JP2002003400A - Mixed inactivated vaccine against iridovirus infection, streptococcus infection, and their complication in fishes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing a mixed inactivated vaccine, and to provide the vaccine capable of giving a countermeasure against infections in cultured fishes, because the cultured fishes frequently suffer from various infections and their complications caused by diversification of kinds of the cultured fishes and further suffer from drug resistant bacteria caused by frequently using chemotherapeutics, so that quality of the cultured fishes is deteriorated, production costs are increased due to disposal of died and diseased fishes, and therefore the countermeasure becomes one of the most important problems for the aquaculture industry. SOLUTION: This method for preparing the mixed inactivated vaccine comprises mixing at least two kinds of antigens the one of which comprises an inactivated antigen derived from a pathogen of the iridovirus infection in the fishes, and the other comprises an inactivated antigen derived from a pathogen of streptococcus infection. Thus, prevention of the above infection and complication is rapidly achieved in a labor-saving manner from geographical and temporal viewpoints, and further achieved at a low cost due to saved labors and expenses.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a fish, for example, a fish belonging to the order Perch, Pteratophyta, Platypus, etc.
Combinational inactivation that is effective in preventing both infectious diseases caused by iridovirus and streptococci, which are known to be infected and developed in amberjack, larch, mackerel, mackerel, tiger pufferfish, pheasant grouper, flounder, and kue, and their complications The present invention relates to a vaccine and a method for producing the vaccine.

[0002]

2. Description of the Related Art Iridovirus infections in fish and vaccines are disclosed in Japanese Patent Application Laid-Open No. 9-176043.
A prophylactic agent for fish streptococcal infection is disclosed in "JP-A-8-2
31408 "respectively. Although vaccines for each of these infectious diseases are already on the market, the completion of a mixed vaccine with a broad spectrum of immunogenicity that is effective against both infectious diseases is not yet known.

[0003]

Over the past decade, along with the rise of the aquaculture industry and the diversification of aquaculture fish species, infectious diseases of fish, for example, iridovirus (I) such as red sea bream, yellowtail, amberjack, etc.
Ridovirus) infection, Lactococcus
Garvieae Streptococcal Infection, Paste
Nodular disease caused by urella picicida, Vibrio infection and the like and these complications frequently occur. As a result, a business crisis
Severe damage has occurred frequently, such as the death of farmed fish and the disposal of diseased fish due to poor quality. Thus, prevention of fish infectious diseases is now the most important issue in aquaculture and related industries. Furthermore, the use of chemotherapeutic agents under such circumstances induces drug-resistant bacteria, makes it difficult to control infectious diseases, narrows the options, and raises aquaculture costs.
Vaccines for fish as prophylactic agents are gaining increasing importance. Although vaccines for iridovirus infection and streptococcal infection have already been put into practical use, the immunogenicity of each vaccine does not extend to these complications.
Such complications have apparently reduced the protective effects of the vaccine. Therefore, a vaccine with a broad immunogenic spectrum that is effective not only for these infectious diseases but also for complications is expected, and its practical use is now an urgent issue.

[0004]

