JPH11332558A - Vaccine for enterococcal disease of fishes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new strain capable of producing a vaccine for significantly preventing occurrence of enterococcal disease, useful for sea water cultivation of yellowtails, etc., by an extremely thin capsule or having no capsule at all. SOLUTION: This vaccine is obtained by culturing Lactococcus garvieae [Enterococcus seriolisida] in a glucose-containing heart infusion agar medium while varying temperature in a range of at least 5-25 deg.C and has an extremely thin capsule or no capsule at all. The strain is deposited as Lactococcus garvieae SWEAT 93,003 attenuated strain (FERM P-16,714). Preferably a vaccine for enterococcal disease is prepared by including a microbial cell inactivated by treating the strain with formaldehyde.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a vaccine useful for long-term prevention of disease damage caused by enterococcal infections in fish, especially sea-cultured fish mainly including yellowtail. Furthermore, the present invention relates to a novel strain belonging to Lactococcus garbiae [Enterococcus cerioricida] useful for the preparation of the vaccine.

[0002]

2. Description of the Related Art Enterococcus celioricida ( En
Enterococcal disease (α-hemolytic streptococcal disease) caused by terococcus seriolicida ) is one of the three most important diseases of fish as well as bacterial diseases, such as tuberculosis and iridovirus infection. Although this disease occurs mainly in the summer season, it is almost anniversary, and unlike other diseases, it is one of the most common diseases of cultured yellowtail due to the fact that large-sized fish such as 1-year-old fish and 2-year-old fish that have passed over the year are severely affected. The damage has been severe.

[0003] For this reason, in order to reduce the spread of outbreaks and the appearance of dead fish at the aquaculture sites, measures using antibacterial drugs such as antibiotics have recently been taken. However, the target yellowtail requires a large amount of pharmaceuticals due to the large size of the fish body, which puts a great deal of management pressure on the aquaculture companies, and the emergence of antibiotic-resistant bacteria due to continuous use has led to the emergence of drugs. At present, there are various problems, such as the case where the method does not work.

[0004] Under these circumstances, the importance of preventive measures against enterococci has been pointed out, and development of vaccines against the disease has been expected.

[0005] In the study of vaccines for the prevention of enterococci, about 5 g / day of freeze-dried cells or 20 mg / day of wet bacteria were applied to about 100 g of formalin-inactivated bacteria per test fish. Administered orally with feed supplementation, or immersed in a 1 mg / ml vaccine solution for 3 minutes, or injected 1 mg intraperitoneally for up to 16 days, and died several weeks after inoculation with live bacteria by intraperitoneal injection As a result of the examination of the rate, it was reported that the protective effect was observed in the peritoneal cavity and the fish immersed 4 weeks after the vaccine administration, and that the protective effect was observed up to 2 weeks after oral administration even if the vaccine administration days were long. (Iida et al., Fish Disease Research: 16 (4), 201-206 (1982)). The formalin-inactivated bacterium was injected intraperitoneally into yellowtail, and after 4 weeks, the serum antibody titer, antibacterial activity, macrophage phagocytic activity, and protection against live bacterial infection were examined. There is a study report that there is a possibility of vaccine prevention because there is a difference in antibacterial activity and infection protective property compared to that of Kusuda et al. (Summary of 1993 Spring Meeting of the Japanese Society of Fisheries Science, 197
page).

Based on such research, an inactivated oral vaccine for enterococci in yellowtail (α-hemolytic streptococci) has been put to practical use.

[0007] However, in the case of the oral vaccine, it is necessary to administer a large amount by continuous administration for at least 5 days, and the effect is only confirmed for several months, and it is economical and effective. The situation is not yet satisfactory. The target is 100g of average weight
There is a problem that it is limited to 300 g of yellowtail. In addition, the need for large doses requires farmers to establish facilities for refrigerated storage of large amounts of vaccine.

On the other hand, the vaccine for injection is expected to have a relatively long-lasting effect with a single small dose, but still exerts a long-lasting effect for six months to one year, causing the occurrence of enterococci in fish. In fact, there is no technology that can prevent this.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to provide a vaccine for preventing the development of enterococcal infections in fish, particularly sea-cultured fish, mainly yellowtail, which is effective for a long period of time when administered in small amounts. The purpose is to provide a highly practical vaccine. A further object of the present invention is to provide a novel strain of Lactococcus garviae [Enterococcus cerioricida] used for the production of such a highly practical vaccine, and a method for producing a vaccine using the same.

[0010]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies on the background, and found that Lactococcus
Inactivating and inactivating a novel attenuated strain (hereinafter also referred to as "producing strain" or "capsule ^- strain") which belongs to Garbiae (Enterococcus cerioricida) and has substantially lost its capsule. When the bacterial cells are intraperitoneally administered to fish, inactivated bacterial cells of a highly toxic Lactococcus garvieae [Enterococcus cerioricida] bacterium having a capsule (hereinafter, also referred to as "protozoa" or "capsule ⁺ strain") Was found to be able to exert protection against live bacterial infection (enterococcus) for a longer period of time as compared with the case of administering. The present inventors have found that by further studies, the production strain ( "capsular ^- strain") itself is inactivated bacteria ( "capsular ^-
Strains), which are 100 times more protective (vaccine effect) than the above strains, and the attenuated bacteria are extremely useful and practical vaccines capable of shortening the production process and further reducing the dose. (Live vaccine).

Hitherto, strains used for the preparation of vaccines have been required to enhance or maintain toxicity by preliminarily passing through living organisms or avoiding subculture in order to exhibit the effect as a vaccine. (JP-A-8-2
No. 31408). In view of such conventional common sense, the finding of the present invention that the attenuated bacterium itself or its inactivated bacterium is effective as a vaccine is surprising. Also, according to the conventional common sense described above, it is necessary to enhance or maintain the toxicity of bacterial cells as a pre-stage of vaccine production, which has been one of the factors of inefficient vaccine production. According to the present invention, there is also an advantage that such pretreatment is unnecessary and vaccine production can be efficiently performed.

