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WO2018155612A1 - Composition for prevention and/or treatment of infection relating to salmonella including bacillus amyloliquefaciens strain and/or treated product of said strain - Google Patents

Composition for prevention and/or treatment of infection relating to salmonella including bacillus amyloliquefaciens strain and/or treated product of said strain Download PDF

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WO2018155612A1
WO2018155612A1 PCT/JP2018/006627 JP2018006627W WO2018155612A1 WO 2018155612 A1 WO2018155612 A1 WO 2018155612A1 JP 2018006627 W JP2018006627 W JP 2018006627W WO 2018155612 A1 WO2018155612 A1 WO 2018155612A1
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strain
salmonella
bacillus amyloliquefaciens
present
feed
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PCT/JP2018/006627
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French (fr)
Japanese (ja)
Inventor
元一郎 瀬尾
佐藤　直樹
和夫 福井
太樹夫 稲富
智英 増田
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東亜薬品工業株式会社
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Publication of WO2018155612A1 publication Critical patent/WO2018155612A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • 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
    • 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
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry

Definitions

  • the present invention uses a composition for preventing, controlling, suppressing, reducing, alleviating, and / or treating infectious diseases related to Salmonella containing a Bacillus amyloliquefaciens strain and / or a processed product of the strain, and the composition.
  • the present invention relates to a method for treating an infection related to Salmonella.
  • Patent Document 1 discloses a “Bacillus amyloliquefaciens strain with deposit number NITE ABP-01844”. However, Patent Document 1 does not disclose or suggest that the Bacillus amyloliquefaciens strain is effective for infectious diseases related to Salmonella.
  • Patent Document 2 discloses “an intestinal Salmonella bacterium reducing agent for the addition of avian feed, which contains live cells of Bacillus subtilis C-3102 (Mikokenjoyo No. 1096) as an active ingredient”. ing. However, it does not disclose or suggest the Bacillus amyloliquefaciens strain having the deposit number NITE ABP-01844, which is a strain according to the present invention.
  • An object of the present invention is to provide a composition effective for infectious diseases related to Salmonella and a method for treating infectious diseases related to Salmonella.
  • the Bacillus amyloliquefaciens strain has an effect on Salmonella typhimurium, which is a causative agent of Salmonella infectious diseases.
  • the present invention is as follows.
  • a composition for preventing, controlling, suppressing, reducing, alleviating, and / or treating infectious diseases related to Salmonella containing a Bacillus amyloliquefaciens strain and / or a processed product of the strain 2. 2. The composition according to item 1, wherein the infectious disease is an infectious disease in livestock. 3. 3. The composition according to item 1 or 2, wherein the Salmonella is Salmonella typhimurium. 4). 3. The composition according to item 1 or 2, wherein the Salmonella is Salmonella enteritidis. 5). 5. The composition according to any one of items 1 to 4, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844. 6).
  • a veterinary drug, feed, feed additive, or drinking water comprising the composition according to any one of 1 to 5 above. 7). Salmonella characterized by administering the veterinary drug, feed, feed additive or drinking water according to item 6 and / or the composition according to any one of items 1 to 5 to an animal other than a human. A method of rearing an animal while preventing, controlling, suppressing, reducing, alleviating, and / or treating an infectious disease. 8). A method for breeding livestock while preventing, controlling, suppressing, reducing, alleviating, and / or treating an infection related to Salmonella, comprising a step of administering a Bacillus amyloliquefaciens strain and / or a processed product of the strain to an animal. 9.
  • a method for preventing, controlling, suppressing, reducing, alleviating, and / or treating an infection related to Salmonella comprising a step of administering a Bacillus amyloliquefaciens strain and / or a processed product of the strain to an animal. 10. 10. The method according to item 8 or 9, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844. 11. A Salmonella typhimurium and / or Salmonella enteritidis control agent comprising a Bacillus amyloliquefaciens strain and / or a processed product of the strain. 12 12.
  • Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
  • Salmonella typhimurium comprising a step of adding a Bacillus amyloliquefaciens strain and / or a processed product thereof to a place where Salmonella typhimurium and / or Salmonella enteritidis are bred or bred, and / Or Salmonella Enteritidis extermination method.
  • 14 14. The method according to item 13 above, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
  • composition, breeding method or treatment method of the present invention had at least one of the following effects.
  • Immunostimulatory effect in chicken serum lysozyme concentration increasing effect, macrophage phagocytosis increasing effect, macrophage chemotaxis increasing effect, and small intestine total IgA concentration increasing effect).
  • the comparison result of the organic acid production ability of the strain based on this invention and a well-known strain The measurement result of Salmonella typhimurium microbe count of Example 2.
  • FIG. The calculation result of the detection rate of Salmonella typhimurium of Example 2.
  • FIG. The measurement result of the Salmonella typhimurium microbe count of the infected individual
  • FIG. The calculation result of Salmonella typhimurium detection rate of the living individual of Example 3.
  • the composition for preventing, controlling, suppressing, reducing, alleviating and / or treating infectious diseases related to Salmonella of the present invention is at least a Bacillus amyloliquefaciens strain. And / or a processed product of the strain.
  • at least a Bacillus amyloliquefaciens strain and / or a processed product of the strain is preferably used for animals (particularly livestock). Is administered orally or with drinking water.
  • Bacillus amyloliquefaciens strain Bacillus amyloliquefaciens according to the present invention (Bacillus amyloliquefaciens, Bacillus amyloliquefaciens) strains, can utilize known strains, particularly preferred strains, deposited as accession number NITEABP-01844 Is TOA5001 stock.
  • the mycological properties of the strain (bacteria) according to the present invention are as follows. Gram-positive, Neisseria gonorrhoeae, aerobic, spore-forming ability, motility. It is low in auxotrophy and can grow on various media such as gravy medium, SCD medium, brain heart infusion medium. On the agar plate medium, the periphery is rough and yellowish white. Furthermore, depending on the type of the medium (Brain Heart Infusion Medium), a raised colony having viscosity inside is formed.
  • the strain according to the present invention is not limited to the strain deposited under the deposit number NITE ABP-01844, but belongs to Bacillus amyloliquefaciens, and Salmonella (particularly Salmonella, which is the causative agent of Salmonella infection). As long as it has an effect on Tifumilium and Salmonella enteritidis), it can be used as a strain according to the present invention. That is, the Bacillus amyloliquefaciens strain of the present invention is intended to include not only the deposited strain itself but also mutants and progeny that are strains having the above-mentioned mycological properties.
  • the mutant preparation method can be obtained by conventionally known irradiation and chemical substance treatment such as nitrosoguanidine, ethylmethanesulfonic acid, methylmethanesulfonic acid and the like. Furthermore, the strain according to the present invention is not toxic even if it is administered in vivo (particularly in livestock), as shown in the Examples below.
  • the strain according to the present invention is obtained by inoculating a nutrient medium containing a carbon source and a nitrogen source and, if necessary, inorganic salts, followed by culturing under aerobic conditions (for example, shaking culture, aeration and agitation culture), Can be obtained.
  • aerobic conditions for example, shaking culture, aeration and agitation culture
  • the culture solution it is effective against the causative bacteria of the infectious disease (for example, Salmonella typhimurium and Salmonella enteritidis) in the unconcentrated culture solution, and the culture solution after filter sterilization Or they have equivalent activity even after freeze-drying or spray-drying.
  • an infectious disease is caused by using the solid itself or a mixture thereof (for example, a contact or mixture with another solid or a liquid in which the strain-growing solid according to the present invention is immersed). It can have an effect on the causative bacteria.
  • the solid may be any material as long as the strain according to the present invention can grow, and means, for example, a plant body that is a raw material for agar medium, silage, natto and the like, and curd represented by yogurt.
  • Any carbon source may be used as long as the strain according to the present invention can be used.
  • Glucose, sucrose, starches, cellulose mixtures, and other carbohydrates are preferably used.
  • the nitrogen source may be any as long as the strain according to the present invention can be used, but preferably peptone, yeast extract, gravy extract and the like are used.
  • the strain according to the present invention can be grown in a liquid medium or a plate culture inoculated on a commercially available medium, for example, tryptosy medium, brain heart infusion medium, or gravy medium.
  • the culture When culturing in a small amount in liquid culture, shaking culture using a test tube or flask is preferred. When culturing in large quantities, it is preferable to culture with aeration and agitation as in the case of other fermentation products.
  • culture When culture is performed in a large tank, it is preferable to first inoculate and culture the strain according to the present invention in a relatively small amount of medium as a preculture, and then transfer the culture to a large production tank for production culture.
  • the composition of the medium used for the preculture and the medium used for the production culture may be the same, or may be changed if necessary.
  • the culture conditions can be appropriately changed within the range in which the strain according to the present invention grows. Usually, the culture is performed at 15 to 45 ° C., preferably 25 to 37 ° C. under aerobic conditions.
  • the culture time varies depending on the culture conditions and the culture volume, but is usually about 1 day to 1 week.
  • the strain according to the present invention can be recovered from the culture solution obtained by the above culture by centrifugation and / or membrane concentration.
  • the collected bacterial cells may be used as they are, or dried bacterial cells that have been subjected to freeze-drying or spray-drying as necessary.
  • the treated product of the strain according to the present invention is various treatments on the cultured strain (washing, far-dehydration, heating, gamma ray or neutron irradiation, spray drying, freeze drying, sterilization, enzyme, surface activity Agent, grinding, grinding, etc.).
  • composition of the present invention includes at least the strain (including cells) according to the present invention and / or a processed product of the strain, and prevents, controls, suppresses, reduces, alleviates, and / or treats an infection related to Salmonella. And, in some cases, have a control, elimination, and / or cure effect.
  • the infection related to Salmonella of the present invention is not particularly limited as long as it is an infection caused by Salmonella.
  • Salmonella Salmonella Typhimurium , Salmonella Typhimurium , Salmonella Typhi ), Salmonella Agona , Salmonella Saintpaul , Salmonella Heidelberg , Salmonella Choleraesuis , Salmonella Infantis , Salmonella Infantis , Salmonella Infantis (Salmonella Thompson), Salmonella Enteritidis (Salmonella Enteritidis), can be exemplified Salmonella Dublin (Salmonella Dublin) and the like.
  • the target of treatment for infectious diseases is animals including humans (particularly mammals).
  • livestock cattle, pigs, chickens
  • pets more specifically pigs, dogs, cats, fish
  • birds ⁇ chicken Especially, chicks, broilers, egg-laying chickens
  • fattening cattle such as dairy cows, beef cattle, F1 (Holstein and Wagyu crossbreed), milking cows, horses, mice and the like
  • milking cows horses, mice and the like
  • mice and the like can be exemplified, but not particularly limited.
