Microorganisms that inhibit the growth of cold water disease bacteria in fish

Abstract

To provide an agent for inhibiting the growth of cold water disease bacteria, and an agent for preventing cold water disease, which are safe for human and fish.SOLUTION: The present invention discloses Bosea sp. OX14 strain deposited in International Patent Organism Depositary of National Institute of Technology and Evaluation (NITE) and with the accession number NITE P-03290, Flavobacterium sp. GL7 strain deposited in International Patent Organism Depositary of National Institute of Technology and Evaluation (NITE) and with the accession number NITE P-03289, or bacteria that are related to the strain and have the action of inhibiting the growth of cold water disease bacteria, conditioned media of these bacteria, and an agent for inhibiting the growth of cold water disease bacteria and an agent for preventing cold water disease, each containing these bacteria or conditioned media.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the inhibition of cold water disease in fish. In particular, the present invention relates to a bacterium capable of inhibiting the growth of cold water disease bacteria, a culture supernatant thereof, and uses thereof. [Background technology]

[0002] The global aquaculture industry is steadily expanding. Freshwater aquaculture mainly involves salmonid fish such as rainbow trout and coho salmon, as well as sweetfish, but economic losses are high due to fish diseases caused by cold water pathogens.

[0003] Coldwater disease is caused by the coldwater disease bacterium (Flavobacterium psychrophilum) and is characterized by bleeding under the gill cover and ulcers and other holes on the body surface. Originally a North American disease, infections in salmon, rainbow trout, and sweetfish have been confirmed in Japan since the 1980s (Non-Patent Document 1).

[0004] Antibiotics are primarily used to treat coldwater disease bacteria, but in recent years there has been concern around the world about the spread of antibiotic-resistant bacteria, and methods that do not use antibiotics are being sought.

[0005] Vaccines are effective against various fish diseases. Vaccine development against coldwater disease bacteria is also being actively pursued (Patent Documents 1, 2, 3, 4, 5), but there is currently no commercially available vaccine against coldwater disease bacteria (Non-Patent Document 2).

[0006] Due to the restrictions on the use of antibiotics and the lack of available vaccines, it is expected that microorganisms with antibacterial activity against coldwater disease bacteria will be used as an alternative method, and many antibacterial microorganisms have been explored. For example, Carnobacterium sp. (Non-Patent Document 3), Pseudomonas sp. (Non-Patent Documents 4, 5, 6, 7), Enterobacter sp. (Non-Patent Document 8), Luteimonas sp., Rhodococcus sp., Microbacterium sp., Sphingopyxis sp., Dietzia sp. (Non-Patent Document 9), etc. are known.

[0007] Among the above-mentioned microorganisms with antibacterial activity, Pseudomonas sp. and Enterobacter sp. have been experimentally shown to reduce the mortality rate caused by coldwater disease bacteria when administered as feed (Non-Patent Documents 4 and 8). However, none of them have been commercially used.

[0008] The method of administering microorganisms as feed has the problem that the administered microorganisms do not easily establish in the intestine (Non-Patent Document 10).

[0009] In recent years, it has become clear that intestinal bacteria and epithelial cells in fish are separated by a chitinous membrane, unlike in mammals (Non-Patent Document 11), and it has become possible that the use of probiotics targeting intestinal bacteria as feed, which has been traditionally done, may not be as effective as in mammals. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Patent Publication No. 2017-088532 [Patent Document 2] Patent Publication No. 2008-220262 [Patent Document 3] Patent Publication No. 2008-137933 [Patent Document 4] Patent Publication No. 2004-352690 [Patent Document 5] Special Opening No. 2004-210769

Non-licensed documents

[0011]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patent document 5

Non-patent document 6

Non-patent document 7

Non-patent document 8

Non-patent document 9

Non-patent document 10

[0012] When coldwater disease occurs, sulfisozole is used as a treatment, but the treatment effect is not sufficient, and as mentioned above, no preventive agent effective against the coldwater disease bacteria has been developed to date.

[0013] Although administration of probiotic bacteria as feed has been carried out at the experimental level, it has not been put to practical use, and as mentioned above, it is becoming clear that the effect of probiotic bacteria in improving the intestinal flora may not be as strong in fish as it is in mammals.

