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WO2018236085A1 - Nouveau bactériophage d'aeromonas hydrophila aer-hyp-1 et son utilisation pour inhiber la croissance d'aeromonas hydrophila - Google Patents

Nouveau bactériophage d'aeromonas hydrophila aer-hyp-1 et son utilisation pour inhiber la croissance d'aeromonas hydrophila Download PDF

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WO2018236085A1
WO2018236085A1 PCT/KR2018/006573 KR2018006573W WO2018236085A1 WO 2018236085 A1 WO2018236085 A1 WO 2018236085A1 KR 2018006573 W KR2018006573 W KR 2018006573W WO 2018236085 A1 WO2018236085 A1 WO 2018236085A1
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bacteriophage
eromonas
hyp
aer
composition
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Korean (ko)
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윤성준
전수연
권안성
송현민
강상현
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Intron Biotechnology Inc
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    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention relates to a method for preventing or treating a disease caused by eromonas hydrofilm using a bacteriophage isolated from nature capable of killing eromonas hydrofilm by infection with eromonas hydrofilm and a composition containing the same as an effective ingredient And more particularly, to a method for isolating Mio viridis versus bacteriophage Aer-HYP-1, which has the ability to kill eromonas hydrofilm and has the genome of SEQ ID NO: 1 No. KCTC 13257BP), and a method for preventing or treating a disease caused by eromonas hydrofilm using the composition comprising the bacteriophage as an active ingredient.
  • Aeromonas hydrophila belonging to the genus Eromonas is a motile gram-negative bacterium and widely distributed in river water and freshwater fish. It is widely used as aquatic organisms such as catfish, freshwater fish such as rainbow trout, It is well known as a cause of serious disease in fish.
  • the serotypes of eromonas hydrofilas include Thermo-stable (O antigen), Thermo-labile (K antigen), and Flagella (H antigen) And it is known that most of the pathogenic eromonas hydrofilas known to date have thermostable bacterial antigens.
  • Eromonas hydrofilas can cause aeromonas diseases such as motile aeromonas septicemia or skin ulcers in a variety of fishes and can cause massive deaths in severe cases It is a cause of significant economic loss in the aquaculture industry of freshwater fish. Therefore, it is urgent to develop a method that can be used to prevent infection of eromonas hydrofilas and further to treat infection.
  • Bacteriophage is a very small microorganism that infects bacteria, usually called phage.
  • the bacteriophage has the ability to kill bacterial cells by infecting the bacteria inside the cells after infecting the bacteria and destroying the cell wall of the host bacteria when the progeny bacteriophages come out of the bacteria after the proliferation.
  • the bacterium infection method of bacteriophage is highly specific, and the types of bacteriophages that can infect specific bacteria are limited to some.
  • certain bacteriophages can infect only a specific category of bacteria, and thus certain bacteriophages can provide an antibacterial effect only for certain bacteria. Due to the bacterium specificity of these bacteriophages, bacteriophage provides an antimicrobial effect only on the bacteria of interest and does not affect the environment or bacteria in the environment. Conventional antibiotics, which have been widely used for bacterial treatment, have simultaneously influenced several kinds of bacteria. This has caused problems such as environmental contamination and disturbance of normal flora of animals. In contrast, bacteriophages operate only on specific bacteria, so that the use of bacteriophages does not cause a total disturbance in the body. Therefore, the use of bacteriophage is very safe as compared with the use of antibiotics, and the possibility of side effects caused by use is relatively low.
  • Bacteriophage is a British bacteriologist Twort 1915 became discovered while conducting research on Staphylococcus aureus (Micrococcus) melting the colonies are transparent by any developer.
  • the French bacteriologist d'Herelle discovered that there was an effect of dissolving Shigella dysenteriae in the filtrate of heterozygous patients.
  • bacteriophage Due to the special ability to kill bacteria, bacteriophage has been expected to be effective as a countermeasure against bacterial infection since its discovery.
  • the discovery of penicillin by Fleming the spread of antibiotics has become common, and studies of bacteriophage have been limited to some Eastern European countries and the Soviet Union.
  • the limitations of existing antibiotics have appeared due to the increase of antibiotic resistant bacteria, and bacteriophages have been attracting attention as an anti - bacterial agent due to the possibility of development as a substitute for existing antibiotics.