  • any concentration may be used as long as the desired effect can be exerted.
  • the strain according to the present invention is 1 kg. It is preferable to feed the feed added so as to be 10 5 to 10 9 cfu (Colony forming unit).
  • the administration time, administration method, and dose of the composition of the present invention are not particularly limited, but it may be usually added to a commercial feed or mixed diet.
  • the amount of administration (feeding, feeding) when blended with commercial feed or administered with mixed feeding (feeding, feeding) is 0.005% to 10%.
  • the dose of the composition of the present invention is not particularly limited. For example, it is desirable to add 0.005% to 10% to a commercial feed.
  • composition of this invention is as follows, for example.
  • Composition for prevention, control, suppression, reduction, alleviation, and / or treatment of infectious diseases related to Salmonella (2) Feed for animal (particularly livestock), feed additive and breeding water
  • Prevention, control, suppression, reduction and alleviation of infectious diseases related to Salmonella characterized by administering veterinary drugs, feeds, feed additives or drinking water and / or the composition of the present invention to animals other than humans And / or methods of raising animals while being treated are also of interest.
  • the present invention is also directed to a method of combating Salmonella typhimurium and Salmonella enteritidis.
  • the Salmonella typhimurium and / or Salmonella enteritidis control agent of the present invention contains at least a Bacillus amyloliquefaciens strain and / or a processed product of the strain.
  • the pesticide of the present invention relates to Salmonella by adding it to a place where Salmonella typhimurium and / or Salmonella enteritidis are breeding or expected to breed (particularly a breeding facility, aquarium, etc.). It is possible to prevent, suppress, reduce, etc. the occurrence of infectious diseases.
  • the Salmonella typhimurium and / or Salmonella enteritidis extermination method of the present invention comprises at least a Bacillus amyloliquefaciens strain and / or a processed product of the strain as Salmonella typhimurium and / or Salmonella enteritidis. Is added to the place where breeding or breeding (especially breeding facilities, aquariums, etc.).
  • the composition of the present invention widely includes veterinary drugs, foods and drinks, feed additives, feeds (formulated feeds, mixed feeds), and breeding water having the above uses.
  • the composition of the present invention is blended with additives such as starch, potato starch, lactose, soybean protein and the like, excipients, binders, disintegrants, lubricants, stabilizers, suspending agents and the like. These can be formulated into powders, tablets, granules, capsules, ointments, liquids and the like by known methods.
  • the organic acid producing ability of the strain according to the present invention was compared with the known strain organic acid producing ability. Details are as follows.
  • the strain according to the present invention and the known strain, B. subtilis reference strain were cultured under the same culture conditions. Specifically, each strain was inoculated into a separate brain heart infusion liquid medium (manufactured by BBL) and cultured with shaking at 37 ° C. for 24 hours. After the culture, the liquid phase and the solid phase cells were separated from the culture solution by cooling centrifugation (10000 ⁇ g, 10 minutes).
  • the obtained liquid phase part was sterilized and filtered through a 0.45 ⁇ m membrane filter.
  • the filtered liquid was used as a culture supernatant for organic acid measurement by the following HPLC.
  • About 0.2 ml of the culture supernatant of each strain was collected in a 1.5 mL tube, and 50 ⁇ L of 500 mM trans-crotonic acid was added as an internal standard substance. Then, it extracted twice with 0.6 mL of 0.25% ammonia as an extract.
  • To the obtained supernatant 0.3 times the amount of 10% HClO 4 was added to precipitate and remove the protein.
  • the obtained supernatant was filtered through a 0.45 ⁇ m membrane filter to obtain an HPLC sample.
  • HPLC was performed under the following conditions.
  • HPLC system manufactured by Waters Column: Organic Acid Column (7.8 ⁇ 300 mm) and guard column (6.0 ⁇ 50 mm) Column temperature and post-column reaction temperature: 60 ° C Flow rate: 0.8mL / min Mobile phase: 0.08% HClO 4 Reaction phase: 0.2 mM BTB, 5.2 mM NaOH, 15 mM Na 2 HPO 4 Detection wavelength: 445 nm
  • the comparison result of the organic acid production ability computed from the result of HPLC is shown in FIG.
  • the acetic acid producing ability of the strain according to the present invention is at least 2.7 times (48.7 / 4) higher than the acetic acid producing ability of the known Bacillus subtilis reference strain. 17.9). From the above results, the strain according to the present invention has higher organic acid producing ability than known strains. More specifically, since organic acids including acetic acid have antibacterial action, it was confirmed that the strain according to the present invention has higher antibacterial activity than known strains.
  • the day of gavage administration was fed on a commercial feed for adult chickens (SDL No. 4 [manufactured by Nippon Formulad Feed Co., Ltd.)] for all wards, and was reared for 28 days.
  • SDL No. 4 manufactured by Nippon Formulad Feed Co., Ltd.
  • test group 1 feed after culturing for 2 to 3 days under the same culture conditions as in Example 1, the cells recovered by centrifugation were mixed with rice bran oil cake and air-dried to obtain 1 ⁇ 10 8 cfu / g dry powder.
  • the known Bacillus subtilis strain was added to a 2.0 ⁇ 10 5 cfu / g feed.
  • test group 2 feed After culturing for 2 to 3 days under the same culture conditions as in Example 1, the cells recovered by centrifugation were mixed with rice bran oil cake and air-dried to obtain 1 ⁇ 10 8 cfu / g dry powder.
  • the strain of the present invention was added to 2.0 ⁇ 10 5 cfu / g feed.
  • 14th and 28th days after the start of the test 5 birds were sacrificed in each group, the contents of the cecum were collected, and the number of Salmonella typhimurium bacteria (log cfu / g) was measured. Furthermore, the detection rate (%) was calculated from the presence or absence of detection of Salmonella typhimurium in the cecal contents of 5 individuals in each section 3 days, 14 days and 28 days after the start of the test.
  • the measurement result of Salmonella typhimurium bacteria count is shown in FIG. As is clear from FIG. 2, in test group 2, the number of Salmonella typhimurium in the cecum decreased by 28.4% between 3 and 28 days after the start of the test, whereas in test group 1 The number of Salmonella typhimurium in the cecum decreased by 2.2% over 28 days, and the number of Salmonella typhimurium in the cecum increased by 12.3% over 28 days in the control group. The calculation result of the detection rate of Salmonella typhimurium is shown in FIG. As apparent from FIG.
  • the strain of the present invention has an effect of suppressing the growth of Salmonella typhimurium in adult chickens. Moreover, it confirmed that it had the Salmonella typhimurium inhibitory effect superior to the well-known Bacillus subtilis strain
  • test group 1 diet was added a known Bacillus subtilis strain was the same dry powder as in Example 2 so as to be 2.0 ⁇ 10 5 cfu / g forage.
  • strain of the present invention in the form of a dry powder was added in the same manner as in Example 2 so as to be 2.0 ⁇ 10 5 cfu / g feed.
  • FIG. 4 shows the measurement results of the number of cecal Salmonella typhimurium bacteria (log cfu / g) in infected individuals.
  • the number of Salmonella typhimurium bacteria in the chicken chick was 25.7% lower than that in the control group and 8% lower than that in the test group 1. Therefore, it was confirmed that the strain of the present invention has an effect of suppressing the growth of Salmonella typhimurium in the chick chick.
  • FIG. 5 shows the result of the infection status ⁇ detection rate of Salmonella typhimurium (%) ⁇ in the cecum of the chicken] for the lived individual. As is clear from FIG.
  • the strain of the present invention has an effect of preventing Salmonella typhimurium infection in chicken chicks. Moreover, it confirmed that it had the Salmonella typhimurium infection prevention effect superior to the well-known Bacillus subtilis strain
  • the strain of the present invention in the form of a dry powder was added in the same manner as in Example 2 so as to be 2.0 ⁇ 10 5 cfu / g feed.
  • the temperature inside the poultry house was 30 ° C. for 1-7 days of age, 28-25 ° C. for 8-21 days, 24-22 ° C. for 22-35 days, and 20 ° C. after 36 days.
  • immune indices serum lysozyme concentration, macrophage phagocytosis, macrophage chemotaxis, and small intestine total IgA concentration
  • Serum lysozyme concentration was measured using ELISA measurement kit (manufactured by SIGMA-ALDRICH) according to the manufacturer's manual. Macrophage phagocytosis rate and macrophage chemotaxis can be determined by immunological function analysis manual ([online], September 29, 2016, National Livestock Improvement Center Hyogo Farm Special Breeding Subcommittee, [Search February 8, 2017], ⁇ Http://www.nlbc.go.jp/hyogo/tishiki/boueki/boueki/boueki/c21bd718afed5f0d1adee9349fd0b4ffe9b5c9cb.pdf>).
  • the small intestine IgA concentration was measured according to the manufacturer's manual using an ELISA measurement kit (Bethel Laboratories, Inc.).
  • the results of serum lysozyme concentration are shown in FIG. As is clear from FIG. 6, in the test group 2, the serum lysozyme concentration ( ⁇ g / mL) was higher than that in the control group and the test group 1.
  • the result of macrophage phagocytosis is shown in FIG. As is clear from FIG. 7, the macrophage phagocytosis rate (%) in test group 2 was higher than that in control group and test group 1.
  • the results of macrophage chemotaxis are shown in FIG. As is clear from FIG. 8, macrophage chemotaxis (%) was higher in test group 2 than in control group and test group 1.
  • the results of the small intestine total IgA concentration are shown in FIG. As is clear from FIG.
  • the test group feed was added strains of the present invention which was the same dry powder as in Example 2 so as to be 2.0 ⁇ 10 6 cfu / g forage.
  • the feed start date of the feed is day 0 (before feeding), 10 days, 12 days, 14 days, 16 days, 21 days, and 35 days.
  • the detection rate (%) was calculated from the presence or absence of detection of Salmonella enteritidis bacteria in the cecum contents.
  • the calculation result of the detection rate of Salmonella enteritidis is shown in FIG. As is clear from FIG. 11, in the control group, the detection rate was 100% (15/15 birds) at 0 days, 10 days, 12 days, 14 days, and 35 days after the start of salary, In the test area, the detection rate decreased to 33.3% (5/15) on the 35th. From the above, it was confirmed that the strain of the present invention has an effect of inhibiting the growth of Salmonella enteritidis in adult chickens.
  • Example 3 (Salmonella enteritidis infection prevention test in chicks) The procedure was the same as in Example 3 except that Salmonella enteritidis wild strain was forcibly administered orally instead of Salmonella typhimurium wild strain.
