Inactivated vaccine preparation and method for preventing infectious disease

Abstract

[Problem] To isolate and identify novel Lactococcus gasseri, and to provide an effective means for preventing diseases in which said Lactococcus gasseri is used as a pathogenic bacterium. [Solution] Provided is an inactivated vaccine preparation and the like against fish streptococcosis that uses Lactococcus gasseri as a pathogenic bacterium, said inactivated vaccine preparation containing inactivated cells of Lactococcus gasseri that are negative in agglutination against type I serum and type II serum in a viable state. It is possible to effectively prevent the occurrence, propagation, and propagation of novel streptococcosis that cannot be prevented by conventional inactivated vaccines against Lactococcus gasseri.

Description

Technical Field

[0001] The present invention relates to an inactivated vaccine preparation for fish streptococcal disease caused by a novel serotype of Lactococcus garvieae (scientific name "Lactococcus garvieae"; the same shall apply hereinafter), a method for preventing fish streptococcal disease, a method for producing an inactivated vaccine preparation, and the like. More specifically, the present invention relates to an inactivated vaccine preparation containing inactivated Lactococcus garvieae cells that are negative for agglutination against type I and type II sera in a live state, a method for preventing fish streptococcal disease, and a method for producing an inactivated vaccine preparation. Background Art

[0002] Streptococcal disease in fish is a common disease, particularly in farmed fish, and can cause significant economic losses. Streptococcal disease in fish includes alpha-hemolytic streptococcal disease, caused by Lactococcus garvae, and beta-hemolytic streptococcal disease, caused by Streptococcus iniae (scientific name: Streptococcus iniae).

[0003] Among them, fish streptococcosis, caused by Lactococcus gasseri, is common in amberjacks such as yellowtail, greater amberjack, and yellowtail amberjack, but also occurs in marine fish such as red sea bream, blood sea bream, and striped trevally, as well as eels and rainbow trout. In amberjacks, symptoms include cloudy / bulging eyes, deformed trunks, redness of the inner gill cover, pericarditis, and water sluicing, with the disease becoming more severe during seasons with high water temperatures.

[0004] Traditionally, when streptococcal disease, caused by Lactococcus garvais, broke out on farms, the disease was treated by fasting the animals for at least a week to quell the outbreak and by using antibiotics such as erythromycin. However, the use of antibiotics, in particular, raises concerns about the emergence of drug-resistant bacteria and the risk of contamination in food. Consequently, vaccines have been developed in recent years as a preventative measure against this disease and are now commercially available.

[0005] Currently, as vaccine preparations for Lactococcus garvais targeting fish, single vaccine preparations, two-way mixed vaccine preparations, and three-way mixed vaccine preparations are used, such as inactivated vaccines inactivated with formalin, inactivated vaccines inactivated and concentrated with formalin, and inactivated vaccines in which a culture solution is enzyme-treated and then inactivated with formalin.

[0006] It is known that Lactococcus garvei has two serotypes: KG- and KG+. KG- is a strain that agglutinates with anti-KG- serum but not with anti-KG+ serum, possesses a capsule, and is highly pathogenic. Meanwhile, KG+ is a strain that agglutinates with both anti-KG- and anti-KG+ serum, lacks a capsule, and is less pathogenic than KG- (see Non-Patent Documents 1 and 2).

[0007] In addition, in recent years, a bacterial strain that does not agglutinate the anti-KG-type serum used for diagnosis and whose serotype is non-KG-type, non-KG+ type has been isolated from farmed fish etc. (with reference to patent documentation 1), and its infection has been continuously spreading in fish farms of aquaculture yellowtail etc., and has become a problem. Therefore, at present, the lactococcus gasseri that agglutinates the diagnostic antiserum in the past is classified as type I, and the bacterial strain that does not agglutinate is classified as type II (with reference to non-patent documentation 3). That is, type I is the KG-type bacterial strain and KG+ type bacterial strain in the past, and type II is the non-KG-type, non-KG+ type bacterial strain that appeared in recent years. Type II is diagnosed by having no agglutination to the diagnostic antiserum (anti-KG-type serum) in the past and having agglutination to the antiserum made with type II bacterial strain.

[0008] As fish vaccines related to Lactococcus garvei, for example, Patent Document 1 discloses a vaccine formulation containing inactivated cells of non-KG- and non-KG+ Lactococcus garvei. Patent Document 2 discloses a vaccine for fish enterococcal disease containing inactivated cells of a strain of Lactococcus garvei characterized by an extremely thin or absent podocyte. Patent Document 3 discloses a vaccine for the prevention and treatment of streptococcal disease in fish using a novel strain. Furthermore, Non-Patent Document 4 describes a method for distinguishing between types I and II using PCR.

[0009] Patent Document 1:

[0010] Japanese Patent No. 6355512

[0011] Patent Document 2:

[0012] Japanese Patent Application Laid-Open No. 11-332558

[0013] Patent Document 3:

[0014] Japanese Patent Application Laid-Open No. 2001-103961

[0015] Non-patent document 1:

[0016] Yoshida,T.,Eshima,T.,Wada,Y.,Yamada,Y.,Kakizaki,E.,Sakai,M.,Kitao,T.and Inglis,V.(1996) "Phenotypic variation associated with an anti-phagocyticfactor in the bacterial fish pathogen Enterococcus seriolicida." Dis AquatOrgan 25,81-86.

[0017] Non-patent document 2:

[0018] M.Kawanishi, T.Yoshida, M.Kijima, K.Yagyu, T.Nakai, S.Okada, A.Endo, M.Murakami, S.Suzuki and H.Morita; "Characterization of Lactococcus garvieaeisolated from radish and broccoli sprouts that exhibited a KG+phenotype, lack of virulence and absence of a capsule"; Letters in Applied Microbiology 44(2007)481-487.

[0019] Non-patent document 3:

[0020] Teruyoshi Yoshida, "Hydrococcal Infection"; Fish Pathology, 51(2), 44-48, 2016.6

[0021] Non-patent document 4:

[0022] Ohbayashi, K., Oinaka, D., Hoai, TD, Yoshida, T. and Nishiki, I.; "PCR-mediated Identification of the Newly Emerging Pathogen Lactococcus garvieaeSerotype II from Seriola quinqueradiata and S.dumerili; Fish Pathology, 52(1), 46-49, 2017.3 Summary of the Invention

[0023] Problems to be solved by the invention

[0024] The present invention aims to classify and identify new Lactococcus garvais and to provide effective preventive measures for diseases caused by this bacterium.

[0025] Solutions used to solve problems

[0026] The present inventors independently isolated a novel strain of Lactococcus gasseri from fish farms in Shizuoka and Miyazaki prefectures, Japan, from spotted scad, greater amberjack, and yellowtail that developed suspected alpha-streptococcal disease despite administration of conventional inactivated vaccines against Lactococcus gasseri. They discovered for the first time that this strain differed from known serotypes (types I and II). Furthermore, they successfully identified this strain as negative for agglutination against both type I and type II serotypes in its viable state. Furthermore, they successfully developed an inactivated vaccine against this novel streptococcal disease and verified its effectiveness.

