Combination vaccine composition for preventing vibriosis and cutaneous lesions in salmonid fish, method for producing the same, and method for administering a vaccine for preventing vibriosis and cutaneous lesions in salmonid fish using the same

Abstract

The present invention relates to a mixed vaccine composition for preventing vibrio disease and ulcer disease in fish comprising a Vibrio anguillarum strain, an Aeromonas salmonisida subspecies salmonisida strain and an Aeromonas salmonisida subspecies masousida strain, a method for preparing the same, and a method for preventing vibrio disease and ulcer disease in fish using the same. More specifically, the invention relates to a mixed vaccine composition for preventing vibrio disease and ulcer disease in fish comprising an inactivated Vibrio anguillarum 23FBVib0271 strain and an inactivated Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain; and an inactivated deposited Aeromonas salmonisida subspecies masousida 23FBAer0174 strain, a method for preparing the same, and a method for preventing vibrio disease and ulcer disease in fish using the same. The mixed vaccine composition according to the present invention effectively prevents Vibrio disease and carbuncleosis in fish, particularly Atlantic salmon, simultaneously without immune interference or side effects, thereby significantly reducing mortality caused by both diseases. Consequently, the amount of antibiotics used and associated costs can be reduced, and the survival rate and productivity of fish can be improved. Therefore, the present invention enables the stable production of farmed fish, contributing to the economic viability and sustainable development of the aquaculture industry.

Description

Technology Field

[0001] The present invention relates to a mixed vaccine composition for preventing vibrio disease and carbunclement disease in fish comprising a Vibrio anguillarum strain, an Aeromonas salmonisida subspecies salmonisida strain, and an Aeromonas salmonisida subspecies masousida strain, a method for preparing the same, and a method for preventing vibrio disease and carbunclement disease in fish using the same. More specifically, the invention relates to an inactivated Vibrio anguillarum 23FBVib0271 strain, an inactivated Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain; and an inactivated deposited Aeromonas salmonisida subspecies masousida The present invention relates to a mixed vaccine composition for preventing vibriosis and ulcer disease in fish containing the 23FBAer0174 strain, a method for manufacturing the same, and a method for preventing vibriosis and ulcer disease in fish using the same. Background Technology

[0003] Salmon is widely consumed globally as a health food, and demand for it continues to rise. Consequently, the salmon aquaculture industry is growing rapidly as an alternative to overcome the limitations of natural catches and secure a stable supply. Large-scale farming is being conducted primarily in countries such as Norway, Chile, and Canada, and recently, South Korea has also entered the industry in earnest to expand domestic consumption and exports. Recently, offshore cage farming and Recirculating Aquaculture Systems (RAS) have been introduced, mainly in East Coast regions such as Dangjin in Chungcheong Province, establishing a foundation for domestic salmon production.

[0004] Among them, the Atlantic salmon is the most commercially valuable salmon species, highly favored worldwide due to its tender flesh and richness in omega-3 fatty acids. Its natural habitat is limited to the North Atlantic coast and parts of the Arctic Ocean, and it is known as an anadromous species that returns to rivers to spawn. However, wild populations have plummeted due to the effects of climate change, overfishing, and habitat destruction, and most Atlantic salmon currently available on the market is supplied through artificial farming. For this reason, the Atlantic salmon is considered to have the greatest economic and industrial significance among farmed fish species worldwide, and the importance of disease prevention and vaccine development against diseases occurring in high-density rearing environments is particularly emphasized.

[0005] However, the salmon farming industry is vulnerable to various infectious diseases. Among them, salmon vibriosis is a representative bacterial disease affecting farmed salmon that inhibits growth and causes mortality, resulting in enormous damage to the aquaculture industry. In particular, Vibrio anguillarum, known as the main causative agent of vibriosis, Vibrio anguillarum The bacteria is a causative agent of hemorrhagic sepsis (vibriosis) and infects various fish species, including salmonid fish. The bacteria are present in seawater and mainly cause infection when wounds are created due to careless handling during sorting or transport, or cause secondary infection from other diseases.

[0006] Meanwhile, 'Ulcerative Disease' is a representative bacterial disease that causes massive economic losses to the entire fish farming industry. Ulcerative Disease induces ulcerative lesions in skin and muscle tissues, leading to stunted growth, increased mortality rates, and reduced marketability in infected individuals, thereby negatively impacting both the survival rate and economic value of farmed fish. In particular, high-density rearing in aquaculture environments accelerates the spread of pathogens, further intensifying the spread of the disease. The main causative agent of ulcerative disease is *Aeromonas salmonisida*, a subspecies of *Aeromonas salmonisida* ( Aeromonas salmonicidasubsp. salmonicida ) and Aeromonas salmonisida subspecies masoushida ( Aeromonas salmonicida subsp. masoucida It causes typical and atypical carbuncles, respectively, belongs to the Gram-negative rod-shaped bacteria, and primarily infects cold-water fish. This strain possesses various virulence factors and has a high ability to evade host immunity due to biofilm formation and mutations in outer membrane proteins. Furthermore, since the pathogenicity of the strain may vary depending on the region, water temperature, and host fish species, the development of vaccines that consider subspecies specificity is essential for effective control.

[0007] To date, antibiotic administration and commercial vaccination have been attempted to control ulcer disease and Vibrio disease. However, antibiotics do not serve as a long-term solution due to the emergence of antibiotic resistance and residue issues, while existing vaccines exhibit limited protective effects against only certain strains. Furthermore, preventive efficacy can be further reduced if there is no proper match between the diversity of vaccine antigens and the host immune response. Therefore, the discovery of more precise and highly efficient vaccines or novel antigen candidates capable of effectively preventing and treating ulcer disease and Vibrio disease simultaneously has emerged as an urgent task. In particular, it is necessary to isolate pathogens specialized for the domestic salmon farming environment and establish customized immunological defense strategies based on this.

[0008] Under the aforementioned background technology, the inventors isolated strains of *Aeromonas salmonisida* subspecies *salmonisida* and *Aeromonas salmonisida* subspecies *masousida*, which cause carbuncles, from Atlantic salmon acclimated to seawater in the Goseong region of Gangwon-do, and isolated and analyzed strains of *Vibrio anguillarum*, which causes vibriosis, from rainbow trout being raised in seawater at an aquaculture farm in the Goheung region of Jeollanam-do. As a result of the analysis, the inventors confirmed that the isolated strains exhibited high pathogenicity in Atlantic salmon and that the inactivated mixed vaccine composition had excellent preventive effects against carbuncles and vibriosis, thereby completing the present invention.

[0009] The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to those skilled in the art to which the present invention belongs. The problem to be solved

[0011] The object of the present invention is (1) the inactivated strain Vibrio anguillarum 23FBVib0271 deposited under accession number KCTC16476BP; (2) the inactivated strain Aeromonas salmonisida subspecies salmonisida 17FBASa0016 deposited under accession number KCTC16475BP; and (3) the inactivated strain Aeromonas salmonisida subspecies masoucidah The present invention provides a mixed vaccine composition for preventing vibriosis and carbuncles in fish, comprising the 23FBAer0174 strain as an antigen.

[0012] Another object of the present invention is a) the strain Vibrio anguillarum 23FBVib0271 deposited under accession number KCTC16476BP; the strain Aeromonas salmonisida subspecies salmonisida 17FBASa0016 deposited under accession number KCTC16475BP; and the strain Aeromonas salmonisida subspecies masoucidase deposited under accession number KCTC16474BP The present invention provides a method for preparing a mixed vaccine composition for preventing vibriosis and carbuncles in fish, comprising the steps of: a) inactivating a 23FBAer0174 strain; and b) mixing the inactivated strain from step a).

[0013] Another objective of the present invention is to provide a method for preventing vibriosis and carbuncles in fish, comprising the step of administering the mixed vaccine composition to fish. means of solving the problem

[0015] To achieve the above objective, the present invention comprises (1) an inactivated Vibrio anguillarum 23FBVib0271 strain deposited under accession number KCTC16476BP;

[0016] (2) Inactivated strain of Aeromonas salmonisida subspecies salmonisida 17FBASa0016 deposited under accession number KCTC16475BP; and

[0017] (3) Aeromonas salmonicida subspecies masoucida deposited under deactivated accession number KCTC16474BP A mixed vaccine composition for preventing vibriosis and carbuncles in fish is provided, comprising the 23FBAer0174 strain as an antigen.

[0018] The present invention also comprises: a) the strain Vibrio anguillarum 23FBVib0271 deposited under accession number KCTC16476BP; the strain Aeromonas salmonisida subspecies salmonisida 17FBASa0016 deposited under accession number KCTC16475BP; and Aeromonas salmonisida subspecies masoucidase deposited under accession number KCTC16474BP. A method for preparing a mixed vaccine composition for preventing vibriosis and carbuncles in fish is provided, comprising the steps of: a) inactivating a 23FBAer0174 strain; and b) mixing the inactivated strain from step a).