  • the strain of the present invention has an effect of suppressing the growth of Salmonella enteritidis in chicken chicks. Moreover, it confirmed that the strain of this invention has the Salmonella enteritidis infection prevention effect of a chicken chick. Furthermore, it was confirmed that it has a better Salmonella enteritidis infection prevention effect than the known Bacillus subtilis strain.
  • compositions, breeding method or treatment method of the present invention had the following effects.
  • Immunostimulatory effect in livestock serum lysozyme concentration increasing effect, macrophage phagocytosis increasing effect, macrophage chemotaxis increasing effect, and small intestine total IgA concentration increasing effect).
  • the present invention can provide a composition for preventing and / or treating infectious diseases related to Salmonella containing a Bacillus amyloliquefaciens strain and / or a processed product of the strain.

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Abstract

Provided is a composition effective for salmonella-related infection. The discovery was made that the Bacillus amyloliquefaciens strain is effective on Salmonella typhimethylium which is a causative bacterium of salmonella-related infection.

Description

バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を含むサルモネラ菌に関する感染症予防及び／又は治療用組成物Bacillus amyloliquefaciens strain and / or composition for prevention and / or treatment of infectious disease related to Salmonella containing treated product of the strain
　本発明は、バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を含むサルモネラ菌に関する感染症予防、防除、抑制、軽減、緩和、及び／又は治療用組成物、並びに該組成物を使用したサルモネラ菌に関する感染症の治療方法に関する。
　本出願は、参照によりここに援用されるところの日本出願特願2017-034085号優先権を請求する。 The present invention uses a composition for preventing, controlling, suppressing, reducing, alleviating, and / or treating infectious diseases related to Salmonella containing a Bacillus amyloliquefaciens strain and / or a processed product of the strain, and the composition. The present invention relates to a method for treating an infection related to Salmonella.
This application claims the priority of Japanese Patent Application No. 2017-034085, which is incorporated herein by reference.
（現在の畜産におけるサルモネラ菌に関する感染症の問題）
　現在の農産畜産業において、サルモネラ菌に関する感染による問題は深刻である。
　特に、鶏はサルモネラに対して感受性が高いこと、同一の系統や日齢のものが集団で飼育されておりかつ飼育規模が大きいこと、介卵感染が成立し、サルモネラが保菌種鶏から雛に伝播し、さらに感染した雛が広範囲に輸送されて汚染が拡大すること等が挙げられる。 (Infectious diseases related to Salmonella in current livestock)
In the current agricultural and livestock industry, problems caused by infection with Salmonella are serious.
In particular, chickens are highly sensitive to Salmonella, the same strains and ages are kept in groups and the scale of breeding is large, egg-borne infection is established, and Salmonella is transferred from a carrier-type chicken to chicks. And infected chicks are transported over a wide area and the contamination is expanded.
（先行技術）
　特許文献１では、「寄託番号NITE ABP-01844のバチルス・アミロリキファシエンス菌株」を開示している。しかしながら、特許文献１では、バチルス・アミロリキファシエンス菌株がサルモネラ菌に関する感染症に効果があることを開示又は示唆をしていない。
　特許文献２では、「バチルス・ズブチリスＣ－３１０２（微工研条寄第１０９６号）の生菌体を有効成分として含有する鳥類飼料添加用腸内サルモネラ（Salmonella)属細菌減少剤」を開示している。しかし、本発明に係る菌株である寄託番号NITE ABP-01844のバチルス・アミロリキファシエンス菌株を開示又は示唆をしていない。 (Prior art)
Patent Document 1 discloses a “Bacillus amyloliquefaciens strain with deposit number NITE ABP-01844”. However, Patent Document 1 does not disclose or suggest that the Bacillus amyloliquefaciens strain is effective for infectious diseases related to Salmonella.
Patent Document 2 discloses “an intestinal Salmonella bacterium reducing agent for the addition of avian feed, which contains live cells of Bacillus subtilis C-3102 (Mikokenjoyo No. 1096) as an active ingredient”. ing. However, it does not disclose or suggest the Bacillus amyloliquefaciens strain having the deposit number NITE ABP-01844, which is a strain according to the present invention.
特開２０１６－１１６４６６号公報Japanese Unexamined Patent Publication No. 2016-116466 特許２５２８０５５号公報Japanese Patent No. 2528055
　本発明は、サルモネラ菌に関する感染症に効果のある組成物並びにサルモネラ菌に関する感染症の治療方法を提供することを課題とする。 An object of the present invention is to provide a composition effective for infectious diseases related to Salmonella and a method for treating infectious diseases related to Salmonella.
　本発明者らは、上記課題に鑑み鋭意検討した結果、バチルス・アミロリキファシエンス菌株がサルモネラ菌に関する感染症の原因菌であるサルモネラ・ティフィミリウムに対して効果を有することを見出して、本発明を完成した。
　すなわち、本発明は以下の通りである。 As a result of intensive studies in view of the above problems, the present inventors have found that the Bacillus amyloliquefaciens strain has an effect on Salmonella typhimurium, which is a causative agent of Salmonella infectious diseases. Was completed.
That is, the present invention is as follows.
　１．バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を含むサルモネラ菌に関する感染症予防、防除、抑制、軽減、緩和、及び／又は治療用組成物。
　２．前記感染症は、家畜での感染症である前項１に記載の組成物。
　３．前記サルモネラ菌は、サルモネラ・ティフィミリウムである前項１又は２に記載の組成物。
　４．前記サルモネラ菌は、サルモネラ・エンテリティディスである前項１又は２に記載の組成物。
　５．前記バチルス・アミロリキファシエンス菌株は、寄託番号ＮＩＴＥ　ＡＢＰ－０１８４４を有する菌株である前項１～４のいずれか１に記載の組成物。
　６．前項１～５のいずれか１に記載の組成物を含む動物用医薬品、飼料、飼料添加物、又は飲料水。
　７．前項６に記載の動物用医薬品、飼料、飼料添加物又は飲料水、並びに／又は、前項１～５のいずれか１に記載の組成物を、ヒトを除く動物に投与することを特徴とするサルモネラ菌に関する感染症を予防、防除、抑制、軽減、緩和、及び／又は治療しながら動物を飼育する方法。
　８．バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を動物に投与する工程を含むサルモネラ菌に関する感染症予防、防除、抑制、軽減、緩和、及び／又は治療しながら家畜を飼育する方法。
　９．バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を動物に投与する工程を含むサルモネラ菌に関する感染症予防、防除、抑制、軽減、緩和、及び／又は治療する方法。
　１０．前記バチルス・アミロリキファシエンス菌株は、寄託番号ＮＩＴＥ　ＡＢＰ－０１８４４を有する菌株である前項８又は９に記載の方法。
　１１．バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を含むサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディス駆除剤。
　１２．前記バチルス・アミロリキファシエンス菌株は、寄託番号ＮＩＴＥ　ＡＢＰ－０１８４４を有する菌株である前項１１に記載の駆除剤。
　１３．バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物をサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディスが繁殖している又は繁殖する場所に添加する工程を含むサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディス駆除方法。
　１４．前記バチルス・アミロリキファシエンス菌株は、寄託番号ＮＩＴＥ　ＡＢＰ－０１８４４を有する菌株である前項１３に記載の方法。 1. A composition for preventing, controlling, suppressing, reducing, alleviating, and / or treating infectious diseases related to Salmonella containing a Bacillus amyloliquefaciens strain and / or a processed product of the strain.
2. 2. The composition according to item 1, wherein the infectious disease is an infectious disease in livestock.
3. 3. The composition according to item 1 or 2, wherein the Salmonella is Salmonella typhimurium.
4). 3. The composition according to item 1 or 2, wherein the Salmonella is Salmonella enteritidis.
5). 5. The composition according to any one of items 1 to 4, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
6). A veterinary drug, feed, feed additive, or drinking water comprising the composition according to any one of 1 to 5 above.
7). Salmonella characterized by administering the veterinary drug, feed, feed additive or drinking water according to item 6 and / or the composition according to any one of items 1 to 5 to an animal other than a human. A method of rearing an animal while preventing, controlling, suppressing, reducing, alleviating, and / or treating an infectious disease.
8). A method for breeding livestock while preventing, controlling, suppressing, reducing, alleviating, and / or treating an infection related to Salmonella, comprising a step of administering a Bacillus amyloliquefaciens strain and / or a processed product of the strain to an animal.
9. A method for preventing, controlling, suppressing, reducing, alleviating, and / or treating an infection related to Salmonella, comprising a step of administering a Bacillus amyloliquefaciens strain and / or a processed product of the strain to an animal.
10. 10. The method according to item 8 or 9, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
11. A Salmonella typhimurium and / or Salmonella enteritidis control agent comprising a Bacillus amyloliquefaciens strain and / or a processed product of the strain.
12 12. The pesticide according to 11 above, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
13. Salmonella typhimurium comprising a step of adding a Bacillus amyloliquefaciens strain and / or a processed product thereof to a place where Salmonella typhimurium and / or Salmonella enteritidis are bred or bred, and / Or Salmonella Enteritidis extermination method.
14 14. The method according to item 13 above, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
　本発明の組成物、飼育方法又は治療方法は、少なくとも以下のいずれか１以上の効果を有することを確認した。
（１）ニワトリの生体内に投与しても毒性がない。
（２）高い抗菌活性。
（３）ニワトリ体内におけるサルモネラ・ティフィミリウム及びサルモネラ・エンテリティディスの生育を抑制する効果。
（４）ニワトリにおけるサルモネラ・ティフィミリウム及びサルモネラ・エンテリティディス感染予防効果。
（５）ニワトリにおける免疫賦活効果（血清リゾチーム濃度上昇効果、マクロファージ貪食率上昇効果、マクロファージ走化性上昇効果、及び小腸総ＩｇＡ濃度上昇効果）。
（６）ニワトリにおける生存率向上効果。 It was confirmed that the composition, breeding method or treatment method of the present invention had at least one of the following effects.
(1) There is no toxicity even if it is administered in vivo in chickens.
(2) High antibacterial activity.
(3) The effect of suppressing the growth of Salmonella typhimurium and Salmonella enteritidis in the chicken.
(4) Prevention of Salmonella typhimurium and Salmonella enteritidis infection in chickens.
(5) Immunostimulatory effect in chicken (serum lysozyme concentration increasing effect, macrophage phagocytosis increasing effect, macrophage chemotaxis increasing effect, and small intestine total IgA concentration increasing effect).
(6) Effect of improving survival rate in chickens.