[0027] Therefore, the present invention provides an inactivated vaccine preparation for fish streptococcal disease caused by Lactococcus garvieae, etc., which contains inactivated Lactococcus garvieae cells that are negative for agglutination against type I serum and type II serum in a live cell state.

[0028] For example, by administering this inactivated vaccine preparation to fish, etc., it is possible to effectively prevent the occurrence, transmission, and spread of fish streptococcal disease caused by Lactococcus garvei whose serotype does not belong to either of the known types I and II, that is, a new type of streptococcal disease that cannot be prevented by conventional inactivated vaccines against Lactococcus garvei.

[0029] In addition, for example, by administering a mixed inactivated vaccine preparation to fish, it may be possible to effectively prevent both known streptococcal diseases and new streptococcal diseases that are different from known streptococcal diseases at the same time. The mixed inactivated vaccine preparation contains: an inactivated vaccine containing inactivated bacterial bodies of Lactococcus gasseri that are negative for agglutination against type I serum and type II serum in a live bacterial state; and an inactivated vaccine containing inactivated bacterial bodies of Lactococcus gasseri serotype I (KG-type and / or KG+ type) and / or type II (non-KG-type, non-KG+ type).

[0030] Effects of the Invention

[0031] The present invention can prevent the occurrence, spread and expansion of fish streptococcal disease caused by Lactococcus garvae, which is negative for agglutination against type I serum and type II serum in a live bacterial state. DETAILED DESCRIPTION

[0032] About Lactococcus garvais LG21S and LG21M strains

[0033] The present inventors independently isolated a total of eight new strains of Lactococcus garvieae from farmed striped scad or greater amberjack suspected of developing alpha-streptococcal disease despite administration of conventional inactivated vaccines against Lactococcus garvieae at farms in Shizuoka and Miyazaki prefectures, Japan. These strains were successfully identified as negative for agglutination against both type I and type II sera in their viable state. One of these isolated and identified strains, isolated from farmed striped scad in Shizuoka Prefecture, was named Lactococcus garvieae LG21S, while the strain isolated from farmed greater amberjack in Miyazaki Prefecture was named Lactococcus garvieae LG21M.

[0034] The morphological properties of the LG21S and LG21M strains are consistent with those of typical Lactococcus garvae, exhibiting the shape of facultative anaerobic Gram-positive streptococci. They are non-motile and do not form spores. In culture, they form white colonies on commonly used meat extract agar plates, casein-soy peptone agar plates, and other media. They also proliferate by shaking culture in commonly used meat extract liquid media, casein-soy peptone liquid media, and other media. The culture temperature is preferably 20-30°C.

[0035] The biochemical properties of the LG21S and LG21M strains are shown below.

[0036] (1) Gram staining: Gram positive

[0037] (2) Nitrate reduction:

[0038] (3) Denitrification reaction:

[0039] (4) VP test: +

[0040] (5) Formation of indole:

[0041] (6) Generation of hydrogen sulfide:

[0042] (7) Utilization of citric acid:

[0043] (8) Pigment production:

[0044] (9) Urease:

[0045] (10) Oxidase:

[0046] (11) Alanine-phenylalanyl-proline arylamidase activity: +

[0047] (12) Pyroglutamate arylamidase activity:

[0048] (13) N-acetyl-β-glucosaminidase activity: +

[0049] (14) Glycyl-tryptophan-arylamidase activity:

[0050] (15) Growth range: pH 4.5-9.5, temperature 10-45°C

[0051] (16) Attitude towards oxygen: Facultative anaerobic

[0052] (17) Availability of carbon sources: D-ribose: +, D-mannitol: +, D-sorbitol: -, lactose: -, D-trehalose: +, D-raffinose: -, sucrose: -, L-arabinose: -, D-arabitol: -, cyclodextrin: +, glycogen: -, pullulan: -, maltose: +, D-melibiose: -, melezitose: -, tagatose: +

[0053] (18) Decomposition of carbohydrates: β-glycosidase activity: +, β-glucuronidase activity: +, β-galactosidase activity: -, α-galactosidase activity: -, β-mannosidase activity: -

[0054] (19) Decomposition of hippuric acid:

[0055] (20) Decomposition of arginine:

[0056] (21) Hemolytic: α hemolytic type

[0057] (22) Anti-KG-antiserum:

[0058] (23) Anti-KG+ antiserum:

[0059] The LG21S and LG21M strains of Lactococcus gasseri have been deposited as patent microorganisms (depository institution: National Institute of Technology for Product Evaluation, Japan Patent Organism Depository, location: 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan, deposit number: NITE BP-03560 and NITE BP-03561, acceptance date: November 19, 2021, strains collected from Japan).

[0060] It should be noted that the present invention is not narrowly limited to the use of the LG21S or LG21M strains as long as inactivation can effectively prevent new streptococcal infections caused by Lactococcus garvais, which is negative for agglutination against type I and type II sera in a live state.

[0061] <Regarding the Lactococcus gasseri strain of the present invention>

[0062] The present invention includes all Lactococcus garvais strains that are negative for agglutination against type I serum and type II serum in a viable state.

[0063] The bacterial cell of the present invention is a Lactococcus garvei that is negative for agglutination against type I serum and type II serum in a viable state, that is, a novel Lactococcus garvei that does not belong to either type I or type II. For example, it can be isolated and used from fish that have developed α-streptococcal disease even after being administered with a conventional inactivated vaccine against Lactococcus garvei. The isolated bacteria can be cultured and proliferated using a known solid culture medium, liquid culture medium, such as a meat extract agar plate medium, a casein-soybean mixed peptone agar plate medium, a meat extract liquid culture medium, a casein-soybean mixed peptone liquid culture medium, etc. It should be noted that the "viable bacterial state" in the present invention refers to a state in which the bacteria are alive or a state in which the three-dimensional shape of the bacterial antigen is maintained as when the bacteria are alive.

[0064] The Lactococcus garvei of the present invention includes bacteria that are negative for agglutination against type I and type II sera in the live state, as well as heat-killed bacteria that are positive for agglutination against type I sera. The fact that the bacteria are negative for agglutination against type I sera in the live state but positive for agglutination against type I sera after heat treatment suggests that, due to mutation, the recognition site for type I sera is covered by a capsule-like structure in the live state, and that this structure is removed by heat treatment, allowing the anti-type I sera to reach the recognition site. This may also support the reason why previous vaccines against type I or type II Lactococcus garvei are ineffective against novel streptococcal diseases.

[0065] In addition to the aforementioned Lactococcus garvei strains, the present invention also includes heat-killed strains that exhibit positive agglutination against KG- and KG+ sera. In this case, the causative agent of the novel streptococcal disease may be a mutant strain of type I, KG+ Lactococcus garvei.

[0066] The Lactococcus garvei of the present invention broadly encompasses bacteria containing the sequence described in SEQ ID NO: 1 in or near the glxR coding region of their genome. Compared to the base sequences of conventional type I or type II Lactococcus garvei, the sequence described in SEQ ID NO: 1 exhibits mutations at three bases (a mutation from adenine to cytosine at position 62 in SEQ ID NO: 1, a deletion between bases 118 and 119, and a deletion between bases 141 and 142). These mutations may contribute to the changes in the serological characteristics of the novel Lactococcus garvei compared to conventional Lactococcus garvei.