[0012] The present inventors, based on the above findings, further capsular ^- aggregation antibody titer in serum of the fish treated with inactivated cells of the strain, confirms phagocytic activity and the like, also capsular ^- viable cell strains After confirming the efficacy as a vaccine and its safety, the present invention was developed.

That is, the present invention is a novel strain belonging to Lactococcus garbiae (Enterococcus cerioricida) listed in any of the following (1) to (3): (1) The capsule is extremely thin or the capsule is extremely thin Lactococcus galbiae, characterized by having no Lactococcus
garvieae ) [Enterococcus celioricida ( Entero
coccusseriolisida )]]. (2) whether the capsule is extremely thin, obtained by culturing Lactococcus garbiae [Enterococcus cerioricida] in a heart-infusion agar medium supplemented with glucose while changing the temperature at least in the range of about 5 to 25 ° C. Alternatively, a strain belonging to Lactococcus garbiae (Enterococcus cerioricida) having no capsule. (3) Lactococcus garbiae MS93003 attenuated strain (FERM P-16714).

Further, the present invention is an inactivated vaccine for enterococci in fish, which is listed in the following (4) or (5): (4) The capsule according to any one of (1) to (3) is A vaccine for enterococci in fish, comprising an inactivated cell of a novel strain of Lactococcus garbiae (Enterococcus cerioricida) which is extremely thin or does not have a capsule; (5) the inactivated cell is , (1) to (3), obtained by treating a strain belonging to Lactococcus garbiae (Enterococcus cerioricida) having a very thin or non-capsular form with formaldehyde. (4) The vaccine for enterococci in fish according to (4).

The present invention also provides a live vaccine for enterococci in fish, which is described in the following (6): (6) The capsule according to any one of (1) to (3) is extremely thin or has a capsule A vaccine for enterococci in fish, comprising a cell of a novel strain of Lactococcus garbiae (Enterococcus cerioricida) having no membrane; the present invention also provides an inactivated or live bacterial vaccine thereof. Is provided in the form of an injection.

The present invention is directed to Lactococcus garbiae [Enterococcus celioricida].
Is a bacterium first isolated from cultured yellowtail around 1974 and originally reported in scientific journals as α-hemolytic Streptococcus sp. From a bacteriological classification. Then, based on further studies of bacteriological properties,
It was renamed Enterococcus ceriolicida in 991, but was recently found to belong to existing lactococci, and again Lactococcus garbiae
garvieae) (International Journal
of Systematic Baceriology, July 1996, p.664-66
8). However, since the names Enterococcus celioricida are still generally used among related industries, both names of Enterococcus celioricida and Lactococcus garbiae are used in this specification.

The name of the disease caused by the bacterium is also called enterococci with the name change of the bacterium in 1991. The term streptococcal disease is generally accepted.

[0018] In the present invention, the term "fish" broadly refers to fish in general, including yellowtails, and the yellowtails include, for example, yellowtail (S. quinqueradiata) and yellowtail (S. aureovittat).
a), fish belonging to the genus Seriola such as amberjack (S. purpurascens) and the like are used for the purpose of broadly encompassing juvenile fish to adult fish.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The strain of the present invention is characterized in that the capsule is extremely thin or has no capsule, Lactococcus garbiae (Enterococcus cerioricida).
It is a novel strain belonging to

The strain of the present invention is prepared by subjecting Lactococcus garvieae [Enterococcus seriolisida] (protozoa), which is a causative agent of enterococcal infections in seawater-cultured fish, especially fish, mainly to yellowtail, to a certain degree, It can be prepared by substantially losing its capsule. In addition,
Lactococcus garbiae (Enterococcus celioricida), which is the protozoan, can be isolated and obtained from fish infected with enterococcal infection by a conventional method, and it belongs to Lactococcus garbiae (Enterococcus celioricida). It doesn't matter if the stock is different.

The method for treating the protozoa is not particularly limited as long as it can prepare Lactococcus garbiae (Enterococcus cerioricida) having an ultrathin capsule or not having a capsule. For example, a method of culturing in a heart infusion agar medium (Eiken Chemical Co., Ltd.) containing glucose, preferably about 0.5% (w / v) glucose, at least in a temperature range of about 5 to 25 ° C. Can be mentioned.

As a more specific culture method, a Lactococcus garvieae [Enterococcus cerioricida] protozoa is inoculated on a sterile heart-infusion agar medium supplemented with glucose, and is inoculated at least in the range of about 5 to 25 ° C., preferably about 5 to 25 ° C. Under conditions where the temperature can fluctuate in the range of 7 to 23 ° C., about 1 to 10 days, more specifically 215
For example, a method in which the culture is allowed to stand for about 225 hours, preferably about 220 hours.

The temperature may fluctuate by about 5 to 25 ° C. within a predetermined time.
The temperature is not particularly limited as long as the low-temperature culture and the high-temperature culture in the range described above are alternately performed to give a temperature stimulus to the cultured bacteria.
A simple example is a temperature fluctuation mode in which a cycle of low temperature → high temperature → low temperature is repeated within 24 hours in accordance with the fluctuation of the atmospheric temperature.

In order to obtain a strain having substantially lost the capsule, the present inventors carried out Lactococcus garbiae [Enterococcus cerioricida] protozoa in an EF agar medium (EF agar medium) prior to the above-mentioned temperature-stimulated culture. Nissui ": Nissui Pharmaceutical Co., Ltd.) at 27 ° C for 57 days (once)
Cultivation, followed by 2-37 in heart infusion agar medium (27 ° C) supplemented with 0.5% (w / v) glucose.
Days was performed four more times passaged cultures, but capsule of the object by any of the culture ^- was not possible to obtain a strain.

