Vaccine preparation, its manufacturing method, and method for preventing bacterial infections in fish

Abstract

To provide a manufacturing method that forms a biofilm that is one of the forms such as survival, adhesion, proliferation, and infection, of a bacterium, which is a fish disease bacterium isolated and cultured in a normal general culture medium, in cases where there are few or almost no infection protective antigens; and to provide a more effective vaccine for fish using the same.SOLUTION: By a method for culturing a bacterial strain that is isolated from fish by a normal general culture medium and that hardly forms a biofilm, with a medium containing a bacterial density sensing substance, a medium containing stress substance, or a medium containing iron ions and activated carbon powders, it becomes possible to produce a biofilm and the components produced during biofilm formation, maturation, and disintegration processes; and moreover, by immersing or injecting fish with a solution containing the biofilm and its produced components as antigens, the effectiveness of a vaccine can be greatly improved.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a vaccine preparation and a method for producing the same, a method for preventing bacterial infections in fish, etc. More specifically, the present invention relates to a method for preventing bacterial infections in fish by culturing an isolated bacterial strain that hardly forms a biofilm (BF) in a liquid medium containing a low concentration of a quorum-sensing substance, a stress substance, or a substance that adsorbs metal ions and the quorum-sensing substance, thereby forming a BF again, and then culturing the BF in the medium to obtain BF components including structures of the BF, such as polysaccharides, nucleic acids, proteins, quorum-sensing substances, extracellular polymeric substances (EPS), membrane vesicles produced inside and outside the BF under BF-forming conditions and substances contained inside the vesicles, components produced / excreted during the BF formation, maturation, and breakdown processes, and bacterial cells (adherent cells, membrane-forming cells, persister cells) on and inside the BF. The present invention relates to a culture medium capable of producing a solution containing BF outer membranes and components excreted outside the BFs, a method for preventing bacterial infection in fish in which the solution obtained by culturing the culture medium is immersed or injected into fish, vaccine preparations for immersion and injection, and methods for producing vaccine preparations for immersion and injection. [Background technology]

[0002] Aquaculture of industrial aquatic organisms is widespread because it can increase yields and provide a stable supply at a relatively low cost. However, the cultivation of these aquatic organisms is prone to disease due to cost-conscious farming practices such as overcrowding, and large amounts of antimicrobial agents are used to combat this. For this reason, the use of vaccines is essential to prevent disease outbreaks.

[0003] The use of antibacterial agents is an effective method for combating diseases in fish farming. For example, sulfisozole (Patent Document 1) and florfenicol are approved as veterinary drugs and are used when coldwater disease occurs in sweetfish (Ayu). However, because farmed organisms are raised in the tens of thousands to hundreds of thousands, large amounts of antibacterial agents are used. Furthermore, if deaths do not subside even after administration of the above-mentioned treatment, cases occur in which antibacterial agents are overdosed or administered for a longer period than prescribed in the dosage instructions. Furthermore, because most bacterial microorganisms in aquaculture water have the opportunity to come into contact with antibacterial agents, even bacteria that are harmless to farmed organisms can develop resistance in various bacterial microorganisms, and when the water is discharged, these resistant bacteria can have an impact on the human environment. For this reason, a method that does not rely on antibacterial agents has been developed for coldwater disease in ayu by heating the breeding water to eliminate the coldwater disease bacteria (Patent Documents 2 and 3). However, this method has significant disadvantages, such as being cost-effective, so it is highly desirable to prevent disease in aquaculture using vaccines rather than antibacterial agents.

[0004] Given this background, there is a strong demand in the field for the development of vaccines against diseases of aquatic organisms.For example, vaccines that have been developed against coldwater disease in sweetfish include one that is formulated by immersing the fish in formalin-killed cells (FKC) (hereinafter abbreviated as "FKC"), which are inactivated by adding formalin to a culture solution containing the coldwater disease bacteria in the logarithmic growth phase (Patent Document 4), a coldwater disease vaccine that activates the sweetfish's immune system by combining rabbit-derived red blood cells (Patent Document 5), a coldwater disease vaccine that is formulated by immersing the fish in a collagenase solution, which is a toxoid produced by the coldwater disease bacteria, and FKC of the coldwater disease bacteria (Patent Document 6), a vaccine that is administered orally (Patent Document 7), and a vaccine that is immersed in an enzyme-treated solution that improves antigen uptake by enzymatically treating the culture solution (Patent Documents 8 and 9).However, none of these vaccines have been commercially available to date due to problems such as the inability to achieve sufficient effectiveness when immersed, injected, or orally administered. Furthermore, there are very few examples of vaccines being marketed against other aquatic organism diseases.

[0005] Meanwhile, traditional research into various fish pathogenic bacteria has typically used commercially available media primarily composed of mammalian muscle and offal extracts (containing various enzymes), casein, whey, soybeans, grains, yeast-derived components, and components obtained by enzymatic digestion of these. Alternatively, "homemade media" prepared by mixing multiple commercially available media in a specified ratio has been used to study fish pathogenic bacteria isolated and cultured in these media. In other words, the various fish pathogenic bacteria that can be isolated and / or cultured using these media are strains isolated using these media, and these isolated strains are used to study the pathogenicity of the bacteria against aquatic organism diseases and various phenotypic traits. Therefore, when conventional culture of various fish pathogenic bacteria results in low efficacy as vaccines, many studies have been conducted to enhance their efficacy as vaccines by mixing them with immunostimulants such as oil adjuvants, aluminum hydroxide, aluminum phosphate, fucoidan, alums, killed tuberculosis bacteria, and Gerbu adjuvant. Furthermore, for example, in the case of coldwater disease, when the state of the virus existing in nature before isolation is significantly different from the state of the virus when isolated using the above-mentioned culture medium, research has been conducted using a culture medium containing host meat extract (Patent Document 10) with the aim of maintaining the phenotype using host muscle components, but this has not yet been put on the market as a vaccine.

[0006] One reason for the lack of progress in the development of vaccines against fish disease bacteria is that, for example, it is known that the pathogenicity of coldwater disease bacteria strains isolated from sweetfish is significantly reduced in sweetfish. When comparing the antigens recognized by specific antibodies in the serum of sweetfish administered with an FKC injection vaccine made by conventionally culturing coldwater disease bacteria strains with those in the serum of sweetfish that have naturally fallen ill in aquaculture farms, etc., the serum of sweetfish administered with FKC produces many antibodies to the lipopolysaccharide of the coldwater disease bacteria, while the antibodies in the serum of naturally infected sweetfish hardly react to lipopolysaccharide at all (Kintsuji et al., Fish Pathology, 42 (2007) 159-161). Therefore, it is thought that FKC produced by conventional culture has few protective antigens (antigens that can prevent infection) or that specific immunity responds preferentially to lipopolysaccharides that are highly immunogenic (easily recognized by the immune system). As with vaccines for other fish, the delay in vaccine development using FKC produced by conventional culture methods is also thought to be one of the reasons for the lack of progress.

[0007] On the other hand, fish vaccines already on the market are limited to streptococcosis (a Gram-positive bacterium) and pseudotuberculosis and vibriosis (a Gram-negative bacterium belonging to the Vibrionaceae family), and no vaccines exist for bacterial diseases of fish other than these. As mentioned above, most vaccines developed for other bacterial diseases of fish that are not on the market use bacteria that have been isolated and cultured in a conventional manner in commercially available media, or improve their effectiveness by adding adjuvants. Therefore, in order to develop a vaccine for fish that is not on the market, it is necessary to develop a new medium that can produce and contain other antigen groups that cannot be obtained by conventional culture in the culture of fish disease bacteria. A vaccine that can contain a large number of new protective antigens is thought to be highly effective. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent Publication No. 2000-191551 [Patent Document 2] Patent Publication No. 2005-287303 [Patent Document 3] Patent Publication No. 2005-245318 [Patent Document 4] Patent Publication No. 2004-210769 [Patent Document 5] Patent Publication No. 2004-352690 [Patent Document 6] Patent No. 6709395 [Patent Document 7] Patent Publication No. 2008-137933 [Patent Document 8] Patent No. 6012013 [Patent Document 9] Patent Publication No. 2021-168607 [Patent Document 10] Patent Publication No. 2008-220262 Summary of the Invention [Problem to be solved by the invention]

[0009] As described above, when developing a vaccine against bacterial infections in fish using the immersion and injection methods with FKC, if the causative bacterium produces few protective antigens even when cultured using conventional methods, it is necessary to use a vaccine produced by a culture method using a medium that produces a culture solution rich in protective antigens. Therefore, an object of the present invention is to provide a culture method using a new medium that enables the production of large amounts of protective antigens against bacteria that cause bacterial infections in fish, and to provide more effective immersion and injection vaccines against bacterial infections in fish using this culture method. [Means for solving the problem]

[0010] The inventors have used bacterial strains isolated from bacteria that cause bacterial infections in fish, and by culturing them in a medium supplemented with a low concentration of a bacterial quorum-sensing substance, by culturing them in the presence of a stress substance, by culturing them in the presence of excess metal ions, by culturing them in a medium containing a substance that adsorbs the bacterial quorum-sensing substance, or by culturing them in a medium supplemented with any or all of these in combination, have been able to extract BFs, components produced / excreted during the BF formation, maturation and decay processes, and bacterial cells, such as polysaccharides, nucleic acids, proteins, bacterial quorum-sensing substances, EPS, membrane vesicles produced inside and outside BFs under BF-forming conditions and substances contained within these vesicles, components produced / excreted during the BF formation, maturation and decay processes, and bacterial cells on / inside BFs (including adherent cells, membrane-forming cells, eternal cells, proliferating cells, stationary cell phase cells, dead cells and components eluted from dead cells), as well as the components thereof. The researchers discovered a new production method using the newly developed medium described above that enables the production of large quantities of BF outer membranes and components excreted outside the BF (polysaccharides, nucleic acids, proteins, bacterial quorum-sensing substances, EPS, membrane vesicles produced inside and outside the BF under BF formation conditions and substances contained within these vesicles, components produced / excreted during the BF formation, maturation, and breakdown processes, bacterial cells on / inside the BF [including adherent cells, membrane-forming cells, eternity cells, proliferating cells, stationary cell-phase cells, dead cells, and components eluted from dead cells], as well as BF outer membranes and components excreted outside the BF; hereafter abbreviated as "BFs, components produced / excreted during the BF formation, maturation, and breakdown processes, and bacterial cells"). They also discovered that the effectiveness of BFs as a vaccine can be significantly improved by immersing or injecting fish in a solution containing these components as inactivated antigens.