[0067] The LG21S strain or the LG21M strain is suitable as the bacterial cell of Lactococcus garvais used in the present invention because it can be used as a vaccine by inactivation and has high efficacy as a vaccine even without concentration or enzyme treatment.

[0068] <Regarding the inactivated vaccine preparation of the present invention>

[0069] The present invention encompasses all inactivated vaccine formulations for fish streptococcal disease caused by Lactococcus garvieae, comprising any of the above-mentioned inactivated bacterial cells. Furthermore, the present invention is not narrowly limited to those containing inactivated bacterial cells as active ingredients and broadly encompasses, for example, inactivated vaccine formulations containing, as active ingredients, bacterial cells and bacterial solutions obtained by inactivating a culture of Lactococcus garvieae that is negative for agglutination against type I and type II sera in a live state. In other words, inactivated bacterial cells and culture solutions are used without separation, or are concentrated without separation, thereby containing inactivated bacterial cells and bacterial components other than the inactivated bacterial cells.

[0070] Inactivated bacteria obtained by inactivating any of the novel Lactococcus gasseri strains, for example, a Lactococcus gasseri strain that is negative for agglutination against type I and type II sera in a live cell state, are useful as inactivated vaccine preparations for preventing novel fish streptococcal diseases.

[0071] As the inactivated bacterial cell, for example, one obtained by culturing and growing any of the aforementioned novel Lactococcus garvais strains and then inactivating the culture solution can be used.

[0072] Inactivation of bacteria can be achieved, for example, by subjecting the culture solution to physical treatment (ultraviolet irradiation, X-ray irradiation, heat treatment, ultrasonic treatment, etc.), chemical treatment (treatment with organic solvents such as formalin and chloroform, acid treatment with weak acids such as acetic acid, treatment with alcohol, chlorine, mercury, etc.), etc.

[0073] For example, formalin inactivation can be achieved by adding formalin to a culture solution at a volume concentration of 0.001 to 2.0%, more preferably 0.01 to 1.0%, and sensitizing the culture solution at 4 to 30°C for 1 to 10 days. For example, the inactivated cells can be washed with a buffer solution to remove the inactivating agent, such as formalin, or neutralized by adding a neutralizing agent to the inactivated cells. Alternatively, the inactivated cells can be recovered by membrane filtration, centrifugation, or the culture solution can be concentrated without separating the cells from the culture solution.

[0074] The amount of inactivated bacteria contained in the inactivated vaccine preparation is not particularly limited. For example, the amount of bacteria before inactivation is preferably 10 3 ~10 11 The range of CFU / mL is more preferably 10 7 ~1011 The range of CFU / mL.

[0075] The inactivated vaccine preparation of the present invention contains an adjuvant, that is, it can be a preparation containing at least the above-mentioned inactivated bacterial cells and an adjuvant as active ingredients.

[0076] Adjuvants that can be used are widely known. For example, animal oils (such as squalene) or their hydrogenated oils, vegetable oils (such as palm oil and castor oil) or their hydrogenated oils; oil-based adjuvants including dehydrated mannitol oleate, liquid paraffin, polybutene, caprylic acid, oleic acid, and higher fatty acid esters; water-soluble adjuvants such as PCPP, saponin, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymers, methacrylic acid copolymers, maleic anhydride copolymers, alkenyl derivative polymers, oil-in-water emulsions, and cationic lipids containing quaternary ammonium salts; precipitating adjuvants such as aluminum hydroxide (alum) and sodium hydroxide; microbial toxin components such as cholera toxin and Escherichia coli pyrogenic toxin; and bentonite, muramyl dipeptide derivatives, and interleukins. Mixtures of these may also be used.

[0077] The present invention may include a mixed inactivated vaccine formulation comprising: an inactivated vaccine comprising the aforementioned inactivated vaccine, for example, an inactivated vaccine comprising inactivated bacterial cells of Lactococcus garrisonii that are negatively agglutinating against type I and type II sera in a live state; and an inactivated vaccine comprising inactivated bacterial cells of type I (KG-type and / or KG+ type) and / or type II (non-KG-type, non-KG+ type) Lactococcus garrisonii. Administration of this mixed inactivated vaccine formulation to fish may effectively prevent both known streptococcal diseases and novel streptococcal diseases that differ from known streptococcal diseases.

[0078] Alternatively, it may be a mixed vaccine preparation containing, in addition to an inactivated vaccine containing inactivated bacteria of Lactococcus gasseri that is negative for agglutination against type I serum and type II serum in a live bacterial state, or a mixed inactivated vaccine containing the inactivated vaccine and inactivated bacteria of type I (KG-type and / or KG+ type) and / or type II (non-KG-type, non-KG+ type) Lactococcus gasseri, any one or more vaccines for other diseases, such as an inactivated vaccine for beta-hemolytic streptococcal disease, an inactivated vaccine for vibriosis, an inactivated vaccine for iridovirus disease, an inactivated vaccine for sarcoidosis, an inactivated vaccine for dysgalactiae Streptococcus infection, and the like.

[0079] Furthermore, a buffer, an isotonic agent, a pain-relieving agent, an antiseptic, an antibacterial agent, an antioxidant, and the like may be appropriately added depending on the purpose and application.

[0080] Preferred examples of the buffer include buffers such as phosphate, acetate, carbonate, and citrate.

[0081] Preferred examples of the isotonic agent include sodium chloride, glycerol, and D-mannitol.

[0082] Preferred examples of the pain-relieving agent include benzyl alcohol.

[0083] Preferred examples of drugs for the purpose of preservation include various preservatives such as thimerosal, parabens, phenoxyethanol, chlorobutanol, benzyl alcohol, phenylethyl alcohol, dehydroacetic acid, and sorbic acid, antibiotics, and synthetic antimicrobial agents.

[0084] Preferred examples of antioxidants include sulfites and ascorbic acid.

[0085] In addition, the drug may contain auxiliary ingredients, such as light-absorbing pigments (riboflavin, adenine, adenosine, etc.) as preservation and efficacy aids, chelating agents / reducing agents for stabilization (vitamin C, citric acid, etc.), carbohydrates (sorbitol, lactose, mannitol, starch, sucrose, glucose, dextran, etc.), casein digests, various vitamins, etc.

[0086] The vaccine preparation may be formulated in any known form without particular limitation. For example, the vaccine may be used as a liquid formulation or may be freeze-dried and then mixed into feed.

[0087] <Regarding the method for producing the inactivated vaccine preparation of the present invention>

[0088] The present invention includes all methods for producing an inactivated vaccine preparation for fish streptococcal disease with Lactococcus garvei as the pathogenic bacteria, including the step of inactivating the cells of any of the above-mentioned Lactococcus garvei.

[0089] As described above, for example, by inactivating the cells of Lactococcus garvieae that is negative for agglutination against type I serum and type II serum in a live state, an inactivated vaccine preparation effective against a novel fish streptococcal disease in which Lactococcus garvieae that is negative for agglutination against type I serum and type II serum in a live state is the causative agent can be produced.