[0019] The present invention also provides a method for preventing vibriosis and carbuncles in fish, comprising the step of administering the mixed vaccine composition to fish. Effects of the invention

[0021] The mixed vaccine composition according to the present invention effectively prevents Vibrio disease and carbuncleosis in fish, particularly Atlantic salmon, simultaneously without immune interference or side effects, thereby significantly reducing mortality caused by both diseases. Consequently, the amount of antibiotics used and associated costs can be reduced, and the survival rate and productivity of fish can be improved. Therefore, the present invention enables the stable production of farmed fish, contributing to the economic viability and sustainable development of the aquaculture industry. Brief explanation of the drawing

[0023] Figure 1 shows the growth curve of the Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain according to various culture temperatures and NaCl concentrations in BHIB. It is a graph showing optical density (A) and bacterial count (B) under various culture conditions. Figure 2 shows Aeromonas salmonisida subspecies masoucidae according to various culture temperatures and NaCl concentrations in BHIB. This shows the growth curve of the 23FBAer0174 strain. It is a graph showing optical density (A) and bacterial count (B) under various culture conditions. Figure 3 shows the growth curves of the Vibrio anguillarum 23FBVib0271 strain according to various culture temperatures and NaCl concentrations in BHIB. It is a graph showing optical density (A) and bacterial count (B) under various culture conditions. Figure 4 shows the results of comparing protein expression under various culture conditions through Western blot. A to C are the results of culturing the Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain in BHIOB, TSB, and LB&LB+G media, respectively. Figure 5 shows the results of comparing protein expression under various culture conditions via Western blot. D–F: Aeromonas salmonisida subspecies masoucida, respectively. This is the result of culturing the 23FBAer0174 strain in BHIB, TSB, and LB&LB+G media. Figure 6 shows the results of comparing protein expression under various culture conditions via Western blot. G~J: Results of culturing the Vibrio anguillarum 23FBVib0271 strain in BHIB, TSB, LB, and LB&LB+G media, respectively. Figure 7 is a figure confirming the presence of DNase (left) and hemolytic activity (right). Figure 8 is a graph showing the cumulative mortality rate of Atlantic salmon infected with Aeromonas salmonisida subspecies salmonisida 17FBASa0016. Fig. 9 is the subspecies Aeromonas salmonicidae masoushida This is a graph showing the cumulative mortality rate of Atlantic salmon infected with 23FBAer0174. Figure 10 is a graph showing the cumulative mortality rate of Atlantic salmon infected with Vibrio anguillarum 23FBVib0271. Fig. 11 shows Aeromonas salmonicida subspecies salmonicida 17FBASa0016(A) and Aeromonas salmonicida subspecies masoucida This is a photograph showing internal and external epidermal lesions accompanied by hemorrhage in Atlantic salmon inoculated with 23FBAer0174(B) and Vibrio anguillarum 23FBVib0271(C). Figure 12 is a graph showing the cumulative mortality rate of rainbow trout infected with Aeromonas salmonisida subspecies salmonisida 17FBASa0016. Fig. 13 is the subspecies Aeromonas salmonisidae masoushida This is a graph showing the cumulative mortality rate of rainbow trout infected with 23FBAer0174. Figure 14 is a graph showing the cumulative mortality rate of rainbow trout infected with Vibrio anguillarum 23FBVib0271. Fig. 15 shows Aeromonas salmonicida subspecies salmonicida 17FBASa0016(A) and Aeromonas salmonicida subspecies masoucida This is a photograph showing internal and external epidermal lesions accompanied by hemorrhage in rainbow trout inoculated with 23FBAer0174(B) and Vibrio anguillarum 23FBVib0271(C). Figure 16 shows the sequencing results in which the strain of the present invention was inactivated via sequencing, and then the absence of strain contamination was confirmed and the species identified through nucleotide sequencing. Figure 16a is the 16S_F sequencing result of Vibrio anguillarum 23FBVib0271, Figure 16b is the 16S_R sequencing result of Vibrio anguillarum 23FBVib0271, Figure 16c is the 16S_F sequencing result of Aeromonas salmonicida subspecies salmonicida 17FBASa0016, Figure 16d is the 16S_R sequencing result of Aeromonas salmonicida subspecies salmonicida 17FBASa0016, and Figure 16e is the 16S_R sequencing result of Aeromonas salmonicida subspecies masoucida. This is the 16S_F sequencing result of 23FBAer0174, and Fig. 16f is Aeromonas salmonisidase subspecies masoucidase. 16S_R sequencing results of 23FBAer0174, and Fig. 16g is the result of BLASTing the sequencing results. Figure 17 is a photograph confirming whether the vaccine was inactivated. Figure 18 is a graph showing the cumulative mortality rate of fish administered with the Vibrio Anguillarum 23FBVib0271 strain. In the graph, P+V represents the control group induced by artificial infection after inoculating with PBS, and V+V represents the control group induced by artificial infection after inoculating with the inactivated Vibrio Anguillarum 23FBVib0271 vaccine. Figure 19 is a graph showing the cumulative mortality rate of fish administered with Aeromonas salmonicida subspecies salmonicida 17FBASa0016. In the graph, P+S represents the control group, which was artificially infected after inoculating with PBS, and S+S represents the control group, which was artificially infected after inoculating with the inactivated Aeromonas salmonicida subspecies salmonicida 17FBASa0016 vaccine. Fig. 20 is the subspecies Aeromonas salmonisidae masoushida This is a graph showing the cumulative mortality rate of fish administered 23FBAer0174. In the graph, P+M represents the control group artificially infected after inoculation with PBS, and M+M represents inactivated Aeromonas salmonicida subspecies masoushida. It was artificially infected after receiving the 23FBAer0174 vaccine. Figure 21 shows the cumulative mortality rate of fish administered (A) Vibrio anguillarum 23FBVib0271, (B) Aeromonas salmonicida subspecies salmonicida 17FBASa0016, and (C) Aeromonas salmonicida subspecies masoucida 23FBAer0174 after mixed vaccination. Figure 22 is a graph confirming weight gain after vaccination with a combination vaccine. Figure 23 is a graph comparing serological indicators before and 6 weeks after the combination vaccine administration. Figure 24 shows the H&E staining results of liver tissue before mixed vaccine administration. Figure 25 shows the H&E staining results of intra-abdominal tissue before mixed vaccine administration. Figure 26 shows the H&E staining results of spleen tissue before mixed vaccine administration. Figure 27 shows the H&E staining results of liver tissue 6 weeks after the combination vaccine inoculation. Figure 28 shows the H&E staining results of intra-abdominal tissue 6 weeks after the mixed vaccine inoculation. Figure 29 shows the H&E staining results of spleen tissue 6 weeks after the mixed vaccine inoculation. Specific details for implementing the invention

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0026] Mixed vaccine composition for preventing bacterial diseases in fish

[0027] In one embodiment of the present invention, Vibrio anguillarum 23FBVib0271 strain isolated from rainbow trout, Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain isolated from Atlantic salmon and Aeromonas salmonisida subspecies masoucida After inactivating the 23FBAer0174 strain, a mixed vaccine composition containing it as an antigen was inoculated into fish, and it was confirmed that it exhibited excellent preventive effects against both Vibrio anguillarum and Aeromonas salmonisida infections and could prevent vibrio disease and carbuncles. Vibrio anguillarum causes vibrio disease, and Aeromonas salmonisida subspecies salmonisida and Aeromonas salmonisida subspecies masoucida cause typical and atypical carbuncles, respectively.

[0028] Accordingly, in one aspect, the present invention comprises (1) an inactivated Vibrio anguillarum 23FBVib0271 strain deposited under accession number KCTC16476BP;

[0029] (2) Inactivated strain of Aeromonas salmonisida subspecies salmonisida 17FBASa0016 deposited under accession number KCTC16475BP; and

[0030] (3) Aeromonas salmonisida subspecies masoucida deposited under deactivated accession number KCTC16474BP The present invention relates to a mixed vaccine composition for preventing vibriosis and carbuncles in fish, comprising the 23FBAer0174 strain as an antigen.

[0031] In the present invention, the Vibrio anguillarum 23FBVib0271 strain is a strain deposited at the Korean Collection for Type Culture on September 10, 2025, and assigned the accession number KCTC16476BP.

[0032] In the present invention, the strain Aeromonas salmonisida subspecies salmonisida 17FBASa0016 is a strain deposited at the Korean Collection for Type Culture on September 10, 2025, and assigned the accession number KCTC16475BP.

[0033] In the present invention, the Aeromonas salmonicida subspecies masoucida Strain 23FBAer0174 is a strain deposited with the Korean Collection for Type Culture on September 10, 2025, and has been assigned the accession number KCTC16474BP.

[0034] In the present invention, the inactivated mixed vaccine composition comprises (a) an inactivated Vibrio anguillarum 23FBVib0271 strain deposited under accession number KCTC16476BP;

[0035] (b) inactivated strain Aeromonas salmonisida subspecies salmonisida 17FBASa0016 deposited under accession number KCTC16475BP; and

[0036] (c) Aeromonas salmonisida subspecies masoucida deposited under deactivated accession number KCTC16474BP It may be characterized by including the 23FBAer0174 strain as an antigen.

[0037] In the present invention, the vaccine may include one or more additional antigens.

[0038] In this invention, the term “vaccine” refers to an antigen used for immunity to prevent infectious diseases, which may be manufactured using inactivated or attenuated microorganisms or viruses. It signifies the administration of weakened or killed pathogenic microorganisms into an individual to artificially acquire immunogenicity against a specific infectious disease. Upon stimulation by the vaccine, the immune system within the individual is activated to produce antibodies. By maintaining this susceptibility and effectively generating antibodies within a short period in the event of reinfection, the disease can be overcome.

