JP2007326794A - Inactivated vaccine using fish streptococcus dysgalactiae as antigen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide inactivated fish S. dysgalactiae infection-preventing monovalent and polyvalent vaccines preventing fish S. dysgalactiae infections in fishes of the genus Seriola and to provide methods for producing them. <P>SOLUTION: A monovalent vaccine using inactivated fish Streptococcus dysgalactiae as the antigen and a trivalent vaccine using a mixture of inactivated fish Streptococcus dysgalactiae, inactivated Lactococcus garvieae, and Vibrio anguillarum as the antigen are made. By inoculating them into fishes of the genus Seriola by an appropriate method, the fishes can be protected against fish Streptococus dysgalactiae infections or against fish Streptococcus dysgalactiae infections, Lactococcus garvieare infections, and Vibrio anguillarum infections. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a vaccine for preventing fish Streptococcus dysgalactiae (hereinafter referred to as S. dysgalactiae) infection in fish classified into the genus Buri, and a method for producing the same. In addition, the present invention relates to a multivalent vaccine capable of preventing various bacterial infections and viral infections in a fish belonging to the genus S. dysgalactiae as at least one antigen, and a method for producing the same.

  Amberjacks and yellowtails belong to the genus Yellowtail, and are cultivated actively mainly in Kyushu and the Shikoku region as the main fish to be cultivated in Japan. On the other hand, various infectious diseases have occurred in such sea surface aquaculture sites, causing serious damage to the aquaculture industry.

  Infectious diseases that occur in the aquaculture site of the genus Buri and cause economic damage include fish S. dysgalactiae, Lactococcus garvieae (hereinafter referred to as L. garvieae), Vibrio anguillarum, (Hereinafter referred to as V. anguillarum), Nocardia seriolae (hereinafter referred to as N. seriolae), red sea bream iridovirus, photobacterium damselae subsp. Piscicida, bacterial jaundice causing fungus ( Bacterial hemolytic jaundice), viral ascites-causing virus (Yellowtail ascites virus), and other infectious diseases.

  Among these pathogens, the fish S. dysgalactiae is a Gram-positive cocci that has emerged most recently, and infections caused by this bacterium mainly occur in shipment-sized amberjack in August-October when the water temperature rises, The damage in the amberjack aquaculture industry is very serious because it not only significantly reduces the commercial value of infected fish but also kills infected fish. Recently, the yellowtail has been damaged by the same infectious diseases.

  Of the infectious diseases that are prevalent in the fish species of the genus Buri, effective vaccines have been developed and distributed on the market for infections caused by L. garvieae, V. anguillarum, and red sea bream virus. However, there is no scientific knowledge yet about vaccines against fish S. dysgalactiae infection, and not only vaccines for marketing purposes but also vaccines at the research level have not been developed at all. Therefore, an effective vaccine for the prevention of fish S. dysgalactiae infections is eagerly desired from the place of aquaculture of yellowtail fish.

  In mammals, for example, S. dysgalactiae infections such as pigs, cattle, and birds are known, but as mentioned above, fish S. dysgalactiae has emerged most recently and has been an academic finding. However, the fish S. dysgalactiae is not classified as the same bacterium as the mammalian S. dysgalactiae, and the bacterial taxonomics are unidentified and details are unknown.

  Therefore, although various vaccines against mammalian S. dysgalactiae infection have been studied (for example, Japanese Translation of PCT International Publication No. 2006-503803), in addition to the unidentified bacterial taxonomy as described above, in the case of fish Since the main humoral immunity is IgM and lacks the ability to produce IgG, which is the main humoral immunity in mammals, mammalian vaccines cannot be applied.