本発明に係る菌株と公知の菌株との有機酸産生能の比較結果。The comparison result of the organic acid production ability of the strain based on this invention and a well-known strain. 実施例２のサルモネラ・ティフィミリウム菌数の測定結果。The measurement result of Salmonella typhimurium microbe count of Example 2. FIG. 実施例２のサルモネラ・ティフィミリウム菌の検出率の算出結果。The calculation result of the detection rate of Salmonella typhimurium of Example 2. FIG. 実施例３の感染個体のサルモネラ・ティフィミリウム菌数の測定結果。The measurement result of the Salmonella typhimurium microbe count of the infected individual | organism | solid of Example 3. FIG. 実施例３の同居個体のサルモネラ・ティフィミリウム検出率の算出結果。The calculation result of Salmonella typhimurium detection rate of the living individual of Example 3. FIG. 実施例４の血清リゾチーム濃度の結果。Results of serum lysozyme concentration of Example 4. 実施例４のマクロファージ貪食率の結果。Results of macrophage phagocytosis rate of Example 4. 実施例４のマクロファージ走化性の結果。Results of macrophage chemotaxis in Example 4. 実施例４の小腸総ＩｇＡ濃度の結果。Results of small intestinal total IgA concentration in Example 4. 実施例４の生存率の結果。Results of survival rate of Example 4. 実施例５のサルモネラ・エンテリティディス菌の検出率の算出結果。The calculation result of the detection rate of Salmonella enteritidis bacteria of Example 5. FIG.
　以下、本発明をさらに詳細に説明する。
　本発明のサルモネラ菌に関する感染症予防、防除、抑制、軽減、緩和、及び／又は治療用組成物（以後、「本発明の組成物」と称する場合がある）は、少なくともバチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を含む。
　本発明のサルモネラ菌に関する感染症予防、防除、抑制、軽減、緩和、及び／又は治療する方法は、少なくともバチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を動物（特に、家畜）に好ましくは経口又は飲水投与する。 Hereinafter, the present invention will be described in more detail.
The composition for preventing, controlling, suppressing, reducing, alleviating and / or treating infectious diseases related to Salmonella of the present invention (hereinafter sometimes referred to as “the composition of the present invention”) is at least a Bacillus amyloliquefaciens strain. And / or a processed product of the strain.
In the method for preventing, controlling, suppressing, reducing, alleviating and / or treating an infection related to Salmonella according to the present invention, at least a Bacillus amyloliquefaciens strain and / or a processed product of the strain is preferably used for animals (particularly livestock). Is administered orally or with drinking water.
（バチルス・アミロリキファシエンス菌株）
　本発明に係るバチルス・アミロリキファシエンス（バチルス・アミロリケファシエンス、Bacillus amyloliquefaciens）菌株は、自体公知の菌株を利用することができるが、特に好ましい菌株は、寄託番号ＮＩＴＥＡＢＰ－０１８４４として寄託されているＴＯＡ５００１株である。 (Bacillus amyloliquefaciens strain)
Bacillus amyloliquefaciens according to the present invention (Bacillus amyloliquefaciens, Bacillus amyloliquefaciens) strains, can utilize known strains, particularly preferred strains, deposited as accession number NITEABP-01844 Is TOA5001 stock.
　本発明に係る菌株（細菌）の菌学的性質は、以下の通りである。
　グラム陽性、桿菌、好気性、芽胞形成能、運動性あり。
　栄養要求性は低く、肉汁培地、SCD 培地、ブレインハートインフュジョン培地等の多様な培地に生育できる。
　寒天平板培地上においては、周辺がラフの円形、黄白色を呈する。さらに、培地の種類（ブレインハートインフュジョン培地）によっては内部に粘性を持つ隆起したコロニーを形成する。 The mycological properties of the strain (bacteria) according to the present invention are as follows.
Gram-positive, Neisseria gonorrhoeae, aerobic, spore-forming ability, motility.
It is low in auxotrophy and can grow on various media such as gravy medium, SCD medium, brain heart infusion medium.
On the agar plate medium, the periphery is rough and yellowish white. Furthermore, depending on the type of the medium (Brain Heart Infusion Medium), a raised colony having viscosity inside is formed.
　本発明に係る菌株は、寄託番号ＮＩＴＥ ＡＢＰ－０１８４４として寄託されている菌株に限定されるものではなく、バチルス・アミロリキファシエンスに属し、サルモネラ菌に関する感染症の原因菌であるサルモネラ菌（特に、サルモネラ・ティフィミリウム及びサルモネラ・エンテリティディス）に対して効果を有する限り、本発明に係る菌株として利用可能である。
　すなわち、本発明のバチルス・アミロリキファシエンス菌株は、寄託菌株自体はもちろん、上記した菌学的性質を有する菌株である変異体及び子孫まで意図する。なお、変異体作成方法は、従来公知の放射線照射及びニトロソグアニジン、エチルメタンスルホン酸、メチルメタンスルホン酸などの化学物質処理によって得ることができる。
　さらに、本発明に係る菌株は、下記の実施例で示されたように、生体内（特に、家畜の体内）に投与されても毒性がない。 The strain according to the present invention is not limited to the strain deposited under the deposit number NITE ABP-01844, but belongs to Bacillus amyloliquefaciens, and Salmonella (particularly Salmonella, which is the causative agent of Salmonella infection). As long as it has an effect on Tifumilium and Salmonella enteritidis), it can be used as a strain according to the present invention.
That is, the Bacillus amyloliquefaciens strain of the present invention is intended to include not only the deposited strain itself but also mutants and progeny that are strains having the above-mentioned mycological properties. The mutant preparation method can be obtained by conventionally known irradiation and chemical substance treatment such as nitrosoguanidine, ethylmethanesulfonic acid, methylmethanesulfonic acid and the like.
Furthermore, the strain according to the present invention is not toxic even if it is administered in vivo (particularly in livestock), as shown in the Examples below.
（本発明に係る菌株の培養）
　本発明に係る菌株は、炭素源及び窒素源、さらに必要に応じて無機塩類を含む栄養培地に接種後、好気条件下で培養（例えば、振盪培養、通気撹拌培養）することにより、菌体を得ることができる。
　培養液の場合は、濃縮させていない培養液のままで感染症の原因菌（例えば、サルモネラ・ティフィミリウム及びサルモネラ・エンテリティディス）に対して効果を有し、フィルター除菌後の培養液又はこれらの凍結乾燥若しくは噴霧乾燥後においても同等の活性を有する。
　固体培養の場合は、本発明に係る菌株が生育した固体そのもの又はその混合物（例えば、別の固体との接触又は混合物、又は本発明に係る菌株生育固体を浸漬した液体）を用いることで感染症の原因菌に対して効果を有することができる。ここで、固体とは、本発明に係る菌株が生育できるものならばいずれでもよいが、例えば、寒天培地、サイレージ、納豆等の原料となる植物体、ヨーグルトに代表される凝乳を意味する。 (Culture of the strain according to the present invention)
The strain according to the present invention is obtained by inoculating a nutrient medium containing a carbon source and a nitrogen source and, if necessary, inorganic salts, followed by culturing under aerobic conditions (for example, shaking culture, aeration and agitation culture), Can be obtained.
In the case of the culture solution, it is effective against the causative bacteria of the infectious disease (for example, Salmonella typhimurium and Salmonella enteritidis) in the unconcentrated culture solution, and the culture solution after filter sterilization Or they have equivalent activity even after freeze-drying or spray-drying.
In the case of solid culture, an infectious disease is caused by using the solid itself or a mixture thereof (for example, a contact or mixture with another solid or a liquid in which the strain-growing solid according to the present invention is immersed). It can have an effect on the causative bacteria. Here, the solid may be any material as long as the strain according to the present invention can grow, and means, for example, a plant body that is a raw material for agar medium, silage, natto and the like, and curd represented by yogurt.
　炭素源としては、本発明に係る菌株が利用できるものならばいずれでもよく、好ましくはグルコース、シュークロース、デンプン類、セルロース混合物、その他の炭水化物を使用するのがよい。
　窒素源としては、本発明に係る菌株が利用できるものならばいずれでもよいが、好ましくはペプトン、酵母エキス、肉汁エキスなどを使用するのがよい。
　さらに、必要に応じて、例えば、ナトリウム塩、カリウム塩、マグネシウム塩、カルシウム塩、硫酸塩、リン酸塩等の無機塩類を培地に添加してもよい。
　なお、本発明に係る菌株は、市販培地、例えばトリプトソイ培地、ブレインハートインフュジョン培地、肉汁培地に接種しての液体又は平板培養で生育可能である。
　液体培養において少量で培養する場合は、試験管又はフラスコを用いる振盪培養が好ましい。大量に培養する場合は、他の発酵生産物の場合と同様に、通気撹拌培養するのが好適である。また、培養を大きなタンクで行う場合、はじめに前培養として比較的少量の培地に本発明に係る菌株を接種培養した後、次に培養物を大きな生産タンクに移してそこで生産培養するのが好ましい。この場合、前培養に使用する培地及び生産培養に使用する培地の組成は、両者同一であってもよいし、必要ならば両者を変えてもよい。
　培養条件としては、本発明に係る菌株が生育する範囲内で適宜変更しうるが、通常は好気条件下にて１５～４５℃、好ましくは２５℃～３７℃で培養するのがよい。培養時間は、培養条件や培養量によっても異なるが、通常は約１日～１週間である。 Any carbon source may be used as long as the strain according to the present invention can be used. Glucose, sucrose, starches, cellulose mixtures, and other carbohydrates are preferably used.
The nitrogen source may be any as long as the strain according to the present invention can be used, but preferably peptone, yeast extract, gravy extract and the like are used.
Furthermore, you may add inorganic salts, such as a sodium salt, potassium salt, magnesium salt, calcium salt, a sulfate, a phosphate, to a culture medium as needed.
The strain according to the present invention can be grown in a liquid medium or a plate culture inoculated on a commercially available medium, for example, tryptosy medium, brain heart infusion medium, or gravy medium.
When culturing in a small amount in liquid culture, shaking culture using a test tube or flask is preferred. When culturing in large quantities, it is preferable to culture with aeration and agitation as in the case of other fermentation products. When culture is performed in a large tank, it is preferable to first inoculate and culture the strain according to the present invention in a relatively small amount of medium as a preculture, and then transfer the culture to a large production tank for production culture. In this case, the composition of the medium used for the preculture and the medium used for the production culture may be the same, or may be changed if necessary.
The culture conditions can be appropriately changed within the range in which the strain according to the present invention grows. Usually, the culture is performed at 15 to 45 ° C., preferably 25 to 37 ° C. under aerobic conditions. The culture time varies depending on the culture conditions and the culture volume, but is usually about 1 day to 1 week.