  • bacteriophages are highly specific for bacteria. Because of the high specificity of these bacteriophages to bacteria, bacteriophages often exhibit antibacterial effects only on some strains, even if they belong to the same species (Species). In addition, the intensity of the antibacterial activity of bacteriophages exhibited according to the target bacterial strain may be different. For this reason, it is necessary to secure various kinds of useful bacteriophages in order to obtain an effective control method for a certain kind of bacteria.
  • the present inventors have developed a composition that can be used for the prevention or treatment of diseases caused by eromonas hydrofilm using bacteriophages isolated from nature capable of killing eromonas hydrofilm, and In an attempt to develop a method for preventing or treating a disease caused by eromonas hydrofilm using this composition, a suitable bacteriophage is isolated from nature, and the separated bacteriophage is distinguished from other bacteriophages to be identified. It is possible to develop a composition containing the bacteriophage as an effective ingredient after securing the sequence information of a genome and then effectively using the composition for the purpose of preventing or treating a disease caused by eromonas hydrofilm By checking, It was completed.
  • an object of the present invention is to provide a Myoviridae bacteriophage Aer-HYP-1 strain isolated from nature, which has the ability to specifically kill eromonas hydrofilm and has a genome represented by SEQ ID NO: 1 (accession number KCTC 13257BP).
  • the present invention relates to a microorganism isolated from nature, which has the ability to specifically kill eromonas hydrofilas and has a genome represented by SEQ. ID. NO. 1, which comprises the microorganism v. Bacteriophage Aer-HYP-1 (Accession No. KCTC 13257BP ), And a method for preventing or treating a disease caused by eromonas hydrofilm using the composition comprising the same as an active ingredient.
  • Bacteriophage Aer-HYP-1 was deposited with the Korean Resource Center for Biotechnology (Accession No. KCTC 13257BP) on May 10, 2017, after being isolated by the present inventors.
  • the present invention provides a bathing agent and a feed additive comprising bacteriophage Aer-HYP-1 as an active ingredient, which can be used for preventing or treating diseases caused by eromonas hydrofilm.
  • the composition of the present invention can be utilized for the purpose of preventing or treating diseases caused by eromonas hydrofilm.
  • &quot prevent " or " prophylaxis " refer to (i) prevention of infection of eromonas hydrofilas; And (ii) inhibiting development into diseases caused by eromonas hydrofilament infection.
  • " treatment " or " treatment " refers to (i) inhibition of a disease caused by eromonas hydrofilas; And (ii) alleviating the pathological condition of the disease caused by eromonas hydrofilas.
  • the terms “separate”, “isolated”, or “separated” refer to the separation of bacteriophages from the natural state using various experimental techniques and the securing of features that can identify the bacteriophages of the invention by distinguishing them from other bacteriophages
  • the present invention also includes propagating the bacteriophage of the present invention industrially so as to utilize it by biotechnology.
  • the pharmaceutically acceptable carriers to be contained in the composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no.
  • the composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, preservatives, etc. in addition to the above components.
  • the composition of the present invention contains bacteriophage Aer-HYP-1 as an active ingredient.
  • the bacteriophage Aer-HYP-1 contained therein is contained at a concentration of 1 ⁇ 10 1 pfu / ml to 1 ⁇ 10 30 pfu / ml or 1 ⁇ 10 1 pfu / g to 1 ⁇ 10 30 pfu / g, 10 4 pfu / ml to 1 x 10 15 pfu / ml or 1 x 10 4 pfu / g to 1 x 10 15 pfu / g.
  • composition of the present invention may be prepared in a unit dose form by being formulated using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. It may be manufactured by inserting it into a multi-capacity container.
  • the formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
  • the composition of the present invention may be embodied as a bathing agent and a feed additive, depending on the manner of application, but not limited thereto.
  • Bacteriophages capable of providing an antimicrobial activity against other bacterial species may be added to the composition of the present invention in order to increase efficiency in such a utilization purpose.
  • other kinds of bacteriophages having antimicrobial activity against eromonas hydrofilm can also be added. Even bacteriophages having antimicrobial activity against eromonas hydrofilaments are different from each other in terms of the strength of antimicrobial activity and the range of antimicrobial activity, so that a proper combination thereof can maximize the effect.