As a result of intensive investigations to achieve prevention of fish S. dysgalactiae infection, the present inventors have demonstrated that an inactivated fish S. dysgalactiae antigen as a vaccine prevents fish S. dysgalactiae infection in fish species It has been found that it has an effect, and the fish S. dysgalactiae infection prevention vaccine of the present invention has been developed.
JP-T-2006-503803

The present invention has been made to overcome the obstacles and problems described in the background art described above, and has not yet been developed at all. The fish S. dysgalactiae infection which is a deadly infection in the genus Buri It aims at providing the unit price vaccine to prevent and its manufacturing method.
The present invention also relates to fish S. dysgalactiae, L. garvieae, L. garvieae, and V. anguillarum that can confer sufficient protective immunity against fish S. dysgalactiae infection, L. garvieae infection, and V. anguillarum infection simultaneously. It is an object to provide a multivalent inactivated vaccine and a method for producing the same.

  The infectious disease preventive vaccine of the present invention is a vaccine capable of preventing fish S. dysgalactiae infection in yellow fish by inoculating yellow fish, and contains inactivated fish S. dysgalactiae as an antigen It is characterized by.

  The vaccine production method of the present invention includes a step of uniformly suspending sterilized purified water as a solvent to avoid aggregation of fish S. dysgalactiae cells, and provides a vaccine of a dosage form having both practicality and stability It is characterized by that.

  That is, the method for producing a vaccine of the present invention generally reduces the osmotic pressure in a vaccine-vaccinated individual so that it can be used as an antigen for a vaccine without aggregating inactivated and precipitated fish cells of S. dysgalactiae. It is a unique method for producing a vaccine to prevent the infection of S. dysgalactiae fish, which incorporates into the production process the preparation of the final dosage form using sterile purified water that is not used because of concerns about the instability of antigens and antigens. Is.

  Further, another infection prevention vaccine of the present invention is a fish S. dysgalactiae, L. which can simultaneously confer sufficient protective immunity against fish S. dysgalactiae infection, L. garvieae infection, and V. anguillarum infection. It is a multivalent inactivated vaccine using garvieae and V. anguillarum as antigens. The multivalent vaccine of the present invention includes Nocardia seriolae, red sea bream iridovirus, photobacterium damselae subsp. Piscicida, bacterial jaundice (Bacterial hemolytic jaundice), One or more types of viral ascites virus (Yellowtail ascites virus) may be further contained as an antigen.

  The fish S. dysgalactiae infection preventive vaccine of the present invention can prevent fish S. dysgalactiae infection, which is a lethal infection of yellowtail fish that could not be prevented so far, and the economic effect in the aquaculture industry is It is large and has a very high social contribution.

  In addition, the trivalent inactivated vaccine with the antigens of fish S. dysgalactiae, L. garvieae, and V. anguillarum of the present invention can be used for fish S. dysgalactiae infection, L. garvieae infection, and Since V. anguillarum infection can be prevented at the same time, the complexity of vaccination expected at the farming site when these three diseases are prevented, that is, bivalent antigens with L. garvieae and V. anguillarum as antigens. The contribution to the farmer is very high in that it eliminates the complexity of having to inject both the commercial vaccine and the inactivated fish S. dysgalactiae vaccine of the present invention.

  In the production of a vaccine, an isotonic solution is usually used in consideration of problems of stability and osmotic pressure. However, the inventors of the present invention have never been reported so far that bacterial cells rapidly aggregate when attempting to prepare a vaccine using the fish S. dysgalactiae inactivated with isotonic solution as an antigen. No event was encountered.

  That is, the bacterial mass formed by the fish S. dysgalactiae does not dissolve in solutions containing salts such as culture media, buffers, and physiological saline, which are widely used as vaccine solvents, and is used when an inactivated vaccine is injected into fish. In a continuous syringe fitted with a fish needle (for continuous syringe: Fujihira Kogyo Co., Ltd.) of φ0.4 (27G) x 3 mm or φ0.5 (25G) x 4 mm It could not be used as an antigen for vaccines.

  Therefore, as a result of intensive studies to solve this problem, the present inventors have suspended the bacterial cells uniformly in sterilized purified water that is not normally used as a solvent for vaccine production. The present inventors have invented a method for producing a fish S. dysgalactiae infectious disease vaccine that can overcome the phenomenon of bacterial aggregation that has become an obstacle when using fish S. dysgalactiae and can be used commercially.