[0090] The inactivated vaccine preparation of the present invention can be prepared, for example, by growing live Lactococcus garvais cells that are negative for agglutination against type I and type II sera, followed by a step of inactivating the cells. Furthermore, after the inactivation step, a step such as adding an adjuvant to the inactivated cells can be appropriately added.

[0091] The bacterial cells used and the method for inactivating the bacterial cells are as described above. In addition, the above-mentioned adjuvants, buffers, isotonic agents, pain-relieving agents, preservatives, antioxidants, etc. may be appropriately added according to the purpose and application.

[0092] <Use of bacteria for the production of inactivated vaccine preparations>

[0093] The present invention broadly includes the use of any of the above-mentioned bacteria for producing an inactivated vaccine preparation against streptococcal disease with Lactococcus gordonii as the causative agent.

[0094] For example, in order to produce an inactivated vaccine preparation for streptococcal disease in which the causative agent is Lactococcus garvae which is negative for agglutination against type I serum and type II serum in a live state, the above-mentioned Lactococcus garvae which is negative for agglutination against type I serum and type II serum in a live state can be widely used.

[0095] <Preventive Method for Fish Streptococcal Disease of the Present Invention>

[0096] The invention broadly includes a method for preventing fish streptococcal disease in which Lactococcus garvais is the causative agent, which comprises administering the above-mentioned inactivated vaccine preparation or the above-mentioned mixed inactivated vaccine preparation.

[0097] By administering the above-mentioned inactivated vaccine formulation to fish, it is possible to effectively prevent the occurrence, spread, and spread of new types of fish streptococcal diseases that cannot be prevented by conventional inactivated vaccines. Furthermore, by administering the above-mentioned mixed inactivated vaccine formulation to fish, it is possible to effectively prevent both known streptococcal diseases and new types of streptococcal diseases that are different from known streptococcal diseases.

[0098] Examples of fish that can be used include fish that are susceptible to streptococcal disease caused by Lactococcus garvall, such as yellowtail fish (seriformes, greater amberjack, yellowtail amberjack, etc.), red sea bream, blood sea bream, flatfish, striped trevally, bamboo bream, and trevally. Fish, mackerel, tuna and other sea fish, eel, rainbow trout, etc.

[0099] Examples of methods for administering inactivated vaccine preparations include injection, dipping, and oral administration.

[0100] In the case of injection, for example, 0.05 to 3.0 mL of the inactivated bacterial cell volume is adjusted to 10 3 ~10 11 CFU / mL range of inactivated vaccine preparations. That is, the amount of bacteria before inactivation is 10 3 ~10 11 An inactivated vaccine preparation having a CFU / mL and a single administration dose of 0.05 to 3.0 mL, administered intramuscularly or intraperitoneally at this dose, is effective against novel fish streptococcal diseases that cannot be prevented by conventional inactivated vaccines against Lactococcus garvais.

[0101] In the case of the immersion method, for example, the target fish is adjusted to contain 10 3 ~10 11CFU / mL range of inactivated vaccine preparations for 0.05 to 48 hours. 3 ~10 11 An inactivated vaccine preparation with a CFU / mL and a immersion time of 0.05 to 48 hours is effective for preventing a new type of fish streptococcal disease that cannot be prevented by conventional inactivated vaccines against Lactococcus garvais.

[0102] In the case of oral administration, for example, the fish are allowed to freely ingest a mixture of the bacteria and the amount of bacteria before inactivation is adjusted to 10 3 ~10 11 CFU / mL range of inactivated vaccine preparations and continuous administration for 1 to 20 days. 3 ~10 11 CFU / mL, and an inactivated vaccine preparation administered orally for 1 to 20 consecutive days is effective in preventing new streptococcal diseases that cannot be prevented by conventional inactivated vaccines against Lactococcus garvei.

[0103] Among them, intraperitoneal administration by injection is most preferred because of its high infection prevention effect and long duration of immunity.

[0104] Regarding the frequency of administration of the inactivated vaccine preparation, a single administration may be sufficient as long as the effect persists, or multiple administrations may be performed, for example, at intervals of 7 to 365 days, depending on the size of the target fish, the degree of vaccine efficacy, etc. Furthermore, the inactivated vaccine preparation may be administered to the target fish by combining multiple administration methods as appropriate.

[0105] [Example 1]

[0106] In Example 1, attempts were made to isolate and identify the pathogenic bacteria from striped scad and greater amberjack suspected of developing α-streptococcal disease even after administration of a conventional mixed inactivated vaccine against Lactococcus garrisons types I and II.

[0107] From around July 2021, at the striped scad farms in Shizuoka Prefecture, Japan, and the striped scad and greater amberjack farms in Miyazaki Prefecture, Japan, several individuals suspected of having alpha-hemolytic streptococcal disease appeared in the farmed fish that had been administered the previous mixed inactivated vaccine against L. garvieae type I and type II. Therefore, six of them were fished out and used as specimens.

[0108] Among the autopsy findings of each subject, obvious findings during external examination included cloudiness or redness of the eyeballs, redness around the eyeballs, redness and ulceration at the base of the tail fin, defects in the dorsal and pectoral fins, and redness of the pectoral, pelvic, and anal fins. Internal findings included redness of the inner side of the gill cover, discoloration of the gills, pericarditis, and splenomegaly.

[0109] The kidney, spleen, or brain of the subject was punctured and streaked onto SCDb agar (a culture medium consisting of casein-soybean peptone broth supplemented with agar). The culture was then incubated at 25°C for 24 hours. A single colony was then picked and inoculated onto a fresh plate of SCDb agar for single colony isolation. This procedure resulted in the isolation of eight strains of bacteria that form white S-shaped colonies: six strains from farmed striped scad in Shizuoka Prefecture, one strain from farmed striped scad in Miyazaki Prefecture, and one strain from farmed greater amberjack in Miyazaki Prefecture.

[0110] Microscopic examination of the isolated bacteria revealed that all of the isolated bacteria had the same morphology as the known L. garvieae gram-positive streptococci.

[0111] The isolated bacteria were inoculated onto Columbia 5% sheep blood agar and cultured. The results showed that all the isolated strains formed an incomplete hemolytic ring similar to the known L. garvieae, indicating α-hemolytic activity.

[0112] For the isolates, a slide agglutination test was performed in a live bacteria state. Among the isolates, one strain isolated from farmed striped scad in Shizuoka Prefecture, one strain isolated from farmed greater amberjack in Miyazaki Prefecture, and five strains as comparative examples, type I·KG-type (KS-7M strain), type I·KG+ type (YT-3 strain), and type II (LG13E strain), were picked out and suspended in diagnostic rabbit antiserum. As anti-type I sera in the diagnostic rabbit antiserum, three types of anti-KG-type serum, anti-KG+ type serum, and anti-type II serum (anti-non-KG- / KG+ serum) were used. The results are shown in Table 1. As shown in Table 1, the agglutination of any isolate to any antiserum was negative.

