JP2023165053A - Composition for treatment, treatment method, method for evaluating feces, diagnostic method, and prognosis evaluation method - Google Patents
Composition for treatment, treatment method, method for evaluating feces, diagnostic method, and prognosis evaluation method Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A61P37/08—Antiallergic agents
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
Description
本発明は、主にイヌの疾患に対する治療用組成物、及び治療方法に関する。 TECHNICAL FIELD The present invention mainly relates to therapeutic compositions and methods for treating diseases in dogs.
従来より、腸内細菌やこれらの代謝物が、腸管の炎症抑制や免疫調整に重要な役割を果たしていることは広く知られているところであり、糞便微生物叢移植法（ｆｅｃａｌ ｍｉｃｒｏｂｉｏｔａ ｔｒａｎｓｐｌａｎｔａｔｉｏｎ:ＦＭＴ）の可能性が示唆されている。特許文献１には、ヒトの腸管急性移植片対宿主病の予防又は治療のために、特定の糞便微生物叢を含む組成物を移植することが記載されている。また、非特許文献１には、急性下痢のイヌの治療法として、ＦＭＴが有効であったことが記載されている。しかしながら、イヌのアトピー性皮膚炎とＦＭＴとの関連性は明らかにされていない。 It has been widely known that intestinal bacteria and their metabolites play an important role in suppressing inflammation and regulating immunity in the intestinal tract. The possibility is suggested. Patent Document 1 describes transplanting a composition containing a specific fecal microflora for the prevention or treatment of acute intestinal graft-versus-host disease in humans. Furthermore, Non-Patent Document 1 describes that FMT was effective as a treatment for dogs with acute diarrhea. However, the relationship between canine atopic dermatitis and FMT has not been clarified.
本発明は、特にイヌのアトピー性皮膚炎等の疾患に対する予防又は治療用組成物、治療方法を提供することを目的とする。本発明は、上記に鑑みてなされたもので、特定の細菌を含む組成物をイヌに投与することにより、上記疾患を予防又は治療することに成功し、本発明を完成するに至った。 The present invention particularly aims to provide a preventive or therapeutic composition and a therapeutic method for diseases such as atopic dermatitis in dogs. The present invention was made in view of the above, and the present invention was completed by successfully preventing or treating the above-mentioned diseases by administering a composition containing specific bacteria to dogs.
本発明によれば、Fusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物を含有する、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎の予防又は治療用組成物、及び当該組成物を投与することによるイヌの治療方法が得られる。 According to the present invention, the genus Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium, Prevotella, Blautia , Lachnospiraceae NK4A136 group, Erysipelatoclostridium genus, Collinsella genus, and Holdemanella genus. A composition for preventing or treating hypercorticism, intervertebral disc herniation, mitral regurgitation, or pancreatitis, and a method for treating dogs by administering the composition are obtained.
本発明によれば、イヌの各種疾患を予防又は治療することが可能な治療用組成物及び治療方法を提供することができる。 According to the present invention, it is possible to provide therapeutic compositions and treatment methods that can prevent or treat various diseases in dogs.
以下に、本発明の治療用組成物、治療方法、糞便の評価方法、診断方法、及び予後評価方法の実施形態を説明する。なお、本実施形態により本発明が限定されるものではない。 Embodiments of the therapeutic composition, treatment method, fecal evaluation method, diagnostic method, and prognosis evaluation method of the present invention will be described below. Note that the present invention is not limited to this embodiment.
本発明は、例えば、以下のような構成を備える。
［項目１］
Fusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物を含有する、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の予防又は治療用組成物。
［項目２］
Fusobacterium属、Collinsella属、Megamonas属、Blautia属からなる群から選択される少なくとも１つの属に属する微生物を含有する、項目１に記載の組成物。
［項目３］
イヌの糞便微生物叢を含む、項目１又は２に記載の組成物。
［項目４］
イヌの糞便又はその処理物を含む、項目１又は２に記載の組成物。
［項目５］
イヌのアトピー性皮膚炎の予防又は治療用である、項目１～４のいずれかに記載の組成物。
［項目６］
Fusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物を含有する組成物を投与することを特徴とする、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の予防方法又は治療方法。
［項目７］
Fusobacterium属、Collinsella属、Megamonas属、Blautia属からなる群から選択される少なくとも１つの属に属する微生物を含有する、項目６に記載の方法。
［項目８］
イヌの糞便微生物叢を含む、項目６又は７に記載の方法。
［項目９］
イヌの糞便又はその処理物を含む、項目６又は７に記載の方法。
［項目１０］
イヌのアトピー性皮膚炎の予防又は治療用である、項目６～９のいずれかに記載の方法。
［項目１１］
糞便中の細菌叢に含まれるFusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物の存在量を解析するステップと、
前記微生物の存在量に基づいて、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の予防又は治療のためのドナー糞便としての有用性を評価するステップとを含む、糞便の評価方法。
［項目１２］
イヌのアトピー性皮膚炎の予防又は治療のためのドナー糞便としての有用性を評価する、項目１１に記載の評価方法。
［項目１３］
糞便中の細菌叢に含まれるFusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物の存在量を解析するステップと、
前記微生物の存在量に基づいて、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の罹患可能性を診断するステップと、を含む診断方法。
［項目１４］
イヌのアトピー性皮膚炎の罹患可能性を診断する、項目１２に記載の診断方法。
［項目１５］
イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の治療後の予後を評価する方法であって、
前記イヌの少なくとも治療前と治療後の糞便中におけるFusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物の存在量を解析するステップと
前記微生物の前記治療前と前記治療後における前記存在量を比較するステップと、
を含む、予後評価方法。
The present invention includes, for example, the following configuration.
[Item 1]
Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium, Prevotella, Blautia, Lachnospiraceae NK4A136 group, Erysipelatoclostridium Canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral discs, containing microorganisms belonging to at least one genus selected from the group consisting of the genus Collinsella, genus Holdemanella, and the genus Holdemanella. A composition for preventing or treating at least one disease selected from hernia, mitral regurgitation, and pancreatitis.
[Item 2]
The composition according to item 1, containing a microorganism belonging to at least one genus selected from the group consisting of Fusobacterium, Collinsella, Megamonas, and Blautia.
[Item 3]
The composition according to item 1 or 2, comprising dog fecal microflora.