[0014] As described above, there is a strong demand for new infection prevention methods that do not rely on antibiotics or vaccines. The present invention focuses on the action of the skin, which is the gateway of infection, rather than the intestines, and aims to provide a skin bacterium that has an inhibitory effect on the proliferation of coldwater disease bacteria, its culture supernatant, and an anti-coldwater disease bacteria substance produced by the bacterium. [Means for solving the problem]

[0015] In order to solve the above problems, the present inventors have searched for bacteria that are useful for preventing coldwater disease. In the search, the conditions set were that the bacteria should be normally present on the skin of fish, should not be pathogenic to humans, and should have an inhibitory effect on the proliferation of coldwater disease bacteria.

[0016] From the above viewpoint, the present inventors obtained 174 strains of skin resident bacteria from the skin mucus of rainbow trout owned by the present inventors. When the growth inhibitory effect of these culture supernatants on coldwater disease bacteria was examined, it was found that Bosea sp. OX14 strain (hereinafter sometimes referred to as "OX14 strain") and Flavobacterium sp. GL7 strain (hereinafter sometimes referred to as "GL7 strain") had a growth inhibitory effect, and the present invention was completed. Note that Bosea bacteria are not included in the BSL level list of pathogenic bacteria by the Japanese Society for Bacteriology, and their pathogenicity to humans has not been confirmed so far, so they are considered to be non-pathogenic. As for the Flavobacterium genus, F. branchiophilum, F. columnare, F. devorans, F. johnsoniae, F. mizutaii, F. psychrophilum, etc. are considered to be opportunistic pathogens, but the GL7 strain has less than 98% identity in the 16S rRNA gene with these species and is considered to be a different species, so it is highly likely that it is not a pathogenic bacteria to humans. In addition, the GL7 strain cannot grow at 36°C, and is therefore considered to be non-pathogenic to humans.

[0017] That is, the present invention provides the following. (1) Bacteria of the genus Bosea or Flavobacterium that have an inhibitory effect on the growth of coldwater disease bacteria. (2) A bacterium having an inhibitory effect on the growth of coldwater disease bacteria, the base sequence of which is: (a) the 16S rRNA sequence of the OX14 strain, or (b) Nucleotide sequence of 16S rRNA of the GL7 strain A bacterium with 98% or more identity to (3) OX14 strain. (4) GL7 strain. (5) A culture supernatant of the bacterium according to any one of (1) to (4), which has antibacterial activity against coldwater disease bacteria. (6) A method for inhibiting the proliferation of coldwater disease bacteria, comprising applying the bacterium according to any one of (1) to (4) or the culture supernatant according to (5) to the coldwater disease bacteria. (7) A growth inhibitor of coldwater disease bacteria, comprising the bacterium according to any one of (1) to (4) or the culture supernatant according to (5). (8) A method for preventing cold water disease, comprising applying the bacterium according to any one of (1) to (4) or the culture supernatant according to (5) to fish. (9) A preventive agent for cold water disease, comprising as an active ingredient the bacterium according to any one of (1) to (4) or the culture supernatant according to (5). Effect of the Invention

[0018] The present invention provides novel OX14 and GL7 strains having a growth inhibitory effect against coldwater disease bacteria, which is a problem in the culture of salmonid fish and sweetfish in various countries around the world, as well as their closely related species, their culture supernatants, and growth inhibitors produced by them. By using these, coldwater disease bacteria can be prevented without using conventional antibiotics, vaccines, etc. Moreover, since the OX14 and GL7 strains according to the present invention are at least naturally occurring bacteria on the skin of fish, they have the advantage of being highly safe for both fish and humans who eat them. [Brief description of the drawings]