[0113]

Table 1

[0114]

[0115] Next, a slide agglutination test was performed on the heat-killed isolates. Among the isolates, one strain isolated from farmed striped scad in Shizuoka Prefecture, one strain isolated from farmed greater amberjack in Miyazaki Prefecture, and four strains, type I·KG-type (KS-7M strain) and type I·KG+ type (YT-3 strain) as comparative examples, were picked up from the colonies and suspended, heated at 100°C for 5 minutes, and then suspended in diagnostic rabbit antiserum. As anti-type I sera in the diagnostic rabbit antiserum, three types of anti-KG-type serum, anti-KG+type serum, and anti-type II serum (anti-non-KG- / KG+ serum) were used. The results are shown in Table 2. As shown in Table 2, the two isolates had different agglutination conditions from those in the live bacterial state. When anti-type I serum was used, the agglutination was positive in both the anti-KG-type serum and the anti-KG+type serum. It should be noted that when anti-type II serum (anti-non-KG- / KG+ serum) was used, agglutination was negative.

[0116]

Table 2

[0117]

[0118] Genetic testing of the isolates was performed. Using the primers for L. garvieae type I / II discrimination described in Non-Patent Document 4, PCR was attempted to detect genes specific to type I or type II. The results showed that amplification of L. garvieae type I-specific genes was confirmed in all isolates, but no amplification of type II-specific genes was confirmed.

[0119] Drug sensitivity tests were performed, and the results showed that the bacteria isolated from the brain formed inhibition zones to erythromycin, doxycycline, thiamphenicol, florfenicol, and oxytetracycline.

[0120] Virus testing was performed. DNA was extracted from the spleen of the subjects and the detection of the red sea bream iridovirus (RSIV)-specific gene by real-time PCR was attempted. However, no amplification of the RSIV-specific gene was confirmed in any of the subjects.

[0121] Acid-fast bacteria testing was performed. The kidneys of each subject were punctured, streaked onto 1% Ogawa medium, and cultured at 28°C for 7 days to attempt bacterial isolation. No acid-fast bacteria were observed growing on the 1% Ogawa medium.

[0122] The isolated bacteria were tested for biochemical properties. Using the Gram-positive cocci identification kit "rapid ID32STREP (bioMérieux, Japan)," the test was conducted on a total of three strains, the same two used in the slide agglutination test and isolated from farmed striped scad in Miyazaki Prefecture. The results are shown in Tables 3 and 4. It should be noted that the results for the three strains tested were identical. Furthermore, the "positive rate" in Tables 3 and 4 represents the positive rate for known L. garvieae strains as described in the instructions provided with the kit.

[0123]

Table 3

[0124]

[0125]

Table 4

[0126]

[0127] The same biochemical properties tests were also conducted on L. garvieae type I, KG-type (KS-7M strain) and type II (LG13E strain) as comparative examples, and the results were compared. In comparisons with L. garvieae type I and KG-type (KS-7M strain), the strain tested negative for D-ribose and tagatose fermentation / oxidation ability, whereas all the isolated strains tested positive. In comparisons with L. garvieae type II (LG13E strain), the strain tested negative for βNAG, whereas all the isolated strains tested positive. It was difficult to determine whether the isolated strains tested positive or negative for lactose fermentation / oxidation ability. Regarding other items, L. garvieae type I, KG-type (KS-7M strain), type II (LG13E strain), and three isolated strains, a total of five strains, were identical.

[0128] As described above, the live bacteria showed negative agglutination against both L. garvieae type I sera (anti-KG- and anti-KG+ sera) and L. garvieae type II sera, while heat-killed bacteria showed positive agglutination against L. garvieae type I sera. This indicates that eight new strains, each with serologically distinct from previously identified types I and II, were successfully isolated and identified, including six from farmed striped scad in Shizuoka Prefecture, one from farmed striped scad in Miyazaki Prefecture, and one from farmed greater amberjack in Miyazaki Prefecture. Among these, the strain isolated from farmed striped scad in Shizuoka Prefecture was designated LG21S and deposited with the National Institute of Technology for Product Evaluation (NITEBP-03560). In addition, a strain isolated from aquacultured greater amberjack in Miyazaki Prefecture was designated as LG21M strain and deposited in the National Institute of Technology for Product Evaluation (NITE) Patent Organism Depository (Deposit Number: NITE BP-03561).

[0129] [Example 2]

[0130] In Example 2, the pathogenicity of the strain isolated in Example 1 was confirmed.

[0131] As test fish, 60 each of artificial yellowtail (imported in February 2021, with an average weight of about 25g), artificial greater amberjack (imported in February 2021, with an average weight of about 28g), and artificial striped trevally (imported in February 2021, with an average weight of about 29g) were prepared.

[0132] For the LG21S strain isolated in Example 1 and another strain (LC2115 strain) isolated from aquacultured striped scad in Shizuoka Prefecture, each strain stored frozen at -80°C was streaked on SCDb agar medium and cultured at 25°C for about 24 hours. The grown colonies were suspended in PBS and adjusted to McFaland No. 3-4. 1 mL of the suspension was inoculated into 9 mL of SCDb liquid medium and 10-fold serial dilutions were performed in the same manner to dilute to 10 -10 After incubation at 25°C for approximately 24 hours, select the sample at the dilution stage before the turbidity peak and dilute it with PBS to 10, 100, and 1,000 times, respectively, as the test bacterial solution.

[0133] For artificial yellowtail, artificial greater amberjack, and artificial striped scad, each group was divided into six groups of 10, with three groups each for inoculation with the LG21S strain and three groups for inoculation with the LC2115 strain. Under anesthesia, 0.1 mL of the bacterial solution prepared at three stages of 10-fold concentration was injected into the abdominal cavity of each fish. After injection, the fish were raised at 25°C for 14 days, and the mortality rate was compared.

[0134] As a result, the LG21S strain (1.3×10 5 ~10 7 CFU / fish) after 14 days, the cumulative mortality rate was 50, 80, and 90% for yellowtail, 100, 90, and 100% for greater amberjack and striped scad, respectively, from the lowest concentration. 5 ~10 7 The cumulative mortality after 14 days at the lowest concentration of 100 μg / L was 40%, 50%, and 100% for yellowtail, 90%, 100%, and 100% for greater amberjack, and 100% for striped trevally.

[0135] In the greater amberjack that survived the inoculation with each bacterial solution, characteristic lesions of α-hemolytic streptococcal disease were observed.

[0136] Bacteria re-isolated from the organs of the dead fish showed characteristics similar to those of the strains used for infection (LG21S or LC2115). Furthermore, slide agglutination tests on the re-isolated bacteria showed that, similar to the strains used for infection (LG21S or LC2115), agglutination was positive using anti-type I serum only when heat-treated.

[0137] In summary, this example shows that the strain isolated in Example 1 is highly pathogenic to various fish species and causes symptoms similar to those seen in various fish farms.

[0138] [Example 3]

[0139] In Example 3, an inactivated vaccine for alpha-streptococcal disease caused by a new serotype of L. garvieae as the causative agent was prepared and its efficacy as a vaccine was verified. The new serotype refers to a serotype that shows negative agglutination against either of L. garvieae type I sera (anti-KG-type sera and anti-KG+ type sera) and anti-L. garvieae type II sera in the live state, and shows positive agglutination against L. garvieae type I sera when heat-killed.

