【００１４】[0014]

【発明の効果】本発明の製剤は、哺乳動物の***炎感染
防御効果にすぐれ、経口または経鼻投与用粘膜ワクチン
であることから極めて実用的である。INDUSTRIAL APPLICABILITY The preparation of the present invention is extremely practical since it is a mucosal vaccine for oral or nasal administration, which is excellent in the effect of preventing mastitis infection in mammals.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ａ６１Ｋ 39/09 Ａ６１Ｋ 39/09 39/108 39/108 Ａ６１Ｐ 15/14 １７１ Ａ６１Ｐ 15/14 １７１ Ｆターム(参考） 2B150 AA01 AA02 AA03 AB10 DD11 4C085 AA03 BA10 BA13 BA14 BA21 BA26 BA31 BA33 BB11 CC07 DD01 DD03 DD08 DD86 EE06 GG08 JJ03 JJ05 JJ11 4C087 AA01 AA02 BC28 BC34 BC36 BC61 BC62 BC71 BC72 CA09 CA11 MA01 MA02 MA23 MA24 MA34 MA52 MA59 ZB05 ZB09 ZC62 ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 7 Identification code FI theme code (reference) A61K 39/09 A61K 39/09 39/108 39/108 A61P 15/14 171 A61P 15/14 171 F term ( (Reference) 2B150 AA01 AA02 AA03 AB10 DD11 4C085 AA03 BA10 BA13 BA14 BA21 BA26 BA31 BA33 BB11 CC07 DD01 DD03 DD08 DD86 EE06 GG08 JJ03 JJ05 JJ11 4C087 AA01 AA02 BC28 BC05 MA02 MA28B05 MA02 MA05 MA23 MA23 MA23 MA02 MA02 MA23 MA23 MA23 MA02 MA02 MA23 MA23 MA23 MA01 MA23

Claims (7)

【特許請求の範囲】[Claims] 【請求項１】不活化された菌体もしくは菌体の外壁蛋白
を含む菌体溶出処理物を含有する、哺乳動物の***炎を
予防するための経口または経鼻投与用製剤。1. A preparation for oral or nasal administration for preventing mastitis in a mammal, which comprises a microbial cell elution-treated product containing inactivated microbial cells or outer wall proteins of the microbial cells. 【請求項２】不活化された菌体もしくは菌体の外壁蛋白
を含む菌体溶出処理物が、菌体もしくは菌体の外壁蛋白
を含む菌体溶出処理物のホルマリン処理により得られる
請求項１記載の製剤。2. The cell-eluted product containing inactivated cells or outer wall proteins of cells is obtained by formalin treatment of the cell-eluted product containing cells or cell outer wall proteins. The described formulation. 【請求項３】不活化された菌体の外壁蛋白を含む菌体溶
出処理物が、菌体の加熱処理により得られる請求項１記
載の製剤。3. The preparation according to claim 1, wherein the cell elution-treated product containing the inactivated outer wall protein of the cell is obtained by heat treatment of the cell. 【請求項４】不活化された菌体の外壁蛋白を含む菌体溶
出処理物が、不活化された菌体の溶菌処理により得られ
る請求項１記載の製剤。4. The preparation according to claim 1, wherein the microbial cell elution-treated product containing the outer wall protein of the inactivated microbial cell is obtained by lysing treatment of the inactivated microbial cell. 【請求項５】さらに、アジュバントを含有してなる請求
項１記載の製剤。5. The preparation according to claim 1, which further comprises an adjuvant. 【請求項６】(1)脂溶性物質および腸溶性高分子化合物
から選ばれる被覆剤で被覆された製剤、(2)油中水型乳
化液剤または(3)リポソーム製剤である請求項１記載の
製剤。6. The preparation according to claim 1, which is (1) a preparation coated with a coating agent selected from a fat-soluble substance and an enteric polymer, (2) a water-in-oil emulsion or (3) a liposome preparation. Formulation. 【請求項７】不活化された菌体もしくは菌体の外壁蛋白
を含む菌体溶出処理物を含有してなる家畜用飼料。7. A livestock feed comprising a cell elution-treated product containing inactivated cells or outer wall proteins of the cells.
JP30911699A 1999-10-29 1999-10-29 Mucosal prophylactic for mastitis Pending JP2003171291A (en)

Priority Applications (3)

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PCT/JP2000/007445 WO2001032205A1 (en) 1999-10-29 2000-10-24 Mucosal preventives for mastitis
AU79564/00A AU7956400A (en) 1999-10-29 2000-10-24 Mucosal preventives for mastitis

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JP30911699A JP2003171291A (en) 1999-10-29 1999-10-29 Mucosal prophylactic for mastitis

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Family Cites Families (5)

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Publication number Priority date Publication date Assignee Title
GB1182555A (en) * 1967-10-16 1970-02-25 Saphar Lab Ltd An Anti-Mastitis Vaccine
IE51174B1 (en) * 1980-04-14 1986-10-29 Merck & Co Inc Group b streptococcal capsular polysaccharides
JPH0356426A (en) * 1989-07-24 1991-03-12 Gifu Meneki Kenkyusho:Kk Bovine mastitis preventive and treating antibody-containing material
TW448185B (en) * 1993-11-05 2001-08-01 Lilly Co Eli Vaccine design and production
GB9326174D0 (en) * 1993-12-22 1994-02-23 Biocine Sclavo Mucosal adjuvant

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