From the above, the characteristics of the strain of the present invention (capsule)
^- ) Is considered to have been found by performing the above-mentioned temperature stimulation. However, prior to such temperature stimulation culture, subculture at the same temperature, for example, culturing in EF agar medium and 0.5%
(W / v) Subculture on a glucose-added heart infusion agar medium may be performed.

Preferably, culturing on an EF agar medium (about 2
7 ° C.) and 0.5% (w / v) glucose, and subcultured several times on a heart infusion agar medium (about 27 ° C.), followed by temperature fluctuation culture on the same agar medium (about 5 to 5 ° C.). (25 ° C.).

Capsule of [0027] the present invention ^- strain, 0.5% (w /
v) It has the property that it grows faster than the original strain (capsule ⁺ strain) under culture conditions using a heart-infusion agar medium supplemented with glucose at about 5 to 25 ° C, preferably about 7 to 23 ° C. doing. Thus, capsule of the present invention ^- strain, after cultivation under the temperature fluctuations described above, about 5 to 25 ° C. with the same medium, preferably growth under culture conditions in the range of about 7 to 23 ° C. By selecting and collecting early colonies, they can be separated and obtained.

The capsule of the invention that are produced in this way ^-
The strain was sent to the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry, located at 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture.
Indication of microorganism on March 18, 998: (Indication for identification by depositor) Deposited as "MS93003 attenuated strain" and (Accession number) "FERM P-16714". Regarding the MS93003 attenuated strain, the International Journal of Systematic Bacteriology (International Journal of Systematic Bacteriology)
Bacteriology), 16 (3), 313-340, 1960, the following bacteriological properties were examined. (1) Morphological properties: The results of observing the colonies of the MS93003 attenuated strain grown on a heart infusion agar medium supplemented with 0.5% (w / v) glucose at 27 ° C. for 24 hours using an electron microscope are shown below. (I) Cell shape: ovoid cocci (ii) Cell size: about 1.4 in diameter × about 0.7 μm in short diameter (iii) Cell polymorphism: linked (iv) Motility: None (v) Presence or absence of capsule: No capsule was observed by observation with a transmission electron microscope (magnification × 50,000) (see FIG. 1). From this, it is judged that the MS93003 attenuated strain does not have a capsule or is extremely thin even if it does. (2) Growth state in various media: using various media,
Table 1 shows the state of each colony grown at 27 ° C. for 11 days. The classification color names are displayed based on “Standard Color Chart A, 1981, supervised by Japan Color Research Institute”.

[0029]

[Table 1]

Table 2 shows that the MS93003 attenuated strain (capsule ^- strain) of the present invention and the MS93003 strain (source strain of the produced strain of the present invention: capsular ⁺ strain) were 0.5% (w /
v) shows the results of comparing the growth status at different temperatures in a glucose-added heart infusion agar medium (HIg medium).

[0031]

[Table 2]

The results of observing the state of colonies obtained by culturing the MS93003 original strain (strongly virulent strain) and the MS93003 attenuated strain at 10 ° C. in the above medium and observing them with a stereoscopic microscope are shown in FIGS. 2 and 3, respectively. Show. (3) Physiological properties: Physiological properties of the MS93003 attenuated strain are as follows.

For properties other than the following 1), 7) and 10), using Eiken Chemical Co., Ltd. Biotest No. 1 “Eiken” and Nippon BioMérieux Biotech Co., Ltd. API20E (API20E), The study was conducted according to the method recommended by the supplier.

1) Gram staining Gram-positive 2) Nitrate reduction -3) VP (acetoin production) test + 4) Indole production -5) Hydrogen sulfide production -6) Citric acid utilization -7) Pigment production -8) Urease -9) Oxidase-10) Can grow at a growth temperature range of about 5-45 ° C 11) β-galactosidase activity-12) IPA test-13) Lysine decarboxylation-14) Ornithine decarboxylation-15) Arginine hydrolyzability + 16) Malonic acid utilization ability -17) Oxidase -18) Tryptophan deaminase -19) Gelatinase-(4) Availability of carbon source: arabinose-glucose + rhamnose-maltose + sucrose-sorbitol-mannitol + inositol-adnit-melibiose-amygdalin + Moreover, capsule of the present invention ^- strain, the original bacteria It is characterized in that it is an attenuated strain with reduced toxicity compared to. MS93
003 (capsule ⁺ strain) and MS93003 of the present invention
The toxin-producing ability of the attenuated strain (capsule ^- strain) is shown in Tables 3 and 4 as the virulence that occurs 10 days after inoculation of yellowtail, respectively.

The water temperature of the yellowtail breeding aquifer at the time of the attack was 2
At 4 ° C, the amount of dissolved oxygen was 7.22 mg / l, the water temperature during the test period was 22 to 24.3 ° C (average 23.6 ° C), and the amount of dissolved oxygen was 6.5 to 7.5 (average 6.5). 99) mg / l.

[0036]

[Table 3]

As can be seen from Table 3, the yellowtail inoculated with the MS93003 protozoa (capsule ⁺ strain) was 10% from the challenge.
Almost died on the day.

[0038]

[Table 4]

As can be seen from Table 4, the MS93 of the present invention
Yellowtail inoculated with the 003 attenuated strain (capsule ^- strain) did not die even 10 days after the challenge, and the number of deaths was 0.

[0040] From the above it, producing strains (capsular of the present invention ^-
Strain) was confirmed to be an attenuated strain with significantly reduced pathogenicity to fish.

The capsule of the present invention ^- strain, was suspended in 10% skimmed milk added saline, can be cryopreserved and lyophilized also suspended in a suitable medium such as Todd diffuser It Broth liquid culture It becomes cloudy and can be stored frozen at -80 ° C or lower.

