JP2001103961A - Vaccine for fish diseases - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel vaccine for treating the chain coccus diseases in fishes. SOLUTION: The vaccine that is prepared from a chain coccus in Enterococcus that does not loose the capsule, particularly Enterococcus seriolicida KG 9408 strain (FERM BP-6749) has excellent vaccine effect against the chain coccus diseases in fishes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a vaccine for preventing or treating streptococcal disease in fish. More particularly, the present invention relates to a novel strain of the genus Enterococcus to which a causative organism of fish streptococcal disease belongs, and a vaccine and a vaccine composition for preventing and treating streptococcal disease in fish using the novel strain.

[0002]

BACKGROUND OF THE INVENTION Streptococcal disease in fish was first reported in a yellowtail farm in Kochi Prefecture in 1974, and since then it has been nationwide every year from summer to autumn, when the water temperature is high. In addition to yellowtail, it has been confirmed to occur in horse mackerel, amberjack, and the like. Enterococcus celioricida (Enterococcu
s seriolicida), beta-hemolytic Streptococcus
Streptococcus iniae and Streptococcus eximilis, Streptococcus equisimilis, and gamma-hemolytic Streptococcus sp.
ccus sp.) have been reported.

[0003] Among the diseases caused by these bacteria, the present invention relates to
The present invention relates to streptococci caused by Enterococcus cerioricida in fish. This bacterium was conventionally treated as Streptococcus, but was classified into Enterococcus in 1991 and named Enterococcus celioricida. Furthermore, in 1996, the synonym of Lactococcus garvieae (Syn
onym) (Teixeira, LM et
al., International J. Systematic Bacteriol., 46,
664-668, 1996), still often referred to as Enterococcus celioricida. From such circumstances, although multiple names are mixed in the present bacterium and diseases caused by the present bacterium, it is often referred to in the present specification as Enterococcus celioricida and streptococci. I do.

It is known that this bacterium has two serotypes depending on the reactivity with the KG antigen. A KG + -type bacterium that has no capsule and the KG antigen is exposed on the surface of bacterial cells is called a KG + type bacterium (hereinafter sometimes referred to as KG +). Type (hereinafter sometimes referred to as KG-) bacteria. The wild type is usually of the KG-type, but by subculturing it in an artificial medium, the capsule disappears between the first and tenth generations and changes to KG +. KG-type strains have a capsule that has been reported to be involved in virulence (Yoshida, T., et al., Disease Aquati
c Organism, 25, 81-86, 1996; Yoshida, T., et al., D
isease Aquatic Organism, 29, 233-235, 1997).

[0005] Streptococci in fish are roughly classified into general types and encephalitis types. The general type presents symptoms and symptoms such as enlargement and protrusion of the eyeball, redness of the inside of the operculum, abscess of the fin base and operculum, and pericarditis. The encephalitis type has few symptoms as seen in the general type, but is characterized by crazy swimming with rollover and turning. Macrolide and tetracycline antibiotics are effective in the early stages of the disease, but there is no effective treatment after the middle stage. In many cases, the use of antibiotics is limited due to the problem of persistence in the fish, and the use of antibiotics causes resistant bacteria by repeated administration. Therefore, it is necessary to refrain from using antibiotics as much as possible. It has already been reported that bacteria that have acquired resistance to these antibiotics have been isolated (Introduction to Fish Diseases, edited by Kiyokuni Takaga, Koseisha Koseikaku, p.
58-61, 1996). Therefore, it is considered that preventive treatment by vaccination will be increasingly important in the future as a measure against streptococcal disease in fish.

[0006] As a currently marketed streptococcal disease vaccine, there is a type in which inactivated streptococci are mixed with a feed (feed) and orally administered to yellowtail. In addition, there is a problem in economics because it must be used in large quantities. Therefore, there has been proposed a method for reducing the dose by using a preventive agent for enterococcal infections in fish containing an inactivated and eluted product of Enterococcus bacterium containing an outer cell wall protein (Japanese Patent Application Laid-Open No. H08-08380). 231408) has not yet been put to practical use. On the other hand, administration methods by injection or immersion are also conceivable. The injection method is labor-intensive because it is necessary to pick up and administer fish for each individual, but has the advantage that the effect is highly reliable and the amount of vaccine used is small. On the other hand, the immersion method is a method in which fish are allowed to swim for a certain period of time in breeding water in which a vaccine solution is suspended, and the same vaccine suspension is used several times to several tens of times by replacing fish. In terms of being able to do so, it can be said that both labor and economy are intermediate between the above two methods.