[Item 4]
The composition according to item 1 or 2, comprising dog feces or a processed product thereof.
[Item 5]
The composition according to any one of items 1 to 4, which is used for preventing or treating atopic dermatitis in dogs.
[Item 6]
Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium, Prevotella, Blautia, Lachnospiraceae NK4A136 group, Erysipelatoclostridium Canine atopic dermatitis, chronic kidney disease, cystitis, characterized by administering a composition containing a microorganism belonging to at least one genus selected from the group consisting of the genus Collinsella, genus Holdemanella, and the genus Holdemanella. , hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis.
[Item 7]
The method according to item 6, comprising a microorganism belonging to at least one genus selected from the group consisting of Fusobacterium, Collinsella, Megamonas, and Blautia.
[Item 8]
8. The method according to item 6 or 7, comprising dog fecal microbiota.
[Item 9]
The method according to item 6 or 7, comprising dog feces or a processed product thereof.
[Item 10]
The method according to any one of items 6 to 9, which is for preventing or treating atopic dermatitis in dogs.
[Item 11]
Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium, Prevotella, which are included in the fecal flora. Analyzing the abundance of microorganisms belonging to at least one genus selected from the group consisting of Blautia genus, Lachnospiraceae NK4A136 group, Erysipelatoclostridium genus, Collinsella genus, and Holdemanella genus;
At least one disease selected from canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis based on the abundance of the microorganisms. A method for evaluating feces, comprising the step of evaluating its usefulness as donor feces for the prevention or treatment of feces.
[Item 12]
The evaluation method according to item 11, which evaluates the usefulness as donor feces for the prevention or treatment of atopic dermatitis in dogs.
[Item 13]
Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium, Prevotella, which are included in the fecal flora. Analyzing the abundance of microorganisms belonging to at least one genus selected from the group consisting of Blautia genus, Lachnospiraceae NK4A136 group, Erysipelatoclostridium genus, Collinsella genus, and Holdemanella genus;
At least one disease selected from canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis based on the abundance of the microorganisms. A diagnostic method comprising the step of diagnosing the possibility of being affected by.
[Item 14]
The diagnostic method according to item 12, for diagnosing the possibility of a dog suffering from atopic dermatitis.
[Item 15]
A method for evaluating the prognosis after treatment of at least one disease selected from canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis. And,
Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium in the feces of the dog at least before and after treatment. analyzing the abundance of microorganisms belonging to at least one genus selected from the group consisting of the genus Prevotella, the genus Blautia, the Lachnospiraceae NK4A136 group, the genus Erysipelatoclostridium, the genus Collinsella, and the genus Holdemanella; and the step of analyzing the abundance of the microorganisms before the treatment of the microorganisms. and comparing the abundance after the treatment;
Prognosis evaluation methods, including.
本発明は、特にイヌのアトピー性皮膚炎などの疾患に対する予防又は治療用組成物、及び治療方法を提供することを目的とする。 The present invention particularly aims to provide a preventive or therapeutic composition and a treatment method for diseases such as canine atopic dermatitis.
本発明の治療用組成物は、以下の特定の属に分類される細菌を含有することを特徴とする。本発明の治療用組成物は、Fusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物を含有する。また、特に、Fusobacterium属、Collinsella属、Megamonas属、Blautia属、Sutterella属、Prevotella属、Bacteroides属からなる群から選択される少なくとも１つの属に属する微生物を含有することが好ましい。また特に、Fusobacterium属、Collinsella属、Megamonas属、Blautia属からなる群から選択される少なくとも１つの属に属する微生物を含有することがより好ましい。 The therapeutic composition of the present invention is characterized by containing bacteria classified into the following specific genera. The therapeutic composition of the present invention can be applied to the genus Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium, Prevotella. , Blautia genus, Lachnospiraceae NK4A136 group, Erysipelatoclostridium genus, Collinsella genus, and Holdemanella genus. In particular, it is preferable to contain a microorganism belonging to at least one genus selected from the group consisting of Fusobacterium, Collinsella, Megamonas, Blautia, Sutterella, Prevotella, and Bacteroides. In particular, it is more preferable to contain a microorganism belonging to at least one genus selected from the group consisting of the genus Fusobacterium, the genus Collinsella, the genus Megamonas, and the genus Blautia.
本発明の治療用組成物は、上記特定の細菌を含有する糞便自体、または糞便の処理物を含んでもよい。糞便の処理物は、採取された糞便を適当な水性液体（例えば生理食塩水、緩衝液等）に懸濁した懸濁液の他、当該懸濁液を適当なふるい、ガーゼ、フィルターなど（例えば孔径０．１ｍｍ～０．５ｍｍ）に通してろ過させたもの、あるいは遠心分離後の沈殿物などが含まれる。さらに、これらの組成物を冷凍庫又は液体窒素により凍結したり、凍結乾燥又は噴霧乾燥などを施したものでもよい。当該水性液体を用いて懸濁液とする場合は、糞便１ｇあたり１．５～３．０ｍｌの液体にて懸濁すればよい。懸濁液を作成後、遠心して細菌を抽出する場合には、その菌量１ｇに対して０．５～０．６ｍｌの液量に再度懸濁する。 The therapeutic composition of the present invention may contain feces itself containing the above-mentioned specific bacteria, or a processed product of feces. The processed fecal material includes a suspension of the collected feces in an appropriate aqueous liquid (e.g., physiological saline, buffer solution, etc.), and a suspension of the collected feces by passing the suspension through an appropriate sieve, gauze, filter, etc. (e.g., This includes those filtered through a pore size of 0.1 mm to 0.5 mm) or the precipitate after centrifugation. Furthermore, these compositions may be frozen using a freezer or liquid nitrogen, or may be freeze-dried or spray-dried. When making a suspension using the aqueous liquid, 1.5 to 3.0 ml of liquid may be used per 1 g of feces. After creating a suspension, if the bacteria are extracted by centrifugation, the suspension is resuspended in a liquid volume of 0.5 to 0.6 ml per 1 g of bacteria.
凍結又は凍結乾燥の際には、各種糖（スクロース、フルクトース、ラクトース、マンニトール等）、グリセロール、ポリエチレングリコール（ＰＥＧ）、トレハロース、グリシン、グルコース、デキストラン、エリスリトールなどの凍結保護剤及び／又は凍結乾燥保護剤を添加することもできる。 When freezing or freeze-drying, use cryoprotectants and/or freeze-dry protection such as various sugars (sucrose, fructose, lactose, mannitol, etc.), glycerol, polyethylene glycol (PEG), trehalose, glycine, glucose, dextran, erythritol, etc. It is also possible to add agents.
