JP5962892B2 - Sea surface aquaculture fish vaccine preparation and infectious disease prevention method - Google Patents

Description

  The present invention relates to an inactivated vaccine preparation containing an inactivated cell of the bacterium against Streptococcus agalactiae infection in a sea surface cultured fish, a method for preventing Streptococcus agalactia infection in a sea surface cultured fish, and the like.

  Sea surface aquaculture is widespread in many marine fishes such as yellowtail, red sea bream, amberjack, flounder, striped horse mackerel, sea mackerel, flounder, etc. due to the fact that the yield can be increased and it can be stably supplied at a relatively low cost. .

  On the other hand, in the case of sea farming, compared to natural fish, the breeding density is high and the environmental conditions are likely to deteriorate, so infectious diseases are likely to occur and spread. In addition, due to the fact that sea temperature rise due to global climate change has been observed, and sea-cultured fish are often moved artificially from isolated areas, There is a possibility that diseases that did not become apparent will newly occur and spread in sea farmed fish.

  Vaccine administration is effective as one of the preventive measures against infectious diseases of sea surface cultured fish. Currently, as a vaccine for sea-cultured fish, α-hemolytic streptococcal disease inactivated vaccine (causal bacterium: Lactococcus garvieae), vibrio disease inactivated vaccine (causal bacterium: Vibrio anguillarum), and iridovirus infection inactivated Vaccine (causal virus: Red seabream iridovirus), tuberculosis inactivated vaccine (causal bacterium: Photobacterium damselae subsp piscicida), β-hemolytic streptococcal disease inactivated vaccine such as flounder (causal bacterium: Streptococcus iniae), Streptococcus dysgalactie infection Disease inactivated vaccine (causal bacteria: Streptococcus dysgalactiae) and the like have been put into practical use (see Patent Documents 1 to 5, etc.). The α-hemolytic streptococcal inactivated vaccine of the fish of the genus Buri is an oral or injection vaccine, the vibrio disease inactivated vaccine is an immersion or injection vaccine, and the others are all injection vaccines.

  In addition, Streptococcus phocae vaccine (Streptococcus phocae) vaccine, Edwardsiella tarda, Streptococcus iniae, inactivated mixed vaccine of Streptococcus parauberis, etc. have been reported as fish vaccines against streptococci (see Patent Documents 6 and 7).

  Here, Streptococcus agalactier will be described below as a related matter of the present invention.

  Streptococcus agalactier is also called group B streptococcus and is one of the causative bacteria such as bovine mastitis. In humans, it is resident in female genitals, etc., and it may infect newborns at the time of childbirth and become a pathogenic fungus for neonatal meningitis and neonatal sepsis. Many are β-hemolytic, but others are α-hemolytic and non-hemolytic (γ-hemolytic).

  Regarding fish, Streptococcus agalactiae infection of the freshwater farmed fish tilapia (Oreochromis aura, Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss) in a farm in Israel in 1984 in a Kuwaiti farm in 2000. Each has been reported (see Non-Patent Documents 1 to 3).

Further, a vaccine against fresh water aquaculture fish tilapia, which is composed of a completely sterilized cell of β-hemolytic group B streptococcus and a concentrated extract of a culture solution of β-hemolytic group B streptococcus ( (See Patent Document 8 and Non-Patent Document 4).
JP-A-8-231408 Japanese Patent Laid-Open No. 9-176043 JP 2006-1849 A International Publication WO2008 / 23453 JP 2007-326794 A International Publication WO2005 / 53716 JP 2008-50300 A International Publication WO2005 / 89447 Elder, A. et al, 1995. Experimental streptococcalmeningo-encephalitis in cultured fish. Vet. Microbiol. 43, 33-40. Duremdez, R. et al, 2004. Isolation of Streptococcus agalactiae fromcultured silver pomfret, Pampus argenteus (Euphrasen), in Kuwait.J. Fish Dis, 27, 307-310. Joyce J. Evans et al. First report of Streptococcus agalactiae and Lactococcus garvieae from a wild bottlenose dolphin (Tursiops truncates) .Journal of Wildlife Diseases, 42 (3), 2006, pp.561-569 Joyce J. Evans et al. Efficacy of Streptococcus agalactiae (group B) vaccine in tilapia (Oreochromis niloticus) by intraperitoneal and bathimmersion administration.Vaccine 22 (2004) 3769-3773.