The present invention is a vaccine useful for preventing enterococci characterized by using the attenuated bacterium (capsule ^- strain) of Lactococcus garbiae [Enterococcus cerioricida] thus obtained. .

[0043] Vaccines of the present invention, the capsular First ^- a inactivated vaccines prepared by inactivating the strain.

[0044] Capsular ^- inactivation treatment of strains, capsule of the present invention ^- pathogenic strains, namely fish, not particularly limited as long as particular process to eliminate the infectivity for yellowtail acids, formaldehyde and chloroform Any inactivation treatment used for preparing an inactivated vaccine, such as treatment with an organic solvent, acid treatment with acetic acid, or the like, can be used. Preferably, it is a formaldehyde treatment, and specifically, usually 0.2 to 0.4%, preferably 0.25 to 0.35%,
More preferably, there is a method of treating cells at 4 to 6 ° C. for 2 days using about 0.3% formaldehyde. The inactivated bacterial cells are removed with an inactivating agent such as an organic solvent or an acid, washed with a physiological saline solution or a buffer, and then recovered as an insoluble precipitate by centrifugation.

Further, the vaccine of the present invention, capsular second to the present invention ^- is a viable vaccine was used as strain.

After washing the strain with a suitable solvent such as physiological saline or buffer solution, ^- [0046] The bacteria vaccine capsule of the present invention
The cells can be prepared by directly utilizing the cells recovered by precipitation by centrifugation.

The inactivated treated cells, live cells or their dried products thus obtained are used as vaccines for enterococci in cultured fish (inactivated vaccines, live cells, etc.) as specifically described in Examples. Vaccine).

The form of the vaccine is not particularly limited, and may be any of an injection, an oral preparation, and an immersion preparation, but is preferably an injection preparation which has a long-lasting effect over a long period of time when administered in a small amount. .

An injectable vaccine can be prepared by suspending the above-mentioned cells or a dried product thereof in sterilized physiological saline for fish or the like. The vaccine for injection includes, in addition to bacterial cells and physiological saline, a suspending agent, a stabilizer, an emulsifier, a buffer, a preservative, a solubilizing agent, or any other appropriate agent usually used for the injection. Additives can also be included.

Conventionally, adjuvants have been used to improve the immunity and the like of vaccines. The vaccine of the present invention can provide a sufficient immunity without using such an adjuvant, and is characterized by that point. However, the present invention does not limit the use of the adjuvant at all, and an adjuvant may be added in addition to the above components, if desired.

The amount of the inactivated treated cells or the dried product thereof contained in the inactivated vaccine which is the first vaccine of the present invention is not particularly limited, but preferably, the absorbance at a wavelength of 620 nm is 0.8. ~ 1.2, more preferably about 1.

On the other hand, the live vaccine, which is the second vaccine of the present invention, has a viable count of about 1/100 of the number of inactivated bacteria contained in the inactivated vaccine and is equivalent to the inactivated vaccine. Can exert its effect. Therefore, the live bacterial vaccine of the present invention contains, but is not limited to, live bacteria or a dried product thereof prepared so that the absorbance at a wavelength of 620 nm is 0.8 to 1.2, more preferably about 1. It can be prepared by further diluting the solution to about 1/100.

The dose (m
Since the effective amount can be appropriately adjusted by increasing or decreasing l), the inactivated vaccine and the live bacterial vaccine of the present invention are not limited to the content of the above-mentioned cells at all.

The vaccine of the present invention is intraperitoneally administered to fish,
The dose for achieving the object of the present invention varies depending on various factors such as the season and water temperature to be administered, the type of fish, the age and the body weight, and cannot be specified unconditionally.
For example, an inactivated vaccine was prepared by using a vaccine having an absorbance of about 1 at a wavelength of 620 nm, and a live bacterial vaccine was prepared by further diluting the vaccine having an absorbance of about 1 at a wavelength of 620 nm by 100 times. The vaccine is used to produce 0.1 to 0.l.
A method of intraperitoneally injecting about 5 ml according to body weight can be mentioned.

The vaccine of the present invention can be administered to fish expressing an immune response without any particular limitation on the weight and age of the fish. For example, the weight is not limited, but is usually 20 g or more, specifically, 20 g to 5 k.
g, preferably 30 g to 1 kg, more preferably 35 g
It can be used for fish of up to 150 g, especially yellowtail.

The vaccine of the present invention has a duration of effect of about 300 days or more, preferably about 1 year or more, in a single administration of both the inactivated vaccine and the live bacterial vaccine.
It can exert a protective effect on infection of yellowtail enterococci over a long period of time.

In addition, when a conventional oral vaccine is administered to about 20,000 juvenile yellowtail juveniles of 100 g size, a minimum of 100 L of vaccine is required (see the “Use of Pharmaceuticals for Fisheries”, the 12th report, issued by the Fisheries Agency). Therefore, it is necessary to provide a separate refrigerated storage space. On the other hand, according to the vaccine of the present invention, 10
In the case of a fish of 0 g size, the dose per fish is 0.5 ml, so that about 10 L of vaccine is sufficient for about 20,000 fish and does not hurt the storage space.
In particular, the live bacterial vaccine of the present invention can exert the same effect at 1 / 100th of the inactivated vaccine, so that it is extremely useful for further reducing the dose and saving storage space. is there.

Hereinafter, the contents of the present invention will be specifically described with reference to Examples and Experimental Examples. However, the present invention is not limited to these.

[0059]

Example 1 A 0-year-old yellowtail fish infected with enterococci was isolated and subcultured on a heart infusion (HIg) agar medium supplemented with 0.5% glucose (manufactured by Eiken Chemical Co., Ltd.). One strain of Lactococcus garbiae [Enterococcus cerioricida] (MS93003 strain) was inoculated on an EF agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and cultured at 27 ° C. in static. After cultivation for 57 days, the grown colonies were fished and the presence or absence of a capsule was confirmed, but no bacteria having lost the capsule were observed.