[0019] [Figure 1] Figure 1 shows the OX14 strain, which had been grown on an agar medium in advance, inoculated vertically with the coldwater disease pathogen KU190628-78 strain and cultured for five days. The control shows the KU190628-78 strain inoculated on an agar medium on which the OX14 strain had not been grown and cultured for five days. [Diagram 2] Figure 2 is a graph showing the growth inhibitory effect of culture supernatants from the mid-logarithmic, late-logarithmic, and stationary phases of OX14 strain on the growth of coldwater disease pathogen KU190628-78 strain. The control is growth when the same amount of medium is added. The turbidity measurement value at this time was set to 100%. * indicates a statistically significant decrease (p<0.05). [Diagram 3]Figure 3 is a graph showing the growth inhibitory effect of the culture supernatant of the OX14 strain on the coldwater disease pathogen KU190628-78 strain by heat treatment, the fractions of less than 3 kDa, more than 3 kDa, the fraction passing through the anion exchange column, the protein fraction eluted with a high salt concentration buffer, and the low molecular weight fraction eluted with methanol containing 1% formic acid. The control is growth when the same amount of medium is added. The turbidity measurement value at this time was set to 100%. * indicates a statistically significant decrease (p<0.05). [Figure 4] Figure 4 shows the GL7 strain, which had been grown on an agar medium in advance, inoculated vertically with the coldwater disease bacterium NCIMB 1947 strain and cultured for two days. The control shows the agar medium on which the GL7 strain had not been grown, inoculated with the coldwater disease bacterium NCIMB 1947 strain in the same manner, and cultured for two days. [Diagram 5] Figure 5 is a graph showing the growth inhibitory effect of culture supernatants of the GL7 strain at mid- and late-logarithmic growth phases, as well as at stationary phase, on the growth of the coldwater disease pathogen NCIMB 1947 strain. The control was the growth when the same amount of medium was added. The turbidity measurement value at this time was set to 100%. * indicates a statistically significant decrease (p<0.05). [Figure 6] Figure 6 is a graph showing the growth inhibitory effect of the following fractions of the culture supernatant of the GL7 strain: fractions of less than 3 kDa, more than 3 kDa, the anion exchange column pass-through fraction, the protein fraction eluted with a high salt buffer, and the low molecular weight fraction eluted with methanol containing 1% formic acid on the growth of the coldwater disease pathogen NCIMB 1947 strain. The control is growth when the same amount of medium was added. The turbidity measurement value at this time was set to 100%. * indicates a statistically significant decrease (p<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In one aspect, the present invention provides a bacterium of the genus Bosea or Flavobacterium having an inhibitory effect on the growth of coldwater disease bacteria.

[0021] In a further aspect, the present invention relates to a bacterium having a growth inhibitory effect against coldwater disease bacteria, the base sequence of which is: (a) the 16S rRNA sequence of the OX14 strain, or (b) Nucleotide sequence of 16S rRNA of the GL7 strain The present invention provides a bacterium having an identity of 98% or more to

[0022] That is, in the above embodiment, a species closely related to the OX14 strain and a species closely related to the GL7 strain are provided. As described above, the present inventors have searched for useful bacteria for preventing coldwater disease, and obtained the OX14 strain and the GL7 strain, which have the effect of inhibiting the proliferation of coldwater disease bacteria. These bacteria have been deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation, and are, respectively, Contract Number NITE P -03290 and NITE P -03289. It is believed that closely related species of these bacteria also have the effect of inhibiting the growth of coldwater disease bacteria. Whether or not they are closely related species is generally determined by the identity of the 16S rRNA base sequence. The 16S rRNA of the bacteria of the above-mentioned embodiment of the present invention has an identity of 98% or more, preferably 99% or more, to the 16S rRNA base sequence of the OX14 strain or the 16S rRNA base sequence of the GL7 strain. The identity of the 16S rRNA base sequence can be examined using known means such as DNA extraction, PCR (Polymerase Chain Reaction) using primers commonly used for bacteria, sequence analysis of the 16S rRNA gene amplified by PCR, the homology search service provided by the National Institute of Genetics, and homology search of base sequences using BLAST.

[0023] As described in the examples, the nucleotide sequences of the 16S rRNA genes of OX14 strain and GL7 strain show 99.6% and 98.9% identity with the nucleotide sequences of the 16S rRNA genes of Bosea lupini and Flavobacterium tructae, respectively.Therefore, examples of closely related species of OX14 strain and GL7 strain include bacteria of the genus Bosea and bacteria of the genus Flavobacterium, respectively, and preferably bacteria of the species Bosea lupini and bacteria of the species Flavobacterium tructae.