  An object of the present invention is to isolate and identify a causative bacterium of a disease that has newly occurred in a sea surface cultured fish or the like, and to provide effective preventive means for the disease.

  The present inventors have isolated the causative bacteria of yellowtail that has developed symptoms such as cloudiness, necrosis, body bending, and skinnyness of unknown eyeballs at a farm in Kagoshima Prefecture, Japan, and the bacteria are Streptococcus agalactier and In addition to identification, we have successfully developed an inactivated vaccine.

  Therefore, the present invention provides an inactivated vaccine preparation containing inactivated cells of the bacterium against Streptococcus agalactier infection of sea surface cultured fish.

  Streptococcus agaractie infection, which is an infectious disease targeted by this inactivated vaccine preparation, may be widely spread and spread in the fish farms of the genus Buri.

  In addition, as mentioned above, rising seawater temperature due to global climate change has been observed, and sea surface aquaculture fish are often moved artificially from isolated areas. Streptococcus agaractie infections may also be widely spread to sea surface aquaculture fish other than the yellowtail.

  On the other hand, for example, by administering this inactivated vaccine preparation to sea surface cultured fish, it is possible to effectively prevent the occurrence, spread, and spread of Streptococcus agaractier infection in sea surface cultured fish.

  According to the present invention, it is possible to prevent the occurrence, spread, and spread of Streptococcus agaractie infection newly generated in sea surface cultured fish and the like.

<About Streptococcus agalactier SAG1001 strain>
The present inventors succeeded in isolating the causative bacteria from yellowtail that was found to be abnormal in a farm in Kagoshima Prefecture, Japan, and identified the bacteria as Streptococcus agalactier. No infectious diseases of the same bacteria have been confirmed in sea surface cultured fish near Japan, the infectious diseases of this species have not been reported in the fishes of the genus Buri, and the symptoms of field-developed fish have been reproduced in infection experiments with isolates From these facts, it was judged that this bacterium was a new strain of Streptococcus agalactier having pathogenicity to sea surface cultured fish, and the isolated strain was named SAG1001 strain.

  The morphological characteristics of SAG1001 strain form several linkages with facultative anaerobic gram-positive cocci. Has capsular cilia, no motility and no spore formation. As a culture property, a white colony is formed by using a meat extract agar plate medium generally used, a casein / soybean mixed peptone agar plate medium, or the like. In addition, it grows by shaking culture in a commonly used meat extract liquid medium, casein / soybean mixed peptone liquid medium, or the like. The culture temperature is preferably 25-30 ° C.

The biochemical properties of SAG1001 strain are shown below.
(1) Gram staining: Gram positive
(2) Reduction of nitrate: +
(3) Denitrification reaction: −
(4) MR test: +
(5) VP test: +
(6) Production of indole: −
(7) Production of hydrogen sulfide: −
(8) Starch hydrolysis:-
(9) Use of citric acid-Koser medium:-, Christensen medium:-
(10) Use of inorganic nitrogen source:-
(11) Production of pigment: −
(12) Urease:-
(13) Oxidase: −
(14) Catalase:-
(15) Range of growth: pH 4.5-9.5, temperature 10-40 ° C
(16) Attitude toward oxygen: facultative anaerobic
(17) OF test: F
(18) Presence or absence of acid / gas generation from saccharides: L-arabinose:-, maltose:-, lactose:-, D-trehalose:-, D-sorbitol:-, D-ribose:-, D-mannitol:- , D-raffinose: −, D-arabitol: −, D-melibiose: −, saccharose: +, tagatose: −
(19) Decomposition of sugars: β-glucosidase activity: −, β-glucuronidase activity: +, β-galactosidase activity: −, α-galactosidase activity: −
(20) Hippuric acid degradation: +
(21) Decomposition of arginine: +
(22) Coagulase: −
(23) Hemolytic: Non-hemolytic type (γ type)

  Deposited patent microorganism for Streptococcus agalactie SAG1001 (Depositor: National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, Location: 1-1-1 Tsukuba Center, Tsukuba City, Ibaraki, Japan, Accession Number FERM P-22063, date of commission: strain collected in Japan on February 4, 2011).