[0007]

As described above, various methods for preventing or treating fish streptococcal disease by vaccine have been proposed as described above, but none of them can satisfy the effects and economics. I can't say. Accordingly, an object of the present invention is to provide a novel vaccine for preventing or treating streptococcal disease, which exhibits a higher vaccine effect than conventionally known vaccines and is excellent in safety and economy.

[0008]

Means for Solving the Problems The present inventors collected field yellowtail that developed streptococcal disease in the sea area of Higashi-cho, Izumi-gun, Kagoshima Prefecture, and isolated bacteria from the yellowtail. The bacterium was identified in light of the ninth edition of the manual of Bergey, DH and was found to be Streptococcus Enterococcus seriolicida. However, this strain has a unique feature that the capsule does not disappear even after 60 passages, and it has cilia that are considered to be involved in pathogenicity. The strain was named. Furthermore, the present inventors, when using the Enterococcus ceriolicida KG9408 strain, compared to a vaccine prepared from a conventionally known streptococcus in which the capsule is lost by repeated passages against streptococci in fish. The present inventors have found that they have a far superior and high vaccine effect, and completed the present invention based on this finding.

That is, the present invention relates to a streptococcus belonging to the genus Enterococcus which does not lose its capsule even after being passaged, a novel streptococcus enterococcus celioricida KG9408 (FERM BP-6749) which is a representative strain thereof, and Fish streptococcal vaccine containing streptococci belonging to the genus Enterococcus in which the capsule does not disappear, a fish streptococcal vaccine composition comprising the vaccine and a pharmacologically acceptable carrier,
It is a method for preventing and treating streptococci comprising administering the vaccine or vaccine composition to fish. In addition, the streptococcus KG9408 strain according to the present invention was deposited on June 10, 1999, with the Research Institute of Biotechnology and Industrial Technology, and the deposit number is FERM BP-6749. In the present invention, a streptococcus belonging to the genus Enterococcus which does not lose its capsule even after subculture is defined as one which does not lose its capsule even after being subcultured for at least 45 passages in a normal medium, more preferably 60 passages. It means that the capsule is not lost even after subculture.

The streptococcus enterococcus according to the present invention
The mycological properties of Serio Ricida KG9408 strain are as follows. (1) Gram-positive cocci, forming several chains (2) Facultative anaerobic (3) Spore formation:-(4) Capsule: + (5) Cilia: + (6) Motility:-(7) Nitrate reduction: + (8) MR test: + (9) VP test: + (10) Indole production:-(11) Hydrogen sulfide production:-(12) Starch production:-(13) Citric acid utilization (Koser Medium):-(14) Citric acid utilization capacity (Christensen medium):-(15) Inorganic nitrogen source (nitrate and ammonium salt):-(16) Pigment formation:-(17) Oxidase:-(18) Catalase:- (19) Growth pH: 4.5 to 9.6, temperature: 10 to 45 ° C (20) OF test: F (21) Gas production from sugar: D-arabinose:-, D-xylose:-, D-glucose: +, D-mannose:
+, D-fructose: +, D-galactose: +, maltose: +, sucrose:-, lactose:-,
Trehalose: +, D-sorbitol: +, D-mannitol: +, inositol:-, glycerin:-, starch:-, D-raffinose:-(22) Decomposition of esculin: + (23) Decomposition of cellulose:-( 24) Decomposition of hippuric acid:-(25) Decomposition of arginine: + (26) Coagulase:-(27) Hemolysis: α-type (28) Sodium chloride resistance: 0.0-6.5% (29) Acid resistance:-(30 ) Growth in medium supplemented with 40% bile acid: + (31) Growth in medium supplemented with 0.1% methylene blue: +

[0011]