[0011] Therefore, the present invention provides a method for producing a vaccine containing an active ingredient selected from the group consisting of BFs, components produced / excreted during the formation, maturation, and breakdown of BFs, and bacterial cells, by culturing in a newly developed medium in which BFs are formed by adding low concentrations of bacterial quorum-sensing substances or stress substances to the medium, or by adsorbing excess bacterial quorum-sensing substances that promote BF formation to a low concentration and adding excess substances that function as adhesion substrates and metal ions to the medium, and also provides a method for preventing bacterial infections in fish, which includes a step of injecting or immersing fish in a solution containing these.

[0012] Therefore, when administering FKC prepared by conventional culture as an injection or immersion vaccine (including those using adjuvants) against bacteria that cause fish bacterial infections has low effectiveness, the effectiveness of immersion or injection vaccines can be significantly improved by using a solution prepared by culturing in a newly developed medium, when the protective antigen is BF, components produced / excreted during the formation, maturation, and breakdown of BF, or the bacterial body.

[0013] Furthermore, the antigen components developed in this invention are composed of a variety of antigens, including those that cannot be obtained by conventional culture. For example, the membrane vesicles (including their surface and internal inclusions) in BF formation have different properties from those secreted during the logarithmic growth phase and stationary phase of the bacterial cell (SR Schooling et al., J. Bacteriol. 5945-5947 (2006)). Therefore, if these are protective antigens, they may be applicable to the development of vaccines for other fish bacterial diseases.

[0014] Furthermore, the method of using FKC produced by culturing in the newly developed medium as an antigen uses a medium made from inexpensive materials and procedures similar to those used in conventional culture, with almost no change in the labor and cost required to produce the antigen. Furthermore, because this antigen can be used as a simple, low-stress immersion vaccine, the present invention also enables effective vaccination of small fish and fry, which is difficult to achieve by injection or other methods.

[0015] In addition, it will be possible to provide the above-mentioned antigens for injectable vaccines against bacterial fish diseases that have not been commercially available until now, contributing as one method for improving the effectiveness of vaccines for the above-mentioned diseases.

[0016] Furthermore, the newly developed medium of the present invention allows bacteria to form BFs easily, in large quantities, and inexpensively in flasks, and therefore contributes to research into the characteristics and industrial applications of bacteria that have not been observed to form BFs or that have only weak BF formation.The present invention provides the following vaccine formulations, methods for producing the same, and methods for preventing bacterial infections in fish.

[0017] [1] A vaccine preparation for immersion or injection used to prevent bacterial infections in fish, which contains at least one active ingredient selected from the group consisting of biofilms (BF) derived from fish bacterial infection bacteria, components produced during the formation, maturation and breakdown of BF, and bacterial bodies. [2] The vaccine preparation according to [1], wherein the bacterial cells are selected from the group consisting of inactivated and killed bacteria of bacteria that cause bacterial infections in fish. [3] A method for producing an immersion or injection vaccine preparation used to prevent bacterial infections in fish, comprising culturing a pathogenic bacterium for bacterial infections in fish by adding a quorum-sensing substance to the pathogen, and forming a biofilm and / or components produced during the formation, maturation, and breakdown of the biofilm. [4] The method for producing a vaccine preparation according to [3], wherein the medium further contains at least one substance selected from the group consisting of metal ions and substances that adsorb quorum-sensing substances. [5] The method for producing a vaccine preparation according to [4], wherein the substance that adsorbs the quorum-sensing substance is activated carbon. [6] A method for preventing bacterial infections in fish, comprising applying or injecting into fish a solution containing a biofilm derived from bacteria that cause bacterial infections in fish, components produced during the biofilm formation, maturation, and collapse process, and / or inactivated or killed bacteria of the bacteria that cause bacterial infections in fish. [7] The method for preventing fish bacterial infections according to [6], wherein the causative bacterium is a bacterium that can be isolated and cultured in a general medium for isolating and culturing bacteria. [8] The method for preventing fish bacterial infections according to [6] or [7], wherein the causative bacterium of fish bacterial infections is a bacterium of the Bacteroidetes phylum, to which coldwater disease and coldwater disease bacteria belong. [9] The method for preventing bacterial infections in fish described in [6], wherein the causative bacteria of bacterial infections in fish are inactivated causative bacteria. [Effects of the Invention]

[0018] The present invention makes it possible to administer vaccines effectively against bacterial infections in fish by injection and immersion methods. [Brief explanation of the drawings]

[0019] [Figure 1A] This photograph shows the visual observation of the biofilm formation state after culturing the SG150804 strain of the coldwater disease pathogen, Bacillus subtilis, in a 24-well plate for 24 hours in the culture medium containing the commercially available quorum-sensing substance C4-AHL, in Example 1. C4-AHL: N-butyryl-DL-homoserine lactone [Figure 1B] 1 is a graph showing the amount of biofilm formed when two strains of the coldwater disease pathogen were used in Example 1 and cultured for 24 hours with a bacterial quorum-sensing substance added to the medium, compared with that when cultured normally. [Figure 1C] 1 is a graph showing the amount of biofilm formed when two strains of the coldwater disease pathogen were used in Example 1 and cultured for 48 hours with a bacterial quorum-sensing substance added to the medium, compared with that when cultured normally. [Figure 2]In Example 2, in order to verify whether a biofilm would form when the coldwater disease bacteria was cultured under stress, ethanol was added to the medium as a stress substance, and the culture solution was stained with crystal violet to confirm the formation of a biofilm. This photograph shows the morphology of the culture solution. [Figure 3] In Example 3, this graph shows the survival rate of ayu fish from Lake Biwa after challenge when the fish were immersed in a solution in which coldwater disease bacteria had been cultured and inactivated in a medium containing a bacterial quorum-sensing substance in an amount that promotes biofilm formation. [Figure 4A] In Example 4, a photograph showing the morphology of the culture medium in which coldwater disease bacteria were cultured in a medium containing iron ions and activated carbon powder was stained with crystal violet to confirm the artificial formation of a biofilm. [Figure 4B] In Example 4, Figure 4B is a photograph confirming whether or not the bacterial masses forming a biofilm in the culture medium of the coldwater disease fungus in Figure 4A have an outer membrane, which is evidence of the formation of a biofilm. [Figure 5] In Example 5, this is a graph showing the survival rate of ayu fish after challenge when the fish were immunized by immersing them in a solution in which coldwater disease bacteria had been cultured and inactivated in a medium containing iron ions and activated carbon powder. [Figure 6] In Example 7, a graph showing the survival rate of ayu fish after challenge when a solution of inactivated coldwater disease bacteria cultured in a medium containing iron ions and activated carbon powder, and a solution of inactivated bacteria cultured in the normal manner, were mixed with an oily adjuvant and injected into ayu fish from Lake Biwa for immunization. DETAILED DESCRIPTION OF THE INVENTION

[0020] <Regarding the vaccine preparation for immersion according to the present invention> The present invention is a medium capable of obtaining large amounts of either BF of bacteria causing bacterial infections in fish, components produced / excreted during the formation, maturation, and breakdown of BF, or bacterial bodies, and includes all immersion and injectable vaccine preparations containing any of the ingredients obtained by culturing in this medium as an active ingredient.

[0021] The bacterial quorum-sensing substance may be any substance that controls the formation of BFs, and may be any known substance, without any particular limitation. Examples include N-3-oxo-butyryl (butyryl: C4, hereinafter abbreviated as "C4")-DL-Homoserine Lactone (Homoserine Lactone: HSL, hereinafter abbreviated as "HSL"), N-3-Oxo-Pentanoyl (Pentanoil: C5, hereinafter abbreviated as "C5")-DL-HSL, N-3-Oxo-Hexanoil (Hexanoil: C6, hereinafter abbreviated as "C6")-DL-HSL, N-3-Oxo-Heptanoil (Heptanoil: C7, hereinafter abbreviated as "C7")-DL-HSL, and N-Oxo-Oct N-3-oxo-decanoyl (octanoyl: C8, hereinafter abbreviated as "C8")-DL-HSL, N-3-oxo-decanoyl (decanoyl: C10, hereinafter abbreviated as "C10")-DL-HSL, N-3-oxo-dodecanoyl (dodecanoyl: C12, hereinafter abbreviated as "C12")-DL-HSL, N-3-oxo-(tetradecanoyl: C14, hereinafter abbreviated as "C14")-DL-HSL SL, N-3-Oxo-Hexadecanoil(Hexadecanoil:C-16, hereinafter abbreviated as "C16")-DL-HSL, N-3-hydroxy(Hydoroxy:OH, hereinafter "OH") )-C4-DL-HSL, N-3-OH-C8-DL-HSL, N-3-OH-C10-DL-HSL, N-3-OH-C12-DL-HSL, N-3-OH -C14-DL-HSL, N3-OH-C16-DL-HSL, N-C4-DL-HSL (N-C4-DL-HSL:C4-AHL, hereinafter abbreviated as "C4-AHL"), N-C6-HSL, N-C7-DL-HSL, N-C8-DL-HSL, N-C10-DL-HSL, N-C12-DL-HSL, N-3-C14-DL-HSL, N-3-C16-DL-HSL, N-C4-DL-Homocysteine ​​Thiolactone (Homocysteine ​​Thiolactone:HCT, hereinafter abbreviated as "HCT"), N-C6-DL-HCT, N-C7-DL-HCT, N-C8-DL-HCT, N-C12DL-HCT, furanosyl borate diester, and the like.Furthermore, bacteria that produce these bacterial quorum-sensing substances may be co-cultured with fish pathogens or cultured in isolation with a membrane through which the quorum-sensing substances pass.