  It should be noted that the present invention only needs to be able to effectively prevent Streptococcus agalactie infections of sea surface cultured fish by inactivation, and is not limited to a narrow case only when this SAG1001 strain is used.

<About an inactivated vaccine preparation according to the present invention>
The present invention includes all inactivated vaccine preparations containing at least the inactivated bacterial body of the bacterium against Streptococcus agalactie infection of sea surface cultured fish. The present invention also provides an inactivated vaccine preparation containing, as an active ingredient, a bacterial solution obtained by inactivating the bacterial culture solution, for example, without separating the bacterial cells and the culture solution. In addition to microbial cells, the microbial cell-derived component contains a wide range by concentrating the microbial cells and the culture solution without separation.

  The cells to be used for inactivation may be used after being separated from fish that have developed Streptococcus agaractie infection, for example. The isolated bacteria can be cultured and propagated in a known solid medium / liquid medium, for example, meat extract agar plate medium, casein / soybean mixed peptone agar plate medium, meat extract liquid medium, casein / soybean mixed peptone liquid medium, etc. it can.

  The SAG1001 strain can be used as a vaccine by being inactivated, and because it is compatible with an adjuvant and has a synergistic vaccine effect by using a mixture of the inactivated and adjuvant. It is most suitable as a cell of Streptococcus agalactier used in the present invention.

  Inactivated cell bodies are, for example, physical treatment (ultraviolet irradiation, X-ray irradiation, heat treatment, ultrasonic treatment, etc.), chemical treatment (organic solvent treatment with formalin / chloroform, etc., weak acid such as acetic acid, etc. Acid treatment with alcohol, treatment with alcohol, chlorine, mercury, etc.).

  For example, formalin is added to the cultured bacterial solution at a volume concentration of 0.001 to 2.0%, more preferably 0.01 to 1.0%, and the cultured bacterial solution is sensitized at 4 to 30 ° C. for 1 to 3 days. Inactivation can be performed. For example, the inactivated cells may be washed with a buffer solution to remove inactivating agents such as formalin, or neutralized by adding a neutralizing agent to the inactivated cells. Alternatively, the inactivated cells may be collected by membrane filtration or centrifugation, or the culture solution may be concentrated without separating the cells and the culture solution.

The amount of the inactivated cells contained in the inactivated vaccine preparation is not particularly limited, but for example, the amount of the cells before inactivation is preferably in the range of 10 ³ to 10 ¹¹ CFU / mL, 10 ⁷ to 10 A range of ¹¹ CFU / mL is more preferred.

  The inactivated vaccine preparation according to the present invention may contain an adjuvant, that is, one containing at least the above-described inactivated cells and an adjuvant as active ingredients.

  For example, synergistic vaccine efficacy can be exerted by using a mixture of an inactivated bacterial cell having good affinity with an adjuvant and an adjuvant, such as SAG1001 strain.

  A wide variety of known adjuvants can be used. For example, animal oil (such as squalene) or hydrogenated oil thereof, vegetable oil (such as palm oil, castor oil) or hydrogenated oil thereof, mannitol / oleic anhydride, liquid paraffin, polybutene, caprylic acid, oleic acid, higher fatty acid ester, etc. Oil-based adjuvants, including PCPP, saponin, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymer, methacrylic acid copolymer, maleic anhydride copolymer, alkenyl derivative polymer, oil-in-water emulsion, Derived from microorganisms such as water-soluble adjuvants such as cationic lipids containing quaternary ammonium salts, precipitation adjuvants such as aluminum hydroxide (alum), sodium hydroxide, cholera toxin, and E. coli heat-labile toxin Examples include toxin components, bentonite, muramyl dipeptide derivatives, and interleukins. Moreover, what mixed these may be used.