本発明において、採取された糞便又はその処理物は、糞便採取後又は処理後６～１０時間保存することができる。保存温度は特に限定されるものではないが、冷蔵保存（例えば４℃）であることが好ましい。 In the present invention, the collected feces or the processed material thereof can be stored for 6 to 10 hours after the feces are collected or processed. Although the storage temperature is not particularly limited, refrigerated storage (for example, 4° C.) is preferable.
本発明の組成物は、溶媒、ｐＨ安定剤、酸性化剤、防腐剤、ビタミン、ミネラル、栄養サプリメント、プレバイオティクス及びプロバイオティクス等を含めることができる。また、組成物の形態は任意の粉末、固体又は液体形態とすることができ、これらの粉末、固体又は液体形態をカプセル製剤とすることもできる。カプセル製剤とすることで、投与を受けるイヌの負担を軽減することができる。 Compositions of the invention can include solvents, pH stabilizers, acidifying agents, preservatives, vitamins, minerals, nutritional supplements, prebiotics and probiotics, and the like. Further, the composition can be in any powder, solid or liquid form, and these powder, solid or liquid forms can also be made into capsule formulations. By making it into a capsule formulation, the burden on the dog receiving the drug can be reduced.
本発明による治療方法は、上記治療用組成物を特定の疾患に罹患したイヌに投与することを含む。特に好適には、治療用組成物として特定の細菌を含む糞便又は糞便処理物を使用する糞便微生物叢移植法（ｆｅｃａｌ ｍｉｃｒｏｂｉｏｔａ ｔｒａｎｓｐｌａｎｔａｔｉｏｎ:ＦＭＴ）である。ＦＭＴ法とは、健常個体の便懸濁液を消化管内に投与することで、正常な細菌叢を大量に投与する治療法であり、腸内細菌叢の異常であるｄｙｓｂｉｏｓｉｓが関与する疾患の治療に試みられる。 The therapeutic method according to the present invention includes administering the therapeutic composition described above to a dog suffering from a specific disease. Particularly preferred is the fecal microbiota transplantation (FMT) method, which uses feces or fecal preparations containing specific bacteria as a therapeutic composition. The FMT method is a treatment method in which a large amount of normal flora is administered by administering a fecal suspension from a healthy individual into the gastrointestinal tract, and it is used to treat diseases related to dysbiosis, which is an abnormality in the intestinal flora. will be attempted.
本発明の治療方法における組成物の投与方法は、経口投与であっても非経口投与であってもよく、特に限定されるものではない。例えば、胃十二指腸チューブによる投与、カプセルなどに充填しての内服、大腸ファイバーや高圧浣腸などを用いた大腸内への投与が挙げられる。 The method of administering the composition in the treatment method of the present invention may be oral or parenteral administration, and is not particularly limited. Examples include administration through a gastroduodenal tube, oral administration in a capsule or the like, and administration into the large intestine using a colon fiber or high-pressure enema.
１回の投与量は、液体の場合は、１５０ｍｌ～３００ｍｌであり、１日１回行う。レシピエントの状態に応じて、繰り返し行う場合には、４日～２週間ごとに計２～４回行う。このようにして、本発明の組成物を、特定の疾患に罹患したイヌに投与することによって、当該疾患を地治療することができる。 A single dose is 150 ml to 300 ml in the case of liquid, and is administered once a day. Depending on the condition of the recipient, if the test is to be repeated, it is performed 2 to 4 times in total every 4 days to 2 weeks. In this way, by administering the composition of the present invention to dogs suffering from a particular disease, the disease can be locally treated.
ＦＭＴ法を使用する場合、便移植を受けたイヌが別の疾患や感染症を併発することを避けるために、ドナーとなる糞便の選択が重要である。したがって、糞便を採取するドナー、及び微生物叢を移植するレシピエントの種類に応じて、ドナーから採取した糞便について、各種ウイルスなどの病原体の有無をスクリーニングすることが好ましい。 When using the FMT method, it is important to select the donor feces in order to avoid other diseases or infections in dogs receiving fecal transplants. Therefore, it is preferable to screen the feces collected from the donor for the presence of pathogens such as various viruses, depending on the type of the donor whose feces are collected and the type of recipient to which the microflora is to be transplanted.
また、本発明に係る治療方法においては、Fusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する腸内細菌を含有するドナーを選択することができる。すなわち、ＦＭＴ法を採用する場合、ドナー候補の個体から糞便を採取し、上記細菌属を有することを事前に確認することができる。 In addition, in the treatment method according to the present invention, the genus Fusobacterium, the genus Sutterella, the genus Romboutsia, the genus Phascolarctobacterium, the genus Bacteroides, the genus Intestinimonas, the genus Megamonas, the genus Peptoclostridium, the genus Helicobacter, the genus Allobaculum, the genus Alloprevotella, the genus Butyricicoccus, the genus Catenibacterium, A donor containing enteric bacteria belonging to at least one genus selected from the group consisting of the genus Prevotella, the genus Blautia, the Lachnospiraceae NK4A136 group, the genus Erysipelatoclostridium, the genus Collinsella, and the genus Holdemanella can be selected. That is, when employing the FMT method, it is possible to collect feces from a donor candidate individual and confirm in advance that it contains the above-mentioned bacterial genus.
本発明の治療用組成物及び治療方法は、アトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎に罹患したイヌに適用可能である。 The therapeutic composition and method of the present invention are applicable to dogs suffering from atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis. It is.
ここで、「治療」とは、対象疾患の発症を抑制することができる限り、抑制の程度は限定されるものではない。したがって、「治療」には完全緩解及び部分緩解のいずれも含まれる。また、「予防」とは、上記疾患の発症を事前に抑制することと、すでに発生している疾患の状態がそれよりも悪くなることを防ぐことのいずれも意味する。 Here, "treatment" is not limited to the degree of suppression as long as the onset of the target disease can be suppressed. Therefore, "treatment" includes both complete remission and partial remission. Moreover, "prevention" means both to suppress the onset of the above-mentioned disease in advance and to prevent the condition of the disease that has already occurred from becoming worse.