[0022] The conditions for culturing in a medium containing the aforementioned bacterial quorum-sensing substance may be any conditions that allow sufficient production of BF, components produced / excreted during the formation, maturation, and decay of BF, or bacterial antigens. These conditions can be well-known and are not particularly limited. For example, sufficient antigens can be obtained by culturing under these conditions, determining the concentration and retention period for BF formation for each bacterial quorum-sensing substance, species, and strain. Examples of suitable concentrations of the bacterial quorum-sensing substance in the medium range from 0.00001 to 0.1 μM. For example, in the case of the coldwater disease fungus SG150804 strain, adding C4-AHL to the medium to a concentration of 0.01 μM and culturing the fungus with shaking at 120 rpm for 24 hours ensures sufficient antigenic components and high quality.

[0023] The conditions for adding a stress substance to the medium and culturing the bacteria are not particularly limited as long as they result in sufficient production of BF, components produced / excreted during the formation, maturation, and breakdown of BF, or bacterial antigens. Any known conditions can be used, but the BF-forming concentration and retention period for each stress substance, bacterial species, and strain can be determined in advance, and sufficient antigens can be obtained by culturing the bacteria under those conditions.

[0024] The stress substance may be any component that can form BF, and a wide variety of known stress substances can be used without any particular limitation. Examples of such stress substances include methanol, ethanol, N-propanol, isopropanol, 1-butanol, 2-methyl-1-propanol, 2-butanol, 2-methyl-2-propanol, pentanol and its structural isomers, hexanol and its structural isomers, heptanol and its structural isomers, octanol and its structural isomers, aliphatic alcohols with eight or more carbon chains and their structural isomers, cyclic structures, unsaturated compounds, and polyunsaturated compounds, dihydric or higher alcohols such as ethylene glycol and their structural isomers, cyclic structures, unsaturated compounds, polyunsaturated compounds, and polymeric structures containing four or more carbon atoms, glycerol, fatty acids with a glycerol skeleton and their derivatives, phenols, ammonia, cholic acid, deoxycholic acid, organic acids (acetic acid, citric acid, lactic acid, fatty acids, etc.), Examples of antibacterial agents include inorganic acids, sodium hypochlorite, benzalkonium chloride, chlorhexidine gluconate, acrinol, iodine preparations, hydrogen peroxide, disinfectants, bacteriostatic agents, and antibiotics containing DNA synthesis inhibitors (for example, natural antibacterial agents (antibiotics) such as β-lactams, aminoglycosides, lincomycins, chloramphenicols, macrolides, ketolides, polypeptides, glycopeptides, tetracyclines, peptides, nucleic acids, and polyenes), semi-synthetic antibacterial agents such as doxycycline and minocycline, and synthetic antibacterial agents such as pyridonecarboxylic acids (quinolones), new quinolones, oxazolidinones, and sulfonamides. ), enzymes, chelating agents, reducing agents, preservatives, bacterial toxins, bacterial components, hormones, eukaryotic intercellular communication substances such as cytokines (e.g., interleukins, lymphokines, monokines, chemokines, etc.), eukaryotic inflammation-related substances (e.g., prostaglandins, histamine, lysozyme, leukotrienes, antibodies, complements, etc.), water-soluble substances such as vitamins, metal ions, preferably iron ions, etc. Mixtures of these may also be used.

[0025] The conditions for culturing in a medium containing a metal-containing substance (ionic substance) and a substance that acts as an attachment substrate to adsorb the bacterial quorum-sensing substance and increase the area of ​​BF formation can be any conditions known in the art, as long as sufficient BFs, components produced / excreted during the BF formation, maturation, and decay process, and bacterial antigens are produced. For example, for the coldwater disease pathogen SG150804 strain, iron(III) chloride is preferably used as the metal ion, and powdered activated carbon is preferably used as the attachment substrate to increase the area of ​​BF formation. Specifically, 375 μL of 2 M sodium hydroxide is added to the liquid medium to make it alkaline, and iron (III) chloride is added to 200 mL of the liquid medium to make it 1 mM. 1 g of commercially available activated carbon powder is then added to the above-mentioned medium, and the medium is stirred at 15°C for 48 hours at 200 rpm. This allows sufficient antigen components to be obtained and of good quality.

[0026] The metal ions may be any ion that promotes the formation of BF, and a wide variety of known ions can be used without particular limitation. Examples include metal or metalloid ions such as lithium, sodium, potassium, beryllium, magnesium, aluminum, calcium, strontium, barium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, molybdenum, silver, cadmium, indium, tin, antimony, tungsten, platinum, gold, lead, and bismuth. Mixtures of these ions may also be used. Iron ions, more preferably iron ions of iron(III) chloride, may be used. Iron ions can be supplied to the medium by adding an iron-containing substance to the medium.

[0027] The iron-containing substance may be any substance capable of forming BF, and a wide variety of known substances may be used without any particular limitation. Examples of such substances include iron foil, iron plate, iron powder, iron particles, iron wire, iron sponge, alloys of iron and other substances, such as carbon steel, stainless steel, chromium-molybdenum steel, and high-tensile steel. Also, examples of such substances include iron(III) acetylacetonate, iron acrylate, iron azide, triiron mononitride, yttrium iron oxide, ethylenediammonium ferrous sulfate, iron(II) chloride, iron(III) chloride, iron(III) chlorate, iron(II) perchlorate, iron(II) perchlorate, iron(III) perchlorate, sodium tetracarbonyliron(-II), nanocarbonyliron, pentacarbonyliron, diiron nonacarbonyl, triiron dodecacarbonyliron(II), formate(II), ammonium iron citrate, iron(I) citrate, iron(II) citrate, sodium iron citrate, iron gluconate, iron(III) chromate, and dichloride. Iron(III) phosphate, iron disilicide, iron disilicide, iron(II) acetate, iron(III) acetate, iron(II) oxide, iron(III) oxide, triiron tetroxide, iron(II) cyanide, iron(III) cyanide, iron(II) cyanate, cyclobutadiene iron tricarbonyl, iron(II) thiocyanate, iron(III) thiocyanate, iron(II) tartrate, iron(III) tartrate, ammonium iron oxalate, iron bromide, iron(II) oxalate, iron(III) oxalate, potassium iron(III) oxalate, triammonium iron trioxalate, iron(II) hydroxide, iron(III) hydroxide, iron(III) hydroxide oxide, iron(II) hydride, iron(III) hydride, sugar-containing iron oxides (e.g., sucroferric oxyhydroxide, etc.), iron oligosaccharide compounds (e.g., iron dextran, ferric diisomaltose, etc.).), iron stearate, iron(II) nitrate, iron(III) nitrate, iron sulfamate, iron(II) selenide, iron(III) selenide, iron(II) selenate, triiron monocarbide, iron(II) carbonate, iron(II) carbonate, iron(II) tungstate, iron(II) titanium dioxide, iron(III) titanium dioxide, iron ethoxide(III), ferric acid, barium ferrate, potassium ferrate, iron(II) trifluoromethanesulfonate, iron(III) tris(2-ethylhexanoate), potassium trisoxalatoferrate(III) (potassium ferric oxalate), iron naphthenate, iron nickel oxide, iron lactate, ferrocene, iron(II) fluoride, iron(III) fluoride, iron fumarate, iron(II) hexacyanide, iron(III) hexacyanide, ammonium ferricyanide, iron pentacarbonyl, meso-tetraphenyl phosphate Examples of suitable iron-containing compounds include phenylporphyrin iron chloride, meso-tetraphenylporphyrin iron-μ-oxo dimer, ferrocenium tetrafluoroborate, heme irons (e.g., ferroheme, hemoglobin, myoglobin, hemosiderin, etc.), ferritin, iron manganate, iron molybdate (III), iron iodide (II), iron iodide (III), iron orthoperiodate (II), iron iodate (II), iron iodate (III), iron sulfide, iron disulfide (II), triiron tetrasulfide, ammonium iron (II) sulfate, iron sulfite (II), iron sulfate (II), iron sulfate (III), ammonium iron (II) sulfate, iron diphosphate, iron hypophosphite, iron (II), iron phosphate (III), iron pyrophosphate (II), and ammonium iron pyrophosphate, as well as ores and powders containing these compounds. Mixtures of two or more of these compounds may also be used.

[0028] Any other substance than iron-containing substances that promotes BF formation may be added to the culture medium. A wide variety of known substances can be used, and they are not particularly limited. Examples include substances containing antibacterial agents, other metals, and their ions, ores containing these, their powders, and processed products. Mixtures of these substances are also acceptable.

[0029] The activated carbon used as the substrate for adsorption and attachment of the bacterial quorum-sensing substance for cultured bacteria is a substance that is mostly carbon and also contains oxygen, hydrogen, calcium, etc., and has the ability to adsorb various substances and can form a BF. A wide range of known raw materials can be used. Examples of raw materials include wood, bamboo, coconut shells, walnut shells, sugarcane, grass, coal, petroleum, animal bones, blood, and ore. Mixtures of these materials are also acceptable.

[0030] A wide variety of known activated carbons can be used as long as they are porous substances that are mostly carbon and comprise oxygen, hydrogen, calcium, etc. Examples of activated carbon include those obtained by carbonizing the above-mentioned raw materials at approximately 200 to 800°C in an oxygen-free state, or those obtained by mixing charcoal with, for example, dehydrating salts or acids (calcium chloride, magnesium chloride, zinc chloride, phosphoric acid, sulfuric acid, alkalis such as sodium hydroxide and potassium hydroxide) and firing the charcoal at approximately 500 to 700°C in an oxygen-free state to activate and remove chemicals (chemical activation method), and those obtained by firing the charcoal at 600 to 1,200°C using an oxidizing gas (water vapor, carbon dioxide, etc.) to activate the charcoal (gas activation method). Furthermore, for charcoal, the types include black charcoal (charcoal carbonized using an in-kiln digestion method, with a fixed carbon content of 75% or more and a refinement level of 2 to 8 degrees), white charcoal (charcoal carbonized using an ex-kiln digestion method, with a fixed carbon content of 85% or more and a refinement level of 0 to 3 degrees), binchotan (white charcoal carbonized from Quercus phillyraeoides, which includes oaks, with a fixed carbon content of 90% or more and a refinement level of 0 to 2 degrees), sawdust charcoal (black) (charcoal made by carbonizing sawdust and bark from sawdust light using an in-kiln digestion method, with a fixed carbon content of 70% or more and a refinement level of 2 to 8 degrees), sawdust charcoal (white) (charcoal made by carbonizing sawdust and bark from sawdust light using an ex-kiln digestion method, with a fixed carbon content of 85% or more and a refinement level of 0 to 3 degrees), and other charcoal (bamboo charcoal, mangrove charcoal, coconut shell charcoal, coal, etc.). Furthermore, examples include chemical composites of carbon molecules, such as carbon nanotubes, carbon nanobelts, fullerenes, and carbon nanobats, which have graphene as their basic molecular structure. Other examples include unspecified carbon-containing objects produced by a high-temperature carbonization method in which a material containing unspecified carbon is heated to approximately 800°C to approximately 950°C and carbonized in a gas such as water vapor or air. Furthermore, mixtures of these may also be used.