[0024] In a particular embodiment, the present invention provides the OX14 strain. In another particular embodiment, the present invention provides the GL7 strain.

[0025] The bacteria of the present invention having an inhibitory effect on the proliferation of coldwater disease bacteria can be cultured by known methods.

[0026] The bacterium of the present invention can usually be cultured in a temperature range of about 10° C. to about 30° C. For example, the OX14 strain grows at about 10° C. to about 30° C., with the optimum temperature being about 25° C. The GL7 strain grows at about 10° C. to about 25° C., with the optimum temperature being about 25° C. Those skilled in the art can select an appropriate culture temperature depending on the bacterium to be cultured.

[0027] The medium used for culturing the bacteria of the present invention may be a known medium. For example, the OX14 and GL7 strains may be cultured in FLP medium (Cepeda et al., 2004. Aquaculture 238: 75-82). The composition of FLP medium is as follows: 4.0 g tryptone, 0.5 g glucose, 0.4 g yeast extract, 0.2 g CaCl2·2H2O, 0.5 g MgSO4·7H2O, pH 7.2 in 1000 ml deionized water. Luria-Bertani medium or OXOID CM3 medium may be used for culturing these strains. Those skilled in the art can select an appropriate medium according to the bacteria to be cultured and can change the medium composition.

[0028] The bacteria of the present invention can be cultured until the desired growth inhibitory effect against the coldwater disease bacteria is observed. The growth inhibitory effect against the coldwater disease bacteria can be examined, for example, as follows. After adding a culture supernatant of the bacteria of the present invention to a liquid containing the coldwater disease bacteria and culturing it at an appropriate temperature for an appropriate time (the culture liquid may be sampled over time), the growth of the coldwater disease bacteria can be determined visually or by measuring the turbidity at 600 nm.

[0029] In another aspect, the present invention provides a culture supernatant of the OX14 strain, a species closely related to the OX14 strain, the GL7 strain, or a species closely related to the GL7 strain, which has antibacterial activity against Bacillus subtilis.

[0030] Whether or not the culture supernatant has antibacterial activity against the coldwater disease bacteria can be determined, for example, by examining the growth of the coldwater disease bacteria as described above. The stronger the growth inhibitory effect, the higher the antibacterial activity.

[0031] When using the bacterium of the present invention, a culture solution containing the bacterium of the present invention may be used, or the bacterium of the present invention separated by means of centrifugation, membrane filtration, or the like may be used. A culture supernatant can be obtained by subjecting a culture solution containing the bacterium of the present invention to means of centrifugation, membrane filtration, or the like. The culture supernatant may be used as is, or may be fractionated and / or concentrated by means of ultrafiltration, evaporation, chromatography, precipitation with an organic solvent, or the like. Alternatively, a culture supernatant that has been treated by lyophilization or the like may be used.

[0032] In a further aspect, the present invention provides a method for inhibiting the growth of Bacillus subtilis, which comprises applying the bacterium of the present invention or a culture supernatant thereof to Bacillus subtilis.

[0033] A specific example of a method for applying the bacteria of the present invention or its culture supernatant to coldwater disease bacteria is, for example, introducing the bacteria of the present invention or its culture supernatant into a fish farming tank. Another example is raising fish for a certain period of time in a pool into which the bacteria of the present invention or its culture supernatant has been introduced. Such rearing in a pool may be repeated multiple times. It is preferable to rear the fish until the bacteria of the present invention is established on the skin of the fish. Another specific example of a method for applying the bacteria of the present invention or its supernatant to coldwater disease bacteria is wastewater treatment from a farming tank. For example, wastewater may be stored in a pool, treated by adding the bacteria of the present invention or its culture supernatant, and then discharged into a river or the sea. Another example is passing the wastewater through a layer containing a carrier on which the bacteria of the present invention is immobilized, and continuously treating it. Such treatment can prevent rivers and seas from being contaminated by coldwater disease bacteria.