（本発明に係る菌株の回収方法）
　本発明に係る菌株を、上記培養によって得た培養液からは遠心分離及び／又は膜濃縮によって回収できる。回収した菌体をそのまま用いても良いし、必要に応じて凍結乾燥又は噴霧乾燥にかけた乾燥菌体を用いても良い。 (Method for recovering strain according to the present invention)
The strain according to the present invention can be recovered from the culture solution obtained by the above culture by centrifugation and / or membrane concentration. The collected bacterial cells may be used as they are, or dried bacterial cells that have been subjected to freeze-drying or spray-drying as necessary.
（本発明に係る菌株の処理物）
　本発明に係る菌株の処理物とは、上記記載のように、培養した菌株に各種の処理（洗浄、遠脱水、加熱、ガンマ線や中性子線照射、噴霧乾燥、凍結乾燥、滅菌、酵素、界面活性剤、磨砕、粉砕等）物を意味する。 (Processed strain of the strain according to the present invention)
As described above, the treated product of the strain according to the present invention is various treatments on the cultured strain (washing, far-dehydration, heating, gamma ray or neutron irradiation, spray drying, freeze drying, sterilization, enzyme, surface activity Agent, grinding, grinding, etc.).
（本発明の組成物）
　本発明の組成物は、少なくとも、本発明に係る菌株（菌体も含む）及び／又は該菌株の処理物を含み、サルモネラ菌に関する感染症予防、防除、抑制、軽減、緩和、及び／又は治療効果、さらには、場合によっては、駆除、排除、及び／又は完治効果を有する。 (Composition of the present invention)
The composition of the present invention includes at least the strain (including cells) according to the present invention and / or a processed product of the strain, and prevents, controls, suppresses, reduces, alleviates, and / or treats an infection related to Salmonella. And, in some cases, have a control, elimination, and / or cure effect.
（サルモネラ菌に関する感染症）
　本発明のサルモネラ菌に関する感染症は、サルモネラ菌が原因となる感染症であれば特に限定されないが、例えば、サルモネラ菌として、例えばサルモネラ・ティフィミリウム（ティフィムリウム）（Salmonella Typhimurium）、サルモネラ・ティフィ（Salmonella Typhi）、サルモネラ・アゴナ（Salmonella Agona）、サルモネラ・セイントポール（Salmonella Saintpaul）、サルモネラ・ハイデルベルグ（Salmonella Heidelberg）、サルモネラ・コレラスイス（Salmonella Choleraesuis）、サルモネラ・インファンティス（Salmonella Infantis）、サルモネラ・トンプソン（Salmonella Thompson）、サルモネラ・エンテリティディス（Salmonella Enteritidis）、サルモネラ・ダブリン（Salmonella Dublin）等を例示することができる。
　感染症の治療対象は、ヒトを含む動物（特に、哺乳動物）であるが、例えば、家畜（牛、豚、鶏）、愛玩動物、より詳しくは、豚、犬、猫、魚、鳥類｛鶏（特に、雛、ブロイラー、採卵鶏）｝、乳牛、肉牛、Ｆ１（ホルスタインと和牛との交配種）等の肥育牛、搾乳牛、馬、マウス等を例示することができるが特に限定されない。 (Infectious diseases related to Salmonella)
The infection related to Salmonella of the present invention is not particularly limited as long as it is an infection caused by Salmonella. For example, as Salmonella, Salmonella Typhimurium , Salmonella Typhimurium , Salmonella Typhi ), Salmonella Agona , Salmonella Saintpaul , Salmonella Heidelberg , Salmonella Choleraesuis , Salmonella Infantis , Salmonella Infantis , Salmonella Infantis (Salmonella Thompson), Salmonella Enteritidis (Salmonella Enteritidis), can be exemplified Salmonella Dublin (Salmonella Dublin) and the like.
The target of treatment for infectious diseases is animals including humans (particularly mammals). For example, livestock (cattle, pigs, chickens), pets, more specifically pigs, dogs, cats, fish, birds {chicken (Especially, chicks, broilers, egg-laying chickens)}, fattening cattle such as dairy cows, beef cattle, F1 (Holstein and Wagyu crossbreed), milking cows, horses, mice and the like can be exemplified, but not particularly limited.
（本発明の組成物の投与方法）
　本発明に係る菌体を使用する場合は、所望の効果が発揮できる範囲内の濃度ならばいずれでもよいが、例えば、動物（特に、家畜）への投与において、本発明に係る菌株が１キログラムあたり１０５～１０９ｃｆｕ（Ｃｏｌｏｎｙ　ｆｏｒｍｉｎｇ　ｕｎｉｔ）となるよう添加した飼料の給餌が好ましい。
　本発明の組成物の投与時期、投与方法及び投与量は特に限定されないが、通常、市販餌に配合するか、混餌で投与すればよい。
　例えば、市販餌に配合する場合や混餌で投与（給与、給餌）する場合の投与（給与、給餌）量は０．００５％～１０％である。
　本発明の組成物の投与量は、特に限定されないが、例えば、市販餌へ０．００５％～１０％添加し投与することが望ましい。 (Method of administration of the composition of the present invention)
When using the bacterial cell according to the present invention, any concentration may be used as long as the desired effect can be exerted. For example, in the administration to animals (particularly livestock), the strain according to the present invention is 1 kg. It is preferable to feed the feed added so as to be 10 5 to 10 9 cfu (Colony forming unit).
The administration time, administration method, and dose of the composition of the present invention are not particularly limited, but it may be usually added to a commercial feed or mixed diet.
For example, the amount of administration (feeding, feeding) when blended with commercial feed or administered with mixed feeding (feeding, feeding) is 0.005% to 10%.
The dose of the composition of the present invention is not particularly limited. For example, it is desirable to add 0.005% to 10% to a commercial feed.
（本発明の組成物の用途）
　本発明の組成物の用途は、例えば、以下の通りである。
（１）サルモネラ菌に関する感染症の予防、防除、抑制、軽減、緩和、及び／又は治療用組成物
（２）動物（特に、家畜）用飼料、飼料添加物及び飼育水
　加えて、本発明は、動物用医薬品、飼料、飼料添加物又は飲料水、並びに／又は、本発明の組成物を、ヒトを除く動物に投与することを特徴とするサルモネラ菌に関する感染症を予防、防除、抑制、軽減、緩和、及び／又は治療しながら動物を飼育する方法も対象とする。
　さらに、本発明はサルモネラ・ティフィミリウム及びサルモネラ・エンテリティディスを駆除する方法も対象とする。 (Use of the composition of the present invention)
The use of the composition of this invention is as follows, for example.
(1) Composition for prevention, control, suppression, reduction, alleviation, and / or treatment of infectious diseases related to Salmonella (2) Feed for animal (particularly livestock), feed additive and breeding water In addition, Prevention, control, suppression, reduction and alleviation of infectious diseases related to Salmonella characterized by administering veterinary drugs, feeds, feed additives or drinking water and / or the composition of the present invention to animals other than humans And / or methods of raising animals while being treated are also of interest.
Furthermore, the present invention is also directed to a method of combating Salmonella typhimurium and Salmonella enteritidis.
（本発明のサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディス駆除剤）
　本発明のサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディス駆除剤は、少なくともバチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を含む。本発明の駆除剤は、サルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディスが繁殖している又は繁殖が予想される場所（特に、飼育施設、水槽等）に添加等することにより、サルモネラ菌に関する感染症の発生を予防、抑制、軽減等をすることができる。 (Salmonella typhimurium and / or Salmonella enteritidis control agent of the present invention)
The Salmonella typhimurium and / or Salmonella enteritidis control agent of the present invention contains at least a Bacillus amyloliquefaciens strain and / or a processed product of the strain. The pesticide of the present invention relates to Salmonella by adding it to a place where Salmonella typhimurium and / or Salmonella enteritidis are breeding or expected to breed (particularly a breeding facility, aquarium, etc.). It is possible to prevent, suppress, reduce, etc. the occurrence of infectious diseases.
（本発明のサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディス駆除方法）
　本発明のサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディス駆除方法は、少なくともバチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物をサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディスが繁殖している又は繁殖する場所（特に、飼育施設、水槽等）に添加する工程を含む。 (Salmonella typhimurium and / or Salmonella enteritidis control method of the present invention)
The Salmonella typhimurium and / or Salmonella enteritidis extermination method of the present invention comprises at least a Bacillus amyloliquefaciens strain and / or a processed product of the strain as Salmonella typhimurium and / or Salmonella enteritidis. Is added to the place where breeding or breeding (especially breeding facilities, aquariums, etc.).
　本発明の組成物には、上記用途を有する動物用医薬品、飲食品、飼料添加物、飼料（配合飼料、混合飼料）、飼育水が広く包含される。
　また、本発明の組成物を、デンプン、馬鈴薯澱粉、乳糖、大豆蛋白等の担体、賦形剤、結合材、崩壊剤、滑沢剤、安定剤、懸濁剤等の添加剤を配合して、周知の方法で粉剤、錠剤、顆粒剤、カプセル剤、軟膏、液剤等に製剤化することができる。 The composition of the present invention widely includes veterinary drugs, foods and drinks, feed additives, feeds (formulated feeds, mixed feeds), and breeding water having the above uses.
In addition, the composition of the present invention is blended with additives such as starch, potato starch, lactose, soybean protein and the like, excipients, binders, disintegrants, lubricants, stabilizers, suspending agents and the like. These can be formulated into powders, tablets, granules, capsules, ointments, liquids and the like by known methods.
　以下に具体例を挙げて本発明を詳細に説明するが、本発明はこれらの例に限定されない。 Hereinafter, the present invention will be described in detail with specific examples, but the present invention is not limited to these examples.