[0031] The activated carbon can be in the form of any known charcoal that has the property of adsorbing many carbon-containing substances. Examples include lump charcoal (carbonized unsplit logs), lump charcoal (carbonized split logs), granular lump charcoal, crushed lump charcoal, powdered charcoal, and molded charcoal (such as sawdust charcoal molded from raw materials and carbonized). Mixtures of these are also acceptable.

[0032] The amounts of iron ions and activated carbon added to the culture medium as stress substances for obtaining BF, components produced / excreted during the BF formation, maturation, and decay processes, and bacterial antigens are approximately 0.1 to 10 mM, preferably 1 mM, for iron(III) chloride, and approximately 1 to 10 g / L, preferably 5 g, for activated carbon powder. The culture time is approximately 24 to 96 hours, preferably approximately 48 hours. The culture temperature is approximately 5 to 23°C, preferably 15°C. The concentration of at least one active ingredient selected from the group consisting of BF and / or components produced during the BF formation, maturation, and decay processes and bacterial cells in the vaccine of the present invention cultured under these favorable conditions is, for example, approximately 1 to 2 mg / L for coldwater disease bacteria.

[0033] Materials other than activated carbon may be any attachment substrate component that increases the adsorption of bacterial quorum-sensing substances and the formation area of ​​BFs, and a wide variety of known materials can be used without particular limitation. Materials of animal origin include, for example, secretions of animal fur, bird feathers, and feathers, insects, bacteria, etc., synthetic plant fibers, recycled fibers made from animal and plant components, fibers made from minerals, plastics and fibers made from components contained in petroleum, chemically synthesized substances from such components, and artificially chemically synthesized organic substances, as well as chemically modified products thereof (those with the addition of ion exchange groups, such as carboxymethyl groups and diethylaminoethyl groups), and powders or filaments of these materials. Examples of plant-derived substances include fruits, seeds, tubers, powders of plant stems, leaves, roots, flowers, etc., germinated seeds, powders of outer shells, bran, starches, fibers, components extracted from these plants, chemically altered products thereof (heated products, frozen products, acid-alkali treated products, etc.), or chemically modified products thereof (chemically modified products with the addition of ion exchange groups, such as carboxymethyl groups and diethylaminoethyl groups, or haptenized substances, etc.), etc. Mixtures of two or more of these may also be used.

[0034] The vaccine preparation for immersion or injection obtained by culturing in the above-mentioned medium contains at least one active ingredient selected from the group consisting of BF, components produced / excreted during the formation, maturation and breakdown of BF, and bacterial cells.

[0035] The vaccine formulations for immersion and injection according to the present invention are vaccines in the form of immersion or injection, in which BF, components produced / excreted during the formation, maturation, and decay of BF, and bacterial cells are used as inactivated antigens. They may be culture solutions prepared in a medium to which one or all of a quorum-sensing substance, a stress substance, iron ions, and activated carbon powder have been added, or may be a mixture of these culture solutions. Furthermore, they may be culture solutions obtained by a method that yields BF, components produced / excreted during the formation, maturation, and decay of BF, and bacterial cells, and then purified, concentrated, or otherwise processed. The basic components contained in the medium may be any known and are not particularly limited.

[0036] When a component vaccine antigen containing a culture supernatant is used, it can be obtained, for example, by removing live bacteria by a known method such as filtration sterilization from a culture medium obtained by culturing a pathogen of a fish bacterial infection by the above-mentioned method, or from a solution obtained by dispersing this culture medium by a known method. Alternatively, it can be obtained by removing live bacteria by a known method such as filtration sterilization from a solution obtained by dispersing and lysing the bacterial cells and components surrounding the bacterial cells in the culture medium obtained by culturing by the above-mentioned method and sterilizing them by a known method.

[0037] A wide variety of known methods for inactivating antigens can be employed, and are not particularly limited. For example, antigens can be inactivated by subjecting the prepared antigen-containing solution to physical treatment (ultraviolet irradiation, X-ray irradiation, heat treatment, ultrasonic treatment, etc.) or chemical treatment (treatment with formalin, etc., treatment with organic solvents such as chloroform or alcohol, acid treatment with a weak acid such as acetic acid, treatment with chlorine or mercury, etc.). For example, formalin inactivation can be performed by adding formalin to the prepared antigen-containing solution at a volume concentration of 0.01 to 2.0%, more preferably 0.05 to 1.0%, and sensitizing the antigen-containing solution at 4 to 30°C for 1 to 10 days. Furthermore, after the inactivation treatment, the inactivating agent such as formalin may be removed by washing with a buffer solution or the like, or neutralized by adding a neutralizing agent. Alternatively, a solution obtained by culturing the cells by the above-described method and sterilizing the bacterial cells and the components surrounding the bacterial cells in the culture solution by a known method may be used.

[0038] The immersion or injectable vaccine preparation according to the present invention may contain an adjuvant.

[0039] A wide range of known adjuvants can be used. Examples of adjuvants include animal oils (squalene, lanolin, etc.) or their hydrogenated oils, vegetable oils (palm oil, castor oil, etc.) or their hydrogenated oils, oil-based adjuvants including anhydrous mannitol oleate, oleic acid, polybutene, caprylic acid, liquid paraffin, and higher fatty acid esters, acrylic acid copolymers, alkenyl derivative polymers, PCPP, soluble aluminum acetate, manganese gluconate, calcium gluconate, manganese glycerophosphate, saponin aluminum salicylate, oil-in-water emulsions, maleic anhydride copolymers, methacrylic acid copolymers, and water-soluble adjuvants such as cationic lipids containing quaternary ammonium salts, carboxyvinyl polymers, aluminum hydroxide (alum), sodium hydroxide, calcium phosphate, and aluminum phosphate, microbial toxins such as Mycobacterium tuberculosis, mycobacteria, cholera toxin, and Escherichia coli heat-labile toxin, as well as bentonite, muramyl dipeptide derivatives, and interleukins. Mixtures of these adjuvants are also acceptable.

[0040] Furthermore, this vaccine preparation for immersion may contain, as appropriate, a buffering agent, an isotonicity agent, a preservative, an antibacterial agent, an antioxidant, a pH adjusting agent, a dispersing agent, an aromatic agent, a coloring agent, an antifoaming agent, etc., depending on the purpose and use.

[0041] Suitable examples of the buffering agent include buffer solutions such as citrate, tartrate, acetate, carbonate, trishydroxymethylaminomethane, HEPES, and phosphate.

[0042] Suitable examples of agents for preservative purposes include chlorobutanol, sorbic acid, phenoxyethanol, benzyl alcohol, phenethyl alcohol, thimerosal, dehydroacetic acid, parahydroxybenzoic acid esters, and various other preservatives, antibiotics, and synthetic antibacterial agents.

[0043] Suitable examples of antioxidants that can be used include ascorbic acid, sulfites, erythorbic acid (isoascorbic acid), catechin, and tea extract (green tea polyphenols).

[0044] Suitable examples of pH adjusters that can be used include acids such as hydrochloric acid, citric acid, acetic acid, carbonic acid, boric acid, phosphoric acid, and sulfuric acid; alkali metal hydroxides such as potassium hydroxide, calcium hydroxide, sodium hydroxide, and magnesium hydroxide; alkali metal carbonates or hydrogen carbonates such as sodium carbonate; alkali metal acetates such as sodium acetate; alkali metal citrates such as sodium citrate; bases such as trometamol; diisopropanolamine, monoethanolamine, and ethylenediaminetetraacetic acid (EDTA).

[0045] Suitable examples of the dispersing agent include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, polysorbate 80, and polyvinylpyrrolidone.

[0046] Suitable examples of aromatic agents include citrus flavors of citrus fruits such as orange, lemon, lime, and grapefruit; fruit flavors of non-citrus fruits such as apple, banana, grape, and peach; milk flavors of dairy products such as milk, cream, and butter; beverage flavors of beverages such as coffee, cocoa, black tea, and oolong tea; vanilla flavors such as vanilla; mint flavors such as peppermint and spearmint; spice flavors such as pepper, cinnamon, ginger, nutmeg, and cloves; nut flavors of nuts such as almonds and peanuts; meat flavors of meats such as beef, pork, and chicken, and seafood flavors such as crab and shrimp; seasoning flavors of soups, sauces, soy sauce, matsutake mushrooms, and shiitake mushrooms; and alcoholic beverage flavors such as liqueurs and cocktails.

[0047] Suitable examples of colorants include red cabbage color, red radish color, Acid Red (Red No. 106), annatto color, amaranth (Red No. 2), Allura Red AC (Red No. 40), anthocyanin color, erythrosine (Red No. 3), indigo carmine (Blue No. 2), turmeric color, cacao color, caramel color, carotene color, carotenoid color, gardenia color, sorghum color, cochineal color, saffron color, Sunset Yellow FCF (Yellow No. 5), and perilla color. Examples of pigments that can be used include tartrazine (Yellow No. 4), butterfly pea pigment, chili pepper pigment, copper chlorophyll, sodium copper chlorophyllin, paprika pigment, Fast Green FCF (Green No. 3), grape skin pigment, flavonoid pigment, Brilliant Blue FCF (Blue No. 1), phloxine (Red No. 104), safflower pigment, red koji pigment, purple sweet potato pigment, purple corn pigment, New Coccine (Red No. 102), lac pigment, and rose bengal (Red No. 105).