  • antibiotics when antibiotics are used, common endophytic bacteria are also harmed, resulting in a decrease in immunity of animals and various side effects due to their use.
  • bacteriophage is an antimicrobial agent that can be used for the antibacterial effect of bacteriophage against bacterial strains in terms of the strength of the antimicrobial activity or the antimicrobial range [the various strains belonging to eromonas hydro pilil species, The range in which activity is exercised.
  • bacteriophages can exhibit antibacterial activity against some bacterial strains belonging to the same species. In other words, even if belonging to the same bacterium species, there may be differences in susceptibility to bacteriophages depending on the individual bacterium. Therefore, the present invention provides a differential antibacterial effect compared to other bacteriophages having antimicrobial activity against eromonas hydro- Can be provided. This provides a big difference in its effectiveness when used in industrial settings.
  • Figure 1 is an electron micrograph of bacteriophage Aer-HYP-1.
  • Fig. 2 shows experimental results showing the bacteriophage Aer-HYP-1's eradication ability against eromonas hydrofilm. Based on the center line of the plate medium, the left side shows only the buffer containing no bacteriophage Aer-HYP-1, while the right side shows the solution containing the bacteriophage Aer-HYP-1. The transparent part observed on the right side is the resultant bacterium formed by the action of bacteriophage Aer-HYP-1.
  • Example One Eromonas Hydropila Isolation of bacteriophage that can kill bacteria
  • erotic Pseudomonas dihydro pillar bacteria to 1 / 1,000 of the TSB (T ryptic S oy B roth) inoculated at a rate medium (Casein Digest, 17 g / L; Soy bean Digest, 3 g / L; deck Samples collected in a tube were added together at a concentration of 2.5 g / L; NaCl, 5 g / L; Dipotassium phosphate, 2.5 g / L), followed by shake culture at 25 DEG C for 3-4 hours. After incubation, the supernatant was recovered by centrifugation at 8,000 rpm for 20 minutes.
  • the recovered supernatant was inoculated with eromonas hydrofilm at a ratio of 1 / 1,000 and then shake-cultured again at 25 ° C for 3-4 hours.
  • this procedure was repeated five times in total so that the number of bacteriophages could be sufficiently increased.
  • the culture was centrifuged at 8,000 rpm for 20 minutes. After centrifugation, the recovered supernatant was filtered using a 0.45 ⁇ m filter. The presence of bacteriophages capable of killing eromonas hydrofilm was examined by a conventional spot assay using the thus obtained filtrate.
  • the above drop test was carried out as follows.
  • the TSB medium was inoculated with eromonas hydrofilm at a ratio of 1 / 1,000, and then cultured with shaking at 25 DEG C overnight.
  • TSA T ryptic S oy A gar
  • plate medium Casein Digest, 15 g / L; Soy bean digest, 5 g / L; NaCl, 5 g / L; agar, 15 g / L.
  • the smear medium was allowed to stand in a clean bench for about 30 minutes to allow the smear solution to dry.
  • Pure bacteriophage was isolated by using the filtrate which confirmed the existence of bacteriophage having killing ability against eromonas hydrofilas.
  • a usual plaque assay was used for the separation of pure bacteriophage. To elaborate this, one of the scavengers formed in the leavening assay was recovered using a sterilized tip, and then added to the culture medium of eromonas hydrofilm for incubation at 25 ° C. for 4-5 hours. After incubation, supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. To the obtained supernatant, a culture medium of eromonas hydrofilm was added at a volume of 1/50 and then again cultured at 25 ° C for 4-5 hours.
  • This procedure was performed at least 5 times to increase the number of bacteriophages, and finally the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. The obtained supernatant was used for the analysis of the washing solution. Since the separation of the pure bacteriophage is not normally accomplished by only one step of the above procedure, the former step is repeated again with the use of the agitation blank formed at this time. This procedure was repeated at least five times to obtain a solution containing pure bacteriophage. The separation of pure bacteriophages was usually repeated until the size and shape of the formed lysate were all similar. Finally, it was confirmed by electron microscopic analysis whether the bacteriophage was purely isolated.
  • the solution containing pure bacteriophage identified in this way was subjected to the following purification procedure.