When the Streptococcus enterococcus cerioricida KG9408 strain according to the present invention is used as a vaccine, it may be administered as a live cell, but it may be administered by physical treatment such as heating, ultraviolet irradiation, formalin, or the like. It is preferable to administer the drug after inactivating it using a known method such as a chemical treatment with chloroform, phenol, beta-propiolactone or the like. When used as a live cell, it is desirable that the cell be cultured by a known method such as subculture or high-temperature culture and detoxified or attenuated to such an extent that it does not develop even if infected.
When used in inactivated, sesame oil, vegetable oils such as rapeseed oil, Freund's complete adjuvant, mineral oil such as Freund's incomplete adjuvant, aluminum hydroxide gel, aluminum sulfate gel, and administered together with known adjuvants such as aluminum sulfate gel, vaccine effect Can be increased.
The effect can be further enhanced by adding a known immunopotentiator such as vitamin E, peptidoglycan, levamisole and the like.

When the vaccine of the present invention is administered, the administration route is oral, immersion, intraperitoneal or intramuscular, and is not particularly limited, but is preferably intramuscular, intraperitoneal injection administration or oral administration. This vaccine can be administered prophylactically before the onset, and by administering it after onset, it can be expected to be effective as a therapeutic agent. The dose is 1 × 10 ⁴ to 1 × 10 ¹² CF in the number of bacteria before inactivation.
U / tail, preferably 1 × 10 ⁶ to 1 × 10 ¹⁰ CFU / tail, more preferably 1 × 10 ⁷ to 1 × 10 ⁹ CFU / tail.

The target fish for vaccine administration is not particularly limited as long as it is a fish species suffering from streptococcal disease, such as yellowtail, amberjack, gibberfish, Japanese horse mackerel, Japanese horse mackerel, Japanese flounder, squid, etc. The age of the fish at the time of administration is not particularly limited as long as it has grown to a size that can be technically injected. Generally, it is sufficient that the weight of the fish reaches 10 to 20 g or more regardless of the type of fish. One dose is enough,
It is desirable to re-immunize after another 3 to 12 months.

The method of culturing a streptococcus belonging to the genus Enterococcus, for example, the strain KG9408 of the genus Enterococcus, which does not lose its capsule even after subculturing according to the present invention, is not particularly limited, and a general method for culturing streptococci may be used. I just need. Gravy broth, Mueller Hinton broth, trypticase soy broth, heart infusion broth, Brain broth
It can be cultured on a heart-infusion (BHI) medium, Todd-Hewitt medium, blood agar medium, serum-supplemented medium, etc., but can be performed on a medium supplemented with blood or serum, glucose and seawater (or salt). To develop. The culturing temperature is usually 10 to 45 ° C, preferably 15 to 37 ° C, and more preferably 20 to 30 ° C. The pH during culture is 4.5 to 9.6,
It is preferably 5.5 to 8.5, more preferably 7.0 to 8.0. The culture may be performed under any conditions such as static culture, shaking culture, and stirring culture, but good growth can be obtained by culturing with gentle stirring.

[0015]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. Example 1 Cultivation of Various Streptococcal Strains Twenty-five strains of Streptococcus enterococcus celioricida shown in Table 1 were subcultured on Todd-Hevid agar medium. The bacteria stored at -80 ° C were thawed, and one drop thereof was spread on a plate with a platinum loop and cultured at 25 ° C for 24 hours. Thereafter, the 10 colonies that appeared were reacted with the antiserum. Antigen determination using antiserum was performed every 5 passages. If it has been mutated to KG +, terminate the culture at that passage number.If it has remained KG-, the bacteria have grown sufficiently, and the portion where the colonies are clustered is scraped off with a platinum loop. Similarly Todd
The cells were inoculated on a heavy agar medium with platinum loops, cultured at 25 ° C. for 24 hours, and thereafter the same operation was repeated. As a result, 8 out of 25 strains were mutated to KG + at passage 15, 6 were at passage 30 and 10 were mutated to KG + at passage 45.
-No change was observed in the capsule.

[0016]

[Table 1]

Note 1: Enterococcus celioricida N
G8206, SA9201, MZ9201, KG740
9: Yoshida et al .; Dizzies of Aquatic Organisms; Vol. 25, 81-86, 1996 (Diseases of Aquatic Organisms, 25, 81-86, 1996)
It is described in. Further, Enterococcus cerioricida NG8206, KG7409, and MZ9501 are described in the same journal, Vol. 29, 233-235, 1997.