SUMMARY OF THE INVENTION The present invention relates to a fish, for example, a fish belonging to the order of Perciformes, Pufferfishes, Flatfish, etc., for example, red sea bream, sea bream, ishidai, ishigakidai, suzuki, yellowtail, amberjack, hiramasa, swordfish, It is effective and safe for the prevention of both infectious diseases caused by infectious diseases and streptococci, such as iridovirus and streptococcus, which have been confirmed and infected with mackerel, tiger pufferfish, pheasant grouper, flounder, and quie. Has solved the above-mentioned problem by providing a mixed inactivated vaccine specific to a pathogen and a method for producing the same. That is, according to the present invention, (1) mixing at least two types of inactivated antigens derived from pathogens of fish iridvirus infection and pathogens of streptococcal infection. (2) at least two types of inactivated antigens derived from pathogens of fish iridvirus infection and inactivated antigens derived from pathogens of streptococcal infection; A combined inactivated vaccine prepared by mixing is provided, respectively. Also,
The present invention is directed to iridovirus infections, streptococcal infections,
In addition, the present invention achieves the prevention of the complications of both of them quickly, labor-saving, and at low cost.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Antigens derived from pathogens of iridovirus infection: Fish that have developed or died due to iridovirus infection in fish, such as fish belonging to the order Perch, Pteratophyta, Platypus, etc. , Red sea bream, sea bream, sea bream, sea bream, sea bream, sea bream, yellowtail, amberjack, flatfish, sea squirrel, mackerel, mackerel, tiger pufferfish, pheasant grouper, flounder, quere, etc., such as spleen, heart,
Iridovirus strains isolated from kidney, gill, liver and the like, for example, red sea bream iridovirus Ehime-1 strain can be used as a pathogen. As antigens derived from such pathogens, virions as intact virus particles, incomplete virions, virion components and their post-translationally modified products, and MCP (major capsid) as virion components
protein, virion non-structural protein and its post-translational modification, protective antigen for infection, epitope for neutralization reaction, and the like. As a culture host of an iridvirus for mass production of an antigen, for example, BF-2 (ATCC
No. CCL 58), FHM, CHSE-214, JSK
G, KRE-3, RTG-2, YTF, GF (ATCC N
o. Known cell lines such as CCL 91) can be used. In addition, the vaccine against the iridovirus infection and the method for producing the vaccine are described in detail in Japanese Patent Application Laid-Open No. 9-176043.

(2) Antigen derived from pathogen of streptococcal infection: DNA is currently used as a pathogen of streptococcal infection in fish.
-By DNA hybridization, at least the following five species, Streptococcus iniae
(Synonymous with S. shiloi), Streptococ
us difficile, Lactococcus
garvieae (Enterococcus sel)
Synonymous with ioricida, the pathogen of alpha-hemolytic streptococcal infection), Lactococcus piscium, and Vagococcus salmoninarum are known (Development of Bio).
logical Standardization, v
ol. 90, pp. 153-160, 1997). In the present invention, these pathogens, for example, NCDO 215
5 (ATCC No. 43921), YT-3 (ATC No. 43921)
C No. 49156), S-1477 (ATCC N
o. 49157), α-hemolytic streptococcus No. For example, 43 strains can be used. As antigens derived from such pathogens, whole cells having various phenotypes, protective antigens, epitopes for neutralization reaction, and the like can be used. Regarding the phenotype, for example, Lactococcus garviea
e, two phenotypes, KG- (non-aggregated and capsulated) and KG + (aggregated and non-capsulated), are known to be useful as immunogens (Diseases of
Aquatic Organism, vol. 37, p
p. 121-126, 1999), and in the present invention, cells of these phenotypes can be used as antigens. In the future, antigens derived from pathogens isolated from fish with streptococcal infection can be used in the same manner as described above. As a culture medium for pathogenic bacterial cells for mass production of such antigens,
Known solid and liquid media for culturing bacteria, such as agar media and broth media, can be used.

(3) Preparation of inactivated antigen:
An antigen derived from the above-mentioned pathogen is used in the form of an inactivated antigen. The inactivated antigen is prepared by, for example, causing an inactivating agent to act on virions, bacterial cells, and the like, thereby inactivating the infectivity. This inactivation step is also used to immobilize the antigen and stabilize its three-dimensional structure. As the inactivating agent, for example, formalin, glutardialdehyde, β-propiolactone and the like are added and mixed before or after preparation of the vaccine stock solution. When formalin is used, the amount added is about 0.0004-0.7% (V /
V), the inactivation temperature is about 2-37 ° C, and the inactivation time is about 5
-180 days. However, when antigenicity or immunogenicity is impaired by inactivation, it is necessary to devise an ingenuity to relax the inactivation condition. Such relaxation can be achieved, for example, by reducing the amount of the inactivating agent, adding and mixing a neutral amino acid or a basic amino acid, or lowering the inactivation temperature.
The free formaldehyde remaining in the inactivation step is
If necessary, this can be neutralized by adding an equal amount of sodium bisulfite or removed by dialysis.