  • a culture medium of eromonas hydrofilm was added in a volume of one-half of the total volume of the solution, followed by culturing again for 4-5 hours. After incubation, supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. This process was repeated five times in total to obtain a solution containing a sufficient number of bacteriophages.
  • the supernatant obtained by the final centrifugation was filtered using a 0.45 ⁇ m filter, and then a conventional polyethylene glycol (PEG) precipitation process was performed.
  • PEG polyethylene glycol
  • PEG and NaCl were added to 100 ml of the filtrate to make 10% PEG 8000 / 0.5 M NaCl, and the mixture was allowed to stand at 4 ° C for 2-3 hours, followed by centrifugation at 8,000 rpm for 30 minutes to obtain a bacteriophage precipitate .
  • precipitate bacteriophage buffer Buffer; 10 mM Tris-HCl , 10 mM MgSO 4, 0.1% Gelatin, pH 8.0
  • This is called a bacteriophage suspension or bacteriophage solution.
  • the bacteriophage was named Bacteriophage Aer-HYP-1, and deposited on May 10, 2017 at the BRC (Korea Research Institute of Bioscience and Biotechnology) (Accession No. KCTC 13257BP ).
  • Example 2 Bacteriophage Aer - HYP -1 and genome sequencing
  • the genome of bacteriophage Aer-HYP-1 was isolated as follows.
  • the bacteriophage suspension obtained by the same method as in Example 1 was used.
  • 200 U of DNase I and 200 A of RNase A were added to 10 ml of the bacteriophage suspension to remove the DNA and RNA of eromonas hydrofilm, which may be contained in the supernatant, and then left at 37 ° C for 30 minutes.
  • 500 ⁇ l of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added to remove DNase I and RNase A activity, and the mixture was allowed to stand for another 10 minutes.
  • EDTA ethylenediaminetetraacetic acid
  • the upper layer was taken out of the separated layers, 1.5 parts by volume of isopropyl alcohol was added thereto, and the mixture was centrifuged at 13,000 rpm for 10 minutes The dielectric was precipitated. After the precipitate was collected, 70% ethanol was added to the precipitate, and the precipitate was further washed by centrifugation at 13,000 rpm for 10 minutes. The washed precipitate was recovered, vacuum dried and dissolved in 100 ⁇ l of water. The above procedure was repeated to secure a large amount of the genome of the bacteriophage Aer-HYP-1.
  • the resulting genomes were sequenced using the illumina Mi-Seq instrument from Macrogen, and genome sequence information of bacteriophage Aer-HYP-1 was obtained.
  • the finally analyzed bacteriophage Aer-HYP-1 genome has a size of 54,294 bp and the entire genomic sequence is shown in SEQ ID NO: 1.
  • genomic sequence information of the obtained bacteriophage Aer-HYP-1 Based on the genomic sequence information of the obtained bacteriophage Aer-HYP-1, similarity to the known bacteriophage genome sequence was examined using BLAST on the web. As a result of BLAST, the genomic sequence of bacteriophage Aer-HYP-1 was analyzed to have relatively high homology with the sequence of eromonas bacteriophage pAh6-C (Genbank Accession No. KJ858521.1) (Query coverage / identity: 97% / 96%). However, the number of open reading frames (ORFs) on the genome of bacteriophage Aer-HYP-1 was 76, while bacteriophage pAh6-C was 86, indicating that these were different bacteriophages.
  • ORFs open reading frames
  • bacteriophage Aer-HYP-1 is a novel bacteriophage different from the previously reported bacteriophages.
  • bacteriophage Aer-HYP-1 can provide a different antimicrobial effect from other bacteriophages reported from the fact that the different types of bacteriophages usually provide different levels of antimicrobial activity and antimicrobial range there was.
  • Example 3 Bacteriophage Aer - HYP -1 of Eromonas Hydropila For bacteria Destructiveness Research
  • the ability of the isolated bacteriophage Aer-HYP-1 to eromonas hydrofilas was examined. The extinction ability was investigated by examining whether or not a transparent ring was formed through the drip test as described in Example 1.
  • the eromonas hydrofilas strains used in the study of the killing activity were total 15 weeks, which were isolated by the present inventors and identified as eromonas hydrofilas.