Next, the bacterium was inoculated on a heart infusion (HIg) agar medium (manufactured by Eiken Chemical Co., Ltd.) supplemented with 0.5% glucose, and cultured at 27 ° C. for 2 to 37 days. After three further subcultures, the presence or absence of the capsule was confirmed, but no bacteria having lost the capsule were detected.

Therefore, the bacterium was statically cultivated for 9 days in a clean bench in which the temperature fluctuated in the range of 7 to 23 ° C. in accordance with the fluctuation of the atmospheric temperature (air temperature) using the same medium.
In this culture, colonies that grew faster than the MS93003 protozoa appeared. Therefore, this colony was collected, and the shape of the bacterium was observed with a transmission electron microscope to confirm that the bacterium had lost the capsule. One strain of this strain was attenuated MS93003 strain (FERM P-16714) deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.
It is.

This MS93003 attenuated strain (capsule ^- strain)
Were stored frozen at -80 ° C until used for vaccine production. Example 2 The cryopreserved MS93003 attenuated strain obtained in Example 1 was
After rapid thawing with warm water at 5 to 40 ° C., the mixture was developed on a heart infusion agar medium supplemented with 0.5% glucose, and cultured at 27 ° C. for 24 hours. After confirming that there is no contamination at the time of colony appearance, inoculate several colonies in a commercially available Todd Hewitt broth (Difco),
The cells were shaken at 27 ° C. for 24 hours and cultured in large amounts. In the culture solution,
Formalin was added so that the final concentration of formaldehyde was 0.3%, and the temperature was maintained at 4 ° C. for 1 to 2 days. After that, the bacteria were inactivated by spreading on HIg agar medium. did.

Further, it is left at 30 to 40 ° C. for 24 hours,
Thereafter, the inactivated cells were collected by centrifugation at 3,500 rpm for 20 minutes. The collected precipitated cells were then washed twice with 0.85% sterile saline and centrifuged again.
The obtained precipitate was added to sterile physiological saline at an absorbance of about 1.0.
(Wavelength: 620 nm) to prepare an inactivated vaccine of the present invention.

The inactivated vaccine was refrigerated at 4 to 5 ° C. until administration to fish. Example 3 obtained in Example 1 was cryopreserved MS93003 attenuated strain ^- after the rapid thawed (capsular strain) at 35-40 ° C. warm water, 0.5
The solution was developed on a heart infusion agar medium supplemented with% glucose and cultured at 27 ° C. for 24 hours. When colonies appeared, it was confirmed that there was no contamination with bacteria, and several colonies were inoculated on a commercially available Todd Hewitt broth (manufactured by Difco), shaken at 27 ° C. for 24 hours, and cultured in large amounts.

The bacterial culture was centrifuged (3500 rpm,
20 minutes), the cells were collected, and the collected precipitated cells
After washing twice with sterile saline and centrifuging, the suspension was suspended in sterile physiological saline so as to have an absorbance of about 1.0 (wavelength: 620 nm) to adjust the concentration. This is then further diluted with sterile physiological saline to a concentration of 1/100 (about 2 × 10 ⁶ cfu / ml).
To obtain a live bacterial vaccine of the present invention. In addition, until the said live bacterial vaccine is administered to fish,
Stored refrigerated at 4-5 ° C.

[0066]

[Experimental example] Experimental example 1 Protective effect of inactivated vaccine Healthy yellowtail (S. quinqueradiata) having a fish weight of about 100 g was fried from 96 fish cages and divided into two groups: 68 test groups and 68 control groups. . 0.5 ml of the inactivated vaccine prepared in Example 2 was added to the test group, and 0.5 ml of physiological saline was added to the control group.
1, each was administered intraperitoneally with a sterile syringe or continuous syringe. 20 of each group were used for measurement of a blood agglutination titer in Experimental Example 3 described later.

For the remaining 48 fish in each group, 15
After day after 65 days, after and 295 days 135 days, each 10 fish of each test and control groups, capsule of the present invention ^- the strain of virulent MS93003 original bacteria (capsular ⁺ strains) 0.5 ml
Was intraperitoneally administered to perform live bacterial infection challenge. In addition, 358 days after the administration of the vaccine or saline, the highly virulent Lactococcus garvieae [Enterococcus seriolicida] bacteria (pods) isolated from the yellowtail infected with enterococci in eight fish of each of the test group and the control group were separately isolated. film ⁺ strain) 0.5m
1 was similarly administered intraperitoneally.

Table 5 shows the number of deaths (tail) of yellowtail caused by enterococci two weeks after the challenge with live bacteria and the mortality (%) with respect to the number of test fish in each administration group.

[0069]

[Table 5]

As can be seen from the above results, the vaccine of the present invention can significantly confer protection against enterococci to fish, and its efficacy is maintained for at least about one year by a single intraperitoneal administration. It turned out to be. Experimental Example 2 Healthy amberjack (S. purpurascens) 2 weighing about 50 g fish
As a test group, 0.5 ml of the inactivated vaccine prepared in Example 2 was intraperitoneally inoculated in 5 animals in the same manner as in Experimental Example 1, and as a control group, 25 amberjack (50 g) were intraperitoneally injected with 0.5 ml of physiological saline. It was inoculated internally. On day 216 after inoculation, a concentration of 1.04 × 10 ⁶ cfu / ml for 10 fish in each group,
On day 394, 1.58 × 10 ⁶ cfu for 9 fish in each group
Virulent MS93003 protozoa (capsule ⁺ strain)
0.5 ml of each was intraperitoneally injected and inoculated to infect live bacteria and observed for 2 weeks to determine the protective effect.
Table 6 shows the results.