(4) Preparation of mixed inactivated vaccine: Regarding the amount of inactivated antigen, for example, when the pathogen is an iridovirus, the amount of infectious virus before inactivation TCID50 (M
edian Tissue Culture Infe
active Dose) is logarithmic value log (TCID50)
/ Ml) to be about 4-8, such as saline or medium such as Dulbecco's PBS (phosp
hate-buffered saline), BME
(Basal Medium Eagle) or the like is used to dilute the vaccine stock solution. Pathogen is Lactococcus
In the case of garvieae, the vaccine stock solution can be diluted in the same manner as described above so that the infectious bacterial mass before inactivation, CFU (Colony Forming Unit), becomes approximately 4-9 in logarithmic log (CFU / ml). . That is, by such dilution, the amount of the inactivated antigen is adjusted to be an amount necessary for inducing immunity. Then, the inactivated antigen obtained therefrom, that is, at least one inactivated antigen derived from the pathogen of the iridovirus infection and at least one inactivated antigen derived from the pathogen of the streptococcal infection are mixed. To prepare a combined inactivated vaccine.

It should be particularly noted that mixing of two or more inactivated antigens enhances or amplifies the side effects of vaccines due to such mixing and interference between antigens, for example, antigenicity specific to each antigen before mixing. It is necessary to confirm that none of these phenomena is present after mixing. If not, the mixture or combination is ineligible. In addition, the present invention provides the above-described antigenicity and immunogenicity in a mixture of inactivated antigens derived from each pathogen related to an iridovirus infection and a streptococcal infection, and shows no side effects. It was completed based on the finding that such a mixture of two types is eligible as a vaccine.

[0010] Further, in the preparation of a vaccine, a stabilizer for enhancing its heat resistance and an adjuvant as an adjuvant for improving its immunogenicity can be added and mixed. For example, sugars and amino acids can be used as stabilizers, and mineral oil, vegetable oil, alum, aluminum phosphate, bentonite, silica, muramyl dipeptide derivatives, thymosin, interleukin, etc. can be used as adjuvants. Then, an appropriate volume, for example, about 10
Dispense into 500 ml vials, seal and seal.
Serve as vaccine. Such a vaccine can be used not only as a liquid but also as a dry preparation by freeze-drying after dispensing. Immediately before use, dry products should be completely re-dissolved with the attached sterile solution before use.

[0011] In addition, before the prepared vaccine preparation is used or put on the market, it is necessary to carry out tests for safety and efficacy in order to guarantee the quality. Various tests concerning the tests are conducted by the Pharmaceutical Affairs Law (Showa "Animal Biological Products Standards" based on the 35th Act No. 145), "Madai Iridovirus Inactivated Vaccine Inactivated Vaccine", "Buri α-hemolytic Streptococcus Inactivated Inactivated Oral Vaccine", and other "animal products" Biologic Assay Criteria].

(5) Usage of combined inactivated vaccine: It can be used for fish of any age at risk of infection. However, from the viewpoint of conservation of fish farming, it is desirable to use it for juveniles or juveniles. As the method of use, for example, intraperitoneal, intramuscular, or subcutaneous inoculation, immersion, oral administration and the like are possible. For immunization by inoculation, about 0.05-1.0 m of vaccine per dose
It is preferable to use 1 and the vaccine is diluted about 10 to 10000 times with breeding water or hypotonic breeding water for immunization by immersion. The vaccine is cold and freezing,
For example, store in a cool dark place at about 2-8 ° C.

The combined inactivated vaccine according to the present invention is extremely effective for simultaneous immunization of both iridovirus infection and streptococcal infection in fish and prevention of complications thereof. Hereinafter, embodiments, configurations and effects of the present invention will be specifically described with reference to reference examples and examples. However, the present invention is not limited to these. Reference Example 1 Mass Production of Iridovirus: Red Sea Bream Iridovirus Ehim
Iridovirus (antigen for vaccine) was mass-produced using e-1 / GF14 strain as a seed.

The medium is BME (Basal Medium).
Eagle) was used to inoculate the seed virus into a monosheet of GF cells cultured in five 1-L roux bottles (multiplicity of infection MOI = 0.01) and incubated at 25 ° C. for 14 days.
Stationary culture was performed. Replace with fresh medium for 3 days from the start of culture.
I went every day. However, the medium was not changed between the 10th and 14th days of the culture. CPE (cytopathage)
On day 14, when the nick effect reached about 80% of the cell monosheet, the culture was harvested and centrifuged at low speed (3,0).
(00 rpm, 20 minutes), and the supernatant was recovered to obtain 500 ml of a virus suspension.