  In addition, this inactivated vaccine preparation is a vaccine against other diseases, for example, α-hemolytic streptococcal inactivated vaccine, vibrio disease inactivated vaccine, iridovirus infection inactivated vaccine, nodular disease inactivated vaccine, Streptococcus It may be a mixed vaccine preparation with any or a plurality of inactivated vaccines of D. galactiae infection.

  In addition, a buffering agent, an isotonic agent, a soothing agent, a preservative, an antioxidant, and the like may be appropriately added according to the purpose and use.

  As a suitable example of a buffering agent, buffer solutions, such as a phosphate, acetate, carbonate, citrate, etc. can be used, for example.

  As a suitable example of an isotonizing agent, sodium chloride, glycerol, D-mannitol etc. can be used, for example.

  As a suitable example of the soothing agent, for example, benzyl alcohol or the like can be used.

  Suitable examples of antiseptic agents include, for example, thimerosal, paraoxybenzoates, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, other antiseptics, antibiotics, and synthetic antibacterials. An agent or the like can be used.

  As a suitable example of an antioxidant, a sulfite, ascorbic acid, etc. can be used, for example.

  In addition, this drug includes auxiliary ingredients such as light-absorbing dyes (riboflavin, adenine, adenosine, etc.) that serve as storage and efficacy aids, and chelating / reducing agents (vitamin C, citric acid, etc.) for stabilization. , Carbohydrates (sorbitol, lactose, mannitol, starch, sucrose, glucose, dextran, etc.), casein digests, various vitamins, and the like may be included.

  About the dosage form etc. of a vaccine formulation, a well-known thing can be employ | adopted and it does not specifically limit. For example, it may be used as a liquid preparation, or may be mixed in food after treatment such as lyophilization.

<About the inactivated vaccine preparation manufacturing method according to the present invention>
The present invention includes all methods for producing an inactivated vaccine preparation against the bacterial infection of sea-cultured fish, including at least a step of inactivating Streptococcus agalactier cells.

  As described above, an inactivated vaccine preparation against the bacterial infection of sea surface cultured fish can be produced by inactivating Streptococcus agalactier cells.

  The inactivated vaccine preparation according to the present invention can be performed by, for example, a step of growing Streptococcus agaractie cells, a step of inactivating the cells, a step of adding an adjuvant to the inactivated cells, and the like. it can.

  The cells used and the method for inactivating the cells are as described above. Further, according to the purpose and use, the above-mentioned adjuvant, buffer, tonicity agent, soothing agent, preservative, antioxidant and the like may be appropriately added.

<About the use of bacteria for the production of inactivated vaccine preparations>
The present invention broadly encompasses the use of the bacterium for the production of an inactivated vaccine formulation against Streptococcus agalactie infections in sea surface cultured fish.

  The above-mentioned Streptococcus agalactier can be widely used for the production of inactivated vaccine preparations against Streptococcus agalactier infection of sea surface cultured fish.

<Method for preventing Streptococcus agalactie infection according to the present invention>
It broadly encompasses a method for preventing Streptococcus agalactie infections in sea-cultured fish, which comprises administering the inactivated vaccine preparation described above.

  By administering the inactivated vaccine preparation described above to sea-cultured fish, it is possible to effectively prevent the occurrence, transmission, and spread of Streptococcus agaractie infections in sea-cultured fish.

  The sea surface cultured fish to be applied includes a wide range of seawater fish to be cultured, and is not particularly limited.

  Examples of the sea surface cultured fish belonging to the periaceae family include yellowtail fishes (such as yellowtail, amberjack, kingfish), striped fishes (such as striped horse mackerel), and horsetailed fishes (such as horse mackerel). In addition, mackerel fishes (such as mackerel, bluefin tuna), crocodile fish (such as sea bream), Thai fish (such as red sea bream, chidai, black sea bream), crocodile fish (such as crocodile), crocodile fish (such as crocodile), medinae Fish (such as medina), grouper fish (such as mahata and cued), sea bass (such as perch, cypress, and Chinese perch) are also included in the sea surface aquaculture fish.

  In addition, flounder fishes (such as flounder), puffer fishes (such as tiger puffer fish), cormorant fishes (such as riverfish), scorpionfish scorpion fish (such as sea bream, scorpionfish, blackfish) etc. It is.