[0140] The preparation of the inactivated bacterial solution is carried out as follows. The new serotype LG21S strain, type I·KG-type (KS-7M strain), and type II (LG13E strain) are used as test strains. Each test strain stored frozen at -80°C is streaked on SCDb agar medium and cultured at 25°C for about 24 hours. The grown colonies are suspended in PBS and adjusted to about McFaland No. 3 to 4. 100 μL is inoculated into 100 mL of SCDb liquid culture medium and cultured at 25°C at about 90 rpm for about 24 hours. Formalin is added to the culture solution at 0.5 vol%, and the solution is sensitized by shaking for about 48 hours to prepare the inactivated bacterial solution. After each inactivated bacterial solution is centrifuged at 12,000G for 10 minutes at 4°C and the supernatant is removed, it is resuspended in PBS with 0.2 vol% formalin added, and the antigen amount is adjusted to be the same between strains to serve as the test antigen. It should be noted that the amount of antigen was adjusted to 1.6 × 10 9 CFU / mL.

[0141] As test fish, 144 each of artificial yellowtail (imported in June 2021, with an average weight of about 25g) and artificial greater amberjack (imported in June 2021, with an average weight of about 28g) were prepared.

[0142] Artificial yellowtail and greater amberjack were divided into six groups of 18 each. Under anesthesia, 0.1 mL of the test antigen was injected intraperitoneally into each group. These groups were designated as the LG21S, KS-7M, and LG13E strain-immunized groups, respectively. Following injection, the fish were housed at 25°C for 14 days. A control group was also injected with PBS.

[0143] As the challenge strain, the LG21S strain was selected and prepared for challenge. The strain, which had been frozen at -80°C, was streaked onto SCDb agar medium and cultured at 25°C for approximately 24 hours. The grown colonies were suspended in PBS and adjusted to McFaland No. 3-4. 1 mL of the suspension was inoculated into 9 mL of SCDb liquid medium and serially diluted 10-fold until 10 -10 After static culture at 25°C for approximately 24 hours, select a sample from the dilution stage immediately before the turbidity peak and dilute it 100-fold or 1,000-fold with PBS. The resulting bacterial solution is used as the challenge bacterial solution.

[0144] Fourteen days after immunization, 0.1 mL of the challenge solution was injected into the abdominal cavity of each fish under anesthesia. After challenge, the fish were kept in a water tank set at 25°C for 14 days, and the survival rate was compared.

[0145] The results are shown in Figures 1 to 4 . Figure 1To show that artificial yellowtail was immunized with inactivated bacteria of the new serotype of L. garvieae LG21S strain and then challenged with the LG21S strain at a low concentration (8.0×10 4 CFU / 0.1mL / bar), Figure 2 To show the effect of immunizing artificial yellowtail with inactivated bacteria of the new serotype of L. garvieae LG21S strain and then challenging with a high concentration of LG21S strain (8.0×10 5 CFU / 0.1mL / bar), Figure 3 To show that artificial greater amberjack was immunized with inactivated bacteria of the new serotype L. garvieae LG21S strain and then challenged with the LG21S strain at a low concentration (8.0×10 4 CFU / 0.1mL / bar), Figure 4 To show that artificial greater amberjack was immunized with inactivated bacteria of the new serotype L. garvieae LG21S strain and then challenged with a high concentration of LG21S strain (8.0×10 5 CFU / 0.1 mL / bar). In each figure, the horizontal axis (days post-challenge) represents the number of days after challenge, and the vertical axis (survival rate, unit: %) represents the survival rate after challenge. In this graph, the "LG21S" line represents the survival rate when immunized with inactivated bacteria of the new serotype L. garvieae LG21S strain; the "KS-7M" line represents the survival rate when immunized with inactivated bacteria of L. garvieae type I / KG-type (KS-7M strain) as a comparative example; the "LG13E" line represents the survival rate when immunized with inactivated bacteria of L. garvieae type II (LG13E strain) as a comparative example; and the "control" line represents the survival rate when PBS was administered as a control.

[0146] Regarding the immunization of artificial yellowtail, Figure 1 As shown in the figure, the artificial yellowtail was challenged with the LG21S strain at a low concentration (8.0×10 4 CFU / 0.1mL / fish, intraperitoneal injection), the survival rate 14 days after the attack was 78% for the control group, 78% for the KS-7M immunized group, 100% for the LG13E immunized group, and 100% for the LG21S immunized group. No significant difference was found between the control group and the immunized groups. However, among the fish that survived 14 days after the attack, several individuals that did not die but showed signs of α-hemolytic streptococcal disease were found in the control group, the KS-7M immunized group, and the LG13E immunized group. After subtracting these, the LC2113 immunized group showed a significantly higher normal rate than the control group. In addition, as Figure 2As shown, the LG21S strain was used to challenge artificial yellowtail with a high concentration (8.0×10 5 CFU / 0.1mL / strip, intraperitoneal injection), the survival rate 14 days after the control group was 67%, while the KS-7M strain immunization group was 83%, the LG13E strain immunization group was 83%, and the LG21S strain immunization group was 100%. The LG21S strain immunization group showed a significantly higher survival rate than the control group (p<0.05, Fisher direct probability calculation method, one-sided test).

[0147] Regarding the immunization of artificially raised greater amberjack, Figure 3 As shown, the LG21S strain was used to challenge the artificial greater amberjack with a low concentration (8.0×10 4 The survival rate 14 days after the injection of 100 μg of CFU / 0.1 mL / strip, intraperitoneal injection) was 0% in the control group, 11% in the KS-7M group, 0% in the LG13E group, and 100% in the LG21S group. The LG21S group showed a significantly higher survival rate than the control group (p < 0.05, Fisher's direct probability calculation method, one-sided test). Figure 4 As shown, the results of the experiment on artificial greater amberjack larvae challenged with the LG21S strain at a high concentration (8.0×10 5 The survival rate 14 days after immunization with 100 μg / mL CFU / 0.1 mL / strip, intraperitoneal injection) was 0% for the control group, 0% for the KS-7M strain immunization group, 0% for the LG13E strain immunization group, and 100% for the LG21S strain immunization group. The LG21S strain immunization group showed a significantly higher survival rate than the control group.

[0148] In summary, the inactivated antigen of the new serotype LG21S strain, which has negative agglutination against any of the L. garvieae type I serum (anti-KG-type serum and anti-KG+ type serum) and anti-L. garvieae type II serum in the non-heated live bacteria state and positive agglutination against L. garvieae type I serum by heat-killed bacteria, showed high effectiveness against the attack of homologous strains, while the inactivated antigens of L. garvieae type I·KG-type (KS-7M strain) and L. garvieae type II (LG13E strain) could not defend against this attack. These results indicate that the LG21S strain is L. garvieae with novel antigenicity different from either serotype I or type II. They also demonstrate that an inactivated vaccine preparation comprising "L. garvieae that exhibits negative agglutination in the live state against either L. garvieae type I serum (anti-KG-type serum and anti-KG+ type serum) or anti-L. garvieae type II serum, and that exhibits positive agglutination in the heat-killed form against L. garvieae type I serum" is effective in preventing α-streptococcal disease in which this bacterium is the causative agent.