Determination of Iridovirus content: 10 cells in GF cells
A two-fold serially diluted virus suspension was inoculated and cultured, and the virus infection titer and log (TCID50 / ml) were measured by determining the presence or absence of CPE at each dilution point. As a result, the virus amount (antigen amount) was 6.0.

Preparation of inactivated iridovirus antigen and vaccine stock solution: Formalin was added to 300 ml of the virus suspension to a final concentration of 0.03% (V / V) and mixed.
Inactivated at 25 ° C. for 25 days. After the inactivation, it was stored in a cold dark room at 4 ° C. as an inactivated vaccine stock solution.

Inspection of inactivated vaccine stock solution: 100 ml of the inactivated vaccine stock solution was taken, and "Madai Iridovirus infection" defined in "Standards for Animal Biological Preparations" based on the Pharmaceutical Affairs Law (Act No. 145 of 1960). Inactivation tests, staining tests, formalin content tests, sterility tests, etc. were performed in accordance with the test criteria for "inactivation vaccine". As a result, it was confirmed that this vaccine stock solution was qualified as an inactivated vaccine stock solution. Reference Example 2 Mass production of streptococci: α-hemolytic streptococcus No. Forty-three strains were used as inoculum to mass produce streptococci (antigens for vaccines).

A bouillon medium (1 L) in a 2 L culture vessel
0.5 ml of the above inoculum was inoculated into 500 ml of 17.0 g of tryptone, 3.0 g of soypeptone, 2.5 g of glucose, 2.5 g of dipotassium phosphate and 5.0 g of sodium chloride, respectively. The cells were cultured at 30 ° C. for 24 hours.

After completion of the above culture, the culture solution was separated, serially diluted 10-fold with Dulbecco's PBS, and inoculated on an agar plate having an inner diameter of 6 cm.
After culturing for 4 hours, the bacterial count was calculated by counting the resulting α-hemolytic colonies. As a result, the number of infected cells (amount of antigen) log (CFU / ml) was 8.7. The agar plate contains a blood agar medium (peptone 1 / L).
4.5 g, soy peptone 5.0 g, sodium chloride 5.0 g, growth factor 1.5 g, agar 14.0 g and sheep defibrillated blood 50 ml each).

Preparation of inactivated streptococcal antigen and vaccine stock solution: Formalin was added to 300 ml of the above-mentioned streptococcal culture solution to a final concentration of 0.1% (V / V) and mixed.
Inactivated at 0 ° C. for 5 days. After the inactivation, it was stored in a cold dark room at 4 ° C. as an inactivated vaccine stock solution.

Inspection of inactivated vaccine stock solution: 100 ml of the inactivated vaccine stock solution was taken, and “Buri α-hemolytic chain” defined in “Animal Biological Preparation Standards” based on the Pharmaceutical Affairs Law (Act No. 145 of 1960). Inactivated Oral Vaccine For Streptococcal Infections "
Inactivation tests, staining tests, formalin content tests, sterility tests, etc. were performed in accordance with the examination standards of the above. As a result, it was confirmed that this vaccine stock solution was qualified as an inactivated vaccine stock solution.

[0022]

Example 1 Preparation of mixed inactivated vaccine: 50 ml of each of the vaccine stock solutions obtained in Reference Example 1 and Reference Example 2 was dispensed, and these two were mixed to obtain a mixed inactivated vaccine. The mixture was prepared and named "Irid-lensa mixed inactivated vaccine" (hereinafter referred to as "two-mix").

In addition, 50 ml of each of the above vaccine stock solutions was mixed with P
50 ml of each BS was added and mixed to prepare an inactivated vaccine for ylide (hereinafter, referred to as “irido”) and an inactivated vaccine for lensor (hereinafter, referred to as “lensa”).