  • Bacteriophage Aer-HYP-1 had the ability to kill for 13 weeks out of 15 of the experimental eromonas hydrofilas. Representative experimental results are shown in Fig.
  • the bacteriophage Aer-HYP-1 did not have the ability to kill bacteria of these species.
  • the bacteriophage Aer-HYP-1 has an excellent killing ability against eromonas hydrofilas and can exert an antibacterial effect against many eromonas hydro pilas strains. This means that the bacteriophage Aer-HYP-1 can be used as an active ingredient of a composition for the prevention or treatment of diseases caused by eromonas hydrofilas.
  • Example 4 Bacteriophage Aer - HYP -1 of Eromonas Hydropila For prevention of fungal infection Experimental Example
  • the bacteriophage Aer-HYP-1 of the present invention not only inhibits the growth of eromonas hydrofilm, but also has the ability to kill eromonas hydrofilm. From this, it is confirmed that bacteriophage Aer-HYP-1 Can be utilized as an effective ingredient of a composition for the prevention of diseases caused by eromonas hydrofilas.
  • Example 5 Bacteriophage Aer - HYP -1 Eromonas Hydropila Bacterium-induced Preventive Animal Test for Disease
  • Rainbow trout (average weight: 23.6 g, average length: 15.7 cm) was divided into two groups of 20 rats, and the rats were separated and kept in a water tank for 14 days. The ambient environment of the water tank was controlled, and the temperature of the laboratory with the water tank was kept constant. Feeds containing 1 ⁇ 10 8 pfu / g of bacteriophage Aer-HYP-1 were fed to the rainbow trout of the experimental group (bacteriophage-treated group) from the beginning of the experiment to the experimental period according to a conventional feed feeding method. On the other hand, the rainbow trout of the control group (bacteriophage MIT) received feeds of the same composition without bacteriophage Aer-HYP-1 in the same manner.
  • Body size ulcer size measurement (average) US score (mean) date D9 D10 D11 D12 D13 D14 Control group (female bacteriophage) 0.50 0.55 0.55 0.65 0.75 0.80 Experimental group (bacteriophage administration) 0 0 0 0 0 0 0 0 0
  • the bacteriophage Aer-HYP-1 of the present invention is very effective for the prevention of diseases caused by eromonas hydrofilm.
  • Example 6 Bacteriophage Aer - HYP -1 Eromonas Hydropila For diseases caused by bacteria Treatment example
  • the therapeutic effects of the bacteriophage Aer-HYP-1 on the diseases caused by eromonas hydrofilas were examined.
  • 40 rabbits (average weight: 23.9 g, average length: 15.8 cm) were divided into two groups and divided into two groups.
  • the ambient environment of the water tank was controlled, and the temperature of the laboratory with the water tank was kept constant. From the 5th day after the start of the experiment, the feedstuffs contaminated with eromonas hydrofilm at a level of 1 ⁇ 10 8 cfu / g for 3 days were fed twice a day in a conventional feed feeding manner. From the last day of contaminated feed, eromonas hydrofilas were identified in both tanks with clinical symptoms.
  • the rainbow trout of the experimental group (bacteriophage-treated group) received bacteriophage Aer-HYP-1 (1 ⁇ 10 8 / ml) from the day after the feeding of the contaminated feed of eromonas hydrofilm for 3 days pfu / g) were fed according to a conventional feed feeding method.
  • the rainbow trout of the control group (bacteriophage MIT) received feeds of the same composition without bacteriophage Aer-HYP-1 in the same manner.
  • the incidence of infestation was examined in all test animals on a daily basis. Investigation of the incidence of eruption caused by eromonas hydrofilas was carried out by measuring the ulcer size of the body surface as in Example 5. The results are shown in Table 3.
  • Body size ulcer size measurement (average) US score (mean) date D8 D9 D10 D11 D12 D13 D14 Control group (female bacteriophage) 1.05 1.35 1.55 1.65 1.65 1.60 1.70 Experimental group (bacteriophage administration) 1.00 0.90 0.55 0.55 0.35 0.15 0.10
  • the bacteriophage Aer-HYP-1 of the present invention is very effective for the treatment of diseases caused by eromonas hydrofilas.
  • Bacteriophage Aer-HYP-1 solution was used to prepare a feed additive containing 1 x 10 8 pfu of bacteriophage Aer-HYP-1 per gram of feed additive.