[0039] The vaccine of the present invention preferably relates to an inactivated vaccine comprising an antigen of an inactivated strain produced by inactivating a cultured pathogen with chemicals or heat.

[0040] The above-mentioned inactivated vaccine may be manufactured using methods widely known in the art, and may be inactivated, for example, by formalin treatment, heating, or ultraviolet irradiation, but is not limited thereto.

[0041] In the present invention, the inactivated Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain and the inactivated Aeromonas salmonisida subspecies masoucida included in the mixed vaccine composition. The concentration of the 23FBAer0174 strain may be characterized as being 5 to 20 times or 6 to 18 times, more preferably 7 to 16 times, 8 to 14 times or 9 to 11 times, and most preferably 10 times, based on the concentration of the independently inactivated Vibrio Anguillarum 23FBVib0271 strain.

[0042] In the present invention, the total antigen concentration of the vaccine composition may be characterized as being 1.05 to 500 mg / ml, 1.5 to 500 mg / ml, 2.1 to 500 mg / ml, preferably 2.1 to 450 mg / ml, more preferably 5 to 450 mg / ml, 10 to 450 mg / ml, 15 to 420 mg / ml, even more preferably 21 to 420 mg / ml, 21 to 400 mg / ml, 21 to 350 mg / ml, 21 to 300 mg / ml, 21 to 250 mg / ml, and most preferably 21 to 210 mg / ml, but is not limited thereto.

[0043] The antigen concentration of the inactivated Vibrio anguillarum 23FBVib0271 strain included in the above mixed vaccine composition may be 0.05 to 20 mg / ml, preferably 0.1 mg / ml to 20 mg / ml, 0.3 mg / ml to 20 mg / ml, 0.5 mg / ml to 20 mg / ml, more preferably 0.5 mg / ml to 15 mg / ml, 0.7 mg / ml to 10 mg / ml, and most preferably 1 mg / ml to 10 mg / ml, but is not limited thereto.

[0044] The antigen concentration of the inactivated Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain included in the above mixed vaccine composition may be 0.5 to 200 mg / ml, preferably 1 mg / ml to 200 mg / ml, 3 mg / ml to 200 mg / ml, 5 mg / ml to 200 mg / ml, more preferably 5 mg / ml to 150 mg / ml, 7 mg / ml to 100 mg / ml, and most preferably 10 mg / ml to 100 mg / ml, but is not limited thereto.

[0045] Inactivated Aeromonas salmonisida subspecies masoucida included in the above mixed vaccine composition The antigen concentration of the 23FBAer0174 strain may be 0.5 to 200 mg / ml, preferably 1 mg / ml to 200 mg / ml, 3 mg / ml to 200 mg / ml, 5 mg / ml to 200 mg / ml, more preferably 5 mg / ml to 150 mg / ml, 7 mg / ml to 100 mg / ml, most preferably 10 mg / ml to 100 mg / ml, but is not limited thereto.

[0046] The antigen concentration range of each antigen included in the above-mentioned mixed vaccine composition is preferably selected within a range that satisfies the ratio described above, but is not limited thereto.

[0047] The mixed vaccine composition according to the present invention may further include a pharmaceutically acceptable adjuvant. Any adjuvant known in the art may be used without limitation and may be selected from the group consisting of nonionic surfactants, aluminum hydroxide gel, aluminum phosphate, squalene, mineral oil, the Montanide series, and chitosan, but is not limited thereto.

[0048] The mixed vaccine composition of the present invention may also use sugars, amino acids, mineral oil, vegetable oil, alum, aluminum phosphate, bentonite, silica, amuramyl dipeptide derivatives, thymosin, interleukin, etc. as stabilizers or additives. Carriers, excipients, and diluents that may be included in the vaccine composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0049] The mixed vaccine composition of the present invention may be formulated and used in the form of a sterile injectable solution, such as a suspension or emulsion, according to conventional methods. Preparations for injection administration include sterile aqueous solutions, water-insoluble agents, suspensions, emulsions, and lyophilized preparations. As water-insoluble agents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.

[0050] The above adjuvant can be applied within the concentration range commonly used by a person skilled in the art in the relevant technical field.

[0051] In the present invention, the mixed vaccine composition may be used by dispensing the vaccine solution into a vial of a suitable volume, for example, about 10 to 500 ml, and then sealing it. This vaccine may be used not only in liquid form but also as a dry preparation by lyophilizing it after injection. The dry preparation may be used by completely redissolving the dry material with a bactericidal solution added immediately before use.

[0052] The mixed vaccine composition of the present invention can be formulated and used in the form of a sterile injectable solution, such as a suspension or emulsion, according to conventional methods, and can be prepared in a freeze-dried form if necessary. The finished vaccine can be filled into a vial or ampoule in a sterile state and stored, and can be maintained stably for a long period under these conditions.

[0054] In one embodiment of the present invention, particularly when the mixed vaccine composition was inoculated into fish, a preventive effect against infection by Aeromonas salmonisida strain, the causative agent of typical / atypical carbuncles disease, and infection by Vibrio anguillarum strain, the causative agent of Vibrio disease, was confirmed. Furthermore, the mixed vaccine composition of the present invention exhibited excellent efficacy against salmonid fish, particularly Atlantic salmon.

[0055] In the present invention, the incision may be characterized as being a typical incision and / or an atypical incision.

[0056] In the present invention, the fish is of the salmon family ( Salmonidae It can be characterized as being a fish.

[0057] In the present invention, the salmonid fish is an Atlantic salmon ( Salmo salar ), brown trout( Salmo trutta ), Adriatic trout( Salmo obtusirostris ), Marmorata trout( Salmo marmoratus ), soap trout( Salmo obtusirostris salonitana ), Ohrid trout( Salmo letnica ), Serbian trout( Salmo farioides ), cream trout( Salmo carpio ), Sardinian trout( Salmo cettii ), Armenian trout( Salmo ischchan ), rainbow trout( Oncorhynchus mykiss ), Cherry Salmon( Oncorhynchus masou ), silver salmon( Oncorhynchus kisutch ), sockeye salmon( Oncorhynchus nerka King salmon (Oncorhynchus tshawytscha ), Pink Salmon( Oncorhynchus gorbuscha ), Chamu salmon ( Oncorhynchus keta ), Arctic humpback salmon( Salvelinus alpinus ) and lake trout( Salvelinus namaycush It may be characterized by one or more selected from the group consisting of ). Preferably, the salmonid fish may be characterized as being Atlantic salmon.

[0059] Method for manufacturing an inactivated vaccine for the prevention of Vibrio disease and carbuncles in fish

[0060] In another aspect, the present invention relates to a) the strain Vibrio anguillarum 23FBVib0271 deposited under accession number KCTC16476BP; the strain Aeromonas salmonisida subspecies salmonisida 17FBASa0016 deposited under accession number KCTC16475BP; and Aeromonas salmonisida subspecies masoucidase deposited under accession number KCTC16474BP. Step of inactivating the 23FBAer0174 strain; and

[0061] b) A method for preparing a mixed vaccine composition for preventing bacterial diseases in fish, comprising the step of mixing the inactivated strain of step a).

[0062] In the present invention, the mixed vaccine composition comprises the strain Vibrio anguillarum 23FBVib0271 deposited under accession number KCTC16476BP; the strain Aeromonas salmonisida subspecies salmonisida 17FBASa0016 deposited under accession number KCTC16475BP; and the strain Aeromonas salmonisida subspecies masoucidase deposited under accession number KCTC16474BP. The 23FBAer0174 strain is cultured, inactivated, mixed, and then the concentration is adjusted to prepare the vaccine composition. In one embodiment of the present invention, the strain was cultured, and 2% formalin was added to the cultured bacterial suspension and inactivated by shaking for 3 hours. The bacterial suspension, confirmed to be inactivated, was centrifuged, washed, mixed in a desired ratio, and the concentration adjusted to prepare a mixed vaccine composition. However, the present invention is not limited thereto and may prepare the mixed vaccine composition by utilizing inactivation methods known in the art.

[0063] In the present invention, the inactivation may be performed by formalin treatment, heating, or ultraviolet irradiation, but is not limited thereto.

[0064] In the present invention, step b) involves the inactivated Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain and the inactivated Aeromonas salmonisida subspecies masoucida The mixture may be characterized by such that the concentration of the 23FBAer0174 strain is 5 to 20 times or 6 to 18 times, more preferably 7 to 16 times, 8 to 14 times or 9 to 11 times, and most preferably 10 times, based on the concentration of the independently inactivated Vibrio Anguillarum 23FBVib0271 strain.

[0066] In the present invention, step b) may be characterized by mixing such that the antigen concentration of the inactivated Vibrio anguillarum 23FBVib0271 strain included in the mixed vaccine composition is 0.05 to 20 mg / ml, preferably 0.1 mg / ml to 20 mg / ml, 0.3 mg / ml to 20 mg / ml, 0.5 mg / ml to 20 mg / ml, more preferably 0.5 mg / ml to 15 mg / ml, 0.7 mg / ml to 10 mg / ml, and most preferably 1 mg / ml to 10 mg / ml, but is not limited thereto.