[0149] [Example 4]

[0150] In Example 4, the genetic homology between the eight new serotypes isolated in Example 1 and conventional strains was investigated.

[0151] 16S rRNA sequencing was performed on the standard L. garvieae strains JCM10343 and KS-7M, as well as the eight new serotypes isolated in Example 1, and sequence comparison was performed on the approximately 1,370 bp that could be deciphered. The results showed that the sequences were completely identical across the decipherable region, and no mutations were confirmed in the eight new serotypes.

[0152] For the eight new serotypes isolated in Example 1, the base sequence of the glxR coding region and the surrounding region was amplified by PCR using primers for L. garvieae type I / II discrimination described in Non-Patent Document 4. The PCR product was sequenced to obtain a decipherable 256 bp sequence. The base sequence of this region was identical among all eight strains. The obtained sequence is shown in SEQ ID NO: 1.

[0153] Sequencing of the PCR product of the KS-7M strain, used as a comparative example, was also performed using the same procedure, yielding a decipherable 258-bp sequence. The resulting sequence is shown in SEQ ID NO: 2. The sequence of the KS-7M strain obtained is identical to the base sequence of the corresponding region in the known reference strain Lg2 and the known ATCC 49156.

[0154] For the region amplified in this example, comparison was made with the sequence of the KS-7M strain (refer to sequence number 2), the known standard strain Lg2, and the known ATCC49156, and mutations were confirmed at three bases in the sequence of the new serotype (refer to sequence number 1) (position 62 in the sequence of sequence number 2 mutated from adenine to cytosine, position 119 of the sequence was deleted, and position 143 of the sequence was deleted).

[0155] [Example 5]

[0156] In Example 5, it was verified whether the inactivated vaccine prepared in Example 3 was also effective against the infection of striped scad.

[0157] As test fish, 60 artificially grown mullet (imported in February 2021, average weight 26 g) were prepared. Under anesthesia, 30 of them were intraperitoneally injected with 0.1 mL of the inactivated vaccine prepared in Example 3 to serve as the LG21S strain immunization group. The remaining 30 fish were intraperitoneally injected with 0.1 mL of PBS under the same conditions to serve as the control group. After injection, the fish were raised at 25°C for 14 days.

[0158] As in Example 3, the LG21S strain was selected as the challenge strain and diluted 100-fold or 1,000-fold with PBS by the same procedure as in Example 3 to prepare a bacterial solution for challenge.

[0159] Fourteen days after immunization, 0.1 mL of the challenge solution was injected into the abdominal cavity of each fish under anesthesia. After challenge, the fish were kept in a water tank set at 25°C for 14 days, and the survival rate was compared.

[0160] The results are shown in Figure 5 and Figure 6 . Figure 5 To show that the captive striped scad was immunized with inactivated bacteria of the new serotype of L. garvieae LG21S strain and then challenged with the LG21S strain at a low concentration (1.1×10 5 CFU / bar) of survival rate, Figure 6 To show that the captive striped scad was immunized with inactivated bacteria of the new serotype of L. garvieae LG21S and then challenged with a high concentration of LG21S (1.1×10 4 CFU / cell). In each figure, the horizontal axis (days post-challenge) represents the number of days after challenge, and the vertical axis (survival rate, unit: %) represents the survival rate after challenge. In this graph, the "LG21S" line represents the survival rate when immunized with inactivated bacteria of the new serotype L. garvieae LG21S strain, and the "control" line represents the survival rate when administered with PBS as a control.

[0161] like Figure 5 As shown, the LG21S strain was used to challenge the artificial striped scad with a low concentration (1.1×10 5 The survival rate 14 days after intraperitoneal injection of CFU / cell was 20% in the control group and 87% in the immunized group, and a significant difference was confirmed between the two groups (p < 0.05, Fisher's direct probability calculation method, one-sided test). Figure 6 As shown, the LG21S strain was used to challenge the artificial striped scad with a high concentration (1.1×10 4 The survival rates 14 days after intraperitoneal injection (CFU / cell) were 7% in the control group and 87% in the immunized group, with a significant difference between the two groups (p<0.05, Fisher's direct probability calculation method, one-sided test).

[0162] As described above, this example demonstrates that an inactivated vaccine preparation of "L. garvieae whose agglutination in the live state against any of the antisera including L. garvieae type I serum (anti-KG- type serum and anti-KG+ type serum) and anti-L. garvieae type II serum is negative, and whose heat-killed bacteria have agglutination positive against L. garvieae type I serum" is also effective in preventing α-streptococcal disease in striped scads, the causative agent of which is L. garvieae.

[0163] [Example 6]

[0164] In Example 6, it was confirmed that fish streptococcal disease caused by infection with the strain isolated in Example 1 is an infectious disease that cannot be prevented by conventional vaccines.

[0165] Immunization was performed on artificial yellowtail (imported in June 2021, average weight 26g) and artificial striped scad (imported in June 2021, average weight 29g) using the conventional vaccine preparation "Piscivac Injection 4 (Piscivac is a registered trademark)" according to the instructions and dosage (n = 18). As a positive control, the test antigen prepared using the strain isolated in Example 1 and the procedure described in Example 3 was administered instead. As a negative control, PBS was administered instead.

[0166] As in Example 3, LG21S strain was selected as the challenge strain to prepare the challenge bacterial solution. 14 days after immunization, 0.1 mL of the challenge bacterial solution was injected into the abdominal cavity of each fish under anesthesia for challenge (1.2×10 8 After challenge, the cells were cultured in aquaria set at 25°C for 14 days, and the survival rates were compared.

[0167] As a result, the survival rate was 0% for the negative control and 100% for the positive control. The survival rate of the group immunized with the conventional vaccine preparation was also 0%, similar to the negative control. This result confirms that the novel fish streptococcal disease identified in Examples 1 and 2 is an infectious disease that cannot be prevented by conventional vaccines.

[0168] [Example 7]

[0169] In Example 7, it was verified whether the culture supernatant of the test strain in Example 3 also had immunogenicity.

[0170] The sterilized solution (1.0×10 9 CFU / mL) was centrifuged at 4°C and 12,000G for 10 minutes, and the separated supernatant was passed through a 0.22μm filter to completely remove the bacteria, which was used as the supernatant antigen. In addition, the bacteria remaining after the supernatant was removed were suspended in PBS twice, washed by centrifugation, and resuspended in PBS supplemented with 0.2vol% formalin to obtain the bacterial antigen (1.0×10 9 CFU / mL).

[0171] Captive greater amberjack (imported in June 2021, average weight 41 g) were immunized by intraperitoneal injection of 0.1 mL of supernatant antigen or bacterial antigen, respectively, and then housed at 25°C for 14 days (n = 18). As a positive control, the inactivated bacterial solution prepared in Example 3 was administered instead, and as a negative control, PBS was administered instead.