Example 2 Culture of Streptococcus enterococcus celioricida KG9408 strain (1) Streptococcus KG9408 strain, 1 × 10 ² CFU / ml, was prepared using Todd-Heavid liquid medium (manufactured by Difco) at pH 7.4. At 25 ° C
The culture was stirred for 24 hours. As a result, 1 × 10 ⁹ KG9408 strains
Proliferated to CFU / ml.

Example 3 Culture of Streptococcus enterococcus seriolicida KG9408 Strain (2) Streptococcus KG9408 strain, 1 × 10 ³ CFU / ml, was prepared using Todd-Heavy liquid medium (manufactured by BBL) at pH 7.8. 12 at 25 ° C
Cultured for hours. Thereafter, the number of viable cells was measured by a conventional method, and it was 2 × 10 ⁹ CFU / ml for the KG9408 strain. Example 4 Relationship between viable cell count and absorbance

Streptococcus KG9408 strain, 1 × 10 ³ CFU / ml, was added to Todd-Heavid liquid medium (manufactured by BBL) to adjust pH.
The cells were cultured at 7.4 and 27 ° C for 2, 4, 8, 12 (2 samples), 24 (2 samples), 48 (2 samples) or 96 hours. After adding 0.3% formalin to the culture solution and inactivating at 25 ° C. for 48 hours, the absorbance at 660 nm was measured. As a result, it became clear that the relationship shown in FIG. 1 exists between the viable cell count and the absorbance.

Example 5 Preparation of Vaccine (1) A BHI medium was added to Todd-Heavid liquid medium (manufactured by Difco).
Streptococcus enterococcus celioricida suspended in water
2 × 10 of KG9408 strain⁵Add CFU / ml and incubate at 25 ° C for 24 hours.
Was. Add 0.3% formalin to the culture solution and incubate at 25 ° C for 48 hours
After inactivation, the cells are centrifuged and collected, and the obtained cells are adjusted to 660 nm.
Absorbance value of about 1 (about 1 × 10 ⁹Phase to CFU / ml
Adjusted to be phosphate buffered saline (pH 7.
Resuspended in 2).

Example 6 Preparation of Vaccine (2) 5 × 10 ⁴ CFU / m of Streptococcus KG9408 Strain Suspended in BHI Medium
l was added to a blood agar medium and cultured at 30 ° C. for 48 hours. After adding 0.3% formalin to the culture solution and inactivating at 25 ° C for 48 hours, centrifuging and collecting the cells, the obtained cells were adjusted so that the absorbance at 660 nm was about 1, and the phosphate buffer was adjusted. Saline solution
(pH 7.2).

Example 7 Preparation of vaccine (3) 500 ml of a vaccine solution prepared in the same manner as in Example 5 was used.
And then heat-sterilized at 65 ° C. for 30 minutes to obtain a vaccine preparation.

Example 8 Preparation of vaccine (4) 250 ml of a vaccine solution prepared in the same manner as in Example 6 was used.
And then heat-sterilized at 72 ° C. for 15 minutes to prepare a vaccine preparation.

Example 9 Preparation of vaccine (5) A vaccine solution prepared in the same manner as in Example 5 was freeze-dried to obtain a vaccine preparation. At the time of administration, this formulation was used after being covered with phosphate buffered saline so as to be 1 × 10 ⁹ CFU / ml.

Next, the usefulness of the vaccine according to the present invention will be described.
This will be described in detail below with reference to experimental data. Test Example 1 Immunogenicity in amberjack Forty amberjacks were bred for 5 days in a circulating breeding tank at a water temperature of 23 ± 0.5 ° C., and no abnormalities were observed in the appearance test, and the body weight was 20 g.
The 32 that reached the above were selected. These fish were divided into two groups, a vaccine injection group and a challenge control group, and 16 fish each, so that the average body weights between the groups were almost equal. To the fish in the vaccine injection group, 0.2 ml of the vaccine solution prepared by the method of Example 5 was injected intraperitoneally per fish. The challenge control group was similarly dosed with 0.2 ml of phosphate buffered saline per fish. Thereafter, each was reared in a circulating system at a rearing temperature of 23 ± 0.5 ° C. for 14 days and observed. On the last day, the breeding water temperature was raised to 25 ± 0.5 ° C. over about one day. As a result, during the observation period, no abnormality was observed in all test fish.