[0067] In the present invention, step b) may be characterized by mixing such that the antigen concentration of the inactivated Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain included in the mixed vaccine composition is 0.5 to 200 mg / ml, preferably 1 mg / ml to 200 mg / ml, 3 mg / ml to 200 mg / ml, 5 mg / ml to 200 mg / ml, more preferably 5 mg / ml to 150 mg / ml, 7 mg / ml to 100 mg / ml, and most preferably 10 mg / ml to 100 mg / ml, but is not limited thereto.

[0068] In the present invention, step b) comprises an inactivated Aeromonas salmonisida subspecies masoucida included in the mixed vaccine composition. The mixture may be characterized by having an antigen concentration of the 23FBAer0174 strain of 0.5 to 200 mg / ml, preferably 1 mg / ml to 200 mg / ml, 3 mg / ml to 200 mg / ml, 5 mg / ml to 200 mg / ml, more preferably 5 mg / ml to 150 mg / ml, 7 mg / ml to 100 mg / ml, and most preferably 10 mg / ml to 100 mg / ml, but is not limited thereto.

[0069] In the present invention, step b) comprises: the antigen concentration of the inactivated Vibrio anguillarum 23FBVib0271 strain included in the mixed vaccine composition being 0.05 to 20 mg / ml, preferably 0.08 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, more preferably 0.5 mg / ml to 10 mg / ml, most preferably 1 mg / ml to 10 mg / ml; and the antigen concentration of the inactivated Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain included in the mixed vaccine composition being 0.5 to 200 mg / ml, preferably 0.8 mg / ml to 150 mg / ml, 1 mg / ml to 100 mg / ml, more preferably 5 mg / ml to 100 mg / ml, most preferably 10 mg / ml to 100 mg / ml; Inactivated Aeromonas salmonisida subspecies masoucida included in the above mixed vaccine composition The mixture may be characterized by having an antigen concentration of the 23FBAer0174 strain of 0.5 to 200 mg / ml, preferably 0.8 mg / ml to 150 mg / ml, 1 mg / ml to 100 mg / ml, more preferably 5 mg / ml to 100 mg / ml, and most preferably 10 mg / ml to 100 mg / ml, but is not limited thereto.

[0071] In the present invention, the incision may be characterized as being a typical incision and / or an atypical incision.

[0073] Prevention methods for Vibrio disease and carbuncles in fish

[0074] In another aspect, the present invention relates to a method for preventing bacterial disease in fish, comprising the step of administering the mixed vaccine composition to fish.

[0075] In the present invention, the bacterial disease may be characterized as being Vibrio disease and ulcer disease.

[0076] The preventive method of the present invention can be applied to fish species susceptible to pathogens causing ulcer disease or vibriosis, preferably to fish of the Salmonidae family, more preferably to Atlantic salmon or rainbow trout, but is not limited thereto.

[0077] In the present invention, the fish is preferably of the salmon family ( Salmonidae It can be selected from a group consisting of ) fish.

[0078] In the present invention, the salmonid fish is preferably an Atlantic salmon ( Salmo salar ), brown trout( Salmo trutta ), Marmorata trout( Salmo marmoratus ), soap trout( Salmo obtusirostris salonitana ), Ohrid trout( Salmo letnica ), cream trout( Salmo carpio ), Sardinian trout( Salmo cettii ), Armenian trout( Salmo ischchan ), rainbow trout( Oncorhynchus mykiss ), Cherry Salmon( Oncorhynchus masou ), silver salmon( Oncorhynchus kisutch ), sockeye salmon( Oncorhynchus nerka King salmon (Oncorhynchus tshawytscha ), Pink Salmon( Oncorhynchus gorbuscha ), Chamu salmon ( Oncorhynchus keta ), Arctic humpback salmon( Salvelinus alpinus ) and lake trout( Salvelinus namaycush It may be characterized by being one or more selected from a group consisting of ), but is not limited thereto.

[0079] In the present invention, the administration may be intraperitoneal, intramuscular, or subcutaneous. The administration is preferably about 0.1 to 0.5 mL per fish body, more preferably about 0.1 to 0.3 mL per fish body, and most preferably about 0.1 to 0.2 mL per fish body, but is not limited thereto. The administration is preferably administered one or more times, or two or more times, but is not limited thereto.

[0080] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0082] Examples

[0083] Example 1. Isolation and identification of Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain, Aeromonas salmonisida subspecies 23FBAer0174 strain, and Vibrio anguillarum 23FBVib0271 strain

[0084] Strains causing carbuncles were isolated and identified from Atlantic salmon acclimating to seawater in the Goseong region of Gangwon-do. They were confirmed to belong to the subspecies *Aeromonas salmonisida* and *Aeromonas salmonisida*, respectively, and were deposited at the KCTC, where they were assigned accession numbers KCTC16475BP and KCTC16474BP, respectively.

[0085] In addition, a strain causing vibriosis was isolated and identified from rainbow trout being raised in seawater at a fish farm in Goheung, Jeollanam-do, and it was confirmed to be a strain belonging to Vibrio anguillarum. It was then deposited with KCTC and assigned the accession number KCTC16476BP.

[0087] Example 2. Confirmation of mycological properties of Aeromonas salmonicida subspecies salmonicida 17FBASa0016 strain, Aeromonas salmonicida subspecies masoucida 23FBAer0174 strain, and Vibrio anguillarum 23FBVib0271 strain

[0088] Example 2-1. Bacterial growth characteristics of three strains according to culture conditions

[0089] To investigate the characteristics of three strains—*Aeromonas salmonicida* subspecies *salmonicida* 17FBASa0016*, *Aeromonas salmonicida* subspecies *masousida* 23FBAer0174*, and *Vibrio anguillarum* 23FBVib0271—according to culture media [brain heart infusion broth (BHIB), tryptic soy broth (TSB), Luria-Bertani broth (LB), LB broth+0.25% glucose (LB+G)], culture temperatures (20℃, 25℃), and NaCl concentrations (0.5%, 1.5%, 2.5%, 3.5%), 100 μL of bacterial suspension subcultured in BHIB the day before the experiment was inoculated into 100 mL of media prepared for each culture condition (BHIB, TSB, LB+NaCl 1.5%, 2.5%, 3.5%, etc.). Shaking incubation (150 rpm) was performed for 24 hours, and bacterial count and absorbance (OD600) were measured at 4, 6, 8, 10, 12, and 24 hours. After dispensing the bacterial suspensions of the test strains at each time point into a 96-well plate, serial dilution (10 -1 ~10 -8 ) was performed. 20 μL of the diluted solution was inoculated onto brain heart infusion agar (BHIA) medium and cultured for 24 hours, after which the number of bacterial colonies formed was counted to calculate the viable cell count.

[0090] The strains Aeromonas salmonicida subspecies salmonicida (ASS) 17FBASa0016, Aeromonas salmonicida subspecies masoucida (ASM) 23FBAer0174, and Vibrio anguillarum 23FBVib0271 (VA) were cultured in BHIB, TSB, LB, and LB+G media under varying NaCl concentrations (0.5–3.5%) and culture temperatures (20, 25℃), and changes in medium absorbance and bacterial count were investigated. As a result, ASS and ASM recorded high absorbance during the logarithmic growth phase (10–12 hours) under conditions of a culture temperature of 25℃ and 0.5% NaCl, and the bacterial count was 1×10⁶. 7It was high, exceeding CFU / mL. VA exhibited high absorbance during the logarithmic growth phase under conditions of an incubation temperature of 25℃ and 1.5% NaCl, and the bacterial count was 1×10⁻⁶ 8 It was found to be high, above CFU / mL (Figs. 1 to 3).

[0092] Example 2-2. Investigation of Antigenicity of Three Strains According to Culture Conditions

[0093] To compare protein expression patterns according to culture media (BHIB, TSB, LB, LB+G), culture temperature (20℃, 25℃), and NaCl concentration (0.5%, 1.5%, 2.5%, 3.5%) for three strains—Aeromonas salmonisida subspecies salmonisida (ASS) 17FBASa0016, Aeromonas salmonisida subspecies masoucida (ASM) 23FBAer0174, and Vibrio anguillarum (VA) 23FBVib0271—0.5% formalin was added to the three strains cultured for 24 hours under each experimental condition and inactivated by shaking culture for 12 hours. The inactivated bacterial suspension was centrifuged at 7,000×g for 15 minutes to remove the supernatant, and the pellet was washed three times with PBS. The final product was resuspended in 0.02% sodium azide (in PBS) to a concentration of 100 mg / mL and stored at 4°C. SDS-PAGE and Western blot were performed to compare protein expression and expression levels according to culture media, and the positions and sizes of protein bands were compared.

[0094] As a result of culture, there was no difference in protein expression for all three types of bacteria even when the type of medium was changed under optimal culture conditions (Figs. 4 to 6). Therefore, it was confirmed that mass production of FKC is possible using the inexpensive TSB when manufacturing vaccines.