  In addition, a bivalent vaccine has been developed to prevent L. garvieae infection and V. anguillarum infection at the same time with a single injection, but L. garvieae infection and V. anguillarum infection have been developed with a single injection. A trivalent vaccine that simultaneously prevents fish S. dysgalactiae infection in addition to the disease has not been developed.

  Even though the trivalent vaccine was not developed as such, the problem encountered by the present inventors was inactivated, that is, the inactivated L. garvieae and V. anguillarum were used as antigens. When fish S. dysgalactiae was mixed as the third antigen, inactivated cells of fish S. dysgalactiae were aggregated.

  As a result of intensive studies, the inventors of the present invention, in addition to the inactivated fish S. dysgalactiae cells, also inactivated L. garvieae and V. anguillarum, using sterile purified water as a solvent, To produce a trivalent vaccine that can effectively and stably prevent fish S. dysgalactiae infection in addition to L. garvieae infection and V. anguillarum infection without agglutinating dysgalactiae cells In particular, the present invention has been completed.

  In the production process of the inactivated fish S. dysgalactiae vaccine of the present invention, sterilized purified water can be used as a solvent that does not aggregate the fish S. dysgalactiae used as an antigen, but it does not contain salts that are thought to cause cell aggregation. The solvent is not limited to sterilized purified water as long as it can maintain the stability and immunogenicity of bacterial cells as vaccine antigens.

  In the method for producing the inactivated fish S. dysgalactiae vaccine of the present invention, the step of substituting and suspending the cells of the fish S. dysgalactiae with sterilized purified water is carried out by using a fungus of the fish S. dysgalactiae that has been sunk using a continuous centrifuge. It can be carried out by suspending the body in sterilized purified water, but it can also be carried out by other methods such as a method in which the extracellular fluid is replaced from the culture medium with sterilized purified water using an ultrafilter. it can.

In the inactivated fish S. dysgalactiae vaccine of the present invention, as a method for inactivating fish S. dysgalactiae used as an antigen, a holymarin treatment method can be used, but it is limited to this method as long as it does not impair antigenicity. For example, a binary ethylenimine treatment method or an ultraviolet irradiation method may be used.
Further, in the present invention, the formalin concentration for inactivating fish S. dysgalactiae can be 0.2 to 0.3 vol%, as long as it is a concentration that does not impair antigenicity and can sufficiently inactivate fish S. dysgalactiae. The concentration may be other than 0.2 to 0.3 vol%.

  Sterilized purified water that can be used as a solvent for the inactivated fish S. dysgalactiae vaccine of the present invention is added with 0.1 to 0.3 vol% formalin in order to maintain the sterility of the vaccine during and after the production process. However, for the same purpose, a drug having antibacterial activity other than formalin can be added if it is safe for the vaccinated fish and the person who ate it.

  The effectiveness of the inactivated fish S. dysgalactiae vaccine of the present invention in the fish of the genus Buri is confirmed by the attack method using the fish S. dysgalactiae for attacking the fish classified into the species of the genus Buri such as amberjack and yellowtail injected with the vaccine. can do. That is, for example, a vaccine injection group that injects the vaccine into the peritoneal cavity, and a control group that injects sterile purified water alone or sterile purified water containing 0.2 to 0.3 vol% formalin for attack of an appropriate number of bacteria. The fish S. dysgalactiae strain was challenged by intraperitoneal injection, and then the survival rate of the test fish in both groups was statistically compared and analyzed, and the survival rate of the test fish in the vaccine injection group was compared with that in the control group. When the survival rate of the test fish is significantly higher, it can be confirmed that the inactivated fish S. dysgalactiae vaccine of the present invention is effective in preventing fish S. dysgalactiae infection.