  As the sea surface cultured fish according to the present invention, aziridae fish, Thai fish or flounderfish are preferred, echidaceae fish is more preferred, and yellowtail fishes are most preferred.

  Examples of the administration method of the inactivated vaccine preparation include an injection method, a dipping method, and an oral method.

In the case of the injection method, for example, an inactivated vaccine preparation prepared by adjusting the amount of bacterial cells before inactivation in the range of 10 ³ to 10 ¹¹ CFU / mL is administered intramuscularly or intraperitoneally to 0.05 to 3.0 mL. That is, the inactivated vaccine preparation in which the amount of bacterial cells before inactivation is 10 ³ to 10 ¹¹ CFU / mL and the dose per dose is 0.05 to 3.0 mL, administered intramuscularly or intraperitoneally at that dose The inactivated vaccine preparation is effective in preventing Streptococcus agalactier infection.

In the case of the immersion method, for example, the target fish is immersed for 0.05 to 48 hours in an inactivated vaccine preparation-containing solution prepared in a range of 10 ³ to 10 ⁹ CFU / mL of the bacterial cells before inactivation. That is, the inactivated vaccine preparation in which the amount of bacterial cells before inactivation is 10 ³ to 10 ⁹ CFU / mL and soaked for 0.05 to 48 hours per time is effective in preventing Streptococcus agalactiae infection. .

In the case of the oral method, for example, a feed mixed with an inactivated vaccine preparation prepared in a range of 10 ³ to 10 ¹¹ CFU / mL of the amount of bacterial cells before inactivation is freely fed and continuously administered for 1 to 20 days I do. That is, the inactivated vaccine preparation in which the amount of bacterial cells before inactivation is 10 ³ to 10 ¹¹ CFU / mL and continuous oral administration for 1 to 20 days is effective in preventing Streptococcus agalactiae infection.

  Of these, intraperitoneal administration by the injection method is most preferred because of its high infection prevention effect and long immunity duration.

  The inactivated vaccine preparation may be administered once as long as its action continues, but may be administered multiple times at 1-60 day intervals depending on the size of the target fish, the degree of vaccine effect, and the like.

  In Example 1, pathological evaluation of four yellowtails in which abnormalities were observed in a farm in Kagoshima Prefecture, Japan was performed.

  In September 2010, about 200 of 4,000 fish were drowned in a farm where approximately 4,000 yellowtails (2-year fish, weight 3.5 kg) were bred in a 9m x 9m ginger in Kagoshima Prefecture. Of these, 4 were tested for pathogenicity.

  As for appearance findings, binocular turbidity was observed in all individuals, and fading, redness of the heel and tail were observed in multiple individuals.

  At autopsy, 3 specimens showed epicarditis, mild splenic enlargement, and renal pelvis, and some specimens contained ascites, small white spots in the spleen, or multiple white nodules.

  In the colonization test, we attempted to isolate bacteria from the brain, kidney, spleen, and caudal part of all samples. As a result, a single bacterium forming a microcolony was isolated from almost all tissues of 4 specimens. These colonies were white, smooth and highly viscous. These colonies show a different shape from the colonies formed by the KS-7M strain (test strain) of Lactococcus garvieae, the causative agent of α-hemolytic streptococci in the genus Buri fish, and the isolated bacteria are Lactococcus garvieae It was estimated to be a different bacterium.

  The isolated bacteria were gram-stained and observed, and all were Gram-positive streptococci.

  Using the antiserum of Lactococcus garvieae, Streptococcus iniae, and Streptococcus parauberis, an agglutination test was performed using bacteria isolated from the brain of each specimen as a material. As a result, no agglutination was confirmed in any of the sera, and it was revealed that the isolated bacteria were not these bacteria.

  Using a commercially available test agent for detecting streptococcal groups `` Pastorex Strep (BioRad) '' that can distinguish A, B, C, D, F, and G groups based on classification by C polysaccharides derived from somatic cell walls, Field type tests were conducted. As a result, this isolate was determined to be Group B. In addition, this showed that this isolate was not Streptococcus dysgalactiae classified into C group.