[0172] As in Example 3, LG21S strain was selected as the challenge strain to prepare the challenge bacterial solution. 14 days after immunization, 0.1 mL of the challenge bacterial solution was injected into the abdominal cavity of each rat under anesthesia for challenge (9.7×10 5 After challenge, the mice were kept in a water tank at 25°C for 14 days, and the survival rates were compared.

[0173] The results showed that the survival rate was 0% for the negative control and 100% for the positive control. When the bacterial antigen was administered, the survival rate was 100%, the same as the positive control, while when the supernatant antigen was administered, the survival rate was 0%, the same as the negative control. These results demonstrate that the culture supernatant of the new serotype strain has low immunogenicity and suggest that using a concentrated bacterial culture stock solution can improve vaccine efficacy and production efficiency.

[0174] [Example 8]

[0175] In Example 8, an attempt was made to isolate the pathogenic bacteria isolated and identified in Examples 1 and 2 from yellowtail.

[0176] Initially, it was confirmed that the new α-hemolytic streptococcal disease shown in Example 1 mainly harms greater amberjack and striped scad, but in August 2022, the same suspected α-hemolytic streptococcal disease individuals were also found in a yellowtail farm in Miyazaki Prefecture, Japan, so attempts were made to isolate and identify the causative bacteria.

[0177] The kidneys, spleens, or brains of yellowtail specimens from the farm suspected of being infected were punctured, and isolation and culture attempts were made in the same manner as in Example 1. The strain, originating from yellowtail, was successfully isolated. Hemolytic activity tests and slide agglutination tests using live bacteria were performed on the isolates, and the results were similar to those of the LG21S and LG21M strains.

[0178] In addition, the results of the biochemical properties test of the isolated bacteria were the same as those in Example 1 (Tables 3 and 4). In addition, the PyrA (L-pyroglutamic acid-β-naphthamide, positive rate 74%) and βNAG (6-bromo-2-naphthyl-N-acetyl-β-D-aminoglucosidoside, positive rate 10%) items were also evaluated as negative, just like the LG21S strain.

[0179] This yellowtail isolate was cultured and adjusted using the same procedures as in Example 2. The test bacterial solution was diluted in PBS at three dilution levels of 10, 100, and 1,000. Under anesthesia, 0.1 mL of the three-step dilutions, adjusted to 10-fold, were injected into the abdominal cavity of artificial yellowtail, artificial greater amberjack, and artificial striped scad. After injection, the animals were housed at 25°C for 14 days, and mortality rates were compared (n = 10). As a comparative example, the inactivated bacterial solution of the LG21S strain prepared in Example 3 was injected instead.

[0180] The results showed that the cumulative mortality rate of cultured yellowtail fish injected with the strain isolated from yellowtail was 50% at a 1,000-fold dilution, 56% at a 100-fold dilution, and 80% at a 10-fold dilution 14 days after injection. In contrast, the mortality rate of cultured greater amberjack injected with the strain isolated from yellowtail was 56% at a 1,000-fold dilution, 80% at a 100-fold dilution, and 100% at a 10-fold dilution 14 days after injection. In contrast, the mortality rate of cultured greater amberjack injected with the strain isolated from yellowtail was 100% at a 1,000-fold dilution, 90% at a 100-fold dilution, and 100% at a 10-fold dilution 14 days after injection. In contrast, the mortality rate of cultured greater amberjack injected with the strain isolated from yellowtail was 100% at a 1,000-fold dilution, 80% at a 100-fold dilution, and 100% at a 10-fold dilution 14 days after injection. When farmed striped trevally were injected with the strain isolated from yellowtail, the cumulative mortality rate 14 days after injection was 80% at a 1,000-fold dilution, 70% at a 100-fold dilution, and 80% at a 10-fold dilution. When injected with the inactivated bacterial solution of the LG21S strain, the cumulative mortality rate was 70% at a 1,000-fold dilution, 80% at a 100-fold dilution, and 60% at a 10-fold dilution.

[0181] As described above, this new α-hemolytic streptococcal disease has been confirmed not only in greater amberjack and striped trevally, but also in yellowtail. The causative bacteria exhibit the same properties and pathogenicity as those isolated from greater amberjack and striped trevally. This suggests that the transmission, spread, and spread of this new α-hemolytic streptococcal disease may occur in yellowtail farms in the future, similar to that seen in greater amberjack and striped trevally. Furthermore, Example 3 demonstrates the effectiveness of the vaccine of the present invention in yellowtail, suggesting that the present invention is very useful for preventing this disease in yellowtail farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0182] Figure 1 :

[0183] This is a graph showing the survival rate when artificial yellowtail were immunized with killed bacteria of the new serotype L. garvieae LG21S strain in Example 3 and then challenged with a low concentration of the LG21S strain.

[0184] Figure 2 :

[0185] This is a graph showing the survival rate when artificial yellowtail were immunized with killed bacteria of the new serotype L. garvieae LG21S strain in Example 3 and then challenged with a high concentration of the LG21S strain.

[0186] Figure 3 :

[0187] This is a graph showing the survival rate when artificial greater amberjack were immunized with inactivated bacteria of the new serotype L. garvieae LG21S strain in Example 3 and then challenged with a low concentration of the LG21S strain.

[0188] Figure 4 :

[0189] This is a graph showing the survival rate when artificial greater amberjack were immunized with inactivated bacteria of the new serotype L. garvieae LG21S strain in Example 3 and then challenged with a high concentration of the LG21S strain.

[0190] Figure 5 :

[0191] This is a graph showing the survival rate when artificial striped scads were immunized with inactivated bacteria of the new serotype L. garvieae LG21S strain in Example 5 and then challenged with a low concentration of the LG21S strain.

[0192] Figure 6 :

[0193] This is a graph showing the survival rate when artificial striped scads were immunized with inactivated bacteria of the new serotype L. garvieae LG21S strain in Example 5 and then challenged with a high concentration of the LG21S strain.

Claims

1. An inactivated vaccine preparation for fish streptococcal disease caused by Lactococcus garvieae, comprising inactivated bacteria of Lactococcus garvieae (scientific name "Lactococcus garvieae"; the same shall apply hereinafter) which are negative for agglutination against type I and type II sera in a live cell state.

2. The inactivated vaccine preparation according to claim 1, wherein Contains heat-killed inactivated Lactococcus garvae that is positive for agglutination against type I serum.

3. The inactivated vaccine preparation according to claim 2, wherein A killed Lactococcus garvais cell containing the sequence described in SEQ ID NO: 1 in the glxR coding region and its vicinity in the genome.

4. A mixed inactivated vaccine preparation comprising: The inactivated vaccine according to claim 1; and An inactivated vaccine containing inactivated bacteria of Lactococcus garvais serotypes I and / or II.

5. A method for preventing fish streptococcal disease caused by Lactococcus garvais, comprising administering the inactivated vaccine preparation according to claim 1.

6. A method for producing an inactivated vaccine preparation for fish streptococcal disease caused by Lactococcus garvieae, comprising the step of inactivating live Lactococcus garvieae cells that are negative for agglutination against type I serum and type II serum.

7. A strain of Lactococcus garvais which is negative for agglutination against type I serum and type II serum in a viable state.

8. A Lactococcus garvais strain having an international deposit number of NITE BP-03560 or NITE BP-03561.

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