また、本発明は、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎の罹患可能性を診断する方法に関する。すなわち、本発明の診断方法は、糞便中の細菌叢に含まれるFusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物の存在量を解析するステップと、前記微生物の存在量に基づいて、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の罹患可能性を診断するステップとを含む。特に、前記属に属する微生物の絶対存在量又は相対存在量が少ない場合、または絶対存在量又は相対存在量が少ない前記群に属する細菌属の種類数が多い場合に、前記疾患の罹患可能性が高いと診断することができる。なお、罹患可能性の程度の判断基準とする前記存在量の閾値は適宜決定することができる。 The present invention also relates to a method for diagnosing the possibility of atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis in dogs. That is, the diagnostic method of the present invention can detect genus Fusobacterium, genus Sutterella, genus Romboutsia, genus Phascolarctobacterium, genus Bacteroides, genus Intestinimonas, genus Megamonas, genus Peptoclostridium, genus Helicobacter, genus Allobaculum, genus Alloprevotella, analyzing the abundance of microorganisms belonging to at least one genus selected from the group consisting of Butyricicoccus, Catenibacterium, Prevotella, Blautia, Lachnospiraceae NK4A136 group, Erysipelatoclostridium, Collinsella, and Holdemanella; Based on the abundance of microorganisms, at least one disease selected from canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis. diagnosing the possibility of infection. In particular, when the absolute abundance or relative abundance of microorganisms belonging to the aforementioned genus is low, or when the number of types of bacterial genera belonging to the aforementioned group with low absolute abundance or relative abundance is large, the possibility of contracting the aforementioned disease increases. It can be diagnosed as high. Note that the abundance threshold used as a criterion for determining the degree of disease possibility can be determined as appropriate.
また、本発明は、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎の治療後の予後を評価する方法に関する。すなわち、本発明の評価方法は、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の治療後の予後を評価する方法であって、前記イヌの少なくとも治療前と治療後の糞便中におけるFusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物の存在量を解析するステップと、前記微生物の前記治療前と前記治療後における前記存在量を比較するステップとを含む。特に、前記群に属する微生物の絶対存在量又は相対存在量が治療前に対して治療後で増加していた場合、または治療後において新規に獲得した前記群に属する細菌属の種類数が多い場合に、予後が良好であると評価することができる。なお、本評価方法において、治療の内容は特に制限はなく、糞便移植に限らない。また、予後の良し悪しを判断する基準とする前記存在量の閾値は適宜決定することができる。 The present invention also relates to a method for evaluating the prognosis after treatment of atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis in dogs. That is, the evaluation method of the present invention can be used to evaluate at least one disease selected from canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis. A method for evaluating the prognosis after treatment of the dog, comprising: Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, in the feces of the dog at least before and after treatment. Microorganisms belonging to at least one genus selected from the group consisting of the genus Helicobacter, the genus Allobaculum, the genus Alloprevotella, the genus Butyricicoccus, the genus Catenibacterium, the genus Prevotella, the genus Blautia, the Lachnospiraceae NK4A136 group, the genus Erysipelatoclostridium, the genus Collinsella, and the genus Holdemanella. The method includes analyzing the abundance of the microorganism and comparing the abundance of the microorganism before the treatment and after the treatment. In particular, when the absolute abundance or relative abundance of microorganisms belonging to the above group increases after treatment compared to before treatment, or when the number of types of bacterial genera belonging to the above group newly acquired after treatment is large. Therefore, the prognosis can be evaluated as good. In this evaluation method, there are no particular restrictions on the content of treatment, and it is not limited to fecal transplantation. Further, the threshold value of the abundance amount, which is used as a criterion for determining the good or bad prognosis, can be determined as appropriate.
また、本発明は、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の予防又は治療に用いるドナー糞便としての有用性を評価する方法に関する。すなわち、本発明の評価方法は、ドナー糞便中の細菌叢に含まれるFusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物の存在量を解析するステップと、前記微生物の存在量に基づいて、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の予防又は治療のためのドナー糞便としての有用性を評価するステップとを含む。特に、前記群に属する微生物の絶対存在量又は相対存在量が多い場合、または前記群に属する細菌属の種類数が多い場合に、ドナー糞便としての有用性が高いと評価することができる。なお、有用性の判断基準とする前記存在量の閾値は適宜決定することができる。 The present invention also provides prevention or treatment of at least one disease selected from canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis. This invention relates to a method for evaluating the usefulness of donor feces for use in treatment. That is, the evaluation method of the present invention can detect the genus Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, and Alloprevotella contained in the bacterial flora of donor feces. , analyzing the abundance of microorganisms belonging to at least one genus selected from the group consisting of , Butyricicoccus, Catenibacterium, Prevotella, Blautia, Lachnospiraceae NK4A136 group, Erysipelatoclostridium, Collinsella, and Holdemanella; At least one disease selected from canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis based on the abundance of the microorganisms. and evaluating its usefulness as donor feces for the prevention or treatment of. In particular, when the absolute abundance or relative abundance of microorganisms belonging to the above group is large, or when the number of types of bacterial genera belonging to the above group is large, it can be evaluated that the usefulness as donor feces is high. Note that the threshold value of the amount of abundance used as a criterion for determining usefulness can be determined as appropriate.
以下、実施例により本発明をさらに具体的に説明する。ただし、本発明の範囲はこれらの実施例により限定されるものではない。 Hereinafter, the present invention will be explained in more detail with reference to Examples. However, the scope of the present invention is not limited by these Examples.
・ＦＭＴ
健常な未去勢のビーグルイヌ２頭（雄）が自然排泄した便を採取した（糞便量：平均４８．８ｇ、範囲２５～９０g）。採取後、３０分以内に水道水で溶解（水道水量：平均５６ｍＬ、範囲２０～１００ｍＬ）した。その後、医療用ガーゼで２回濾過し、得られた糞便液を注射用シリンジで吸引し、アトピー性皮膚炎を呈するイヌ１２頭（疾患群）に経口投与した。ドナーとレシピエントの組合せは図１に示した。ドナーＮｏ．７由来の便を７頭のレシピエント犬に移植し、ドナーＶａｎｉｌｌａ由来の便を５頭のレシピエント犬に移植した。
・FMT
Naturally excreted feces from two healthy, unneutered beagle dogs (male) were collected (fecal volume: average 48.8 g, range 25-90 g). After collection, it was dissolved in tap water within 30 minutes (tap water volume: average 56 mL, range 20-100 mL). Thereafter, it was filtered twice through medical gauze, and the resulting fecal fluid was aspirated with an injection syringe and orally administered to 12 dogs exhibiting atopic dermatitis (disease group). The donor and recipient combinations are shown in Figure 1. Donor No. Stool from donor Vanilla was transplanted into seven recipient dogs, and stool from donor Vanilla was transplanted into five recipient dogs.