（本発明に係る菌株と公知の菌株との有機酸産生能の比較）
　本発明に係る菌株（バチルス・アミノリキファシエンス　ＴＯＡ－５００１株：寄託番号ＮＩＴＥ　ＡＢＰ－０１８４４を有する菌株）の有機酸産生能を、公知の菌株有機酸産生能と比較した。詳細は、以下の通りである。
　本発明に係る菌株及び公知の菌株であるバチルス・サブティリス（Ｂ．ｓｕｂｔｉｌｉｓ）基準株を同一の培養条件で培養した。
　具体的には、各菌株をそれぞれ別個のブレインハートインフュージョン液体培地（ＢＢＬ社製）に接種し、３７℃にて２４時間振盪培養した。
　培養後、培養液を冷却遠心分離（１００００×ｇ、１０分間）により、液相と固相菌体を分離した。得られた液相部分を、０．４５μｍのメンブランフィルターにより除菌濾過した。濾過した液体を培養上清として以下のＨＰＬＣによる有機酸測定に供した。
　各菌株の培養上清０．２ｍｌ前後を１．５ｍＬチューブに採取し、内部標準物質として５００ｍＭ trans-クロトン酸５０μＬを添加した。その後、抽出液として０．２５％アンモニア０．６ ｍＬにて２回抽出した。得られた上清に０．３倍量の１０％ＨＣｌＯ４を添加し、タンパク質を沈殿除去した。得られた上清を０．４５μｍのメンブランフィルターにて濾過し、ＨＰＬＣサンプルとした。
　以下の条件でＨＰＬＣを行った。
　ＨＰＬＣシステム：Ｗａｔｅｒｓ社製
　カラム：Ｏｒｇａｎｉｃ　Ａｃｉｄ　Ｃｏｌｕｍｕｎ（７．８×３００ ｍｍ）及びガードカラム（６．０×５０ｍｍ）
　カラム温度及びポストカラム反応温度：６０℃
　流量：０．８ｍＬ／ｍｉｎ
　移動相：０．０８％ＨＣｌＯ４
　反応相：０．２ ｍＭ ＢＴＢ、５．２ｍＭ ＮａＯＨ、１５ ｍＭＮａ２ＨＰＯ４
　検出波長：４４５ ｎｍ (Comparison of organic acid producing ability between the strain according to the present invention and a known strain)
The organic acid producing ability of the strain according to the present invention (Bacillus aminolifaciens TOA-5001 strain: strain having deposit number NITE ABP-01844) was compared with the known strain organic acid producing ability. Details are as follows.
The strain according to the present invention and the known strain, B. subtilis reference strain, were cultured under the same culture conditions.
Specifically, each strain was inoculated into a separate brain heart infusion liquid medium (manufactured by BBL) and cultured with shaking at 37 ° C. for 24 hours.
After the culture, the liquid phase and the solid phase cells were separated from the culture solution by cooling centrifugation (10000 × g, 10 minutes). The obtained liquid phase part was sterilized and filtered through a 0.45 μm membrane filter. The filtered liquid was used as a culture supernatant for organic acid measurement by the following HPLC.
About 0.2 ml of the culture supernatant of each strain was collected in a 1.5 mL tube, and 50 μL of 500 mM trans-crotonic acid was added as an internal standard substance. Then, it extracted twice with 0.6 mL of 0.25% ammonia as an extract. To the obtained supernatant, 0.3 times the amount of 10% HClO 4 was added to precipitate and remove the protein. The obtained supernatant was filtered through a 0.45 μm membrane filter to obtain an HPLC sample.
HPLC was performed under the following conditions.
HPLC system: manufactured by Waters Column: Organic Acid Column (7.8 × 300 mm) and guard column (6.0 × 50 mm)
Column temperature and post-column reaction temperature: 60 ° C
Flow rate: 0.8mL / min
Mobile phase: 0.08% HClO 4
Reaction phase: 0.2 mM BTB, 5.2 mM NaOH, 15 mM Na 2 HPO 4
Detection wavelength: 445 nm
　ＨＰＬＣの結果から算出された有機酸産生能の比較結果を図１に示す。
　図１から明らかなように、本発明に係る菌株の酢酸（ａｃｅｔｉｃ　ａｃｉｄ）産生能は、公知のバチルス・サブティリス基準株の酢酸産生能と比較して、少なくとも２．７倍（４８．７／１７．９）であることを確認した。
　上記結果より、本発明に係る菌株は、公知菌株よりも高い有機酸産生能を有する。より詳しくは、酢酸を初めとする有機酸は抗菌作用を有していることから、本発明に係る菌株は、公知菌株よりも高い抗菌活性を有することを確認した。 The comparison result of the organic acid production ability computed from the result of HPLC is shown in FIG.
As is apparent from FIG. 1, the acetic acid producing ability of the strain according to the present invention is at least 2.7 times (48.7 / 4) higher than the acetic acid producing ability of the known Bacillus subtilis reference strain. 17.9).
From the above results, the strain according to the present invention has higher organic acid producing ability than known strains. More specifically, since organic acids including acetic acid have antibacterial action, it was confirmed that the strain according to the present invention has higher antibacterial activity than known strains.
（ニワトリ成体におけるサルモネラ・ティフィミリウム増殖抑制試験）
　ニワトリ成体において、サルモネラ・ティフィミリウム野生株を感染させた後、本発明の組成物を含む飼料の給与を開始し、サルモネラ・ティフィミリウム増殖に対する効果について調べた。
　６７週齢ニワトリ（ボリスブラウン）６０羽を試験に供した（対照区：２０羽、試験区１：２０羽、試験区２：２０羽）。６７週齢時にサルモネラ・ティフィミリウム野生株を２．７×１０７個／ｍＬを含む菌液（精製水に溶解）を１ｍＬずつ全羽に強制経口投与した。強制経口投与した日を０日目として、全区ともに市販成鶏用配合飼料（ＳＤＬ　Ｎｏ．４［日本配合飼料（株）製］）を０日目から給与し、２８日間飼育した。試験区１飼料には、実施例１と同様の培養条件により２～３日培養後、遠心分離により回収した菌体を米ぬか油粕と混合、風乾して、１×１０８ｃｆｕ／ｇ乾燥粉末とした公知のバチルス・サブチルス株を２．０×１０５ｃｆｕ／ｇ飼料となるように添加した。試験区２飼料には、実施例１と同様の培養条件により２～３日培養後、遠心分離により回収した菌体を米ぬか油粕と混合、風乾して、１×１０８ｃｆｕ／ｇ乾燥粉末とした本発明の菌株を２．０×１０５ｃｆｕ／ｇ飼料となるように添加した。試験開始後３日、１４日、２８日で、各区５羽ずつ屠殺し、盲腸内容物を採取し、サルモネラ・ティフィミリウム菌数（ｌｏｇ　ｃｆｕ／ｇ）を測定した。さらに、試験開始後３日、１４日、２８日目に屠殺した各区５羽の個体の盲腸内容物におけるサルモネラ・ティフィミリウム菌の検出の有無から検出率（％）を算出した。 (Salmonella typhimurium growth inhibition test in adult chickens)
Adult chickens were infected with Salmonella typhimurium wild strain and then fed with a feed containing the composition of the present invention to examine the effect on Salmonella typhimurium growth.
Sixty-six 67-week-old chickens (Boris Brown) were used for the test (control group: 20 birds, test group 1:20, test group 2:20 birds). At 67 weeks of age, 1 ml each of a Salmonella typhimurium wild strain containing 2.7 × 10 7 cells / mL (dissolved in purified water) was forcibly orally administered to all the birds. On the 0th day, the day of gavage administration was fed on a commercial feed for adult chickens (SDL No. 4 [manufactured by Nippon Formulad Feed Co., Ltd.)] for all wards, and was reared for 28 days. In test group 1 feed, after culturing for 2 to 3 days under the same culture conditions as in Example 1, the cells recovered by centrifugation were mixed with rice bran oil cake and air-dried to obtain 1 × 10 8 cfu / g dry powder. The known Bacillus subtilis strain was added to a 2.0 × 10 5 cfu / g feed. In test group 2 feed, after culturing for 2 to 3 days under the same culture conditions as in Example 1, the cells recovered by centrifugation were mixed with rice bran oil cake and air-dried to obtain 1 × 10 8 cfu / g dry powder. The strain of the present invention was added to 2.0 × 10 5 cfu / g feed. On the 3rd, 14th and 28th days after the start of the test, 5 birds were sacrificed in each group, the contents of the cecum were collected, and the number of Salmonella typhimurium bacteria (log cfu / g) was measured. Furthermore, the detection rate (%) was calculated from the presence or absence of detection of Salmonella typhimurium in the cecal contents of 5 individuals in each section 3 days, 14 days and 28 days after the start of the test.
　サルモネラ・ティフィミリウム菌数の測定結果を図２に示す。
　図２から明らかなように、試験区２では、盲腸内のサルモネラ・ティフィミリウム菌数が、試験開始後３日後から２８日後の間で２８．４％減少したのに対し、試験区１では盲腸内のサルモネラ・ティフィミリウム菌数が、２８日間で２．２％減少し、対照区では盲腸内のサルモネラ・ティフィミリウム菌数が、２８日間で１２．３％増加した。
　サルモネラ・ティフィミリウム菌の検出率の算出結果を図３に示す。
　図３から明らかなように、対照区及び試験区１では、試験開始後３日、１４日、２８日で、検出率は１００％（５羽／５羽）であったのに対し、試験区２では、２８日では検出率が６０％（３羽／５羽）まで低下した。
　以上より、本発明の菌株は、ニワトリ成体体内におけるサルモネラ・ティフィミリウムの生育を抑制する効果を有することを確認した。また公知のバチルス・サブチルス株よりも優れたサルモネラ・ティフィミリウム抑制効果を有することを確認した。 The measurement result of Salmonella typhimurium bacteria count is shown in FIG.
As is clear from FIG. 2, in test group 2, the number of Salmonella typhimurium in the cecum decreased by 28.4% between 3 and 28 days after the start of the test, whereas in test group 1 The number of Salmonella typhimurium in the cecum decreased by 2.2% over 28 days, and the number of Salmonella typhimurium in the cecum increased by 12.3% over 28 days in the control group.
The calculation result of the detection rate of Salmonella typhimurium is shown in FIG.
As apparent from FIG. 3, in the control group and test group 1, the detection rate was 100% (5/5 birds) on the 3rd, 14th and 28th days after the start of the test, whereas the test group In 2, the detection rate decreased to 60% (3/5 birds) on the 28th.
From the above, it was confirmed that the strain of the present invention has an effect of suppressing the growth of Salmonella typhimurium in adult chickens. Moreover, it confirmed that it had the Salmonella typhimurium inhibitory effect superior to the well-known Bacillus subtilis strain | stump | stock.