[0096] Example 2-3. Analysis of Biochemical Characteristics of Three Strains

[0097] Three strains, Aeromonas salmonicida subspecies salmonicida (ASS) 17FBASa0016, Aeromonas salmonicida subspecies masoucida (ASM) 23FBAer0174, and Vibrio anguillarum (VA) 23FBVib0271, were inoculated onto TSA medium, and the morphology, size, and color of the colonies were analyzed at 25°C for 1 to 2 days. The presence or absence of DNase production by the strains was measured by incubating on DNase agar supplemented with 0.01% bromophenol blue at 25°C for 24 hours. The hemolytic activity of the strains was confirmed by incubating the bacteria on a blood agar plate supplemented with 5% sheep blood at 25°C for 24 hours.

[0098] Biochemical analysis was performed by inoculating bacteria into the API 20E KIT and observing the results after incubation at 25°C for 24 to 48 hours. Briefly, sterile distilled water was poured into the incubation box tray to prevent the samples from drying out. A single colony was then taken from each strain and suspended in 1× PBS. Using a pipette, the suspension was filled into the cuplets according to the manufacturer's instructions. The strip lids were then closed, and the samples were incubated at 25°C for 24 hours. Subsequently, color changes were recorded, and for some readings, the reaction was observed and recorded using auxiliary reagents.

[0099] As a result, Vibrio anguillarum 23FBVib0271 strain (O1) formed yellow colonies on TCBS, and Aeromonas salmonisida subspecies salmonisida 17FBASa0016 showed brown pigmentation on TSA (Tryptic Soy Agar) medium.

[0100] DNase production was measured on DNase agar supplemented with 0.01% bromophenol blue. As a result, no DNase production was detected in any of the three strains (Fig. 7). Hemolytic activity was measured on blood agar supplemented with 5% sheep blood. As a result, the Vibrio anguillarum strain 23FBVib0271 (O1) exhibited alpha (α) hemolysis, while the Aeromonas salmonicida subspecies salmonicida 17FBASa0016 strain and the Aeromonas salmonicida subspecies masoucida 23FBAer0174 strain exhibited gamma (γ) hemolysis (Fig. 7).

[0101] Vibrio Anguillarum 23FBVib0271 strain (O1) showed positive reactions for Indole production, ONPG, Citrate (Citrate), VP (Sodium pyruvate), ADH (Arginine dihydrolase), Gelatin (Gelatinase), Glucose, D-mannitol, Inositol, D-sorbitol, D-sucrose, and L-arabionse, and negative reactions for LDC (Lysine decarboxylase), ODC (Ornithine decarboxylase), H2S production, URE (Urease), TDA (Tryptophan deaminase), L-rhamnose, D-melibiose, and Amydalin, exhibiting biochemical reactions identical to previously reported Vibrio Anguillarum strains (Table 1, Lim et al., 2023).

[0102] Vibrio anguillarum 23FBVib0271 strain (O1), Aeromonas salmonicida subspecies salmonicida 17FBASa0016 strain and Aeromonas salmonicida subspecies masoucida 23FBAer0174 strain exhibited common biochemical reactions in indole production, ODC (Ornithine decarboxylase), Arginine dihydrolase (ADH), H2S production, URE (Urease), Gelatin (Gelatinase), Glucose, D-mannitol, L-rhamnose, and D-melibiose.

[0103] The strain Aeromonas salmonisida subspecies salmonisida 17FBASa0016 showed positive reactions for indole production, LDC (Lysine decarboxylase), ADH (Arginine dihydrolase), and TDA (Tryptophan deaminase), exhibiting biochemical characteristics different from strains of Aeromonas salmonisida subspecies salmonisida previously found in Korea (Table 2).

[0104] The strain Aeromonas salmonicida subspecies masoucida 23FBAer0174 showed positive reactions for indole production, VP (Sodium pyruvate), LDC (Lysine decarboxylase), ADH (Arginine dihydrolase), TDA (Tryptophan deaminase), Gelatin (Gelatinase), Glucose, D-mannitol, Inositol, D-sorbitol, D-sucrose, and Amydalin. In addition, it showed negative reactions for ONPG, Citrate (Citrate utilization), ODC (Ornithine decarboxylase), H2S production, URE (Urease), L-rhamnose, D-melibiose, and Amydalin (Table 2). In particular, the ASM 23FBAer0174 strain showed positive results for VP (Sodium pyruvate), Inositol, D-sorbitol, and Amydalin, exhibiting different biochemical characteristics compared to ASS. In addition, ASM showed different biochemical reactions when compared to three previously reported domestic isolates of Aeromonas salmonisida subspecies salmonisida, exhibiting positive reactions for indole production, LDC (Lysine decarboxylase), ADH (Arginine dihydrolase), and TDA (Tryptophan deaminase) (Lim and Hong, 2020).

[0105]

[0106]

[0108] Example 3. Confirmation of pathogenicity of Aeromonas salmonicida subspecies salmonicida (ASS) strain 17FBASa0016, Aeromonas salmonicida subspecies masoucida (ASM) strain 23FBAer0174, and Vibrio anguillarum strain 23FBVib0271 according to fish species

[0109] Example 3-1. Method for analyzing pathogenicity in Atlantic salmon and rainbow trout

[0110] Atlantic salmon and rainbow trout were acclimated in a 400L flow-through tank maintained at a water temperature of 12℃±1. Feed was administered daily at 1% of body weight, and the water temperature, water quality, occurrence of fish mortality, and neutralizing agents were recorded daily and monitored for 4 weeks.

[0111] For artificial infection of Atlantic salmon, MS-222 was added to the rearing water to a concentration of 200 ppm, and then strains Aeromonas salmonicida subspecies salmonicida (ASS) 17FBASa0016, Aeromonas salmonicida subspecies masoucida (ASM) 23FBAer0174, and Vibrio anguillarum (VA) 23FBVib0271 were inoculated, respectively, and three concentration groups (1×10 6 , 1×10 5 , 1×10 4 A total of 20 tanks (flow-through, 11±0.5℃) were set up to perform repeated experiments for the CFU / fish inoculation group (inoculation of 100 µl per fish body) and each concentration group. Atlantic salmon (average 5.8 cm, 3.1 g) were transferred from the acclimatization tank to the experimental tank for acclimatization, and 100 µl was inoculated per individual (negative control group received PBS injection), and the cumulative mortality rate was observed for 3 weeks.

[0112] Rainbow trout were similarly anesthetized and inoculated with strains Aeromonas salmonicida subspecies salmonicida (ASS) 17FBASa0016, Aeromonas salmonicida subspecies masoucida (ASM) 23FBAer0174, and Vibrio anguillarum 23FBVib0271, respectively, and three concentration groups (1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 A total of 20 tanks (flow-through, 11±0.5℃) were set up to perform repeated experiments for the CFU / fish inoculation group (inoculation of 100 µl intraperitoneally per fish) and each concentration group. Rainbow trout (average 18.4 cm, 28.1 g) were transferred from the acclimatization tank to the experimental tank for acclimatization, and 100 µl was inoculated per individual (negative control group received PBS injection), and the cumulative mortality rate was observed for 3 weeks.

[0114] Example 3-2. Results of pathogenicity analysis for Atlantic salmon and rainbow trout

[0115] The cumulative mortality rate of Atlantic salmon for Aeromonas salmonicida subspecies salmonicida (ASS) strain 17FBASa0016 and Aeromonas salmonicida subspecies masoucida (ASM) strain 23FBAer0174 is 1×10 6 From the high-concentration inoculation group of CFU / fish to the low 1×10 3 Total mortality was observed up to the CFU / fish inoculation group, and 1×10 2 The cumulative mortality rate over 3 weeks in the CFU / fish group was 40% (Figs. 8, 9). The cumulative mortality rate of Atlantic salmon for Vibrio anguillarum (VA) 23FBVib0271 strain was 1×10 6 ~10 4 In the high-concentration CFU / fish group, there was 100% mortality by the second day after bacterial inoculation, and 1×10 3 The cumulative mortality rate of the CFU / fish group was found to be 50%, and 1×10 2 The cumulative mortality rate of the CFU / fish group showed low results at 10% after 21 days (Fig. 10). LD50 of Aeromonas salmonicida subspecies salmonicida (ASS) 17FBASa0016 strain, Aeromonas salmonicida subspecies masoucida (ASM) 23FBAer0174 strain, and Vibrio anguillarum (VA) 23FBVib0271 strain 50 The values ​​are 6.3 × 10⁻⁶ respectively. 3 , 7.9×10 3 , 1.6×10 4 It was confirmed to be CFU / mL. Specific abdominal and body surface hemorrhage, liver congestion, ocular hemorrhage, and shedding symptoms were observed in dead Atlantic salmon (Fig. 11).