・サンプリング
各レシピエントに対して、経口糞便移植を単回実施後、糞便移植前（Ｐｒｅ）、２８日後（Ｐｏｓｔ１）、および、５６日後（Ｐｏｓｔ２）の臨床スコア（かゆみスコア、ＣＡＤＥＳＩスコア）の記録、および、糞便検体の採取を実施した。かゆみスコアの記録は、ＰＶＡＳ(Ｐｒｕｒｉｔｕｓ Ｖｉｓｕａｌ Ａｎａｌｏｇ Ｓｃａｌｅ)：痒み視覚アナログ尺度に従い、イヌの痒みを飼い主が０～１０で点数化する方法で実施した。また、ＣＡＤＥＳＩスコアの記録は、ＣＡＤＥＳＩ-４(Ｃａｎｉｎｅ Ａｔｏｐｉｃ Ｄｅｒｍａｔｉｔｉｓ Ｅｘｔｅｎｔ ａｎｄ Ｓｅｖｅｒｉｔｙ Ｉｎｄｅｘ－４):イヌアトピー性皮膚炎重症度指数－第４版に従い、全身２０ヶ所における３つの皮膚病変（１．紅斑、２．苔癬化、３．脱毛／表皮剥離）を０～３で点数化し、合計０～１８０で獣医師が皮膚炎を点数化する方法により実施した。糞便検体の採取は、各レシピエントの肛門に指を入れ糞便を直接採取した（用手法）。採取した糞便を保存用チューブに入れ液体窒素で急速冷凍し、糞便入りチューブを－８０℃冷凍庫で保存した。
・Sampling After performing a single oral fecal transplant for each recipient, record clinical scores (itch score, CADESI score) before fecal transplant (Pre), 28 days later (Post 1), and 56 days later (Post 2) , and collection of fecal samples was performed. The itch score was recorded according to the Pruritus Visual Analog Scale (PVAS), in which the dog's owner scored the dog's itch on a scale of 0 to 10. In addition, the CADESI score is recorded according to CADESI-4 (Canine Atopic Dermatitis Extent and Severity Index-4): Canine Atopic Dermatitis Severity Index - 4th edition, based on three skin lesions (1. erythema, 2. lichenification, 3. hair loss/epidermal peeling) were scored on a scale of 0 to 3, and a veterinarian scored dermatitis on a total of 0 to 180. Fecal samples were collected directly by inserting a finger into the anus of each recipient (manual method). The collected feces were placed in a storage tube and quickly frozen with liquid nitrogen, and the tube containing feces was stored in a -80°C freezer.
糞便検体は既知の方法により、１６Ｓ ｒＲＮＡ遺伝子のＶ３－Ｖ４領域のシーケンスによるメタ１６Ｓ解析に供し、腸内細菌叢の解析を実施した。１検体あたりの総リード数は５０００にノーマライズして解析を実施した。 The fecal samples were subjected to meta-16S analysis by sequencing the V3-V4 region of the 16S rRNA gene by a known method, and the intestinal flora was analyzed. The total number of reads per sample was normalized to 5000 and the analysis was performed.
・糞便移植による腸内細菌叢構成の変化
まず、糞便移植による疾患群の腸内細菌叢構成の変化を調べる目的で、各検体間のｕｎｗｅｉｇｈｔｅｄ ＵｎｉＦｒａｃ Ｄｉｓｔａｎｃｅ、および、ｗｅｉｇｈｔｅｄ ＵｎｉＦｒａｃ Ｄｉｓｔａｎｃｅを計算し、主座標分析を実施した（図２、３）。その結果、糞便移植前において（Ｐｒｅ）、疾患群とドナー群はそれぞれ別のクラスターを形成していたが、糞便移植後（Ｐｏｓｔ１、Ｐｏｓｔ２）、疾患群の細菌叢はドナーの細菌叢に有意に近づいた。
・Changes in the intestinal flora composition due to fecal transplantation First, in order to investigate changes in the intestinal flora composition of disease groups due to fecal transplantation, we calculated the unweighted UniFrac Distance and the weighted UniFrac Distance between each sample, and calculated the principal coordinates. The analysis was performed (Figs. 2, 3). As a result, the disease group and donor group formed separate clusters before fecal transplantation (Pre), but after fecal transplantation (Post1, Post2), the disease group's bacterial flora was significantly different from that of the donor. Approaching the.
次に、糞便移植による腸内細菌叢のα多様性（ＡＳＶ数（ユニーク配列数：amplicon sequence variant）、Ｓｈａｎｎｏｎ ｉｎｄｅｘ、Ｐｉｅｌｏｕ’ｓ ｅｖｅｎｎｅｓｓ）の変化について解析を実施した（図４）。その結果、糞便移植後、種の豊富さを示すＡＳＶ数が有意に上昇した。Ｓｈａｎｎｏｎ ｉｎｄｅｘ、および均等度を示すＰｉｅｌｏｕ’ｓ ｅｖｅｎｎｅｓｓには変化はなかった。このことから、疾患群は糞便移植後、腸内細菌叢に新たな細菌を獲得したことが示唆された。 Next, we analyzed changes in α-diversity (number of ASVs (amplicon sequence variants), Shannon index, Pielou's evenness) of the intestinal flora due to fecal transplantation (FIG. 4). As a result, after fecal transplantation, the number of ASV, which indicates species richness, increased significantly. There was no change in the Shannon index or Pielou's evenness. This suggests that the diseased group acquired new bacteria in their intestinal flora after fecal transplantation.