  • the feed additives were prepared by adding maltodextrin to the bacteriophage solution (50%, w / v) followed by lyophilization. And finally pulverized into a fine powder form.
  • the drying process during the manufacturing process may be replaced by vacuum drying, warm drying, or drying at room temperature.
  • a feed additive without bacteriophage was also prepared by using the buffer (Buffer; 10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0) used in the preparation of the bacteriophage solution instead of the bacteriophage .
  • Buffer 10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0
  • Each of the two feed additives was mixed with a 250 - fold amount of raw fish to produce two final feeds.
  • the bath preparation was prepared as follows. Bacteriophage Aer-HYP-1 solution was used to prepare a bathing agent so that 1 ⁇ 10 8 pfu of bacteriophage Aer-HYP-1 per 1 ml of bath was contained.
  • the preparation method of the bathing agent is such that the above bacteriophage Aer-HYP-1 solution is added so as to contain 1 ⁇ 10 8 pfu of bacteriophage Aer-HYP-1 per 1 ml of the buffer used in the production of the bacteriophage solution Respectively.
  • the buffer solution used in the preparation of the bacteriophage solution was used as the non-bacteriophage-free bath solution.
  • the two bath preparations thus prepared were diluted with water at a volume ratio of 1,000 times and used as a final bathing agent.
  • Example 7 and Example 8 Using the feeds prepared in Example 7 and Example 8, and a bathing agent, the improvement of the specification results in rainbow trout breeding was investigated. In particular, the survey was conducted in terms of mortality.
  • a total of 500 rainbow trout were divided into two groups of 250 rats divided into two groups (feed-fed group-A, bath-treated group-B) for four weeks. Each group was subdivided into a group consisting of 125 animals. Each subgroup was divided into a small group (small group-1) with bacteriophage Aer-HYP-1 and a small group (small group -2) without bacteriophage.
  • the rainbow trout that was subject to the test was a 5 - week - old rainbow trout (average weight: 23.4 g, average length: 15.5 cm).
  • Rainbow trout of each test group were kept in separate tanks at regular intervals. Each subgroup is identified and named as shown in Table 4 below.
  • Subgroup classification and display in the rainbow trout specimen test apply Classification and display of small groups Application of bacteriophage Aer-HYP-1 Bacteriophage not applied Group fed with feed A-1 A-2 A group treated with bath B-1 B-2
  • Example 7 In the case of feed feed, the feed prepared in Example 7 was fed according to a conventional feed feeding method according to the classification of Table 4, and in the case of the bath treatment, the feed prepared in accordance with the preparation method of bath preparation described in Example 8 A bath agent was treated according to a conventional bath bath treatment method in which a fish body was immersed in a diluting solution of a bath agent according to Table 4. The results are shown in Table 5.

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Abstract

La présente invention concerne un bactériophage de Myoviridae Aer-HYP-1 (numéro d'accès KCTC 13257BP), isolé du milieu naturel, ayant la capacité de tuer Aeromonas hydrophila et présentant un génome représenté par SEQ ID NO : 1, ainsi qu'un procédé de prévention et de traitement d'une maladie causée par Aeromonas hydrophila au moyen d'une composition contenant ledit bactériophage en tant que principe actif.
PCT/KR2018/006573 2017-06-21 2018-06-11 Nouveau bactériophage d'aeromonas hydrophila aer-hyp-1 et son utilisation pour inhiber la croissance d'aeromonas hydrophila Ceased WO2018236085A1 (fr)

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KR1020170078390A KR101859973B1 (ko) 2017-06-21 2017-06-21 신규한 에로모나스 하이드로필라 박테리오파지 Aer-HYP-1 및 이의 에로모나스 하이드로필라 균 증식 억제 용도

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Cited By (1)

* Cited by examiner, † Cited by third party
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CN110484513A (zh) * 2019-08-23 2019-11-22 中国水产科学研究院黑龙江水产研究所 噬菌体pAhMJG及其在治疗嗜水气单胞菌引发的鱼类疾病中的应用

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KR102639330B1 (ko) 2023-11-09 2024-02-28 대한민국 병원성 에로모나스균의 종 판별을 위한 프라이머 조성물 및 이를 포함하는 진단키트

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