Biochemical characterization of the isolated bacteria was performed using a Gram-positive cocci identification kit "Rapid ID32 Strep (Nippon Biomelieu)". As a control, the same test was performed for the KS-7M strain of Lactococcus garvieae. As shown in Table 1, the isolates were different from Lactococcus garvieae in 9 out of 26 items.

  From the above results, it was found that the bacteria isolated from yellowtail that had an abnormality in the farms in Kagoshima Prefecture were group B streptococci, and none of Lactococcus garvieae, Streptococcus iniae, Streptococcus parauberis, or Streptococcus dysgalactiae .

  In Example 2, the 16S rRNA of the isolated bacterium of Example 1 was analyzed.

  The colony of the isolated bacteria of Example 1 was suspended in 500 μL of sterilized ultrapure water, boiled for 5 minutes, centrifuged, and the supernatant was collected and used as a template. PCR amplification was performed using 16S rRNA universal primers (SEQ ID NOs: 1 and 2).

  The PCR product was purified using “QIAquick PCR Purification Kit (QIAGEN)”, and a plasmid in which the PCR product was inserted into the T vector was prepared using “TOPO TA Cloning Kit For Sequencing (Invitrogen)”. After transforming into E. coli, the plasmid was amplified, the plasmid DNA was purified by “Wizard Plus SV Minipreps DNA Purification System (Promega)”, and then sequence analysis was performed (SEQ ID NO: 3).

  As a result, the 16S rRNA sequence of the isolate showed homology of 100% (1507bp / 1507bp) with the previously reported Streptococcus agalactier (COH1 strain, Accession no.AAJR01000001, 39929-41335bp), in a farm in Kagoshima Prefecture. The fungus isolated from the yellowtail with abnormalities was identified as Streptococcus agalactier. This isolate was named SAG1001 strain.

  In Example 3, the hemolytic property of the isolate SAG1001 strain was examined.

  Streptococcus iniae (BS-1 strain), Lactococcus garvieae (KS-7M strain) and isolates (SAG1001 strain) were seeded on sheep blood agar medium and cultured at 25 ° C. for 2 days.

  As a result, in Streptococcus iniae (BS-1 strain), a transparent hemolysis ring was formed around the colony, and β hemolysis was exhibited.In Lactococcus garvieae (KS-7M strain), a green incomplete hemolysis ring was formed, and α hemolysis was observed. In contrast, the isolate (SAG1001 strain) did not form a hemolytic ring and was found to be non-hemolytic (γ-hemolytic).

  In Example 4, an infection experiment of isolated bacteria (Streptococcus agalactie SAG1001 strain) was performed.

  Streptococcus agalactie strain SAG1001 was inoculated into 0.5% NaCl-added SCD liquid medium (Soybean Casein Digest Broth) and cultured at 28 ° C. for 2 days with shaking.

Thirty-six aquaculture yellowtails and 36 aquaculture amberjacks were divided into three groups of 12 each.Streptococcus agalactier SAG1001 strains were divided into 4.1 × 10 ⁷ CFU / tail (stock solution), 4.1 × 10 ⁵ CFU / tail (each), respectively. (100-fold dilution) and 4.1 × 10 ³ CFU / tail (10,000-fold dilution) were injected intraperitoneally and infected. Each test fish was raised at 28 ° C. for 14 days after infection.

As a result, in the case of yellowtail, in the group injected with 4.1 × 10 ⁷ CFU / tail (stock solution) of Streptococcus agalactier SAG1001 strain, 4 days after infection, in the group injected with 4.1 × 10 ³ CFU / tail (diluted 10,000 times) All animals died by day 6 after infection, and the mortality rate was 100%.

In the case of amberjack, the mortality rate of the group injected with 4.1 × 10 ⁷ CFU / tail (stock solution) of Streptococcus agalactiee SAG1001 was 92%, and the mortality rate of the group injected with 4.1 × 10 ⁵ CFU / tail (100-fold dilution) was 42 %, The death rate of the group injected with 4.1 × 10 ³ CFU / tail (10,000-fold dilution) was 75%.