・腸内細菌叢の変化と臨床スコアの関連
レシピエントが新たに獲得した細菌がドナー由来の細菌かどうかを調べる目的で、レシピエントと各レシピエントに対応するドナーで共通して保有する、即ち、レシピエントとドナーで１００％配列が一致するＡＳＶ数を糞便移植の前後で比較した（図５）。その結果、糞便移植後、レシピエントがドナーと共通して保有するＡＳＶ数は有意に増加し、そのほとんどが５６日後まで維持された。さらに、新規ＡＳＶ獲得数と臨床スコアの相関について解析を行った。スコア改善率は、各スコアについて（Ｐｏｓｔ－Ｐｒｅ）／Ｐｒｅとして計算した。その結果、５６日後において獲得ＡＳＶ数とかゆみスコアの相関係数が０．６８、また獲得ＡＳＶ数とＣＡＤＥＳＩスコアの相関係数が０．７６となり、両スコアに相関が認められた（図６）。以上のことから、糞便移植によりドナー由来の細菌がレシピエントに定着したこと、ドナーから獲得された菌種数が多いほど、臨床症状の改善効果が大きいことが明らかとなった。
・Relationship between changes in intestinal flora and clinical scores In order to investigate whether the bacteria newly acquired by the recipient are donor-derived bacteria, the bacteria that are commonly carried by the recipient and the donor corresponding to each recipient, i.e. , the number of ASVs with 100% sequence identity between recipient and donor was compared before and after fecal transplantation (Figure 5). As a result, after fecal transplantation, the number of ASVs shared by recipients and donors significantly increased, and most of these were maintained until 56 days later. Furthermore, we analyzed the correlation between the number of new ASV acquisitions and clinical scores. Score improvement rate was calculated as (Post-Pre)/Pre for each score. As a result, after 56 days, the correlation coefficient between the number of acquired ASVs and the itch score was 0.68, and the correlation coefficient between the number of acquired ASVs and the CADESI score was 0.76, indicating a correlation between both scores (Figure 6). . From the above, it has become clear that donor-derived bacteria colonize the recipient through fecal transplantation, and that the greater the number of bacterial species acquired from the donor, the greater the effect of improving clinical symptoms.
・臨床スコアと相関する細菌の同定
糞便移植により変動した細菌種を明らかにするために、各ＡＳＶについて、Ｔａｘｏｎｏｍｙ解析を実施した。Ｔａｘｏｎｏｍｙ解析（各ＡＳＶの分類）はＳＩＬＶＡ１３８をリファレンスとして実施した。これにより、ＡＳＶのリード数から各細菌のａｂｕｎｄａｎｃｅ（リード数の合計値）を算出した。ａｂｕｎｄａｎｃｅを１検体あたりの総リード数で割った数値を占有率とした。各検体の門レベルの細菌叢構成を図７、８に示した。図８は、図７の結果を個体ごとにＰｒｅ、Ｐｏｓｔ１、Ｐｏｓｔ２の順番で細菌叢構成を並べかえた図である。ドナー２頭の腸内細菌叢はFirmicutes門、Fusobacteria門、Proteobacteria門、Actinobacteria門で多くを占められていた。疾患群においては、全頭がFirmicutes門およびProteobacteria門を保有していた。一方、Fusobacteria門、および、Actinobacteria門を保有する個体はそれぞれ３頭、７頭であったが、糞便移植によりそれぞれ９頭、１０頭に増加した。
- Identification of bacteria correlated with clinical score In order to clarify the bacterial species that changed due to fecal transplantation, taxonomy analysis was performed for each ASV. Taxonomy analysis (classification of each ASV) was performed using SILVA138 as a reference. Thereby, the abundance (total value of the number of reads) of each bacterium was calculated from the number of reads of ASV. The value obtained by dividing the abundance by the total number of reads per sample was defined as the occupancy rate. The phylum-level bacterial flora composition of each specimen is shown in Figures 7 and 8. FIG. 8 is a diagram in which the bacterial flora composition is rearranged in the order of Pre, Post1, and Post2 for each individual based on the results of FIG. The intestinal flora of the two donors was dominated by the phyla Firmicutes, Fusobacteria, Proteobacteria, and Actinobacteria. In the diseased group, all animals possessed the phyla Firmicutes and Proteobacteria. On the other hand, the number of individuals possessing the Fusobacteria phylum and Actinobacteria phylum was 3 and 7, respectively, but this increased to 9 and 10, respectively, by fecal transplantation.
次に、レシピエントが糞便移植によりドナーから獲得した細菌についてより詳細を明らかにするため、ドナーから獲得したＡＳＶの同定結果とそれを保有する個体の割合について調査した（図９、１１）。糞便移植後、ドナーＮｏ．７の糞便の糞便移植を受けたレシピエントからは新たに４４、ドナーＶａｎｉｌｌａの糞便移植を受けたレシピエントからは新たに６０個のＡＳＶが検出され、このうち２６個のＡＳＶについては両ドナーに共通していた。図９は、ドナーＮｏ．７の糞便の糞便移植を受けたレシピエントから新たに検出された４４個のＡＳＶリストであり、太字は後述する図１１に示すドナーＶａｎｉｌｌａと共通であったＡＳＶを示す。また、Ｐｏｓｔ１（％）、Ｐｏｓｔ２（％）は、当該ＡＳＶを保有する個体の割合を示す。また、図１１は、ドナーＶａｎｉｌｌａの糞便移植を受けたレシピエントから新たに検出された６０個のＡＳＶリストであり、太字は図９に示すドナーＮｏ．７と共通であったＡＳＶを示す。各ＡＳＶの保有個体の割合は１７～１００％のばらつきがあったが、その多くが５６日後まで維持されていた。 Next, in order to clarify the details of the bacteria that the recipient acquired from the donor through fecal transplantation, we investigated the identification results of ASV acquired from the donor and the proportion of individuals carrying it (Figures 9 and 11). After fecal transplantation, donor no. 44 new ASVs were detected in recipients who received fecal transplants from donor Vanilla, and 60 new ASVs were detected from recipients who received fecal transplants from donor Vanilla, of which 26 ASVs were detected in both donors. It was common. FIG. 9 shows donor no. This is a list of 44 ASVs newly detected from a recipient who received a fecal transplant using feces from No. 7, and the bold letters indicate ASVs that were common to the donor Vanilla shown in FIG. 11, which will be described later. Further, Post1 (%) and Post2 (%) indicate the proportion of individuals possessing the ASV. Furthermore, FIG. 11 is a list of 60 ASVs newly detected from recipients who received donor Vanilla's fecal transplant, and the bold characters are the donor numbers shown in FIG. The ASV that was common to 7 is shown. The percentage of individuals carrying each ASV varied from 17% to 100%, but most of them were maintained until 56 days later.