  As shown in the Examples below, as a result of conducting an attack test using the above pathogenic fish S. dysgalactiae on the S. dysgalactiae infection prevention vaccine injection group and control group of the present invention, The survival rate of the test fish in the group was higher than the survival rate of the test fish in the non-vaccinated control group, and there was a statistically significant difference between the survival rates, indicating that the fish of the vaccine S. dysgalactiae A preventive effect against infectious diseases was confirmed.

The minimum effective antigen amount at which the inactivated fish S. dysgalactiae vaccine of the present invention exerts a preventive effect against fish S. dysgalactiae infection is determined from the test results shown in the Examples below, from the number of bacteria before inactivation (colony The formation unit (CFU) was found to be 1.2 × 10 ⁸ CFU / 0.1 ml / dose or less, but the number of bacteria is not limited as long as the number of bacteria can prevent fish S. dysgalactiae infection.

  The inactivated fish S. dysgalactiae vaccine of the present invention can be produced as containing no adjuvant, but may contain an adjuvant as long as it is safe for the fish of the genus Buri fish to be inoculated. Examples of adjuvants that can be used include oil adjuvants blended in salmon and trout vaccines such as ISA-763AVG (manufactured by Seppic) and ISA-708VG (manufactured by Seppic).

  As the fish S. dysgalactiae strain used as an antigen of the inactivated fish S. dysgalactiae vaccine of the present invention, the SD3M strain isolated and identified from the fish S. dysgalactiae-affected amberjack can be used. The strain that can be used is not limited to the SD3M strain, but if it is an S. dysgalactiae fish that exhibits antigenicity that can confer sufficient immunity to prevent fish S. dysgalactiae infection, the SD3M strain Other fish S. dysgalactiae strains may be used.

  The inactivated fish S. dysgalactiae vaccine of the present invention may be a unit price vaccine that uses only fish S. dysgalactiae as an antigen, or may be a multivalent vaccine that simultaneously prevents multiple types of infectious diseases in Brassica good.

  The present inventors have invented a trivalent vaccine using the antigens of fish S. dysgalactiae, L. garvieae, and V. anguillarum as multivalent vaccines, and this multivalent vaccine is similar to the fish S. dysgalactiae unit price vaccine. Further, it can be prepared by a production method in which a step of suspending three kinds of inactivated antigens using sterilized purified water as a solvent is introduced. As described above, the solvent used is a solvent that does not contain salts that are considered to be a cause of bacterial cell aggregation, and that can maintain the stability and immunogenicity of bacterial cells as vaccine antigens. It is not limited.

  The effectiveness of the inactivated fish S. dysgalactiae vaccine to evaluate the preventive effect of the three mixed inactivated vaccines of the present invention against fish S. dysgalactiae infection, L. garvieae infection, and V. anguillarum infection Similarly, it is possible to evaluate by performing an attack test using pathogenic strains of each bacterium as an antigen.

  As fish S. dysgalactiae strain, L. garvieae strain, and V. anguillarum strain used as antigens of the three mixed inactivated vaccines of the present invention, SD3M strain, KS-7M strain and KT-5 strain can be used, respectively. However, it is not limited to these strains, and it has an antigenicity that can confer sufficient immunity to the fish of the genus Pseudomonas, sufficient to prevent fish S. dysgalactiae infection, L. garvieae infection, and V. anguillarum infection. Any strain other than the SD3M strain, KS-7M strain, and KT-5 strain may be used.

  The multivalent vaccine of the present invention can be a trivalent vaccine with the antigens fish S. dysgalactiae, L. garvieae, and V. anguillarum as an antigen. It may be a multivalent vaccine that further contains one or more species such as N. seriolae, muddy iridovirus, nodule-causing bacteria, bacterial jaundice-causing bacteria, and viral ascites-causing virus.

  The inactivated fish S. dysgalactiae prophylaxis unit vaccine and multivalent vaccine of the present invention can be used as an injectable vaccine, but the administration method of the vaccine is not limited to injection, for example, an oral administration method or a vaccine soaked in food An immersion method may be used in which a fish of the genus Buri is immersed in the liquid.