[0024] These vaccines are dispensed in 10 ml aliquots into 20 ml vials, sealed and sealed, and then tested for safety and efficacy as described in Example 2, Examples 3 and 4, Was used for the test described in the above section. Example 2 Safety and Efficacy of Two Mixtures: To confirm the safety and efficacy of the two-mix vaccine prepared in Example 1, juvenile yellowtail (body weight 18.65-28.34 g, body length 12.0- 13.3c
m), and amberjack fry (body weight 17.83-29.52)
g, body length 10.6-12.5 cm), and a test was conducted by the direct attack method using 200 fish each. That is, first, the above two fry were randomly distributed to a total of 8 test plots, 25 fish / test plot, and then a test plot in which two mixed vaccines were inoculated.
And, as a comparative control, a test group in which each vaccine of ylide and lenza prepared in Example 1 was inoculated and an untreated test group in which no vaccine was inoculated were used. Next, 0.1 ml of the vaccine was inoculated into the peritoneal cavity of each fry.
On the day, each fry was directly challenged by inoculating 0.1 ml of the iridvirus (106 TCID50 / tail) or streptococci (106 CFU / tail) into the peritoneal cavity of each fry, and the presence or absence of onset was observed in captivity. The breeding was conducted in an isolated water tank with a water temperature of 25 ± 1 ° C (40L of seawater was placed in a 60L internal water tank.
To this, fresh seawater was introduced at a flow rate of 2 L / min and old water was discharged in an equal amount to continuously exchange breeding seawater. Table 1 shows the results.

The combined inactivated vaccine of ylide and lenza according to the present invention was safe and effective, and the immunogenicity as its effect was specific to each pathogen. The RPS (relative percentage sur) in the table
Vival) is calculated based on the following equation: RPS = [(untreated area mortality rate−inoculation area mortality rate)] / untreated area mortality rate] × 100.

[0026]

[Table 1] Example 3 Effect of two inoculation sites on safety and efficacy: Difference in inoculation site and safety of two-combination vaccine prepared in Example 1.
In order to confirm the correlation with efficacy, 350 yellowtail fry (weight 17.28-26.54 g, body length 11.8-1)
3.1 cm) and a direct attack test was performed. After randomly distributing these fry to 25 fish / test group, a total of 14 test groups, 0.1 ml of the vaccine was inoculated intraperitoneally or intramuscularly to each fry. The setting of the comparison control in the test group inoculated with the two-complex vaccine, the test method by direct attack, the observation of breeding, and the calculation of RPS were performed in the same manner as described in Example 2.

Table 2 shows the results. The combined inactivated vaccine of ylide and lenza according to the present invention was safe and effective by both intraperitoneal and intramuscular inoculation.

[0028]

[Table 2] Example 4 Preventive Effect of Two-combination Vaccine Against Ylide-Linked Combination Infection: The same procedure as described in Example 2 was carried out except for the following points. 50 yellowtail fry (weight 17.13-26.67)
g, body length 11.6 to 13.2 cm) were randomly distributed in a total of 2 groups, a test group and a control group (untreated group), by 25 fish, and then two vaccines were injected intraperitoneally into each fry in the test group. To 0.1
ml was inoculated, and the untreated group was not inoculated with the vaccine. In addition, a direct attack related to mixed infection was observed on the eleventh day after vaccination, with the iridovirus (106 TCID50 /
Tail) and streptococci (106 CFU / tail) were inoculated intraperitoneally with 0.1 ml each. As a result of subsequent observations, the mortality rate was 96% in the untreated plot, 4% in the test plot vaccinated, and 96 in the RPS, indicating that the two mixtures of the present invention are effective in preventing mixed ylide-linkage infection. It was determined that there was.

[0029]

Industrial Applicability The present invention provides a method for preventing iridovirus infection, streptococcal infection, and the complications of both, quickly and labor-saving from a geographical and temporal point of view, and at a low cost in terms of labor and cost. Is achieved. Therefore, it greatly contributes to the improvement of productivity and quality in the aquaculture industry and related industries, and to the improvement of environmental health in aquaculture, and brings the gospel to these industries.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yumiko Aoi 2-9-1, Yawata-cho, Kanonji-shi, Kagawa Pref. CC08 DD02 DD03 DD41 EE03 FF01 FF13 FF14 GG03 GG04 GG06 GG08

Claims (2)

[Claims] 1. A combined inactivated vaccine comprising a mixture of at least two inactivated antigens derived from a pathogen of a fish iridvirus infection and a pathogen of a streptococcal infection. Manufacturing method. 2. A combined inactivated vaccine prepared by mixing at least two inactivated antigens derived from a pathogen of a fish iridvirus infection and a pathogen of a streptococcal infection. .