さらに、レシピエントがドナーから新規に獲得した各ＡＳＶの占有率と臨床スコア、および、臨床スコアの改善率の相関解析を実施した。具体的には、ドナーＮｏ．７から糞便移植を受けたレシピエントのＰｒｅ、Ｐｏｓｔ１、Ｐｏｓｔ２の腸内細菌叢のデータより、図９で検出されている各ＡＳＶのリード数を抽出し、各ＡＳＶのリード数と各スコアの相関係数（Ｓｐｅａｒｍａｎ’ｓ ｒａｎｋ ｃｏｒｒｅｌａｔｉｏｎ ｃｏｅｆｆｉｃｉｅｎｔ）を算出した。なお、スコア改善率は、（Ｐｏｓｔ－Ｐｒｅ）／Ｐｒｅとして計算し、Ｐｒｅのスコア改善率は０とした。ｐ＜０．０５であったＡＳＶを図１０に示した。ドナーＶａｎｉｌｌａから糞便移植を受けたレシピエントに関しても同様に実施した。結果、複数のＡＳＶとの相関が認められた（図１２）。これらのＡＳＶについて属レベルで集計した結果、臨床スコアとの相関がみられた（ｐ＜０．０５、ｒｈｏ＞０．４）細菌は、Fusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属の１９属であった（図１３）。当該結果から、これらの１９属の細菌の存在は、アトピー性皮膚炎の症状改善に寄与する可能性が示された。 Furthermore, a correlation analysis was performed between the occupancy rate of each ASV newly acquired by the recipient from the donor, the clinical score, and the improvement rate of the clinical score. Specifically, donor no. The number of reads for each ASV detected in Figure 9 was extracted from the Pre, Post1, and Post2 intestinal flora data of recipients who received a fecal transplant from 7, and the correlation between the number of reads for each ASV and each score was calculated. Spearman's rank correlation coefficient was calculated. Note that the score improvement rate was calculated as (Post-Pre)/Pre, and the score improvement rate of Pre was set to 0. ASVs with p<0.05 are shown in FIG. 10. The same procedure was performed for recipients who received fecal transplants from donor Vanilla. As a result, a correlation with multiple ASVs was observed (FIG. 12). As a result of aggregating these ASVs at the genus level, the bacteria that showed a correlation with the clinical score (p<0.05, rho>0.4) were Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, and Bacteroides. , Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium, Prevotella, Blautia, Lachnospiraceae NK4A136 group, Erysipelatoclostridium, Collinsella, and Holdemanella. (Figure 13). The results indicated that the presence of these 19 genera of bacteria may contribute to improving the symptoms of atopic dermatitis.
これらの細菌の獲得による二次的な細菌叢の変化を調べる目的で、ドナー由来ではない細菌もすべて含めて、ＰｒｅとＰｏｓｔ１のＬｉｎｅａｒ ｄｉｓｃｒｉｍｉｎａｎｔ ａｎａｌｙｓｉｓ ｅｆｆｅｃｔ ｓｉｚｅ (ＬＥｆＳｅ)解析を実施した。疾患群のＰｒｅとＰｏｓｔ１の腸内細菌叢の属レベルのａｂｕｎｄａｎｃｅについてＬＥｆＳｅ解析を実施した。ＬＥｆＳｅ解析の結果、|ＬＤＡ ｓｃｏｒｅ|＞２、かつｐ＜０．０５ （Ｋｒｕｓｋａｌ-Ｗａｌｌｉｓ ｔｅｓｔ）をカットオフとし、これを満たすものを図１４に記載した。図１４中、上の黒いバーはＰｏｓｔ１よりもＰｒｅで多かった属を示し、下のグレーのバーは、ＰｒｅよりもＰｏｓｔ１で多かった属を示す。また、黒い丸で示した属は、図１３で前述した臨床スコアとの相関がみられた１９の細菌属と共通する属を示す。その結果、糞便移植後、Echerichia Shigella属菌、および、Clostridioides属菌の減少が認められた。また、ドナーから獲得した９属を含む１１属の細菌の増加が認められた。 In order to investigate changes in the bacterial flora secondary to the acquisition of these bacteria, Pre and Post1 linear discriminant analysis effect size (LEfSe) analysis was performed, including all non-donor-derived bacteria. LEfSe analysis was performed on the genus-level abundance of the intestinal flora of Pre and Post1 disease groups. As a result of the LEfSe analysis, |LDA score| > 2 and p < 0.05 (Kruskal-Wallis test) were set as cutoffs, and those satisfying this are shown in FIG. In FIG. 14, the upper black bar indicates genera that were more abundant in Pre than Post1, and the lower gray bar indicates genera that were more numerous in Post1 than Pre. Furthermore, the genera indicated by black circles are common to the 19 bacterial genera that were found to be correlated with the clinical score described above in FIG. As a result, a decrease in Echerichia Shigella and Clostridioides was observed after fecal transplantation. Additionally, an increase in 11 genera of bacteria, including 9 genera acquired from donors, was observed.
・同定した１９属の細菌を含む糞便移植の汎用性
前述の１９属細菌を含む糞便移植のアトピー性皮膚炎以外の疾患への応用可能性について調べるために、過去に解析を行った個体の腸内細菌叢のデータを用いて、以下の複数の疾患に罹患したイヌ（疾患群）と健常なイヌ（健常群）の腸内細菌叢を比較した。健常群は、過去に罹患した疾患がなく、現在罹患中の疾患がない個体をランダムに抽出した。
- Versatility of fecal transplants containing bacteria from the 19 genera identified In order to investigate the applicability of fecal transplants containing bacteria from the 19 genera mentioned above to diseases other than atopic dermatitis, we used the intestines of individuals previously analyzed. Using data on the intestinal flora, we compared the intestinal flora of dogs with the following multiple diseases (disease group) and healthy dogs (healthy group). For the healthy group, individuals with no past or current disease were randomly selected.