[0116] The cumulative mortality rate of rainbow trout for Aeromonas salmonicida subspecies salmonicida (ASS) strain 17FBASa0016 and Aeromonas salmonicida subspecies masoucida (ASM) strain 23FBAer0174 is 1×10 6Starting from the high-concentration CFU / fish inoculation group, cumulative mortality rates were recorded at 30% and 20%, respectively, which are significantly lower than those of Atlantic salmon. 1×10 5 The CFU / fish inoculation group showed a cumulative mortality rate of 10%, whereas no mortality was observed in the lower concentration inoculation groups (Figs. 12, 13). Specific symptoms of surface hemorrhage were observed in morbid fish (Fig. 15). The cumulative mortality rate of rainbow trout against Vibrio anguillarum (VA) 23FBVib0271 strain was 1×10 6 In the high-concentration CFU / fish group, initial mortality was significantly acute (Fig. 14) and clinical symptoms accompanied by hemorrhage on the body surface appeared (Fig. 15), but the cumulative mortality rate was limited to 60%, and 1×10 5 The cumulative mortality rate of the CFU / fish group was also found to be 60%, and 10 4 The cumulative mortality rate of the CFU / fish group was 50%, compared to the lowest concentration group of 1×10 3 The CFU / fish result showed a 20% cumulative mortality rate (Fig. 14). LD50 of Aeromonas salmonicida subspecies salmonicida (ASS) strain 17FBASa0016 and Aeromonas salmonicida subspecies masoucida (ASM) strain 23FBAer0174 50 The values ​​were 1×10⁻⁶, respectively, where the mortality rate of the high-concentration inoculation group was lower than 50%. 7 It was calculated as a value greater than or equal to CFU / mL, and the LD50 of the Vibrio anguillarum 23FBVib0271 strain 50 The value is 6.3×10 5 It was confirmed at CFU / mL. In conclusion, the strain of the present invention exhibited pathogenicity to rainbow trout and Atlantic salmon, and the LD50 for Atlantic salmon. 50The value was more than 100 times lower than that of rainbow trout, which means that the strains of Aeromonas salmonicida subspecies salmonicida (ASS) 17FBASa0016, Aeromonas salmonicida subspecies masoucida (ASM) 23FBAer0174, and Vibrio anguillarum 23FBVib0271 of the present invention exhibit pathogenicity across salmonid fish in general, and are strains that exhibit high pathogenicity in Atlantic salmon in particular, suggesting that the vaccine composition of the present invention may be effective against salmonid fish, especially Atlantic salmon.

[0118] Example 4. Production of inactivated vaccine antigens

[0119] Example 4-1. Preparation of Formalin-Inactivated Vaccine (FKC)

[0120] Vibrio anguillarum 23FBVib0271 strain was subcultured in TSA (sTSA) supplemented with 3% NaCl and cultured at 25°C for 24 hours. Subsequently, a single colony was inoculated into 1 mL of TSB (sTSB) medium supplemented with 3% NaCl and subcultured for 18 hours; afterward, 1 mL was inoculated into 1 L of sTSB medium, and the absorbance (OD) 600 ) were cultured until it reached 0.5. Aeromonas salmonicida subspecies salmonicida (ASS) strain 17FBASa0016 and Aeromonas salmonicida subspecies masoucida (ASM) strain 23FBAer0174 were inoculated into TSA medium and cultured at 20°C for 24 hours. Subsequently, one single colony was inoculated into 1 mL of TSB medium and subcultured for 18 hours; afterward, 1 mL was inoculated into 1 L of sTSB medium and the absorbance (OD) 600 ) cultured until it became 0.5.

[0121] 2% formalin was added to the cultured bacteria and inactivated in a shaking incubator for 3 hours. 100 μL of the inactivated culture was plated onto TSA medium to confirm bacterial inactivation. The inactivated culture was centrifuged at 7,000 × g for 15 minutes to remove the supernatant, and the pellet was washed three times with 1 × PBS. The final product was resuspended to a concentration of 100 mg / mL, 0.01% sodium azide was added, and the sample was stored at 4°C.

[0122] Inactivated Aeromonas salmonisida subspecies salmonisida (ASS) 17FBASa0016 strain and Aeromonas salmonisida subspecies masoucida (ASM) 23FBAer0174 strain were each diluted to 10 mg / mL and Vibrio anguillarum (VA) 23FBVib0271 strain to 1 mg / mL, and a vaccine was prepared by adding 2% aluminum hydrooxide gel (REHYDROGEL, CHEMTRADE, USA) at a 15% concentration.

[0124] Example 4-2. Evaluation of the efficacy of inactivated vaccine (FKC)

[0125] a) Preparation of experimental fish

[0126] Healthy Atlantic salmon (average body weight 18g) were acclimated to a flow-through system in a 400L rectangular tank at a water temperature of 12±1℃. During the experiment, monitoring was conducted by recording daily water temperature, water quality, occurrence of fish mortality, and neutralizing agents.

[0128] b) Preparation of bacteria

[0129] Vibrio anguillarum 23FBVib0271 strain was subcultured into sTSA medium and cultured at 25°C for 24 hours. Subsequently, one single colony was inoculated into 1 mL of sTSB medium and cultured for 18 hours, after which 300 μL was inoculated into 30 mL of sTSB medium and cultured for 18 hours. Aeromonas salmonisida subspecies salmonisida (ASS) 17FBASa0016 strain and Aeromonas salmonisida subspecies masoucida (ASM) 23FBAer0174 strain were subcultured into TSA medium and cultured at 20°C for 24 hours. Subsequently, one single colony was inoculated into 1 mL of TSB medium and cultured for 18 hours, after which 300 μL was inoculated into 30 mL of TSB medium and cultured for 18 hours.

[0130] After 18 hours of incubation, the pellet was centrifuged at 3,000×g for 15 minutes, the supernatant was removed, and the pellet was inverted to dry for 5 minutes. Subsequently, the weight of the pellet was measured, and the Vibrio anguillarum 23FBVib0271 strain was suspended in 1×PBS at a concentration of 4 mg / mL, while Aeromonas salmonisida subspecies salmonisida 17FBASa0016 and Aeromonas salmonisida subspecies masoucida 23FBAer0174 strains were suspended at a concentration of 40 mg / mL. At this time, the bacterial count was approximately 1×10⁶ 9 It is CFU / mL.

[0131] To select the attack experiment concentration, 10 4 , 10 5 , 10 6 Each strain was inoculated intraperitoneally at a concentration of CFU / mL, and the challenge inoculation concentration was selected after observing the cumulative mortality rate. To confirm the exact concentration of the inoculated bacteria, the diluted solution was inoculated onto sTSA or TSA media, and the number of colonies was counted.

[0133] c) Verification of inactivation status and contamination of the produced inactivated vaccine

[0134] 16S rDNA PCR and sequencing were performed on the cultured strains using 1 μL before inactivation to check for contamination (Fig. 16). The three cultured bacteria were inactivated by adding 2% formalin and shaking the culture for 3 hours, and 100 μL of the inactivated culture was plated onto TSA medium to confirm bacterial inactivation (Fig. 17). As a result of BLAST search of the nucleotide sequences of the three strains obtained through sequencing in the NCBI database, Vibrio anguillarum 23FBVib0271 showed more than 99% nucleotide sequence similarity with Vibrio anguillarum, and Aeromonas salmonisida subspecies salmonisida 17FBASa0016 and Aeromonas salmonisida subspecies masousida 23FBAer0174 strains showed more than 99% nucleotide sequence similarity with Aeromonas salmonisida subspecies salmonisida and Aeromonas salmonisida subspecies masousida, respectively, confirming that there was no contamination of the strains.

[0136] (d) Investigation of cumulative mortality rate and relative survival rate through artificial infection

[0137] Six weeks after vaccination, to evaluate the efficacy of the single vaccine, a separate anesthesia tank was prepared and MS-222 was added to the rearing water at a concentration of 200 ppm to induce anesthesia in the experimental fish.

[0138] 10 animals, 20 each for the vaccinated group and the unvaccinated group 5 The cumulative mortality rate was recorded for 3 weeks by artificially infecting with bacteria at a concentration of CFU / mL. Relative percent survival (RPS) was calculated using the formula RPS(%) = 1 - (cumulative mortality rate of the vaccinated group / cumulative mortality rate of the control group) × 100.

[0139] To check for bacterial infection in dead fish, the spleen or whole body was dissected and inoculated onto TSA medium, and Vibrio anguillarum was additionally inoculated onto TCBS medium and cultured to obtain colonies.

[0140] To determine the significant difference (p < 0.05) between the vaccinated group and the control group (PBS vaccinated group) 21 days after challenge inoculation, statistical differences were analyzed using the Log Rank test of the SPSS statistical program.

[0142] Through preliminary experiments to select the challenge inoculation concentration, 10 4 , 10 5 , 10 6 As a result of challenge inoculation at CFU / fish concentrations, a 100% mortality rate was confirmed at all concentrations. Accordingly, 1×10 5 The CFU / fish concentration was selected as the optimal attack inoculation concentration.

[0143] Six weeks after single-dose vaccination, 1×10⁶ of each strain were administered in two replicates with 20 animals per group. 5 100 μL of the vaccine was administered intraperitoneally to the vaccinated group and the control group (PBS vaccinated group) at a CFU / fish concentration, and the cumulative mortality rate was observed for 3 weeks. As a result, the control group (PBS vaccinated group) of all groups showed a mortality rate of 100%.

[0144] In contrast, the Vibrio anguillarum 23FBVib0271 monovaccination group showed a cumulative mortality rate of 0%, the Aeromonas salmonicida subspecies salmonicida 17FBASa0016 monovaccination group showed 25%, and the Aeromonas salmonicida subspecies masoucida 23FBAer0174 monovaccination group showed 5% (Figs. 18, 19, 20).