[0048] Suitable examples of the antifoaming agent include dimethicone, simethicone, silicone emulsion, sorbitan sesquioleate, and nonionic substances.

[0049] In addition to the above, the present formulation may contain auxiliary ingredients such as light-absorbing pigments (riboflavin, adenine, adenosine, etc.) that aid in preservation and efficacy, chelating agents and reducing agents (vitamin C, citric acid, etc.) for stabilization, carbohydrates (glucose, sucrose, sorbitol, dextran, starch, mannitol, lactose, etc.), casein digests, and various vitamins.

[0050] The dosage form of the vaccine preparation is not particularly limited and may be any known form, for example, a liquid preparation.

[0051] Alternatively, the vaccine preparation may be a combination vaccine preparation with one or more vaccines against other diseases (or one or more antigens associated with other diseases).

[0052] <Method for preventing bacterial infection in fish according to the present invention> The present invention is a preventive method using an immersion or injection vaccine preparation for preventing bacterial infections in fish, and includes all of the methods for preventing bacterial infections in fish, including the steps of injecting a solution cultured using a culture method that can obtain BF, components produced / excreted during the formation, maturation, and breakdown of BF, and bacterial cells, into fish, and the steps of immersing fish.

[0053] Regarding bacterial infections in fish, for example, the above-mentioned vaccine preparation can be effectively prevented by placing breeding water in a container, adding the above-mentioned vaccine preparation, and immersing fish in the solution to immunize them, or by injecting the preparation into the abdominal cavity of the fish to immunize them.

[0054] The fish to which the present invention is applicable are not particularly limited as long as they live in water or aquatic areas. For example, fish raised in aquaculture facilities, aquariums, etc. can be the target of application. Furthermore, the immersion formulation of the present invention can also be applied to small individuals such as small fish and fry.

[0055] The method for immersing fish in the solution of the present invention can be a wide variety of known methods and is not particularly limited. For example, a container can be placed in a location around an aquaculture pond that is protected from direct sunlight, and rearing water is added to it. After adding the vaccine preparation for immersion, the fish can be immersed in the solution for 1 to 120 minutes while aerating, and then the fish can be returned to the aquaculture pond to perform immunization. The immersion time can be appropriately set, taking into consideration the time that has minimal impact on the fish and is sufficient to achieve the desired effect.

[0056] In the case of administration by immersion, the number of times is not particularly limited. For example, immersion may be performed once to three times per administration, and depending on the size of the target fish, the degree of vaccine effect, etc., immersion may be performed multiple times at intervals of 1 to 60 days. Furthermore, the immersion time may be adjusted appropriately depending on the intervals and number of immersion times.

[0057] In the case of immersion administration, for example, when the target is small fish such as sweetfish or small individuals such as fry, immersion may be performed once for 30 to 120 minutes at a specified immersion interval, or immersion for a specified period of time may be performed 2 to 4 times at intervals of 10 to 30 days, more preferably 2 to 4 times at intervals of 14 to 28 days, and most preferably 2 to 3 times at intervals of 14 to 21 days.

[0058] The method for immunizing fish by injection can be any known method and is not particularly limited. For example, the above-mentioned vaccine preparation may be mixed with an adjuvant in equal amounts, and 10 to 100 μL of the mixture may be injected intraperitoneally into fish that have been anesthetized with a fish anesthetic, and the fish may then be returned to the aquaculture pond to perform immunization. The injection amount may be appropriately determined, taking into consideration the amount that has minimal effect on the fish and is sufficiently effective.

[0059] In the case of injection administration, the number of times is not particularly limited. For example, depending on the size of the target fish, the degree of vaccine effect, etc., multiple injections may be administered at intervals of 1 to 60 days after the initial immunization.

[0060] In the present invention, the infectious disease to be prevented is not particularly limited as long as it is a bacterial infection that affects fish, and thus broadly includes bacterial infections in fish.

[0061] For example, the bacterial infection may be caused by bacteria isolated and cultured in a commercially available medium or an autologous medium, but the BF-forming ability is reduced or lost, and the effect as a vaccine may not be obtained by normal culture. In the present invention, a solution obtained by culturing the above-mentioned medium, either BF, components produced / excreted during the BF formation, maturation, and decay process, or bacteria, is used as a vaccine, and the solution contains protective antigens that cannot be obtained by normal culture and is administered by injection or immersion, which may result in the formation of more effective and powerful immunity and may be more effective as a vaccine.

[0062] Examples of bacterial infections in fish include Edwardsiella septicemia, Edwardsiella tarda infection, Aeromonas salmonicida infection (furuncle disease), Aeromonas hydrophila infection, Pseudomonas septicemia, red spot disease, red mouth disease, serratiosis, vibriosis, and pseudotuberculosis, which are caused by bacteria belonging to the Gamma Proteobacteria class; gliding bacteriosis, bacterial gill disease, columnaris disease, and cold water disease (in salmonids, sweetfish, carp, crucian carp, etc.) which are caused by bacteria belonging to the Bacteroidetes phylum (Flavobacterium genus); and bacterial kidney disease, nocardiosis, and streptococcosis, which are caused by gram-positive bacteria.

[0063] For example, if the fish bacterial infection is coldwater disease, FKC prepared from a strain of the causative bacterium isolated and normally cultured in a homemade medium made with commercially available ingredients has a low vaccine effect, and since the strain hardly forms BF, the above-mentioned medium, which can obtain BF, the components produced / excreted during the BF formation / maturation / decomposition process, and bacterial cells, may be more effective.

[0064] <Production Method According to the Present Invention> The present invention encompasses all methods for producing immersion and injectable vaccine preparations used to prevent bacterial infections in fish, including methods for producing immersion or injectable vaccine preparations that include the steps of obtaining antigens as BFs, components produced / excreted during the formation, maturation, and breakdown of BFs, and bacterial cells through liquid culture.

[0065] It is possible to manufacture vaccine preparations for immersion and injection containing as active ingredients antigens related to the pathogenicity of fish bacterial infections, components produced / excreted during the formation, maturation and breakdown of BF, and antigen components obtained by culturing using a culture method that forms bacterial cells. [Example]

[0066] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0067] In Example 1, two strains of Flavobacterium psychrophilum (Flavobacterium psychrophilum, hereafter referred to as "coldwater disease bacteria"), the causative bacterium of sweetfish coldwater disease, were cultured by adding a bacterial quorum-sensing substance to the medium so that the concentration of the substance changed from high to low, and it was examined whether a reaction to form or suppress BF was initiated again.

[0068] The coldwater disease pathogen SG150804 and PH0424 strains (isolated in 2015 and 2004, respectively) isolated from sweetfish were inoculated into 200 mL of MCY (Modified Cytofaga Yeast-extract: MCY, hereafter abbreviated as "MCY") liquid medium (1 L of distilled water containing 2 g of peptone, 0.5 g of yeast extract, 0.2 g of meat extract, 0.2 g of sodium acetate, and 0.2 g of calcium chloride, pH 7.0). The resulting culture was then cultured at 15°C for 24 hours at 200 rpm in a 24-well plate (2 cm). 2 After adding 2 mL of each solution to each well (100 μM C4-AHL solution per well), filter-sterilized 100 μM C4-AHL was added at 10, 1, 0.1, and 0.01 μM concentrations. The wells were incubated at 15°C and 40 rpm for 24 or 48 hours with shaking. After incubation, each well was washed four times with 2 mL of distilled water and stained with 2 mL of 1% crystal violet. After staining, each well was washed four times with 2 mL of distilled water to remove excess staining solution. 2 mL of pure ethanol was added to each well and allowed to stand for 30 minutes to dissolve the stained BF. The absorbance of the solution at 585 nm was measured. The measured values ​​for each C4-AHL concentration were compared with the amount of Specific Biofilm Formation (SBF) calculated based on the ((AHL-added group) - No-added group) / Medium-only group.

[0069] The results are shown in Figures 1A, 1B, and 1C. Figure 1A is a visual photograph of SG150804 strain cultured in a 24-well plate at a concentration of 10 μM or 0.01 μM before measuring the amount of BF formed. Figure 1B is a graph showing the amount of BF formed by each strain after 24 hours of culture, and Figure 1C is a graph showing the amount of BF formed by each strain after 48 hours of culture. In Figures 1B and 1C, the horizontal axis represents the C4-AHL concentration, and the vertical axis represents the amount of BF formed (SBF). SG150804 represents the results for the SG150804 strain, and PH0424 represents the results for the PH0424 strain.

[0070] Figure 1A shows the state of the SG150804 strain after culture in C4-AHL-supplemented medium. After 24 hours, the grown cells were dispersed at a high concentration of 10 μM C4-AHL, whereas at a low concentration of 0.01 μM, clumps of bacteria aggregated and formed raised clumps adhering to the bottom of the 24-well plate. Furthermore, gaps between the clumps were observed, obscuring visible bacterial cells. In the control (no addition) group, the grown cells were observed to have grown into spherical bodies that were not attached to the bottom, as well as dispersed bodies. These results indicate that BFs were not formed between bacteria at high concentrations of C4-AHL, as shown by the dispersed grown cells. At 0.01 μM, strong clumping and adhesion were observed, indicating that BFs were formed between bacteria, which could be visually confirmed. The spherical clumps in the control group may have been formed by clumping of bacteria that could not adhere to the bottom. Figures 1B and 1C show the amount of BFs formed by the SG150804 strain. As shown in Figure 1B, after 24 hours, BF formation was suppressed at a high concentration of 10 μM C4-AHL, while it was promoted at low concentrations of 0.1–0.01 μM. As shown in Figure 1C, after 48 hours, BF formation was suppressed at all concentrations. This result indicates that the SG150804 strain was regulated by low concentrations of C4-AHL after 24 hours. Furthermore, after 48 hours, BF formation was lost. This result suggests that at low bacterial concentrations up to 24 hours, BF formation was promoted by the action of added C4-AHL, and that as the bacterial concentration increased, BF formation was suppressed by an increase in the quorum-sensing substance produced by SG150804 itself in the medium. The amount of BF formation by PH0424 is shown in Figures 1B and 2C. As shown in Figure 1B, after 24 hours, BF formation was suppressed at a high concentration of 10 μM C4-AHL, but not at low concentrations. As shown in Figure 1C, after 48 hours, the suppression was maintained at high concentrations, but promotion began at low concentrations of 0.1-0.01 μM. These results suggest that, although the addition of C4-AHL suppresses BF formation in some strains, promotion may require a quorum-sensing substance other than C4-AHL produced by the bacteria themselves.These results also indicate that by culturing the SG150804 or PH0424 strains with shaking for 24 or 48 hours in medium supplemented with C4-AHL at a concentration of 0.01 μM, BF, components produced / excreted during the BF formation, maturation, and breakdown process, or bacterial cells can be obtained. [Example]

[0071] In Example 2, in order to verify whether the coldwater disease pathogen forms a biofilm when cultured under stress, we verified whether the coldwater disease pathogen forms a BF when cultured in a medium containing ethanol as a stress substance.