[0034] The amount of the bacteria of the present invention or its supernatant to be used can be appropriately determined by a person skilled in the art depending on the desired degree of inhibitory effect on the growth of coldwater disease bacteria, the type of fish, the number of fish, the size of the culture tank, the application time, or the amount of wastewater.

[0035] In yet another aspect, the present invention provides an inhibitor of the growth of coldwater disease bacteria, comprising the bacterium of the present invention or a culture supernatant thereof.

[0036] The agent of the present invention can be produced by a known method. For example, the bacterium of the present invention or its culture supernatant can be mixed with a suitable carrier and, if desired, formed into a desired dosage form through steps such as concentration, drying, tableting, and pulverization. The dosage form of the agent of the present invention is not particularly limited, and may be, for example, a liquid, paste, powder, granules, pellets, or block.

[0037] For example, the agent of the present invention may be added to an aquaculture tank. Also, for example, fish may be raised for a certain period of time in a pool into which the agent of the present invention has been added. Also, for example, the agent of the present invention may be added to wastewater from an aquaculture tank stored in a pool. Also, for example, when the agent of the present invention is in the form of granules, pellets, blocks, etc., it may be continuously treated as described above.

[0038] The amount of the agent of the present invention to be used can be appropriately determined by a person skilled in the art depending on the desired degree of inhibitory effect on the growth of coldwater disease bacteria, the type of fish, the number of fish, the size of the culture tank, the application time or the amount of wastewater, etc.

[0039] In a further aspect, the present invention provides a method for preventing cold water disease, which comprises applying the bacterium of the present invention or a culture supernatant thereof to fish.

[0040] The method for applying the bacterium of the present invention or its culture supernatant to fish may be the same as the method described above.

[0041] The target fishes are fishes that may be infected with coldwater disease bacteria, and fishes that are infected with coldwater disease bacteria. The types of such fishes are not particularly limited, and examples include salmonid fish such as salmon and trout, sweetfish, carp fish, eels, etc. Typical examples of the target fishes include salmon, coho salmon, sockeye salmon, chinook salmon, brown trout, brook trout, cherry salmon, rainbow trout, sweetfish, etc.

[0042] In yet another aspect, the present invention provides an agent for preventing cold water disease, comprising the bacterium of the present invention or a culture supernatant thereof as an active ingredient.

[0043] The above explanations regarding the growth inhibitor of coldwater disease bacteria can be applied to the production, dosage form, and administration of the preventive agent of the present invention to fish. The dosage of the preventive agent of the present invention can be determined based on the reduction of the morbidity rate of coldwater disease, preferably to zero, the disappearance of coldwater disease bacteria from wastewater discharged into rivers or the sea, or the establishment of the bacterium of the present invention on the skin of fish, etc.

[0044] Unless otherwise specified, the terms used in this specification are to be interpreted in the sense commonly understood in the fields of fisheries, biology, biochemistry, chemistry, pharmacology, veterinary medicine, and the like.

[0045] The present invention will be described in more detail and specifically below by showing examples, but the scope of the present invention is not limited to these examples. EXAMPLES

[0046] Example 1: Isolation and identification of bacteria from rainbow trout skin mucus that have an inhibitory effect on the growth of coldwater disease bacteria The present inventors searched for useful bacteria in the skin mucus of rainbow trout, with the conditions that they were normal bacteria on the skin of fish, were not pathogenic to humans, and had an inhibitory effect on the growth of coldwater disease bacteria. 174 strains of normal skin bacteria were obtained from the skin mucus of rainbow trout owned by the present inventors. When the inhibitory effect on the growth of coldwater disease bacteria using the culture supernatant of these bacteria was examined, it was found that the OX14 and GL7 strains had an inhibitory effect on the growth of coldwater disease bacteria.

[0047] The mycological properties of the OX14 and GL7 strains were as follows: Analysis of the 16S rRNA gene sequences of the OX14 and GL7 strains revealed that they showed 99.6% and 98.9% identity to the 16S rRNA gene sequences of Bosea lupini and Flavobacterium tructae, respectively, and thus identified them as Bosea sp. OX14 and Flavobacterium sp. GL7, respectively.