（ニワトリ雛におけるサルモネラ・ティフィミリウム感染予防試験）
　ニワトリ雛において、サルモネラ・ティフィミリウム野生株を感染させる前に、本発明の組成物を含む飼料の給与を開始し、感染個体に対する効果及び同居個体に対する効果について調べた。
　１日齢ボリスブラウン雛４５羽を試験に供した（対照区：１５羽、試験区１；１５羽、試験区２：１５羽）。全区ともに市販鶏用配合飼料（ＳＤＬ　Ｎｏ．１［日本配合飼料（株）製］）を１日齢より給与し、１４日間飼育した。試験区１飼料には、実施例２と同様に乾燥粉末状とした公知のバチルス・サブチルス株を２．０×１０５ｃｆｕ／ｇ飼料となるように添加した。試験区２飼料には、実施例２と同様に乾燥粉末状とした本発明の菌株を２．０×１０５ｃｆｕ／ｇ飼料となるように添加した。３日齢時（飼料給与開始後２日目）に各区１５羽中３羽の雛（感染個体）にサルモネラ・ティフィミリウム野生株を２．７×１０７個／ｍＬを含む菌液を１ｍＬずつ強制経口投与し、飼料給与開始後１４日目（強制経口投与後１２日目）に同居個体の盲腸内容物におけるサルモネラ・ティフィミリウム菌の検出の有無から検出率（％）を算出した。感染個体は１５日齢で解剖し、盲腸内容物のサルモネラ・ティフィミリウム菌数を測定した。 (Salmonella typhimurium infection prevention test in chicks)
In chicken chicks, before the wild strain of Salmonella typhimurium was infected, feeding of the feed containing the composition of the present invention was started, and the effect on the infected individual and the effect on the living individual were examined.
45 1-day-old Boris Brown chicks were subjected to the test (control group: 15 birds, test group 1: 15 birds, test group 2: 15 birds). In all wards, commercially available mixed feed for chickens (SDL No. 1 [manufactured by Nippon Formulad Feed Co., Ltd.]) was fed from the age of 1 day and raised for 14 days. The test group 1 diet, was added a known Bacillus subtilis strain was the same dry powder as in Example 2 so as to be 2.0 × 10 5 cfu / g forage. To the test group 2 feed, the strain of the present invention in the form of a dry powder was added in the same manner as in Example 2 so as to be 2.0 × 10 5 cfu / g feed. At the age of 3 days (2 days after the start of feed feeding), 3 chicks (infected individuals) in 15 wards of each ward (1 ml) containing 1 ml of Salmonella typhimurium wild strain 2.7 × 10 7 cells / mL They were forcibly orally administered each time, and the detection rate (%) was calculated from the presence or absence of detection of Salmonella typhimurium in the cecal contents of the cohabiting individuals on the 14th day (12 days after the forcible oral administration) after the start of feed feeding. The infected individuals were dissected at 15 days of age, and the number of Salmonella typhimurium bacteria in the cecum contents was measured.
　感染個体における盲腸内サルモネラ・ティフィミリウム菌数（ｌｏｇ　ｃｆｕ／ｇ）の測定結果を図４に示す。
　図４から明らかなように、試験区２では、ニワトリ雛体内のサルモネラ・ティフィミリウム菌数が、対照区と比較して２５．７％、試験区１と比較して８％低くなった。
　よって、本発明の菌株は、ニワトリ雛体内におけるサルモネラ・ティフィミリウムの生育を抑制する効果を有することを確認した。
　次に、同居個体に対する感染状況｛鶏盲腸内サルモネラ・ティフィミリウム検出率（％）｝の結果を図５に示す。
　図５から明らかなように、試験区２は、サルモネラ・ティフィミリウム感染した個体数（ＳＴ陽性の個体数）が、対照区及び試験区１と比較して、５０％以下であった。
　よって、本発明の菌株は、ニワトリ雛のサルモネラ・ティフィミリウム感染予防効果を有することを確認した。また公知のバチルス・サブチルス株よりも優れたサルモネラ・ティフィミリウム感染予防効果を有することを確認した。 FIG. 4 shows the measurement results of the number of cecal Salmonella typhimurium bacteria (log cfu / g) in infected individuals.
As is clear from FIG. 4, in the test group 2, the number of Salmonella typhimurium bacteria in the chicken chick was 25.7% lower than that in the control group and 8% lower than that in the test group 1.
Therefore, it was confirmed that the strain of the present invention has an effect of suppressing the growth of Salmonella typhimurium in the chick chick.
Next, FIG. 5 shows the result of the infection status {detection rate of Salmonella typhimurium (%)} in the cecum of the chicken] for the lived individual.
As is clear from FIG. 5, the number of individuals infected with Salmonella typhimurium (the number of ST positive individuals) in test group 2 was 50% or less as compared with control group and test group 1.
Therefore, it was confirmed that the strain of the present invention has an effect of preventing Salmonella typhimurium infection in chicken chicks. Moreover, it confirmed that it had the Salmonella typhimurium infection prevention effect superior to the well-known Bacillus subtilis strain | stump | stock.
（免疫賦活効果の確認）
　雌ニワトリ（チャンキー）を試験に供した（対照区：５０羽、試験区１：５０羽、試験区２：５０羽）。各区ともに市販ブロイラー用飼料（前期・後期ともに無薬飼料）を１日齢より給与し、民間養鶏場のセミウインドレス鶏舎（１７羽／ｍ２）にて０～４９日齢まで飼養した。試験区１飼料には、実施例２と同様に乾燥粉末状とした公知のバチルス・サブチルス株を２．０×１０５ｃｆｕ／ｇ飼料となるように添加した。試験区２飼料には、実施例２と同様に乾燥粉末状とした本発明の菌株を２．０×１０５ｃｆｕ／ｇ飼料となるように添加した。鶏舎内温度は、１－７日齢は３０℃、８－２１日齢は２８－２５℃、２２－３５日齢は２４－２２℃、３６日齢以降は２０℃とした。４９日齢時に免疫指標（血清リゾチーム濃度、マクロファージ貪食率、マクロファージ走化性、及び小腸総ＩｇＡ濃度）並びに生存率を評価した。血清リゾチーム濃度は、ＥＬＩＳＡ測定キット（ＳＩＧＭＡ－ＡＬＤＲＩＣＨ社製）を用いて、メーカーのマニュアルに従い測定した。マクロファージ貪食率及びマクロファージ走化性は、免疫機能分析マニュアル（［ｏｎｌｉｎｅ］、２０１６年９月２９日、独立行政法人　家畜改良センター兵庫牧場　特別飼育分科会、［平成２９年２月８日検索］、〈http://www.nlbc.go.jp/hyogo/tishiki/boueki/boueki/c21bd718afed5f0d1adee9349fd0b4ffe9b5c9cb.pdf〉）の方法にて測定した。小腸ＩｇＡ濃度は、ＥＬＩＳＡ測定キット（Ｂｅｔｈｅｌ　Ｌａｂｏｒａｔｏｒｉｅｓ，　Ｉｎｃ社製）を用いて、メーカーのマニュアルに従い測定した。 (Confirmation of immunostimulatory effect)
Female chickens (chunky) were subjected to the test (control group: 50, test group 1:50, test group 2:50). In each ward, commercial broiler feed (non-drug feed in the first and second semesters) was fed from the age of 1 day, and was raised from 0 to 49 days of age in a semi-windless poultry house (17 / m 2 ) in a private poultry farm. The well-known Bacillus subtilis strain | stump | stock made into the dry powder form similarly to Example 2 was added to the test group 1 feed so that it might become 2.0 * 10 < 5 > cfu / g feed. To the test group 2 feed, the strain of the present invention in the form of a dry powder was added in the same manner as in Example 2 so as to be 2.0 × 10 5 cfu / g feed. The temperature inside the poultry house was 30 ° C. for 1-7 days of age, 28-25 ° C. for 8-21 days, 24-22 ° C. for 22-35 days, and 20 ° C. after 36 days. At 49 days of age, immune indices (serum lysozyme concentration, macrophage phagocytosis, macrophage chemotaxis, and small intestine total IgA concentration) and survival were evaluated. Serum lysozyme concentration was measured using ELISA measurement kit (manufactured by SIGMA-ALDRICH) according to the manufacturer's manual. Macrophage phagocytosis rate and macrophage chemotaxis can be determined by immunological function analysis manual ([online], September 29, 2016, National Livestock Improvement Center Hyogo Farm Special Breeding Subcommittee, [Search February 8, 2017], <Http://www.nlbc.go.jp/hyogo/tishiki/boueki/boueki/boueki/c21bd718afed5f0d1adee9349fd0b4ffe9b5c9cb.pdf>). The small intestine IgA concentration was measured according to the manufacturer's manual using an ELISA measurement kit (Bethel Laboratories, Inc.).
　血清リゾチーム濃度の結果を図６に示す。
　図６から明らかなように、試験区２では、血清リゾチーム濃度（μｇ／ｍＬ）が、対照区、試験区１と比較して高かった。
　マクロファージ貪食率の結果を図７に示す。
　図７から明らかなように、試験区２では、マクロファージ貪食率（％）が、対照区、試験区１と比較して高かった。
　マクロファージ走化性の結果を図８に示す。
　図８から明らかなように、試験区２では、マクロファージ走化性（％）が、対照区、試験区１と比較して高かった。
　小腸総ＩｇＡ濃度の結果を図９に示す。
　図９から明らかなように、試験区２では、小腸総ＩｇＡ濃度（μｇ／ｇ）が、対照区、試験区１と比較して、約２倍であった。
　生存率の結果を図１０に示す。
　図１０から明らかなように、試験区２では、生存率（％）が、対照区、試験区１と比較して高かった。
　以上の結果から、本発明の菌株は、ニワトリにおいて免疫を活性化し、生存率の向上をもたらすことを確認した。また公知のバチルス・サブチルス株よりも優れた免疫賦活効果（血清リゾチーム濃度上昇効果、マクロファージ貪食率上昇効果、マクロファージ走化性上昇効果、及び小腸総ＩｇＡ濃度上昇効果）を有することを確認した。 The results of serum lysozyme concentration are shown in FIG.
As is clear from FIG. 6, in the test group 2, the serum lysozyme concentration (μg / mL) was higher than that in the control group and the test group 1.
The result of macrophage phagocytosis is shown in FIG.
As is clear from FIG. 7, the macrophage phagocytosis rate (%) in test group 2 was higher than that in control group and test group 1.
The results of macrophage chemotaxis are shown in FIG.
As is clear from FIG. 8, macrophage chemotaxis (%) was higher in test group 2 than in control group and test group 1.
The results of the small intestine total IgA concentration are shown in FIG.
As is clear from FIG. 9, in test group 2, the small intestine total IgA concentration (μg / g) was about twice that in control group and test group 1.
The results of the survival rate are shown in FIG.
As is clear from FIG. 10, the survival rate (%) in test group 2 was higher than that in control group and test group 1.
From the above results, it was confirmed that the strain of the present invention activated immunity in chickens and resulted in improved survival rate. In addition, it was confirmed that it has an immunostimulatory effect (serum lysozyme concentration increasing effect, macrophage phagocytosis increasing effect, macrophage chemotaxis increasing effect, and small intestinal total IgA concentration increasing effect) superior to known Bacillus subtilis strains.