  As described above, inactivated fish S. dysgalactiae infection preventive unit cost vaccines and multivalent vaccines that can confer sufficient protective immunity against fish S. dysgalactiae infections in the genus Buri fish and prove their effectiveness This is the first time the present invention has been made.

(1) Method for producing vaccine for preventing inactivated fish S. dysgalactiae Infectious disease vaccine isolated from amberjack suspected of being infected with fish S. dysgalactiae and identified as fish S. dysgalactiae by biochemical tests and Lancefield group tests The strain was named SD3M strain and was used as a vaccine production strain for the production of inactivated fish S. dysgalactiae infection prevention vaccine.
Formalin with a final concentration of 0.2 vol% was added to 100 liters of SD3M strain culture medium grown to a bacterial count of 1.2X10 ⁹ CFU / ml in a fermenter at 25 ° C using Soybean Casein Digest (SCD) liquid medium. The strain was slowly stirred at 25 ° C. for 48 hours to inactivate the SD3M strain. Next, after incubating the inactivated SD3M strain solution with a continuous centrifuge to prepare for sedimentation, 28 liters of sterile purified water added with 0.2 vol% formalin was added to the sedimentation to suspend the cells and aliquot 200 ml. did.
Substantial bacterial concentration in this vaccine product is calculated as 6.0X10 because the amount of sterilized purified water when suspending centrifugation was reduced to 1/5 of the culture volume and concentrated 5 times. ⁹ CFU / ml.
The inactivated fish S. dysgalactiae vaccine manufactured in small portions can be confirmed to maintain the homogeneity of the vaccine solution by visual observation at least one year after the production. A continuous syringe equipped with a fish needle (for continuous syringe: Fujihira Kogyo Co., Ltd.) with φ0.4 (27G) x 3 mm and φ0.5 (25G) x 4 mm It was proved that the stability was maintained for a long time because it was possible to accurately inject the test fish in the following efficacy test. .

(2) Method for testing the effectiveness of the vaccine to prevent inactivated fish S. dysgalactiae infection The stock solution of inactivated fish S. dysgalactiae vaccine, diluted 2.5-fold and 5-fold with sterile purified water containing 0.2 vol% formalin A vaccine was used. Further, as a control, sterilized purified water containing 0.2 vol% formalin was used. Divide into 100 groups of 100 amberjack with an average body weight of about 16 g and 25 fish in each group.The test fish in each group is diluted into a stock solution vaccine (stock solution vaccine group), 2.5 times diluted vaccine (2.5 times diluted vaccine group), and 5 times diluted. Safety of this vaccine in the test fish by intraperitoneal injection of 0.1 ml / tail of either vaccine (5-fold diluted vaccine group) or 0.2 vol% formalin-containing sterilized purified water (control group) Sex was judged. Thereafter, 04K01 strain (pathogenic fish S. dysgalactiae strain isolated from fish affected by fish S. dysgalactiae within Kyoritsu Pharmaceutical Co., Ltd.) as an attacking strain of fish S. dysgalactiae in 1 x 10 ⁸ CFU / 0.1 ml / Inoculated intraperitoneally with tail. The test fish of each group attacked in this way were reared and observed for 2 weeks, and the preventive effect of the test vaccine against fish S. dysgalactiae infection was determined.