[0048] The OX14 and GL7 strains grew well in FLP medium, Luria-Bertani medium, and OXOID CM3 medium. The OX14 strain grew at about 10 to about 30°C, with the optimum temperature being about 25°C. The GL7 strain grew at about 10 to about 25°C, with the optimum temperature being about 25°C.

[0049] DNA was extracted from the skin mucus, gills, and intestines of rainbow trout, and the V3-V4 region of the 16S rRNA gene was amplified by PCR and analyzed using a Miseq sequencer to clarify the microbiota of each. Based on the information on the microbiota, the abundance of OX14 and GL7 strains was investigated. As a result, the abundance of OX14 strain was 1.6, 61.4, and 0.0% in the skin mucus, gills, and intestines, respectively, and the abundance of GL7 strain was 0.2, 0.0, and 0.0% in the skin mucus, gills, and intestines, respectively, and both were major members of the resident bacteria on the skin (and gills in the case of OX14 strain).

[0050] In addition, analysis using microbiota data from the skin mucus of American rainbow trout (Lowrey et al., 2015. Applied and Environmental Microbiology 81: 6915-6925.) revealed that the OX14 and GL7 strains were present at 0.03 and 0.05%, respectively, suggesting that these two strains are core microorganisms of rainbow trout skin bacteria.

[0051] The OX14 strain was deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan. Contract Number NITE P The GL7 strain was deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan. Contract Number NITE P -03289 was assigned. EXAMPLES

[0052] Example 2: Inhibitory effect of OX14 strain on the growth of coldwater disease bacteria The OX14 strain was inoculated linearly onto FLP agar medium and cultured for 7 days. The coldwater disease fungus cultured on FLP medium was inoculated linearly and perpendicularly onto the grown OX14 strain and cultured for 5 days.

[0053] As a result, it was shown that the OX14 strain inhibited the proliferation of the KU190628-78 strain, which is a problem in sweetfish. The KU190628-78 strain was isolated from the kidneys of sweetfish at Kinki University. The test results are shown in Figure 1. EXAMPLES

[0054] Example 3: Inhibitory effect of culture supernatant of OX14 strain on the growth of coldwater disease pathogen The OX14 strain was cultured in FLP medium, and the culture fluid was collected at the mid-, late-, and stationary phases of logarithmic growth, and filtered through a 0.2 μm filter to obtain the culture supernatant. 50 μl of the culture supernatant, 190 μl of FLP medium, and 20 μl of the coldwater disease bacteria were added to a 96-well plate and cultured at 16°C for 3 days.

[0055] The results showed that the OX14 strain suppressed the growth of the KU190628-78 strain, which is problematic in sweetfish. The stationary phase culture supernatant was the most effective, suppressing growth by 35% compared to the control with the same amount of medium added. The test results are shown in Figure 2. EXAMPLES

[0056] Example 4: Fractionation of culture supernatant of OX14 strain The OX14 strain was cultured in FLP medium, and the culture medium was collected during the stationary growth phase and filtered through a 0.2 μm pore size filter to obtain the culture supernatant. The culture supernatant was fractionated into substances with a molecular weight of 3 kDa or less and 3 kDa or more through a Centriprep YM-3 column (Merck). In addition, the fraction was passed through an anion exchange column Oasis MAX (Waters) to obtain a pass-through fraction, a protein fraction eluted with a high salt concentration buffer (HEPES 50 mM, NaCl 0.5 M, pH 7.2), and a low molecular weight fraction eluted with methanol containing 1% formic acid. 50 μl of the culture supernatant, 190 μl of FLP medium, and 20 μl of C. moniliforme were added to a 96-well plate and cultured at 16°C for 3 days.

[0057] As a result, it was shown that the fraction of the culture supernatant of the OX14 strain that was 3 kDa or less and the fraction that passed through the anion exchange column inhibited the growth of the KU190628-78 strain. The test results are shown in Figure 3. EXAMPLES

[0058] Example 5: Inhibitory effect of GL7 strain on the growth of coldwater disease bacteria The GL7 strain was inoculated onto FLP agar medium and cultured for 7 days. Since the colony of the GL7 strain has the tendency to expand, a part of it was cut out and embedded in a new FLP agar medium. The part where the GL7 strain cultured on the FLP medium was embedded was inoculated with the coldwater disease fungus vertically and linearly, and cultured for 2 days.