（ニワトリ成体におけるサルモネラ・エンテリティディス増殖抑制試験）
　サルモネラ・エンテリティディス感染を自然発症したニワトリ成体において、盲腸フィステルを装着して、本発明の組成物を含む飼料の給与を開始し、サルモネラ・エンテリティディス増殖に対する効果について調べた。
　３１２～３６９日齢ニワトリ（ボリスブラウン）３０羽について、盲腸フィステルを装着して、試験に供した｛対照区（３１２～３６９日齢、平均日齢３３５．３日齢、平均体重１９８６．５ｇ）：１５羽、試験区（３１２～３６６日齢、平均日齢３３３．３日齢、平均体重２０１０．９ｇ）：１５羽｝。全区ともに市販成鶏用配合飼料（ＳＤＬ　Ｎｏ．４［日本配合飼料（株）製］）を給与し、３５日間飼育した。試験区飼料には、実施例２と同様に乾燥粉末状とした本発明の菌株を２．０×１０６ｃｆｕ／ｇ飼料となるように添加した。当該飼料の給与開始日を０日目として、０日（給与前）、１０日、１２日、１４日、１６日、２１日、３５日で、各区１５羽ずつ、盲腸フィステルより盲腸内容物を採取し、盲腸内容物におけるサルモネラ・エンテリティディス菌の検出の有無から検出率（％）を算出した。 (Salmonella enteritidis growth inhibition test in adult chickens)
In adult chickens that spontaneously developed Salmonella enteritidis infection, a cecal fistula was attached, and feeding of a feed containing the composition of the present invention was started, and the effect on Salmonella enteritidis growth was examined.
Thirty-one 312 to 369 day-old chickens (Boris Brown) were put on the cecal fistula and used for the test {control group (312-369 days old, average age 335.3 days, average weight 1986.5 g) : 15 birds, test section (312 to 366 days old, average age 333.3 days old, average weight 2010.9 g): 15 birds}. All the wards were fed commercial adult chicken formula feed (SDL No. 4 [manufactured by Japan Formula Feed Co., Ltd.]) and reared for 35 days. The test group feed was added strains of the present invention which was the same dry powder as in Example 2 so as to be 2.0 × 10 6 cfu / g forage. The feed start date of the feed is day 0 (before feeding), 10 days, 12 days, 14 days, 16 days, 21 days, and 35 days. The detection rate (%) was calculated from the presence or absence of detection of Salmonella enteritidis bacteria in the cecum contents.
　サルモネラ・エンテリティディス菌の検出率の算出結果を図１１に示す。
　図１１から明らかなように、対照区では、給与開始後０日、１０日、１２日、１４日、３５日で、検出率は１００％（１５羽／１５羽）であったのに対し、試験区では、３５日では検出率が３３．３％（５羽／１５羽）まで低下した。
　以上より、本発明の菌株は、ニワトリ成体体内におけるサルモネラ・エンテリティディスの生育を抑制する効果を有することを確認した。 The calculation result of the detection rate of Salmonella enteritidis is shown in FIG.
As is clear from FIG. 11, in the control group, the detection rate was 100% (15/15 birds) at 0 days, 10 days, 12 days, 14 days, and 35 days after the start of salary, In the test area, the detection rate decreased to 33.3% (5/15) on the 35th.
From the above, it was confirmed that the strain of the present invention has an effect of inhibiting the growth of Salmonella enteritidis in adult chickens.
（ニワトリ雛におけるサルモネラ・エンテリティディス感染予防試験）
　サルモネラ・ティフィミリウム野生株の代わりに、サルモネラ・エンテリティディス野生株を強制経口投与した点以外は、上記の実施例３と同様の手順により試験した。 (Salmonella enteritidis infection prevention test in chicks)
The procedure was the same as in Example 3 except that Salmonella enteritidis wild strain was forcibly administered orally instead of Salmonella typhimurium wild strain.
　本発明の菌株は、ニワトリ雛体内におけるサルモネラ・エンテリティディスの生育を抑制する効果を有することを確認した。
　また、本発明の菌株は、ニワトリ雛のサルモネラ・エンテリティディス感染予防効果を有することを確認した。さらに、公知のバチルス・サブチルス株よりも優れたサルモネラ・エンテリティディス感染予防効果を有することを確認した。 It was confirmed that the strain of the present invention has an effect of suppressing the growth of Salmonella enteritidis in chicken chicks.
Moreover, it confirmed that the strain of this invention has the Salmonella enteritidis infection prevention effect of a chicken chick. Furthermore, it was confirmed that it has a better Salmonella enteritidis infection prevention effect than the known Bacillus subtilis strain.
（結論）
　以上の実施例から、本発明の組成物、飼育方法又は治療方法は、以下の効果を有することを確認した。
（１）動物｛特に、家畜（牛、豚、鶏）｝の生体内に投与しても毒性がない。
（２）公知菌株よりも高い抗菌活性。
（３）家畜体内におけるサルモネラ菌の生育を抑制する効果。
（４）家畜におけるサルモネラ菌感染予防効果。
（５）家畜における免疫賦活効果（血清リゾチーム濃度上昇効果、マクロファージ貪食率上昇効果、マクロファージ走化性上昇効果、及び小腸総ＩｇＡ濃度上昇効果）。
（６）家畜における生存率向上効果。 (Conclusion)
From the above examples, it was confirmed that the composition, breeding method or treatment method of the present invention had the following effects.
(1) There is no toxicity even if administered into the body of an animal {particularly livestock (cattle, pig, chicken)}.
(2) Higher antibacterial activity than known strains.
(3) The effect of suppressing the growth of Salmonella in livestock.
(4) Salmonella infection prevention effect in livestock.
(5) Immunostimulatory effect in livestock (serum lysozyme concentration increasing effect, macrophage phagocytosis increasing effect, macrophage chemotaxis increasing effect, and small intestine total IgA concentration increasing effect).
(6) Survival rate improvement effect in livestock.
　本発明は、バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を含むサルモネラ菌に関する感染症予防及び／又は治療用組成物を提供することができる。 The present invention can provide a composition for preventing and / or treating infectious diseases related to Salmonella containing a Bacillus amyloliquefaciens strain and / or a processed product of the strain.

Claims (14)

  1. 　バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を含むサルモネラ菌に関する感染症予防、防除、抑制、軽減、緩和、及び／又は治療用組成物。 A Bacillus amyloliquefaciens strain and / or a composition for preventing, controlling, suppressing, reducing, alleviating and / or treating infectious diseases related to Salmonella containing treated products of the strain.
  2. 　前記感染症は、家畜での感染症である請求項１に記載の組成物。 The composition according to claim 1, wherein the infectious disease is an infectious disease in livestock.
  3. 　前記サルモネラ菌は、サルモネラ・ティフィミリウムである請求項１又は２に記載の組成物。 The composition according to claim 1 or 2, wherein the Salmonella is Salmonella typhimurium.
  4. 　前記サルモネラ菌は、サルモネラ・エンテリティディスである請求項１又は２に記載の組成物。 The composition according to claim 1 or 2, wherein the Salmonella is Salmonella enteritidis.
  5. 　前記バチルス・アミロリキファシエンス菌株は、寄託番号ＮＩＴＥ　ＡＢＰ－０１８４４を有する菌株である請求項１～４のいずれか１に記載の組成物。 The composition according to any one of claims 1 to 4, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
  6. 　請求項１～５のいずれか１に記載の組成物を含む動物用医薬品、飼料、飼料添加物、又は飲料水。 An animal medicine, feed, feed additive, or drinking water comprising the composition according to any one of claims 1 to 5.
  7. 　請求項６に記載の動物用医薬品、飼料、飼料添加物又は飲料水、並びに／又は、請求項１～５のいずれか１に記載の組成物を、ヒトを除く動物に投与することを特徴とするサルモネラ菌に関する感染症を予防、防除、抑制、軽減、緩和、及び／又は治療しながら動物を飼育する方法。 The veterinary drug, feed, feed additive or drinking water according to claim 6 and / or the composition according to any one of claims 1 to 5 are administered to animals other than humans. A method of raising an animal while preventing, controlling, suppressing, reducing, alleviating, and / or treating an infection related to Salmonella.
  8. 　バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を動物に投与する工程を含むサルモネラ菌に関する感染症予防、防除、抑制、軽減、緩和、及び／又は治療しながら家畜を飼育する方法。 A method of breeding livestock while preventing, controlling, suppressing, reducing, alleviating, and / or treating infectious diseases related to Salmonella, including a step of administering a Bacillus amyloliquefaciens strain and / or a processed product of the strain to an animal.
  9. 　バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を動物に投与する工程を含むサルモネラ菌に関する感染症予防、防除、抑制、軽減、緩和、及び／又は治療する方法。 A method of preventing, controlling, suppressing, reducing, alleviating, and / or treating an infection related to Salmonella, including a step of administering a Bacillus amyloliquefaciens strain and / or a processed product of the strain to an animal.
  10. 　前記バチルス・アミロリキファシエンス菌株は、寄託番号ＮＩＴＥ　ＡＢＰ－０１８４４を有する菌株である請求項８又は９に記載の方法。 The method according to claim 8 or 9, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
  11. 　バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物を含むサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディス駆除剤。 Salmonella typhimurium and / or Salmonella enteritidis control agent containing Bacillus amyloliquefaciens strain and / or processed product of the strain.
  12. 　前記バチルス・アミロリキファシエンス菌株は、寄託番号ＮＩＴＥ　ＡＢＰ－０１８４４を有する菌株である請求項１１に記載の駆除剤。 The pesticide according to claim 11, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
  13. 　バチルス・アミロリキファシエンス菌株及び／又は該菌株の処理物をサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディスが繁殖している又は繁殖する場所に添加する工程を含むサルモネラ・ティフィミリウム及び／又はサルモネラ・エンテリティディス駆除方法。 Salmonella typhimurium comprising a step of adding a Bacillus amyloliquefaciens strain and / or a processed product thereof to a place where Salmonella typhimurium and / or Salmonella enteritidis are bred or bred, and / Or Salmonella Enteritidis extermination method.
  14. 　前記バチルス・アミロリキファシエンス菌株は、寄託番号ＮＩＴＥ　ＡＢＰ－０１８４４を有する菌株である請求項１３に記載の方法。 The method according to claim 13, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
PCT/JP2018/006627 2017-02-24 2018-02-23 Composition for prevention and/or treatment of infection relating to salmonella including bacillus amyloliquefaciens strain and/or treated product of said strain WO2018155612A1 (en)

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