(3) Effectiveness test results of inactivated fish S. dysgalactiae infection vaccine 23 weeks after the attack with the attack strain 04K01, 23 out of 25 fish in the control group died The survival rate was 8%. On the other hand, the survival rates in the test fish of the stock solution vaccine group, 2.5-fold dilution vaccine group, and 5-fold dilution vaccine group were 84%, 48%, and 56%, respectively, compared with the survival rate in the control group There was a high statistically significant difference between the survival rate in the control group and the survival rate in each vaccine group (p <0.05, Fisher's exact calculation).
As a result, it was revealed that the inactivated fish S. dysgalactiae vaccine of the present invention is effective in preventing fish S. dysgalactiae infection. In addition, since this efficacy test was conducted one year after the production of the test vaccine, the effectiveness of the inactivated fish S. dysgalactiae infection vaccine of the present invention has been stably maintained for at least one year. It became clear that
From the results of this efficacy test, the minimum effective antigen amount in the vaccine of a vaccine using the fish S. dysgalactiae SD3M strain as the production strain is the antigen amount (the number of bacteria before inactivation) in the 5-fold diluted vaccine that was confirmed to be effective. It was found to be below 1.2X10 ⁸ CFU / 0.1ml / dose.
On the other hand, no abnormalities in clinical observation or feeding conditions were observed in the test fish during the 2 weeks after the vaccination of the test vaccine, and the inactivated fish S. dysgalactiae infection prevention vaccine of the present invention was It became clear that it was safe.

(4) Method for producing three mixed inactivated vaccines using fish S. dysgalactiae, L. garvieae, and V. anguillarum as antigens Fish S. dysgalactiae SD3M strain, L. garvieae KS-7M strain, and V. anguillarum KT- Five strains were grown in a fermenter at 25 ° C. in 100 liter, 20 liter and 20 liter SCD liquid media, respectively. Subsequently, formalin having a final concentration of 0.2 to 0.3 vol% was added to each of the grown strains, and each strain was inactivated while being stirred at 25 ° C for 24 to 48 hours. Next, each inactivated bacterial solution was centrifuged to make a sedimentation, and then 18 liters, 1 liter, and 1 liter of sterilized purified water were added and suspended in the sedimentation of each bacterial cell. Finally, each bacterial cell suspension was mixed (volume ratio 90: 5: 5), and the mixed bacterial cell suspension was divided into 200 ml portions.
The pre-inactivation concentration of the fish S. dysgalactiae SD3M strain fraction in this subdivided product was 1.2 × 10 ⁹ CFU / ml, but as described above, the solution of sterilized purified water when suspending the centrifugal sedimentation Since the amount was reduced from the amount of the culture solution, the substantial bacterial concentration in the subdivided product was 6.0 × 10 ⁹ CFU / ml. On the other hand, the substantial bacterial concentrations in the subdivided products of L. garvieae KS-7M strain and V. anguillarum KT-5 strain were 2 × 10 ⁹ CFU / ml and 2 × 10 ⁹ CFU / ml, respectively.
The L. garvieae KS-7M strain and V. anguillarum KT-5 strain used as strains for the production of this three-type mixed inactivated vaccine are both commercially available two-type mixed vaccine for yellowtail (Pishibac Vibrio + Rensa, Kyoritsu) It is used as a manufacturing strain of Pharmaceutical Co., Ltd.
This three-type mixed inactivated vaccine has a φ0.4 (27G) x 3 mm and φ0.5 (25G) x 4 mm aquaculture needles (for continuous syringes: Fuji) even when about one year has passed since manufacture. There is no inconvenience that the vaccine solution is clogged in a continuous syringe equipped with Hira Kogyo Co., Ltd., and it is stable for a long time because it was able to accurately inject the test fish in the following efficacy test And proved to maintain the dosage form.

(5) Method for testing efficacy of three mixed inactivated vaccines using fish S. dysgalactiae, L. garvieae, and V. anguillarum as antigens Fish S. dysgalactiae, L. garvieae, and V. anguillarum produced as described above Three types of mixed inactivated vaccines, each of which was used as an antigen, were injected intraperitoneally into 3 groups of amberjack (average body weight of about 20 to 31 g), each group consisting of 10 to 27 tails, and then fed and observed for 2 weeks. The safety of this vaccine in test fish was determined. Then, for each group of test fish, the fish S. dysgalactiae attack strain (04K01 strain), the L. garvieae attack strain (KS-7C strain), and the V. anguillarum attack strain (OK-0301 strain) One of these was inoculated intraperitoneally at 1 × 10 ⁸ CFU / 0.1 ml / tail, 1 × 10 ⁶ CFU / 0.1 ml / tail and 1 × 10 ⁶ CFU / 0.1 ml / tail, respectively. Three groups of test fish attacked in this way were reared and observed for 2 weeks, and the preventive effect of the test vaccine against fish S. dysgalactiae infection, L. garvieae infection, and V. anguillarum infection was determined. As control groups, 3 groups (10 to 27 tails per group, average body weight: about 20 to 31 g) were prepared, and 0.1 ml of 0.3 vol% formalin-containing sterilized purified water was injected intraperitoneally into the amberjack of each control group.