[0071]

[Table 6]

As can be seen from these results, as in Experimental Example 1, the inactivated vaccine of the present invention can significantly impart a protective ability to amberjack against live Lactococcus garvieae infection, The efficacy was found to last for more than one year with a single intraperitoneal administration. Experimental Example 3 The blood agglutinin titer of fish was examined as one of the indicators of the protective ability against enterococci. Specifically, in the experimental system of Experimental Example 1, the test group (20
Blood was collected from each of the 5 groups on the 15th, 65th, 135th and 295th days after the administration for the tail) and the control group (20).

As a comparative experiment, highly toxic MS930 was used.
The inactivated vaccine of the highly virulent MS93003 (capsule ⁺ strain) obtained by inactivating the 03 original strain according to the method of Example 2 was administered to yellowtail in the same manner as in Experimental Example 1, and 15 days after administration. , 65 days, 135 days and 295 days, the blood of the yellowtail was collected.

The hemagglutination titer of the obtained blood (serum)
The method was performed according to Roverson's microtiter method.
niques in Fish Immunology, 1990, SOS Publications, 4
3 DeNormandie Ave. Fair Haven, NJ07704-3303 USA].
Specifically, the experiment was performed in each well containing serum (25 μl) serially diluted in saline.
Add 25 μl of attenuated strain (capsule ^- strain) and mix.
Incubation was carried out overnight at room temperature, and after the agglutination reaction, the titer of each well was measured. As a control experiment, an experiment using normal fish serum (non-immune serum) was performed in the same manner. The agglutination titer (Agglutinating titer: log ₂ ) was evaluated with a score of 1 to 12, with the wells in which the generated aggregation was visually confirmed as the last.

FIG. 4 shows the results. In the figure,-●
- the present invention attenuated strains of sera collected over time from the fish administered inactivated vaccines of the present invention ^- illustrates the aggregation for (capsular strain), - ▲ - the virulent strain of (capsular ⁺ strains) Shows the agglutination ability of serum collected over time from fish to which the inactivated vaccine was administered to the attenuated strain of the present invention (capsule ^- strain);
( ^- ) Indicates the agglutination ability (control) of the serum collected over time from unvaccinated fish to the attenuated strain of the present invention (capsule ^- strain).

From FIG. 4, it can be seen that the inactivated vaccine using the produced strain (capsule ^- strain) of the present invention is the same as the original strain (capsule ⁺ strain).
It was found that the agglutinin titer was significantly higher than that of a vaccine prepared by inactivating the agglutinin. After 295 days after immunization, the vaccine prepared by inactivating the original bacteria (capsule ⁺ strain) showed no significant difference from the control, whereas the vaccine of the present invention The conjugated vaccine (capsule ^- strain inactivated vaccine) maintained a significantly higher agglutinin titer. From this, the inactivated vaccine of the present invention prepared using the capsular ^- strain was more effective and persistent against enterococci than the inactivated vaccine prepared using the conventional capsular ⁺ strain. It is thought to exhibit excellent defense performance in Experimental Example 4 The inactivated vaccine prepared in Example 2 was inoculated intraperitoneally into 70 healthy juvenile yellowtail, which were fried from a cage and weighing about 35 g.
Each 1 ml was inoculated. Twenty-five (Group (1)) 15 days after inoculation and 40 (Group (2)) 63 days after inoculation, highly virulent Lactococcus garbiae (Enterococcus) isolated from yellowtail infected with enterococci. [Serioricida]] (capsule ⁺ strain) was intraperitoneally injected to perform live bacterial infection. Similarly, as a control experiment, the same virulent strain was intraperitoneally injected to the same number of untreated healthy fry as the test group inoculated with the vaccine to carry out live bacterial infection.

Table 7 shows the number of deaths due to enterococci (tail) and the ratio of the number of deaths to the number of test fish (%) two weeks after the infection.

[0078]

[Table 7]

In order to carry out vaccine administration efficiently, the first condition is that an immune response on the fish side develops. In this sense, it is necessary to select a fish size (age) for which an immune response has been established as the administered fish. is there. However, in order to prevent enterococcal infection as early as possible from the stage of fry, it is desirable that the size (age) be as small as possible, and it is necessary to consider the administration cultivation efficiency. According to the above experiment, the inactivated vaccine of the present invention can provide a sufficient protective effect against enterococci (α-hemolytic streptococci) even for about 35 g of juvenile yellowtail fry. Can be significantly prevented at an early stage. Experimental Example 5 Passive Immunity Effect Test In the experimental system of Experimental Example 1, blood was collected from fish on the 135th day after administration of the inactivated vaccine of the present invention, and 3 ml of immunized fish serum prepared therefrom was used for each yellowtail having an average body weight of 45 g. The animals were intraperitoneally administered to 10 fish. As a control group, 10 yellowtails each having an average body weight of 45 g prepared by intraperitoneal administration of physiological saline instead of immune fish serum were prepared.

Twenty hours after each administration, yellowtail of each group was
Lactococcus isolated from yellowtail infected with enterococci
Garbiae (Enterococcus celioricida) bacteria
(Capsule ⁺(Strain: MS9502 strain) 2.45 × 10^Fourcf
u / 0.2 ml intraperitoneally, 10
A day later, the state of death due to enterococci was observed.

As a result, no death was observed in the serum-administered group, whereas six yellowtails died in the control group. As described above, since yellowtail acquired protection ability against enterococci by inoculation with the immunized fish serum, it is considered that passive immunity was established in the serum-administered group. Experimental Example 6 Phagocytosis test on serum-treated bacteria In the experimental system of Experimental Example 1, blood was collected from the fish on day 135 after administration of the inactivated vaccine of the present invention to prepare immune serum.