[0072] The SG150804 strain was inoculated into 200 mL of MCY liquid medium containing 1% ethyl alcohol, and then cultured at 15°C for 48 hours with shaking at 120 rpm. The culture solution was then dropped onto a glass slide, allowed to dry naturally, and stained with 1% crystal violet to prepare a slide for confirmation of BF formation.

[0073] Figure 2 shows a photograph of the BF formation visualized by microscopic observation of a slide prepared by dropping the culture medium onto a glass slide, allowing it to air dry, and then staining it with 1% crystal violet.

[0074] As shown in Figure 2, culturing in MCY liquid medium supplemented with 1% ethanol resulted in the formation of purple-stained BFs that encompassed the bacterial cells. Smaller vesicle-like components smaller than the purple-stained bacterial cells were observed within the BFs. This result indicates that ethanol stressed the bacterial cells, resulting in the formation of BFs in the culture medium to counteract the stress. Therefore, exposure to stressors can artificially produce BFs, components produced / excreted during the BF formation, maturation, and decay process, and bacterial cells. However, in this example, where stress was applied to form BFs, the amount of BF formed was small and there was a large amount of floating bacterial cells. Therefore, to obtain large amounts of BFs, it is desirable to use other methods, such as exposure to more extreme stress. [Example]

[0075] In Example 3, we examined the preventive effect against coldwater disease when ayu (Osmeridae, scientific name: Plecoglossus altivelis) from Lake Biwa was immersed in a solution prepared by inactivating a bacterial solution cultured in a medium containing a low concentration of bacterial quorum-sensing substance (C4-AHL: commercially available product) that causes coldwater disease strains to form BFs, and then immunizing the fish with the solution.

[0076] The SG150804 strain was inoculated into 200 mL of 1 / 2 CGY (Casitone Gelatin Yeast-extract: CGY, hereafter abbreviated as "1 / 2CGY") liquid medium (2.5 g of Casitone, 0.5 g of yeast extract, 1.5 g of gelatin, and 0.147 g of calcium chloride in 1 L of distilled water) containing 0.01 μM C4-AHL and cultured at 15°C and 200 rpm for 24 hours. After 24 hours of agitation, formalin was added to the medium to a concentration of 0.3% to inactivate the strain. For comparison, the same strain was cultured in 200 mL of 1 / 2 CGY liquid medium at 15°C and 200 rpm for 24 hours, and then inactivated with formalin to a concentration of 0.3% to prepare the conventional culture FKC vaccine solution for immersion.

[0077] Next, the test fish were immunized by immersion. The vaccine concentrate was diluted 10-fold with groundwater to a total volume of 2 L, and 10 μL of antifoaming agent KM-72 (antifoaming agent for food additives manufactured by Kurimoto Pharmaceutical Co., Ltd.; hereafter referred to as "antifoaming agent") was added to the solution. Sixty sweetfish from Lake Biwa (average weight 2.0 g) were immersed in the solution under aeration for 30 minutes, then returned to a breeding tank and reared in groundwater at a temperature of 17.5°C for 21 days. A control group was left untreated and reared under the same conditions for the same period.

[0078] Next, a challenge test was conducted. The SG150804 strain of coldwater disease bacteria was cultured in 200 mL of 1 / 2 CGY liquid medium at 15°C and 200 rpm for 48 hours with agitation, and then added to 1 L of 1 / 2 CGY liquid medium and cultured for a further 48 hours at 15°C and 200 rpm with agitation. This was then diluted 4-fold with groundwater to a total volume of 2 L, and 10 μL of an antifoaming agent was added to prepare a challenge solution. 65 test fish from each group were placed in this diluted challenge solution and immersed for 30 minutes to challenge the fish (1.9 × 10 9 After the incubation period, the fish were returned to the breeding tank and kept in groundwater at a temperature of 17.5°C for 21 days.

[0079] The results are shown in Figure 3. Figure 3 is a graph showing the survival rate of ayu fish from Lake Biwa challenged with a vaccine solution in which the culture medium containing 0.01 μM equivalents of C4-AHL, a bacterial quorum-sensing substance, was inactivated by adding 0.3% formalin to the culture medium during cultivation of the coldwater disease pathogen, or with conventionally cultured FKC as a control. The ayu fish were then challenged with the vaccine solution 21 days after immunization. In the figure, the horizontal axis represents the number of days from the day of challenge, and the vertical axis represents the survival rate (%). In the figure, AHL-FKC (AHL Adding Culture-FKC: AHL-FKC, hereafter abbreviated as "AHL-FKC") shows the results of immunization by the immersion method with inactivated antigen that was cultured in 1 / 2 CGY medium containing 0.01 μM C4-AHL, "normal culture FKC" shows the results of immunization by the immersion method with inactivated antigen that was cultured in 1 / 2 CGY liquid medium, and "control" shows the results of no immunization.

[0080] As shown in Figure 3, the survival rates for the untreated control group and those immunized by immersion with the conventional cultured FKC vaccine were 7.8% and 18.2%, respectively. The survival rate for the AHL-FKC vaccine immunization by immersion was significantly higher (43.1%), a significant difference (p<0.01) based on Fisher's exact probability test (one-tailed, p<0.01). There was no significant difference (p=0.07) between the immunization by immersion with the conventional cultured FKC vaccine. These results demonstrate that coldwater disease can be effectively prevented by immersion immunization with an antigen solution prepared by a manufacturing method that involves inactivating culture medium cultured in medium supplemented with 0.01 μM equivalents of C4-AHL, which can yield either BF, components produced / excreted during the BF formation, maturation, and decay process, or bacterial cells. [Example]

[0081] In Example 4, a culture method is shown in which iron ions are added to the culture medium as a stress substance and activated carbon powder is added as an adsorption and adhesion substrate for bacterial quorum-sensing substances to form BFs in a coldwater disease strain isolated from ayu fish from Lake Biwa, thereby forming BFs and significantly expanding the volume of BFs formed.

[0082] The medium for BF formation was iron ion / activated carbon-added 1 / 2 CGY liquid medium, prepared by adding 375 μL of 2 M sodium hydroxide solution to 200 mL of autoclaved 1 / 2 CGY medium with stirring, followed by the addition of 100 mM filter-sterilized iron(III) chloride to a concentration of 1 mM with stirring, and then adding 1 g of commercially available, heat-sterilized activated carbon powder. The SG150804 strain was inoculated into the iron ion / activated carbon-added 1 / 2 CGY liquid medium immediately after preparation and cultured at 15°C and 200 rpm for 48 hours to confirm biofilm formation.

[0083] The results are shown in Figures 4A and 4B. Figure 4A shows a photograph of the BF formation visualized under a microscope after the culture medium was dropped onto a slide, allowed to air dry, and then stained with 1% crystal violet to prepare a preparation. Figure 4B shows a photograph of the BF outer membrane formation visualized under a microscope after the culture medium was dropped onto a gelatin-coated slide, gently heated with a burner to dry the bacterial solution, and then washed with acetone to remove the components of the BF outer membrane, as well as the bacterial cells and other components, and fixed. The resulting preparation was then stained with 1% crystal violet to prepare a preparation.

[0084] As shown in Figure 4A, when cultured in 1 / 2 CGY liquid medium supplemented with iron ions and activated carbon, BFs formed on the activated carbon particles in the culture medium, forming large bacterial masses measuring over 20 μm. Furthermore, some of the bacterial masses connected multiple activated carbon powder particles to form larger particles. This result indicates a significant expansion of the volume of BFs formed. Next, as shown in Figure 4B, the outer membrane of BFs expanded to approximately 130 μm after the contents were washed away can be confirmed. This result clearly indicates that the bacterial masses formed on activated carbon powder particles are BFs with a strong outer membrane. Furthermore, this indicates that 1 / 2 CGY liquid medium supplemented with iron ions and activated carbon is a medium capable of artificially forming large amounts of biofilms.

[0085] Furthermore, when the SG150804 strain was cultured in MCY liquid medium, it hardly formed BFs, but when this bacterium was cultured in 1 / 2 CGY liquid medium supplemented with iron ions and activated carbon for 48 hours, it formed BFs. This suggests that the properties of activated carbon powder adsorb the quorum-sensing substance, keeping its concentration low, and that the stress effect of excess iron ions contributes to BF formation. Furthermore, the presence of activated carbon particles inside the BF bacterial mass may also adsorb the quorum-sensing substance, which continues to be secreted inside the BF bacterial mass, due to the properties of activated carbon powder, leading to a larger BF formation volume. [Example]

[0086] In Example 5, the coldwater disease bacteria was cultured in 1 / 2 CGY liquid medium supplemented with iron ions and activated carbon, and large amounts of BF, components produced / excreted during the formation, maturation, and breakdown of BF, or bacterial bodies were obtained in FKC (BF-FKC: Biofilm Formed FKC, hereafter abbreviated as "BF-FKC") were obtained. The preventive effect against coldwater disease when ayu from Lake Biwa were immersed in the FKC was examined, in comparison with the effect when ayu were injected with or immersed in conventionally cultured FKC.