[0059] As a result, it was shown that the GL7 strain inhibited the growth of the NCIMB 1947 strain, which is a problem in salmon. The NCIMB 1947 strain is a bacterial strain preserved in the National Collections of Industrial, Food and Marine Bacteria, Ltd. in the UK. The test results are shown in Figure 4. EXAMPLES

[0060] Example 6: Inhibitory effect of culture supernatant of GL7 strain on the growth of coldwater disease pathogen The GL7 strain was cultured in FLP medium, and the culture fluid was collected at the mid-, late-, and stationary phases of logarithmic growth, and filtered through a 0.2 μm filter to obtain the culture supernatant. 50 μl of the culture supernatant, 190 μl of FLP medium, and 20 μl of the coldwater disease fungus were added to a 96-well plate and cultured at 16°C for 3 days.

[0061] The results showed that the GL7 strain inhibited the growth of the NCIMB 1947 strain, which is a problem in salmon. The GL7 strain was not effective in the mid-logarithmic growth phase, but an inhibitory effect was observed in the culture supernatant from the late logarithmic growth phase and stationary phase, with the stationary phase culture supernatant being the most effective, suppressing growth by 31% compared to the control to which the same amount of medium was added. The test results are shown in Figure 5. EXAMPLES

[0062] Example 5: Fractionation of culture supernatant of GL7 strain The GL7 strain was cultured in FLP medium, and the culture medium was collected during the stationary phase of the logarithmic growth phase and filtered through a 0.2 μm pore size filter to obtain the culture supernatant. The culture supernatant was fractionated into substances of 3 kDa or less and 3 kDa or more through a Centriprep YM-3 column (Merck). In addition, the pass-through fraction was passed through an anion exchange column Oasis MAX (Waters), as well as a protein fraction eluted with a high salt concentration buffer (HEPES 50 mM, NaCl 0.5 M, pH 7.2), and a low molecular weight fraction eluted with methanol containing 1% formic acid. 50 μl of the culture supernatant, 190 μl of FLP medium, and 20 μl of the coldwater disease bacteria were added to a 96-well plate and cultured at 16 degrees for 3 days.

[0063] As a result, it was shown that the fraction of GL7 strain culture supernatant with a molecular weight of 3 kDa or less and the fraction that passed through the anion exchange column inhibited the growth of NCIMB 1947 strain. The test results are shown in Figure 6. [Industrial Applicability]

[0064] The present invention provides a preventive agent for cold water disease which is highly safe for humans and fish, and is therefore useful in the fisheries, particularly the aquaculture industry, the food industry, and the like. [Accession number]

[0065] Bosea sp. OX14 strain Located at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture Deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (Received: September 24, 2020) , Contract Number NITE P -03290 was assigned. Flavobacterium sp. GL7 strain Located at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture Deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (Received: September 24, 2020) , Contract Number NITE P -03289 was assigned.

Claims

1. The Bosea sp. OX14 strain was deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation and assigned the accession number NITE P-03290.

2. Flavobacterium sp. GL7 strain, which has been deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation and assigned the accession number NITE P-03289.

3. 3. A culture supernatant of the bacterium according to claim 1 or 2, which has antibacterial activity against Flavobacterium psychrophilum.

4. A method for inhibiting the proliferation of coldwater disease bacteria, which comprises applying the bacterium according to claim 1 or 2 or the culture supernatant according to claim 3 to the coldwater disease bacteria.

5. A growth inhibitor for coldwater disease bacteria, comprising the bacterium according to claim 1 or 2 or the culture supernatant according to claim 3.

6. A method for preventing cold water disease, which comprises applying the bacterium according to claim 1 or 2 or the culture supernatant according to claim 3 to fish.

7. 10. A preventive agent for cold water disease, comprising the bacterium according to claim 1 or 2 or the culture supernatant according to claim 3 as an active ingredient.

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