  The appearance of the dead fish showed cloudiness of the eyeball, necrosis, body curvature, and skinnyness, which were similar to those of α-hemolytic streptococci. Autopsy findings showed epicarditis and spleen enlargement. The symptom of an outbreak fish in a farm in Kagoshima Prefecture was reproduced. It was confirmed that the infectious bacteria were reisolated from the brain of the dead fish.

  From the above results, it was found that the present bacterium has high pathogenicity to yellowtail fishes such as yellowtail and amberjack.

  In Example 5, the production of a vaccine against Streptococcus agalactier and the efficacy as a vaccine against fish of the genus Buri were examined.

The colony of Streptococcus agalactiae SAG1001 grown on SCD agar medium with 0.5% NaCl (Soybean Casein Digest Agar Broth, the same shall apply hereinafter) is scraped, suspended in McFarland turbidity standard solution No. 4 with PBS, and diluted 100 times with PBS. Then, 100 μL of the diluted solution was inoculated into 100 mL of 0.5% NaCl-added SCD liquid medium (Soybean Casein Digest Broth, hereinafter the same), and cultured with shaking at 28 ° C. for 2 days. The number of viable bacteria before inactivation was 1.4 × 10 ⁹ CFU / mL.

  0.3% (volume) formalin was added to the culture solution, sensitized at 28 ° C. for 2 days to inactivate the cells, and stored at 4 ° C. In addition, it was confirmed by an inactivation test that the inactivation was completed.

Divide 90 fish yellowtails and 90 fishery amberjacks into 3 groups each, with PBS as the first group (control) and 0.3% formalin-inactivated bacteria solution (1.4 x 10 ⁸ CFU) as the second group / Tail), the third group was mixed with 0.3% formalin-inactivated bacteria solution (4.2 × 10 ⁷ CFU / tail) and adjuvant “Montanide ISA 763 A VG (Seppic)” at 30:70, Intraperitoneally injected and immunized. After immunization, the mice were raised at 25 ° C. for 14 days.

14 days after immunization, Streptococcus agalactie strain SAG1001 was injected with 5.6 × 10 ³ CFU / tail intraperitoneally and challenged. After the challenge, they were further raised at 28 ° C for 14 days.

The results are shown in Tables 2 and 3. Table 2 shows the survival rate of yellowtail, and Table 3 shows the normal rate of amberjack. In the table, “PBS” is the result of the group injected with PBS intraperitoneally at the time of immunization, “Inactivated bacterial solution” is the result of the group injected intraperitoneally with 0.3% formalin inactivated bacterial solution at the time of immunization, “Liquid + Adjuvant” represents the results of groups in which a 30:70 mixed solution of 0.3% formalin-inactivated bacterial solution and adjuvant emulsion was injected intraperitoneally at the time of immunization. "RPS (relative
"percent survival)" represents a value calculated by the following Equation 1. In the case of affected fish, individuals with symptoms such as thinning, necrosis / bleeding of the eyeball, protrusion of the eyeball, and curvature of the spine were counted as affected fish.

  As shown in Tables 2 and 3, when immunizing yellowtail or amberjack with an inactivated bacterial solution, an onset protection effect against Streptococcus agalactier was observed compared to the control. In addition, when a yellowtail or amberjack was immunized with a mixed solution of an inactivated bacterial solution and an adjuvant, a remarkable onset protection effect was observed. Therefore, this inactivated bacterial solution and a mixed solution of this inactivated bacterial solution and an adjuvant are effective as a vaccine against Streptococcus agalactie infections of sea surface cultured fish, particularly fish of the genus Buri.

  In addition, live fish or normal fish of yellowtail and amberjack were randomly selected 5 each, and bacteria were isolated from their brain and spleen. As a result, the bacteria were not re-isolated and the bacteria were completely eliminated.

  In Example 6, the attack method against yellowtail was changed, and the efficacy as a vaccine was examined.