[0087] First, 0.3% formalin was added to a solution of the coldwater disease fungus cultured in the same manner as in Example 4, and the mixture was stirred at 15°C and 200 rpm for 5 days to prepare BF-FKC. Conventional culture FKC prepared in the same manner as in Example 3 was used. Conventional culture FKC for injection was mixed with an equal amount of Freund's Adjuvant (IFA, hereafter abbreviated as "IFA") and used.

[0088] Next, test fish were immunized by immersion or injection. For the BF-FKC immersion vaccine group, 50 ayu (average weight 3.4 g) from Lake Biwa (mean weight 3.4 g) were immersed in a 100-fold dilution of the stock solution with groundwater (to make 2 L) and 10 μL of antifoaming agent added. The vaccine was then immunized by immersion for 120 minutes under aeration. The fish were then returned to a rearing tank and reared in groundwater at 17.5°C for 14 days. The fish were then immunized a second time under the same conditions and reared under the same conditions for another 14 days. For the conventional culture FKC immersion vaccine group, 50 ayu (average weight 3.4 g) from Lake Biwa (mean weight 3.4 g) were immersed in a 10-fold dilution of the stock solution with groundwater (to make 2 L) and 10 μL of antifoaming agent added. The vaccine was then immersed for 30 minutes under aeration. The fish were then returned to a rearing tank and reared in groundwater at 17.5°C for 21 days. For the conventional culture FKC injection vaccine group, a mixture of equal amounts of conventional culture FKC and IFA was injected intraperitoneally at 50 μL per fish, and the fish were reared for 21 days under the same conditions as the conventional culture FKC immersion group. The control group was left untreated and reared under the same conditions as the conventional culture FKC immersion group for the same period.

[0089] Next, a challenge test was carried out. A challenge solution was prepared using the SG150804 strain of coldwater disease bacteria in the same manner as in Example 3. The challenge solution was then diluted 4 times with groundwater to a total volume of 2 L, and 70 test fish from each group were placed in the solution and immersed for 30 minutes to challenge (1.0 × 10 9 After the immunization, the mice were returned to the breeding tank and kept in groundwater at a temperature of 17.5°C for 21 days. The challenge dates for the first and second immunization groups were the same.

[0090] The results are shown in Figure 5. Figure 5 is a graph showing the survival rate of Lake Biwa sweetfish immunized by immersion in a BF-FKC solution prepared with the coldwater disease pathogen SG150804 strain. In the graph, the horizontal axis represents the number of days from the day of challenge, and the vertical axis represents the survival rate (%). In the graph, BF-FKC immersion represents the results of immunization with a 100-fold diluted BF-FKC vaccine twice at 14-day intervals. Conventional culture FKC immersion represents the results of immunization with a 10-fold diluted conventional culture FKC vaccine once. Conventional culture FKC injection represents the results of intraperitoneal injection of a conventional culture FKC injection vaccine mixed with IFA. The control (untreated) represents the untreated case.

[0091] As shown in Figure 5, the survival rate 21 days after challenge was 42.9% in the untreated group. The survival rate was significantly higher at 91.6% when immunized with the 100x diluted BF-FKC vaccine by immersion, a significant difference (p<0.01) was observed by Fisher's exact probability test (one-sided, p<0.01). The survival rate for the conventional cultured FKC immersion vaccine, using the same method, was 55.9%, not significantly different (p=0.09), while the survival rate for the conventional cultured FKC injection vaccine was 90.3%, significantly different (p<0.01). These results indicate that immunization with the BF-FKC solution by immersion resulted in a 1.3% higher survival rate than immunization with the conventional cultured FKC injection vaccine, which is considered to be effective. This indicates that the BF-FKC immersion vaccine is more effective in preventing coldwater disease than the conventional cultured FKC injection vaccine.

[0092] Furthermore, ayu from Lake Biwa immunized with an AHL-FKC immersion vaccine prepared by adding a low concentration of the quorum-sensing substance (commercially available) shown in Example 1 to the culture medium and culturing the vaccine, which had been inactivated with formalin, had a survival rate of 43.1% after 21 days, while ayu from Lake Biwa immunized in the same manner with the BF-FKC immersion vaccine had a survival rate of 91.6% after 21 days, demonstrating a much greater effect than the AHL-FKC immersion vaccine of Example 1. Furthermore, the BF-FKC immersion vaccine, which does not use a commercially available quorum-sensing substance, is far less expensive to produce, and in terms of both effectiveness and production cost, BF-FKC is a more suitable formulation than the AHL-FKC immersion vaccine of Example 1.

[0093] Furthermore, the production of BF-FKC immersion vaccine and conventional culture FKC immersion vaccine can be carried out with almost the same amount of effort, and the effectiveness of BF-FKC immersion vaccine is higher than that of conventional culture FKC injection vaccine. As it is a vaccine administered by immersion, it causes less stress to fish and is more effective than conventional culture FKC injection vaccine. Therefore, BF-FKC immersion vaccine is superior to conventional culture FKC injection vaccine. [Example]

[0094] In Example 6, the stability of the protective effect of the BF-FKC immersion vaccine was examined.

[0095] The BF-FKC immersion vaccine prepared in the same manner as in Example 4 was used.

[0096] Next, to investigate the variability in the efficacy of the BF-FKC immersion vaccine, the following experiment was conducted nine times. First, the BF-FKC immersion vaccine was prepared by diluting the original solution 10 times with groundwater to a total volume of 2 L, to which 10 μL of antifoaming agent was added. 50-70 ayu (average weight 2.1-4.1 g) from Lake Biwa were immunized by immersing them in the vaccine for 30 minutes under aeration. They were then returned to a breeding tank and reared in groundwater at a temperature of 17.5°C for 21 days. A control group was left untreated and reared under the same conditions for the same number of fish as those in the BF-FKC immersion vaccine group, for the same period of time.

[0097] Next, a challenge test was carried out. A challenge solution was prepared using the SG150804 strain of coldwater disease bacteria in the same manner as in Example 3, and sweetfish were immersed in the solution for 30 minutes to challenge the bacteria (1.0 to 2.8 × 10 9 After the incubation period, the fish were returned to the breeding tank and kept in groundwater at a temperature of 17.5°C for 21 days.

[0098] The average survival rate 21 days after challenge in the untreated control group was 39.0% (standard deviation: 12.7%), while in the group immunized by immersion with the BF-FKC vaccine, the survival rate was 83.9% (standard deviation: 7.3%), with a significant difference (p<0.01) compared to the control group in all nine tests. These results indicate that the effectiveness of the BF-FKC immersion vaccine is hardly affected by variations in the immune system development state or rearing environment of the ayu from Lake Biwa that were tested, with an average body weight of at least 2.1g, and provides a stable, high level of preventive effect against coldwater disease. [Example]

[0099] In Example 7, the preventive effects against coldwater disease were compared and verified when BF-FKC and conventional cultured FKC were inoculated by injection into sweetfish from Lake Biwa for immunization.

[0100] BF-FKC and conventional culture FKC were prepared using the coldwater disease fungus SG150804 strain in the same manner as in Example 4, and were mixed with equal amounts of IFA before use.

[0101] Next, ayu (average weight 3.4g) from Lake Biwa were immunized by injection. A vaccine consisting of equal amounts of BF-FKC or conventional cultured FKC mixed with IFA was administered intraperitoneally to each test ayu at 50μL per fish. The fish were then returned to a breeding tank and reared in groundwater at 17.5°C for 21 days. A control group was left untreated and reared under the same conditions for 21 days.

[0102] Next, a challenge test was carried out. The challenge bacterial solution was prepared by culturing the test bacteria in the same manner as in Example 4. The challenge bacterial solution was then diluted 4 times with groundwater to a total volume of 2 L, and the test fish from each group were placed in the solution and immersed for 30 minutes to challenge (1.0 × 10 9After the challenge, the mice were returned to the breeding tank and kept in groundwater at a temperature of 17.5°C for 21 days.

[0103] The results are shown in Figure 6. Figure 6 is a graph showing the survival rate of ayu fish from Lake Biwa after challenge when they were immunized by injection with BF-FKC produced with the coldwater disease pathogen SG150804 strain and conventionally cultured FKC. In the figure, the horizontal axis represents the number of days from the day of challenge, and the vertical axis represents the survival rate (%). In the figure, BF-FKC injection represents the result of immunization by injection of a mixture of equal volumes of BF-FKC and IFA, conventionally cultured FKC injection represents the result of immunization by injection of a mixture of equal volumes of conventionally cultured FKC and IFA, and the control group (untreated) represents the case without treatment.

[0104] As shown in Figure 6, the survival rate 21 days after challenge was 26.9% in the untreated group, while the survival rate was 94.3% in the group immunized with the BF-FKC injectable vaccine, a significantly higher rate, with a significant difference (p<0.01) according to Fisher's exact test (one-tailed, p<0.01). Furthermore, the survival rate in the group immunized with the conventional cultured FKC injectable vaccine was 85.1%, a significant difference (p<0.01). These results indicate that immunization with the BF-FKC injectable vaccine resulted in a 9.2% higher survival rate than immunization with the known effective conventional cultured FKC injectable vaccine, indicating that BF-FKC provides a more effective preventive effect against coldwater disease, even when administered by injection. Therefore, BF-FKC, whether administered by immersion or injection, is more effective than the known effective conventional cultured FKC injectable vaccine, demonstrating its potential for more effective prevention of coldwater disease.