[0145] When calculated as Relative Percent Survival (RPS), the vaccine groups for Vibrio anguillarum 23FBVib0271, Aeromonas salmonisida subspecies salmonisida 17FBASa0016, and Aeromonas salmonisida subspecies masoucida 23FBAer0174 strains reached 100%, 75%, and 95%, respectively, showing a significant difference (P<0.001) compared to the control group (PBS vaccination group).

[0146] Accordingly, the single vaccine containing the inactivated strain of the present invention recorded an RPS of over 75% and showed high protective efficacy (Table 3). This experiment suggests that the inactivated strain antigen of the present invention can protect test fish from major pathogens.

[0147]

[0149] Example 5. Analysis of the efficacy of mixed vaccine administration

[0150] Example 5-1. Preparation of Mixed Formalin Inactivated Vaccine (FKC)

[0151] The strains Vibrio anguillarum 23FBVib0271, Aeromonas salmonisida subspecies salmonisida 17FBASa0016, and Aeromonas salmonisida subspecies masousida 23FBAer0174, produced by the inactivation vaccine manufacturing method, were treated with formalin-inactivated antigens, and the strains inactivated in the order of Vibrio anguillarum 23FBVib0271, Aeromonas salmonisida subspecies salmonisida 17FBASa0016, and Aeromonas salmonisida subspecies masousida 23FBAer0174 were treated with 1, 10, 10 mg / fish (Mix 1, total 21 mg / fish), 0.1, 1, 1 mg / fish (Mix 2, total 2.1 mg / fish), and 0.001, 0.01, 0.01 mg / fish (Mix 3, total After preparing three concentrations of mixed vaccines by mixing them so that they could be administered at a concentration of 0.21 mg / fish, the vaccine was prepared by adding 2% aluminum hydroxide gel (REHYDROGEL, CHEMTRADE, USA) at a concentration of 15% as an adjuvant (antigen adjuvant) (Table 4).

[0152] Preparation and composition of combination vaccines division Mix (1) (based on 400 mg / mL of antigen) Mix (2) (based on 40 mg / mL of antigen) Mix (3) (based on antigen 4 mg / mL) ASS antigen 2.5mL (1,000mg) 2.5mL (100mg) 2.5mL (10mg) ASM antigen 2.5mL (1,000mg) 2.5mL (100mg) 2.5m (10mg) VA Antigen 0.25mL (100mg) 0.25mL (10mg) 0.25mL (1mg) PBS 3,250 μL 3,250 μL 3,250 μL REHYDROGE(Adjuvant) 1.5mL 1.5mL 1.5mL Number of animals vaccinated (vaccination amount) 100 (100µl / fish) 100(100µl / fish) 100(100µl / fish) Mixed vaccine suspension 10mL 10mL 10mL

[0154] Example 5-2. Evaluation of the efficacy of the mixed inactivated vaccine (FKC)

[0155] a) Preparation of experimental fish

[0156] Healthy Atlantic salmon (average weight 18g) that had completed disease testing using the above method were acclimated to a freshwater flow-through system in a 400L rectangular tank at a water temperature of 12±1℃. After vaccination, monitoring was conducted by recording daily water temperature, water quality, occurrence of fish mortality, and neutralizing agents.

[0158] b) Preparation of bacteria

[0159] Vibrio anguillarum 23FBVib0271 strain was subcultured into sTSA medium and cultured at 25°C for 24 hours. Subsequently, one single colony was cultured in 1 mL of sTSB medium for 18 hours, then inoculated into 30 mL of sTSB medium with 300 μL and cultured for 18 hours. Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain and Aeromonas salmonisida subspecies masoucida 23FBAer0174 strain were subcultured into TSA medium and cultured at 20°C for 24 hours. Subsequently, one single colony was cultured in 1 mL of TSB medium for 18 hours, then inoculated into 30 mL of TSB medium with 300 μL and cultured for 18 hours.

[0161] c) Investigation of cumulative mortality rate and relative survival rate through artificial infection

[0162] To evaluate the efficacy of the combination vaccine before and 6 weeks after vaccination, a separate anesthesia tank was prepared, and experimental fish were immersed in rearing water supplemented with MS-222 at a concentration of 200 ppm to induce an anesthetic state. For cultured Aeromonas salmonisida and Vibrio anguillarum, the number of inoculated bacteria at which a cumulative mortality rate of 60% or more (1×10⁻⁶ 5Each strain was artificially infected twice in 20 fish each in the vaccinated group and the control group (PBS inoculated group) using CFU / fish as the challenge dose, and the cumulative mortality rate was recorded for 3 weeks. To check for bacterial infection in the dead fish, internal organs were dissected, and the spleen or whole body was plated with Aeromonas salmonisida on TSA medium and Vibrio anguillarum on TCBS medium. Relative percent survival (RPS) was calculated using the formula RPS(%) = 1 - (cumulative mortality rate of vaccinated group / cumulative mortality rate of control group) × 100.

[0163] To determine the significant difference (p < 0.05) between the vaccinated group and the control group (PBS vaccinated group) 21 days after challenge inoculation, statistical differences were analyzed using the Log Rank test of the SPSS statistical program.

[0164] As a result of the experiment, all control groups (PBS vaccination groups) showed a 100% mortality rate. Among the mixed vaccine vaccination groups, the Mix (1), Mix (2), and Mix (3) vaccination groups for Vibrio anguillarum 23FBVib0271 showed cumulative mortality rates of 0%, 10%, and 20%, respectively, and the RPS were calculated as 100, 90, and 80, respectively. Through this, it was confirmed that the vaccine exhibited excellent protective efficacy, recording a high RPS of over 80% in all vaccination groups (Fig. 21(A)).

[0165] For Aeromonas salmonicida subspecies salmonicida 17FBASa0016, the cumulative mortality rates of the Mix (1), Mix (2), and Mix (3) vaccination groups were observed to be 12.5%, 67.5%, and 100%, respectively. The corresponding RPS were calculated to be 77.5, 32.5, and 0, respectively, and the Mix (3) vaccination group showed no protective efficacy of the vaccine. This suggests that protective efficacy may vary significantly depending on the vaccine concentration (Fig. 21(B)).

[0166] Aeromonas salmonisida subspecies masoushida For 23FBAer0174, the Mix (1), Mix (2), and Mix (3) inoculation groups recorded cumulative mortality rates of 2.5%, 12.5%, and 25%, respectively, and the corresponding RPS were calculated as 97.5, 87.5, and 75, respectively. Excellent protective efficacy was shown at all concentrations, and the highest effect was observed in the Mix (1) inoculation group (Fig. 21(C), Table 5).

[0167]

[0168] The RPS of the vaccinated group showed a significant survival rate (P<0.001) compared to the control group (PBS vaccination group).

[0169] In conclusion, overall, all experimental results showed a tendency for cumulative mortality rates to decrease as vaccine concentrations increased. All combination vaccines demonstrated high protective efficacy against Vibrio anguillarum 23FBVib0271 and Aeromonas salmonisida subspecies masoucida 23FBAer0174, and also demonstrated excellent protective efficacy against Aeromonas salmonisida subspecies 17FBASa0016 at specific vaccine concentrations. This suggests that when the antigen concentrations of Vibrio anguillarum, Aeromonas salmonisida subspecies salmonisida, and Aeromonas salmonisida subspecies masoucida strains are appropriately mixed, excellent protective ability against all of these strains can be simultaneously demonstrated.

[0171] Example 5-3. Safety evaluation of mixed inactivated vaccine

[0172] a) Feed intake and body weight inspection

[0173] Before administering the mixed vaccine, body weight was measured in the vaccinated group and the control group (PBS vaccination group), and daily feed intake was recorded after vaccination. Subsequently, at the end of the experiment, 6 weeks after vaccination, the body weight of the vaccinated group and the control group (PBS vaccination group) was measured. Growth evaluation of the experimental fish was calculated using the formula: weight gain / initial experiment weight × 100, and feed efficiency was calculated using the formula: 100 × (final body weight - initial fish weight) / total cumulative feed amount.

[0174] Before vaccination (Day 0), the weight of Atlantic salmon in each group was measured as 18±0.29g, 19±0.3g, 17.75±0.3g, and 18±0.28g (Median±SD), respectively, in the order of Mix (1), Mix (2), Mix (3), and control group (PBS vaccination group). At 6 weeks (wpv 6) after vaccination with the mixed vaccine, the weight of each group was 20±0.35g for Mix (1), 20±0.24g for Mix (2), 19±0.39g for Mix (3), and 20±0.28g for the control group (PBS vaccination group) (Median±SD), and no significant difference was found between the vaccinated group and the control group (PBS vaccination group).

[0175] As a result of the growth rate evaluation, the values ​​were 111.11%, 105.26%, 107.04%, and 111.11% for Mix (1), Mix (2), Mix (3), and the control group (PBS inoculated group), respectively, and there was no significant difference between the groups. Feed efficiency was measured as 0.92%, 0.46%, 0.57%, and 0.67% for Mix (1), Mix (2), Mix (3), and the control group (PBS inoculated group), respectively, and, similar to the growth rate, there was no significant difference between the groups (Fig. 22).