The immunoserum and the normal (non-immune) yellowtail serum are used as antibodies, and the capsular virulent bacteria having the capsular antigens: MS93003 strains and MS9502 strains, and the attenuated non-capsulated bacteria of the present invention Bacteria: The opsonic activity of the antibody was examined using each of the attenuated MS93003 strains.

Specifically, each of the immune serum and the normal (non-immune) yellowtail serum was combined with MS93003, MS
Each of the 9502 strain and the MS93003 attenuated strain (produced strain) were cultured for 1 hour at 15 ° C., then cultured with glass-attached phagocytic cells (25 ° C., 1 hour), and then non-adhered cells were removed. Thereafter, the cells were fixed with methanol and stained with Giemsa, and the number of phagocytic cells containing 5 or more bacteria or attached to glass was measured for 200 phagocytic cells.

Table 8 shows the results. In the table, “phagocytosis ratio” means the ratio of the number of phagocytic cells containing 5 or more bacteria or adhering to glass in 200 phagocytic cells.

[0085]

[Table 8]

The agglutination titers of the immune sera against the virulent and attenuated strains are 1: 4 and 1:64, respectively.

As can be seen from Table 8, the phagocytosis rate of phagocytic cells treated with [immune serum + potent strain] was significantly higher than that of phagocytic cells treated with [non-immune serum + potent strain]. Was. When non-immune serum was used, [Non-immune serum +
The phagocytosis rate of the [virulent strain] -treated phagocytic cells was about half that of the [non-immune serum + attenuated strain] -treated phagocytic cells, indicating phagocytosis resistance.

[0088] From these facts, believed to phagocytosis in enterococci disease defense mechanism in the immune fish plays an important role, the original bacteria (capsular ⁺ bacteria) of Lactococcus Garubieae [Enterococcus seriolicida] (antigen It has been found that the antibody against the attenuated bacterium of the present invention bound to ()) has an opsonic effect that activates phagocytic cells and directs the antibacterial effect to the antigen. In addition, it was suggested that the protective antigen of Lactococcus garbiae (Enterococcus cerioricida) may be localized not on the capsule but on the surface of the cells. Experimental Example 7 Protective Effect of Live Bacterial Vaccine Healthy yellowtail (S. quinqueradiata) having a fish weight of about 100 g was fried from 80 fish cages and divided into two groups: 40 test groups and 40 control groups. The test group was 0.5 ml of the live bacterial vaccine prepared in Example 3, the control group was 0.5 ml of physiological saline,
Each was intraperitoneally administered with a sterile syringe or a continuous syringe.

At 15, 65, 135 and 295 days after the administration, 10 days each of the test group and the control group were obtained.
The tail, capsule of the present invention ^- a virulent MS93003 original bacteria (capsular ⁺ strains) 0.5 ml of strain administered intraperitoneally, was viable infectious challenge.

Table 9 shows the number of deaths due to enterococci (tail) and the number of deaths (%) with respect to the number of test fish two weeks after the live-cell infection.

[0091]

[Table 9]

As can be seen from these results, according to the live bacterial vaccine of the present invention, 1/100 of the inactivated vaccine of the present invention.
It was found that 100 doses (approximately 10 ⁶ cfu / ml) can significantly confer protection against enterococci in fish, and that the efficacy can be maintained for at least about 300 days by a single intraperitoneal administration. . Experimental Example 8 Fate of bacteria in fish after administration of live bacterial vaccine Live bacterial vaccine (attenuated capsular ^- strain (attenuated MS93003 strain)) prepared according to the method of Example 3: 6.2 bacteria
× 10 ⁶ cfu / ml) 0.5 ml with an average weight of 333
g of yellowtail were inoculated intraperitoneally into 30 tails, three were collected over time, and the brain, blood and spleen were collected, and the number of growing colonies was counted on a Todfuit agar medium to determine the number of bacteria in the blood and organs. Was measured. Table 10 shows the results.

[0093]

[Table 10]

As shown in Table 10, bacteria in the brain and blood could not be detected by day 3, and the spleen was not detected in 1 day.
It became undetectable by the first day. Thus, according to the live bacterial vaccine of the present invention, no bacteria were detected in fish within 11 days after inoculation, and the safety was confirmed. Experimental Example 9 It is known that pathogenic bacteria generally increase their pathogenicity by passing through a host (fish body). Therefore, the live bacterial vaccine of the present invention was artificially passed through fish bodies for 10 generations. And changes in pathogenicity (with or without enhancement) were examined.

Specifically, according to the method of Embodiment 3, M
S93003 attenuated strain (capsule ^- strain) cultured in Todfuit liquid medium (10 ⁸ cfu / m2)
l) 1 ml was inoculated intraperitoneally into yellowtail (primary), and
After a period of time, the spleen of the inoculated fish was excised and then strained from the spleen, and the spleen ground solution was inoculated into the next generation yellowtail. Hereafter, 1
Spleen extraction, inoculation of the inoculated fish, and injection of the spleen trituration solution were repeated in the same manner over generation 0. Actually, since the amount of bacteria in the spleen trituration solution was reduced due to the inherently attenuated bacteria and it became difficult to inoculate the bacteria in the next generation, the bacteria collected from the extirpated spleen of the inoculated fish were supplemented with 0.5% glucose-added heart-in. The culture was enriched on a fusion (HIg) agar medium and used as the cells for the next inoculation.

In this way, the MS93003 attenuated strain (penetrating strain) that passed through the teenage fish and the MS93003 attenuated strain (patient that had not passed through the fish) that did not pass through the fish were each used as a yellowtail having an average body weight of 126 g. Was intraperitoneally inoculated, and observed for 2 weeks to compare the virulence of the two strains to determine whether or not the pathogenicity of the fish-passing strain was enhanced. In addition,
The experiment was performed at 2.39 × 10 ⁶ cf for the fish passage strain.
u / ml and 2.39 × 10 ⁷ cfu / ml, and with respect to strains not passing through the fish body, 3.16 × 10 ⁶ cfu / ml and 3.16 × 10 ⁷ cfu / ml, respectively. The test was carried out by administering 0.5 ml of the bacterial solution to 15 yellowtails.