Claims

1. A vaccine preparation for immersion or injection used to prevent coldwater disease, comprising: a culture medium obtained by culturing Flavobacterium psychrophilum in the presence of a quorum-sensing substance and a substance that adsorbs metal ions and the quorum-sensing substance; A vaccine preparation comprising, as active ingredients, a biofilm derived from Flavobacterium psychrophilum and / or components produced during the process of the formation, maturation and breakdown of the biofilm, and cells of Flavobacterium psychrophilum, The bacterial quorum-sensing substance may be N-3-oxo-butylyl (butylyl: C4, hereinafter abbreviated as "C4")-DL-homoserine lactone (homoserine lactone: HSL, hereinafter abbreviated as "HSL"), N-3-oxo-pentanoil (pentanoil: C5, hereinafter abbreviated as "C5")-DL-HSL, N-3-oxo-hexanoil (hexanoil: C6, hereinafter abbreviated as "C6")-DL-HSL, N-3-oxo-heptanoil (heptanoil: C7, hereinafter abbreviated as "C7")-DL-HSL, N-3-oxo-octanoil (octanoil: C8, hereinafter abbreviated as "C8")-DL-HSL, N-3-oxo-octanoil (octanoil: C9, hereinafter abbreviated as "C9")-DL-HSL, N-3-oxo-octanoil (octanoil: C10, hereinafter abbreviated as "C10")-DL-HSL, N-3-oxo-octanoil (octanoil: C11, hereinafter abbreviated as "C11")-DL-HSL, N-3-oxo-octanoil (octanoil: C12, hereinafter abbreviated as "C12")-DL-HSL, N-3-oxo-octanoil (octanoil: C13, hereinafter abbreviated as "C13")-DL-HSL, N-3-oxo-octanoil (octanoil: C14, hereinafter abbreviated as "C14")-DL-HSL, N-3-oxo-octanoil (octanoil: C15, hereinafter abbreviated as "C15")-DL-HSL, N-3-oxo-octanoil (oct N-3-oxo-decanoil (octanoil: C8, hereinafter abbreviated as "C8")-DL-HSL, N-3-oxo-decanoil (decanoil: C10, hereinafter abbreviated as "C10")-DL-HSL, N-3-oxo-dodecanoil (dodecanoil: C12, hereinafter abbreviated as "C12")-DL-HSL, N-3-oxo-(tetradecanoyl: C14, hereinafter abbreviated as "C14")-DL-HSL SL, N-3-Oxo-Hexadecanoil (Hexadecanoil: C-16, hereinafter abbreviated as "C16") -DL-HSL, N-3-hydroxy (Hydoroxy: OH, hereinafter "OH") )-C4-DL-HSL, N-3-OH-C6-DL-HSL, N-3-OH-C8-DL-HSL, N-3-OH-C10-DL-HSL, N-3-OH -C14-DL-HSL, N3-OH-C16-DL-HSL, N-C4-DL-HSL (N-C4-DL-HSL: C4-AHL, hereinafter abbreviated as "C4-AHL"), N-C6-HSL, N-C7-DL -HSL, N-C8-DL-HSL, N-C10-DL-HSL, N-C12-DL-HSL, N-3-C14-DL-HSL, N-3-C16-DL-HSL, N-C4-DL-Homocysteine at least one selected from the group consisting of thiolactone (homocysteine ​​thiolactone: HCT, hereinafter abbreviated as "HCT"), N-C6-DL-HCT, N-C7-DL-HCT, N-C8-DL-HCT, N-C12DL-HCT, and furanosyl boric acid diester; Vaccine preparations.

2. The vaccine preparation according to claim 1, wherein the Flavobacterium psychrophilum cells include inactivated and / or killed cells.

3. A method for producing an immersion or injection vaccine preparation for use in preventing coldwater disease, comprising the steps of: culturing Flavobacterium psychrophilum in the presence of a quorum-sensing substance and a substance that adsorbs metal ions and the quorum-sensing substance; A method for producing a vaccine preparation, comprising forming a biofilm derived from Flavobacterium psychrophilum and / or components produced during the formation, maturation, and breakdown of the biofilm, The bacterial quorum-sensing substance may be N-3-oxo-butylyl (butylyl: C4, hereinafter abbreviated as "C4")-DL-homoserine lactone (homoserine lactone: HSL, hereinafter abbreviated as "HSL"), N-3-oxo-pentanoil (pentanoil: C5, hereinafter abbreviated as "C5")-DL-HSL, N-3-oxo-hexanoil (hexanoil: C6, hereinafter abbreviated as "C6")-DL-HSL, N-3-oxo-heptanoil (heptanoil: C7, hereinafter abbreviated as "C7")-DL-HSL, N-3-oxo-octanoil (octanoil: C8, hereinafter abbreviated as "C8")-DL-HSL, N-3-oxo-octanoil (octanoil: C9, hereinafter abbreviated as "C9")-DL-HSL, N-3-oxo-octanoil (octanoil: C10, hereinafter abbreviated as "C10")-DL-HSL, N-3-oxo-octanoil (octanoil: C11, hereinafter abbreviated as "C11")-DL-HSL, N-3-oxo-octanoil (octanoil: C12, hereinafter abbreviated as "C12")-DL-HSL, N-3-oxo-octanoil (octanoil: C13, hereinafter abbreviated as "C13")-DL-HSL, N-3-oxo-octanoil (octanoil: C14, hereinafter abbreviated as "C14")-DL-HSL, N-3-oxo-octanoil (octanoil: C15, hereinafter abbreviated as "C15")-DL-HSL, N-3-oxo-octanoil (oct N-3-oxo-decanoil (octanoil: C8, hereinafter abbreviated as "C8")-DL-HSL, N-3-oxo-decanoil (decanoil: C10, hereinafter abbreviated as "C10")-DL-HSL, N-3-oxo-dodecanoil (dodecanoil: C12, hereinafter abbreviated as "C12")-DL-HSL, N-3-oxo-(tetradecanoyl: C14, hereinafter abbreviated as "C14")-DL-HSL SL, N-3-Oxo-Hexadecanoil (Hexadecanoil: C-16, hereinafter abbreviated as "C16") -DL-HSL, N-3-hydroxy (Hydoroxy: OH, hereinafter "OH") )-C4-DL-HSL, N-3-OH-C6-DL-HSL, N-3-OH-C8-DL-HSL, N-3-OH-C10-DL-HSL, N-3-OH -C14-DL-HSL, N3-OH-C16-DL-HSL, N-C4-DL-HSL (N-C4-DL-HSL: C4-AHL, hereinafter abbreviated as "C4-AHL"), N-C6-HSL, N-C7-DL -HSL, N-C8-DL-HSL, N-C10-DL-HSL, N-C12-DL-HSL, N-3-C14-DL-HSL, N-3-C16-DL-HSL, N-C4-DL-Homocysteine at least one selected from the group consisting of thiolactone (homocysteine ​​thiolactone: HCT, hereinafter abbreviated as "HCT"), N-C6-DL-HCT, N-C7-DL-HCT, N-C8-DL-HCT, N-C12DL-HCT, and furanosyl boric acid diester; Methods for manufacturing vaccine formulations.

4. The method for producing a vaccine preparation according to claim 3, wherein the substance that adsorbs the quorum-sensing substance is activated carbon.

5. A method for preventing coldwater disease, comprising applying or injecting a solution containing a biofilm derived from Flavobacterium psychrophilum and / or components produced during the process of said biofilm formation, maturation and breakdown, and inactivated and / or killed bacteria of said Flavobacterium psychrophilum to fish; the solution contains a culture medium obtained by culturing Flavobacterium psychrophilum in the presence of a quorum-sensing substance and a substance that adsorbs metal ions and the quorum-sensing substance; The bacterial quorum-sensing substance may be N-3-oxo-butylyl (butylyl: C4, hereinafter abbreviated as "C4")-DL-homoserine lactone (homoserine lactone: HSL, hereinafter abbreviated as "HSL"), N-3-oxo-pentanoil (pentanoil: C5, hereinafter abbreviated as "C5")-DL-HSL, N-3-oxo-hexanoil (hexanoil: C6, hereinafter abbreviated as "C6")-DL-HSL, N-3-oxo-heptanoil (heptanoil: C7, hereinafter abbreviated as "C7")-DL-HSL, N-3-oxo-octanoil (octanoil: C8, hereinafter abbreviated as "C8")-DL-HSL, N-3-oxo-octanoil (octanoil: C9, hereinafter abbreviated as "C9")-DL-HSL, N-3-oxo-octanoil (octanoil: C10, hereinafter abbreviated as "C10")-DL-HSL, N-3-oxo-octanoil (octanoil: C11, hereinafter abbreviated as "C11")-DL-HSL, N-3-oxo-octanoil (octanoil: C12, hereinafter abbreviated as "C12")-DL-HSL, N-3-oxo-octanoil (octanoil: C13, hereinafter abbreviated as "C13")-DL-HSL, N-3-oxo-octanoil (octanoil: C14, hereinafter abbreviated as "C14")-DL-HSL, N-3-oxo-octanoil (octanoil: C15, hereinafter abbreviated as "C15")-DL-HSL, N-3-oxo-octanoil (oct N-3-oxo-decanoil (octanoil: C8, hereinafter abbreviated as "C8")-DL-HSL, N-3-oxo-decanoil (decanoil: C10, hereinafter abbreviated as "C10")-DL-HSL, N-3-oxo-dodecanoil (dodecanoil: C12, hereinafter abbreviated as "C12")-DL-HSL, N-3-oxo-(tetradecanoyl: C14, hereinafter abbreviated as "C14")-DL-HSL SL, N-3-Oxo-Hexadecanoil (Hexadecanoil: C-16, hereinafter abbreviated as "C16") -DL-HSL, N-3-hydroxy (Hydoroxy: OH, hereinafter "OH") )-C4-DL-HSL, N-3-OH-C6-DL-HSL, N-3-OH-C8-DL-HSL, N-3-OH-C10-DL-HSL, N-3-OH -C14-DL-HSL, N3-OH-C16-DL-HSL, N-C4-DL-HSL (N-C4-DL-HSL: C4-AHL, hereinafter abbreviated as "C4-AHL"), N-C6-HSL, N-C7-DL -HSL, N-C8-DL-HSL, N-C10-DL-HSL, N-C12-DL-HSL, N-3-C14-DL-HSL, N-3-C16-DL-HSL, N-C4-DL-Homocysteine at least one selected from the group consisting of thiolactone (homocysteine ​​thiolactone: HCT, hereinafter abbreviated as "HCT"), N-C6-DL-HCT, N-C7-DL-HCT, N-C8-DL-HCT, N-C12DL-HCT, and furanosyl boric acid diester; How to prevent cold water disease.

6. 6. The method for preventing cold water disease according to claim 5, wherein the Flavobacterium psychrophilum is an inactivated bacterium.

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