[0176] These results suggest that the mixed vaccine of the present invention does not have a negative effect on the growth and feed efficiency of Atlantic salmon and can maintain the normal growth process of the experimental fish. Therefore, the mixed vaccine of the present invention can be evaluated as a safe vaccine that effectively induces an immune response in fish while minimizing its impact on growth and productivity.

[0178] b) Serological tests

[0179] Prior to the combination vaccine administration, blood was collected from the caudal veins of 10 Atlantic salmon per group from the vaccinated group and the control group (PBS vaccination group), and serum was obtained by centrifuging at 3,000×g for 10 minutes. Aspartate transaminase (AST), Alanine transaminase (ALT), Total Cholesterol (TCH), and Glucose (Glu) were measured in the obtained serum using a blood biochemistry analyzer (HITACHI, Japan). To confirm significant differences (p < 0.05) between the vaccinated group and the control group (PBS vaccination group), statistical differences were analyzed using the non-parametric Kruskal-Walis and Mann-Whitney U methods with the SPSS statistical program.

[0180] Blood was collected from 10 Atlantic salmon before vaccination (Day 0) and 10 Atlantic salmon from each group 6 weeks after combination vaccine administration (wpv 6). The samples were centrifuged at 3,000×g for 15 minutes to obtain 100 μL of serum. Using this, the levels of Aspartate transaminase (AST), Alanine transaminase (ALT), Alkaline phosphatase (ALP), and Glucose (Glu) were compared with the control group (PBS vaccination group).

[0181] As a result of the analysis, AST levels in all combination vaccine groups did not show a significant difference from day 0 and the control group (PBS vaccination group), and were measured in the range of 112-256 U / L, which fell within the range of “136.88-555.85 as suggested by Rozas-Serri et al. (2022).”

[0182] ALT levels ranged from 40 to 55 U / L, with no significant difference from day 0 and the control group (PBS vaccination group). Although slightly exceeding the range of 2.7 to 37.38 suggested by Rozas-Serri et al. (2022), the results were similar to those of the control group, suggesting that these results were due to differences in measurement equipment or rearing environment rather than vaccination.

[0183] ALP levels ranged from 80 to 184 U / L, with no significant differences observed between the groups, and the measured values ​​fell within the range of “99.8–255.49 as suggested by Rozas-Serri et al. (2022).”

[0184] Finally, Glu levels were measured in the range of 56-176 mg / dL, and all groups were distributed within the range of “25.22-169.17” as suggested by Rozas-Serri et al. (2022) (Fig. 23).

[0185] In conclusion, the serological values ​​of the combined vaccine group did not show a significant difference from the control group (PBS group) on day 0, suggesting that the combined vaccine does not have a negative effect on the serological status of Atlantic salmon.

[0187] c) Histopathological examination

[0188] Before and 6 weeks after the combination vaccine administration, injection sites and livers were collected from three fish in each group, fixed in a fixative, washed, and dehydrated with alcohol. The dehydrated samples were infiltrated with paraffin, embedded, and quenched once solidified. Tissue sections approximately 3–6 µm thick were then prepared and attached to slide glasses. Subsequently, the sections were dried, deparaffinized, hydrated, washed with distilled water, and stained with Hematoxylin & Eosin (H&E). The prepared tissue specimens were observed under a light microscope to confirm the presence or absence of histological lesions.

[0189] Liver, intraperitoneal injection tissue (IP), and spleen were sampled before the combination vaccine (Day 0) and 6 weeks after vaccination (wpv 6), and histological changes were observed through H&E staining. At Day 0, no lesions were identified in any tissue, and normal tissue structures were observed (Figs. 24, 25, 26).

[0190] Six weeks after vaccination (wpv 6), no lesions were found in the liver or spleen tissues. No lesions were observed in the intraperitoneal injection tissue (ip), and only one of the three animals in the Mix (1) group showed microscopic cell infiltration, while histologically it was within the normal range (Figs. 27, 28, 29).

[0191] In conclusion, no lesions were found in the liver and spleen, and localized cell infiltration was observed in the Mix (1) group, but this is presumed to be a phenomenon related to high antigen concentration.

[0193] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0195] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC16474BP Date of Deposit: 2025-09-10 Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC16475BP Date of Deposit: 2025-09-10 Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC16476BP Date of Deposit: 2025-09-10

Claims

Claim 1 (1) inactivated strain Vibrio anguillarum 23FBVib0271 deposited under accession number KCTC16476BP; (2) inactivated strain Aeromonas salmonisida subspecies salmonisida 17FBASa0016 deposited under accession number KCTC16475BP; and (3) inactivated strain Aeromonas salmonisida subspecies masoucida deposited under accession number KCTC16474BP A mixed vaccine composition for preventing vibriosis, typical carbuncles, and atypical carbuncles in salmonid fish, comprising the 23FBAer0174 strain as an antigen. Claim 2 In claim 1, the inactivated Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain and the inactivated Aeromonas salmonisida subspecies masoucida included in the mixed vaccine composition A mixed vaccine composition characterized in that the concentration of the 23FBAer0174 strain is 5 to 20 times the concentration of the independently inactivated Vibrio Anguillarum 23FBVib0271 strain. Claim 3 In claim 1, the antigen concentration of the inactivated Vibrio anguillarum 23FBVib0271 strain included in the mixed vaccine composition is 0.5 to 20 mg / ml; and the inactivated Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain and the inactivated Aeromonas salmonisida subspecies masoucida A mixed vaccine composition characterized by the antigen concentration of the 23FBAer0174 strain being 5 to 200 mg / ml each. Claim 4 A mixed vaccine composition according to claim 1, further comprising a pharmaceutically acceptable adjuvant. Claim 5 In paragraph 1, the salmonid fish is an Atlantic salmon ( Salmo salar ), brown trout( Salmo trutta ), Adriatic trout( Salmo obtusirostris ), Marmorata trout( Salmo marmoratus ), soap trout( Salmo obtusirostris salonitana ), Ohrid trout( Salmo letnica ), Serbian trout( Salmo farioides ), cream trout( Salmo carpio ), Sardinian trout( Salmo cettii ), Armenian trout( Salmo ischchan ), rainbow trout( Oncorhynchus mykiss ), Cherry Salmon( Oncorhynchus masou ), silver salmon( Oncorhynchus kisutch ), sockeye salmon( Oncorhynchus nerka King salmon (Oncorhynchus tshawytscha ), Pink Salmon( Oncorhynchus gorbuscha ), Chamu salmon ( Oncorhynchus keta ), Arctic humpback salmon( Salvelinus alpinus ) and lake trout( Salvelinus namaycush A mixed vaccine composition characterized by being one or more selected from the group consisting of ). Claim 6 a) Vibrio anguillarum 23FBVib0271 strain deposited under accession number KCTC16476BP; Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain deposited under accession number KCTC16475BP; and Aeromonas salmonisida subspecies masoucidase deposited under accession number KCTC16474BP A method for preparing a mixed vaccine composition for preventing Vibrio disease, typical carbuncles, and atypical carbuncles in salmonid fish, comprising: a step of inactivating a strain of 23FBAer0174; and b) a step of mixing the inactivated strain of step a). Claim 7 A method for preparing a mixed vaccine composition according to claim 6, characterized in that the inactivation is performed by formalin treatment, freezing, heating, or ultraviolet irradiation. Claim 8 In claim 6, step b) comprises in which the antigen concentration of the inactivated Vibrio anguillarum 23FBVib0271 strain included in the mixed vaccine composition is 0.5 to 20 mg / ml, and the inactivated Aeromonas salmonisida subspecies salmonisida 17FBASa0016 strain and the inactivated Aeromonas salmonisida subspecies masoucida A method for preparing a mixed vaccine composition characterized by mixing the antigen concentrations of the 23FBAer0174 strain to be 5 to 200 mg / ml each. Claim 9 A method for preventing vibriosis, typical carbuncles, and atypical carbuncles in salmonid fish, comprising the step of administering a mixed vaccine composition according to any one of claims 1 to 5 to a fish. Claim 10 In paragraph 9, the above salmonid fish is Atlantic salmon ( Salmo salar ), brown trout( Salmo trutta ), Adriatic trout( Salmo obtusirostris ), Marmorata trout( Salmo marmoratus ), soap trout( Salmo obtusirostris salonitana ), Ohrid trout( Salmo letnica ), Serbian trout( Salmo farioides ), cream trout( Salmo carpio ), Sardinian trout( Salmo cettii ), Armenian trout( Salmo ischchan ), rainbow trout( Oncorhynchus mykiss ), Cherry Salmon( Oncorhynchus masou ), silver salmon( Oncorhynchus kisutch ), sockeye salmon( Oncorhynchus nerka King salmon (Oncorhynchus tshawytscha ), Pink Salmon( Oncorhynchus gorbuscha ), Chamu salmon ( Oncorhynchus keta ), Arctic humpback salmon( Salvelinus alpinus ) and lake trout( Salvelinus namaycush A method for preventing Vibrio disease, typical carbuncles, and atypical carbuncles in salmonid fish, characterized by being one or more selected from a group consisting of ). Claim 11 A method for preventing Vibrio disease, typical ulcer disease and atypical ulcer disease in salmonid fish, characterized in that, in claim 9, the administration is intraperitoneal administration, intramuscular administration, or subcutaneous administration.

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