A virulent strain of Streptococcus enterococcus celioricida (KG9408 strain) was cultured for 24 hours in Todd-Hevid's liquid medium, and the bacterial solution was diluted with phosphate buffered saline.
A challenge strain solution was prepared. The bacterial concentration was the concentration at which the mortality was about 80% in the preliminary test. After injecting 0.1 ml of the bacterial solution for challenge into the abdominal cavity of all fish in the vaccine injection group and challenge control group, the breeding water temperature was raised to 27 ± 0.5 ° C. over 4 hours and observed for 14 days. As a result, the mortality rate in the challenge control group was 87.5%, whereas no mortality was observed in the vaccine injection group (Table 2-Experiment 1). When the test was repeated under the same conditions, the mortality rates of the challenge control group and the vaccine injection group were 93.8% and 6.3%, respectively (Table 2-Experiment 2).

[0028]

[Table 2]

Test Example 2 Immunogenicity in Yellowtail (1) An attack test in yellowtail was carried out in substantially the same manner as in Test Example 1. The amount of challenged bacteria was 5.1 × 10 ³ CFU / 0.1 ml / tail.
The vaccine prepared by the method of Example 5 was
Three groups were administered with 0.1 ml per tail, and one group was administered with 0.1 ml per tail of a vaccine solution diluted 2 or 4 times with phosphate buffered saline. The challenge control group received 0.1 ml of phosphate buffered saline instead of the vaccine solution. As a result, the mortality rate of the challenge control group was 87.5%, while the mortality rate of each vaccine group was 0% (Table 3).

[0030]

[Table 3]

Test Example 3 Immunogenicity in Yellowtail (2) An attack test in yellowtail was carried out in the same manner as in Test Example 2. The challenge amount was 5.1 × 10 ⁴ CFU / 0.1 ml / tail. As a result, the mortality rate of the challenge group was 100%, whereas the mortality rate of the vaccine administration group was 0 to 6.3%, indicating a high vaccine effect (Table 4).

[0032]

[Table 4]

Comparative Test Example 1 Effect of Streptococcal NS Strain Vaccine A vaccine solution was prepared in the same manner as in Example 5 using Streptococcus enterococcus cerioricida NSS9310 strain in which the capsule and cilia were lost by passage. Using this vaccine solution, immunogenicity was examined in the same manner as in Test Example 1. As a result, the mortality rate in the two groups set as the challenge control group was 93.8% and 87.5%, whereas the mortality rate in the NS strain vaccine injection group (two groups) was 62.5%.
68.8%, which was much lower than the vaccine prepared using the Enterococcus cerioricida KG9408 strain of the present invention.

[0034]

[Table 5]

[0035]

EFFECT OF THE INVENTION The vaccine of the present invention prepared from a Streptococcus of the genus Enterococcus which does not lose its capsule even by subculturing is susceptible to streptococci such as yellowtail, amberjack, yellowtail, horse mackerel, white horse mackerel, flounder, and lobster. It is highly effective in the prevention and treatment of streptococci in fish species.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between the viable cell count and the absorbance.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:01) C12R 1:01)

Claims (9)

[Claims] 1. A streptococcus belonging to the genus Enterococcus, which does not lose its capsule even after subculture. 2. The streptococcus according to claim 1, wherein the Enterococcus bacterium is Enterococcus seriolicida. 3. A new strain Enterococcus celioricida K.
G9408 (FERM BP-6749). 4. A fish streptococcal disease vaccine containing the Streptococcus of Enterococcus genus according to claim 1. 5. A fish streptococcal vaccine composition comprising the streptococcus of the genus Enterococcus according to claim 1 and a pharmacologically acceptable carrier. 6. The vaccine according to claim 4, wherein the streptococcus is a new strain Enterococcus cerioricida KG9408 (FERM BP-6749). 7. The vaccine composition for a fish streptococcal disease according to claim 5, wherein the streptococcus is a new strain, Enterococcus cerioricida KG9408 (FERM BP-6749). [8] A method for preventing and treating streptococci in fish by administering the vaccine according to any one of [4] and [6]. 9. A method for preventing and treating streptococci in fish by administering the vaccine composition according to any one of claims 5 and 7.