まず、アレルギー性皮膚炎（ｎ＝７）、慢性腎臓病（ｎ＝２６）、膀胱炎（ｎ＝２０）、糖尿病（ｎ＝９）、てんかん（ｎ＝１７）、肝炎（ｎ＝１８）、副腎皮質機能亢進症（ｎ＝２２）、椎間板ヘルニア（ｎ＝５０）、ケンネルコフ（ｎ＝１２）、僧帽弁閉鎖不全症（ｎ＝１１５）、膵炎（ｎ＝３０）、尿路結石症（ｎ＝５５）の１２の疾患に罹患しているイヌの細菌叢と健常（ｎ＝８５）なイヌの細菌叢について、Ｓｈａｎｎｏｎ ｉｎｄｅｘを算出し、比較したところ、７疾患（慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎）で健常なイヌと有意差（ｐ＜０．０５）が認められた。 First, allergic dermatitis (n=7), chronic kidney disease (n=26), cystitis (n=20), diabetes (n=9), epilepsy (n=17), hepatitis (n=18), Hyperadrenocorticism (n = 22), intervertebral disc herniation (n = 50), kennel cough (n = 12), mitral regurgitation (n = 115), pancreatitis (n = 30), urolithiasis ( The Shannon index was calculated and compared for the bacterial flora of dogs suffering from 12 diseases (n = 55) and the flora of healthy dogs (n = 85). Significant differences (p<0.05) were observed between healthy dogs and healthy dogs in the following: hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis.
この健常群と比較して腸内細菌叢のα多様性が低かった７疾患に罹患した疾患群と、健常群の細菌叢についてＬＥｆＳｅ解析を実施した。各疾患群において、ＬＥｆＳｅ解析にて健常群と比較して占有率が低くなっている細菌をスクリーニングした（図１５）。占有率は各細菌のａｂｕｎｄａｎｃｅ（リード数の合計値）を１検体あたりの総リード数で割った数値であり、細菌叢における各細菌の存在割合を示す。その結果、Colinsella属、Fusobacterium属、Megamonas属、および、Blautia属は７疾患、Sutterella属は６疾患、Prevotella属は４疾患、Bacteroides属は３疾患において健常群と比較して占有率が低かった。以上の事から、これらの属は種々の疾患の治療や予後の改善、診断に応用できる可能性があることが示唆された。 LEfSe analysis was performed on the bacterial flora of the healthy group and the disease group affected by 7 diseases in which the α-diversity of the intestinal flora was lower than that of the healthy group. In each disease group, bacteria with a lower occupancy rate than the healthy group were screened by LEfSe analysis (FIG. 15). The occupancy rate is a value obtained by dividing the abundance (total value of the number of reads) of each bacterium by the total number of reads per sample, and indicates the proportion of each bacterium in the bacterial flora. As a result, the occupancy rate was lower in 7 diseases for the genera Collinsella, Fusobacterium, Megamonas, and Blautia, 6 diseases for the genus Sutterella, 4 diseases for the genus Prevotella, and 3 diseases for the genus Bacteroides compared to the healthy group. The above results suggest that these genera may be applicable to the treatment, prognosis improvement, and diagnosis of various diseases.
以上、本実施形態について説明したが、上記実施形態は本発明の理解を容易にするためのものであり、本発明を限定して解釈するためのものではない。本発明は、その趣旨を逸脱することなく、変更、改良され得ると共に、本発明にはその等価物も含まれる。
Although the present embodiment has been described above, the above embodiment is for facilitating understanding of the present invention, and is not for construing the present invention in a limited manner. The present invention may be modified and improved without departing from the spirit thereof, and the present invention also includes equivalents thereof.
Claims (15)
前記微生物の存在量に基づいて、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の予防又は治療のためのドナー糞便としての有用性を評価するステップとを含む、糞便の評価方法。 Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium, Prevotella, which are included in the fecal flora. Analyzing the abundance of microorganisms belonging to at least one genus selected from the group consisting of Blautia genus, Lachnospiraceae NK4A136 group, Erysipelatoclostridium genus, Collinsella genus, and Holdemanella genus;
At least one disease selected from canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis based on the abundance of the microorganisms. A method for evaluating feces, comprising the step of evaluating its usefulness as donor feces for the prevention or treatment of feces.
前記微生物の存在量に基づいて、イヌのアトピー性皮膚炎、慢性腎臓病、膀胱炎、肝炎、副腎皮質機能亢進症、椎間板ヘルニア、僧帽弁閉鎖不全症、膵炎から選択される少なくとも１つの疾患の罹患可能性を診断するステップと、を含む診断方法。 Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium, Prevotella, which are included in the fecal flora. Analyzing the abundance of microorganisms belonging to at least one genus selected from the group consisting of Blautia genus, Lachnospiraceae NK4A136 group, Erysipelatoclostridium genus, Collinsella genus, and Holdemanella genus;
At least one disease selected from canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis based on the abundance of the microorganisms. A diagnostic method comprising the step of diagnosing the possibility of being affected by.
前記イヌの少なくとも治療前と治療後の糞便中におけるFusobacterium属、Sutterella属、Romboutsia属、Phascolarctobacterium属、Bacteroides属、Intestinimonas属、Megamonas属、Peptoclostridium属、Helicobacter属、Allobaculum属、Alloprevotella属、Butyricicoccus属、Catenibacterium属、Prevotella属、Blautia属、Lachnospiraceae NK4A136 group、Erysipelatoclostridium属、Collinsella属、および、Holdemanella属からなる群から選択される少なくとも１つの属に属する微生物の存在量を解析するステップと
前記微生物の前記治療前と前記治療後における前記存在量を比較するステップと、
を含む、予後評価方法。
A method for evaluating the prognosis after treatment of at least one disease selected from canine atopic dermatitis, chronic kidney disease, cystitis, hepatitis, hyperadrenocorticism, intervertebral disc herniation, mitral regurgitation, and pancreatitis. And,
Fusobacterium, Sutterella, Romboutsia, Phascolarctobacterium, Bacteroides, Intestinimonas, Megamonas, Peptoclostridium, Helicobacter, Allobaculum, Alloprevotella, Butyricicoccus, Catenibacterium in the feces of the dog at least before and after treatment. analyzing the abundance of microorganisms belonging to at least one genus selected from the group consisting of the genus Prevotella, the genus Blautia, the Lachnospiraceae NK4A136 group, the genus Erysipelatoclostridium, the genus Collinsella, and the genus Holdemanella; and the step of analyzing the abundance of the microorganisms before the treatment of the microorganisms. and comparing the abundance after the treatment;
Prognosis evaluation methods, including.
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