(6) Effectiveness test results of three mixed inactivated vaccines using fish S. dysgalactiae, L. garvieae, and V. anguillarum as antigens In the effectiveness test of the fish S. dysgalactiae fraction, By the end of 2 weeks after the attack with the 04K01 strain, 27 out of 27 fish in the control group died, and the survival rate was 0%. On the other hand, the survival rate in the group injected with the test vaccine was 78%, which was higher than the survival rate in the control group. The survival rate in the control group and the survival rate in the group injected with the test vaccine were Statistically significant differences were observed between the rates (p <0.05, Fisher's exact probability calculation).
In the effectiveness test of the L. garvieae fraction, 22 out of 23 fish in the control group died by 2 weeks after the challenge with the L. garvieae challenge strain KS-7C. The survival rate was 4%. On the other hand, the survival rate in the group injected with the test vaccine was 100% (23/23 fish), which was higher than the survival rate in the control group, and the survival rate in the control group and the test vaccine were injected. There was a statistically significant difference between the survival rates in the selected groups (p <0.05, Fisher's exact calculation).
In the effectiveness test of the V. anguillarum fraction, 9 out of 10 fish in the control group died by 2 weeks after the attack with the V. anguillarum challenge strain OK-0301. The survival rate was 10%. On the other hand, in the group injected with the test vaccine, only 1 out of 10 animals died, and the survival rate was 90%, which was higher than the survival rate in the control group, and the survival rate in the control group. There was a statistically significant difference between the survival rate in the group injected with the test vaccine (p <0.05, Fisher's exact probability calculation).
As a result, it was revealed that the three-type mixed inactivated vaccine of the present invention is effective in preventing fish S. dysgalactiae infection, L. garvieae infection, and V. anguillarum infection. In addition, since this efficacy test was conducted one year after the production of the test vaccine, it is clear that the effectiveness of the triple mixed inactivated vaccine of the present invention has been stably maintained for at least one year. became.
On the other hand, no abnormalities in clinical observations or abnormal feeding conditions were observed in the test fish during the 2 week breeding period after vaccination with the test vaccine. It became clear that.

  The fish S. dysgalactiae SD3M strain used as the antigen of the vaccine of the present invention was deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology as of April 20, 2006 under the deposit number FERM P-20893. Yes.

Claims (5)

  A vaccine for preventing Streptococcus dysgalactiae infection, which contains inactivated fish Streptococcus dysgalactiae as an antigen.   2. The method for producing a Streptococcus disgalactie infection-preventing vaccine according to claim 1, comprising a step of suspending cells of fish Streptococcus dysgalactiae in purified water.   Infectious disease prevention vaccine containing fish Streptococcus dysgalactiae, Lactococcus garvieae, and Vibrio anguillarum as antigens.   The infectious disease according to claim 3, comprising a step of suspending cells of fish Streptococcus dysgalactiae, Lactococcus garvieae, and Vibrio anguillarum in purified water. A method of producing a prophylactic vaccine.   Nocardia seriolae, red sea bream iridovirus, photobacterium damselae subsp. Piscicida, bacterial jaundice (Bacterial hemolytic jaundice), viral ascites causing virus (Yellowtail) The vaccine for preventing infectious diseases according to claim 1 or 3, further comprising one or more types of ascites virus).