As in Example 5, a colony of Streptococcus agalactiae SAG1001 strain grown on a 0.5% NaCl-added SCD agar medium was scraped, suspended in PBS to about McFarland turbidity standard solution No. 4, diluted 1,000-fold with PBS, 0.5% 100 μL of the diluted solution was inoculated into 100 mL of NaCl-added SCD liquid medium, and cultured with shaking at 28 ° C. for 2 days. The number of viable bacteria before inactivation was 9.6 × 10 ⁸ CFU / mL.

  0.2% (volume) formalin was added to the culture and sensitized at 28 ° C for 3 days to inactivate the cells, and stored at 4 ° C. In addition, it was confirmed by an inactivation test that the inactivation was completed.

Divide 60 aquaculture fish into 30 groups, each with 2 groups, PBS (control) for the first group and 0.2% formalin-inactivated bacteria solution (9.6 × 10 ⁷ CFU / tail) for the second group. Internal injection and immunization. After immunization, the mice were raised at 25 ° C. for 14 days.

After 14 days of immunization, the cells were immersed in breeding water containing 9.3 × 10 ⁵ CFU / mL of Streptococcus agalactiae strain SAG1001 for 30 minutes and attacked. After the challenge, they were further raised at 28 ° C for 14 days.

The results are shown in Table 4. Table 4 shows the survival rate of yellowtail when the yellowtail is attacked by immersion. In the table, “PBS” represents the results of the group injected intraperitoneally with PBS at the time of immunization, and “inactivated bacterial solution” represents the results of the group injected intraperitoneally with 0.2% formalin inactivated bacterial solution at the time of immunization. “RPS (relative percent survival)” represents the value calculated by Equation 1 as in Table 2 or Table 3.

  As shown in Table 4, even when the yellowtail was attacked by immersion, the onset protection effect against Streptococcus agalactier was observed as compared with the control, as in Example 5. Further, even when no adjuvant was added, the onset protective effect against Streptococcus agalactier was observed as in Example 5.

  At the present time, Streptococcus agalactie infections in sea-cultured fish have been rarely observed in sea-cultured fish near Japan. However, in the infection experiment in yellowtail, the symptom is serious, many yellowtails die, and the whitetail has severe symptoms such as cloudiness of the eyeball, necrosis, flexion of the body and skinnyness, and does not retain commercial value. Therefore, the widespread spread of this infection in the cultivated Brassic fish results in very large economic losses. Therefore, it is considered useful in the aquaculture industry to prevent the occurrence, propagation, and spread of this disease according to the present invention.

  In addition, due to the fact that sea temperature rise due to global climate change has been observed, and sea-cultured fish are often moved artificially from isolated areas, Streptococcus agalactiae infections will continue in the future. May spread widely to sea surface aquaculture fish other than yellowtail fish. From that point of view, it is considered useful in the aquaculture industry to prevent the occurrence, propagation, and spread of this disease according to the present invention.

  In addition, the use of vaccine preparations to prevent Streptococcus agaractie infection is more cost-effective than the treatment with antibiotics after the occurrence of Streptococcus agalactie infection, and there are concerns about residual drugs. There is an advantage of less.

Claims (8)

An inactivated vaccine preparation containing an inactivated cell of the bacterium against a non-hemolytic Streptococcus agalactiae infection of a fish of the genus Buri .   The inactivated vaccine formulation of Claim 1 which contains the fungus liquid obtained by inactivating the culture | cultivation liquid of the said bacteria as an active ingredient.   The inactivated vaccine formulation of Claim 1 or Claim 2 containing an adjuvant. The inactivated vaccine according to any one of claims 1 to 3, wherein the amount of bacterial cells before inactivation is 10 ³ to 10 ¹¹ CFU / mL, and the dose per administration is 0.05 to 3.0 mL. Formulation.   The inactivated vaccine formulation as described in any one of Claims 1-4 administered intraperitoneally. A method for preventing non-hemolytic Streptococcus agalactie infections of yellowtail fish , wherein the inactivated vaccine preparation according to any one of claims 1 to 5 is administered. A method for producing an inactivated vaccine preparation against a bacterial infection of a fish of the genus Buri , which comprises a step of inactivating a cell of non-hemolytic Streptococcus agalactie derived from a fish of the genus Buri .   Streptococcus agalactiae strain SAG1001 with accession number FERM P-22063.