[0097] As a result, each of the test group of yellowtail after two weeks has all survived without death, viable cell vaccines of the present invention: ^- at least in the density for (attenuated strain capsular strains), fish pass Did not increase virulence. Also, from these results, the live bacterial vaccine of the present invention showed an effective amount (about 10 ⁶ cf) shown in Experimental Example 7.
u / ml) at least 10-fold concentration did not kill the fish, indicating high safety.

[0098]

According to the results of the above experimental examples, the vaccine of the present invention can be used for both inactivated vaccines and live bacterial vaccines.
It has been found that once administration during the fry period can significantly prevent the occurrence of enterococci caused by Lactococcus garbiae (Enterococcus cerioricida) for at least one year. In particular, it has been found that the live bacterial vaccine of the present invention exerts the same protective ability against enterococcal infection at 1/100 of the dose of the inactivated vaccine. It was found to be useful for further miniaturization.

Therefore, according to the vaccine of the present invention, the protective ability was significantly enhanced or the enterococcal infection was significantly superior to the conventional oral vaccine or the inactivated vaccine (capsule ⁺ strain), or the protection persistence was significantly improved. A prophylactic agent can be provided. Further, according to the vaccine of the present invention, disease damage due to enterococcal infection of fish, especially yellowtail can be reduced, and as a result, the opportunity for administration of antibiotics and the like can be reduced. The decrease in the medication opportunity leads to the suppression of the emergence of drug-resistant bacteria, and the maintenance of the medication effect. Therefore, the vaccine of the present invention can contribute to stabilization of the management of the farmer.

[0100] Furthermore, capsular Lactococcus Garubieae of the present invention used for the manufacture of such useful vaccines [Enterococcus seriolicida] ^- strains are significantly attenuated strain toxicity reduced compared to the original raw bacteria As a result, there is no need to enhance toxicity and maintain as in the prior art, prior to vaccine production. Thus, capsule of the present invention ^- According to strain, it can be manufactured with simple and efficient excellent vaccines enterococcal disease infection protective ability of the foregoing.

[Brief description of the drawings]

FIG. 1 shows transmission electron micrographs (magnification: magnification) of the original strain (capsule ⁺ ) of Lactococcus garbiae (Enterococcus seriocisida) (capsule ⁺ ) (FIG. A) and the novel bacterium of the present invention (capsule ^- strain) (FIG. B). × 50,000). It is observed that the protozoa of Lactococcus garbiae (Enterococcus cerioricida) have a capsule around the cells, but no capsule is observed in the strain of the present invention.

FIG. 2 is a drawing showing the shape of a colony obtained by culturing a protozoan strain of Enterococcus celioricida (capsule ⁺ strain) on a heart infusion agar medium supplemented with 0.5% glycol at 10 ° C. and growing by a stereoscopic microscope photograph. . The magnification is × 63.

FIG. 3: Attenuated strain (capsule ^- strain) of the invention (FIGS. A and B)
1 is a drawing showing a shape of a colony grown by culturing at 10 ° C. on a heart infusion agar medium supplemented with 0.5% glycol using a stereoscopic microscope photograph. The enlargement magnifications in FIGS. A and B are both × 63.

FIG. 4 is a diagram showing the results of Experimental Example 2. In the figure,
Indicates the agglutinating ability of serum collected over time from fish to which the vaccine of the present invention was administered, to the attenuated strain of the present invention (capsule ^- strain), and-▲-indicates the inactivated vaccine of the virulent strain (capsule- ⁺ strain). Shows the agglutination ability of serum collected over time from the administered fish to the attenuated strain of the present invention (capsule ^- strain), and-■-shows the attenuated strain of the present invention of serum collected over time from unvaccinated fish (Capsule ^- strain) Aggregation ability (control). The horizontal axis of the figure is the number of days elapsed since the fish was immunized with the vaccine, and the agglutinin titer (log ₂ ) of the serum collected at that time is shown on the vertical axis.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 39/02 A61K 39/02 (72) Inventor Hiroshi Yasuda 6-16-3 Aoshima, Miyazaki-shi, Miyazaki Miyazaki Pref.

Claims (7)

[Claims] 1. A Lactococcus Garubieae the capsule is characterized in that it does not have a very thin to or capsule (Lactoc
occus garvieae) strain belonging to [Enterococcus seriolicida (Enterococcusseriolisida)]. 2. A method according to claim 1, wherein Lactococcus garbiae (Enterococcus celioricida) is incubated at least about 5 to 25 ° C. in a heart-infusion agar medium supplemented with glucose.
A strain belonging to Lactococcus garbiae (Enterococcus cerioricida) having a very thin or no capsule obtained through a step of culturing while varying the temperature within a range. 3. Lactococcus garbiae MS930
03 attenuated strain (FERMP-16714). 4. The method according to claim 1, wherein the capsule contains an extremely thin or non-capsulated Lactococcus garvieae [Enterococcus cerioricida] strain-inactivated cell. A vaccine for enterococci in fish. 5. An inactivated cell obtained by subjecting a lactococcal garvieae (Enterococcus cerioricida) according to any one of claims 1 to 3 to extremely thin or non-capsular formaldehyde treatment. The vaccine for enterococci in fish according to claim 4, which is obtained. 6. A fish characterized in that the capsule according to any one of claims 1 to 3 contains a live bacterium of an extremely thin or non-capsulated Lactococcus garvieae [Enterococcus cerioricida] strain. Vaccine for enterococci. 7. The vaccine for enterococci in fish according to any one of claims 4 to 6, which is in the form of an injection.