JP7360594B2 - Vaccine preparations and methods for preventing iridovirus infections in fish - Google Patents

Description

The present invention provides a vaccine preparation for fish iridovirus infections such as red sea bream iridovirus disease, and a method for preventing fish iridovirus infections, which contains an inactivated iridovirus as an antigen and administers 10 ^7.0 TCID ₅₀ or more per dose. related to etc.

Aquaculture is widely used for many fish species because it can increase yields and provide a stable supply at a relatively low cost. On the other hand, in the case of aquaculture, compared to wild fish, the breeding density is high and environmental conditions tend to deteriorate, making it easier for infectious diseases to occur and spread.

For example, red sea bream iridovirus disease is known as an iridovirus infection of fish. Red sea bream iridoviral disease is a viral infection caused by Red sea bream iridoviral disease, and is a highly fatal disease with symptoms such as discoloration of the gills due to severe anemia and enlarged spleen. Mortality rates reach 20-60%. The infection was first confirmed in farmed red sea bream in western Japan in 1990, and since then, the infection has spread to various farmed fish, and to date, the infection has been confirmed in more than 30 fish species, mainly perch.

In response, a formalin-inactivated vaccine preparation has been developed as one of the means of controlling this disease, and the vaccine preparation has already been put on the market for red sea bream, yellowtail fish, striped trevally, yaito grouper, brown grouper, mullet grouper, and mackerel grouper. . For example, Patent Document 1 describes an iridovirus infectious disease vaccine for fish prepared by inactivating an iridovirus strain obtained through a specific passage.

On the other hand, it has become clear that the commercially available formalin-inactivated vaccine formulations have low vaccine efficacy for fish species other than those listed above, and there is a need to develop vaccines effective against these fish species. For example, Non-Patent Document 1 states, ``Compared to other fish species, the protective effect of passive immunization is lower in rockfish, and in a passive immunization test using serum from infected resistant fish... there was no significant difference in mortality rate.'' It was mentioned that "humoral immunity alone is not sufficient to protect against infection in stonefish, and the involvement of other defense mechanisms such as cell-mediated immunity is thought to be necessary." In addition, Non-Patent Document 2 mentions that ``vaccine effects on fish species of the genus Ishidae such as Ishida bream and Ishigaki bream are low'' and that ``we should try to develop effective vaccines against Ishida bream and Ishigaki bream''. ing.
Japanese Patent Application Publication No. 2007-197454 Tomomasa Matsuyama et al., "Effect of passive immunization against red sea bream iridovirus disease in five marine fish species", Fish Pathology, 51(1),32-35, 2016,3 Yasuhiko Kawato et al., "Red sea bream iridovirus disease", Fish disease research Fish Pathology, 52(2),57-62, 2017,6

As mentioned above, for example, red sea bream iridovirus disease, which is one of the iridovirus infections of fish, has been confirmed to occur in more than 30 fish species, mainly from the order Perciformes. However, the effectiveness of formalin-inactivated vaccine preparations is low for fish species other than red sea bream, yellowtail fish, striped mackerel, yaito grouper, white grouper, mullet snail, and mackerel, and it is currently difficult to effectively control the disease in these fish species. .

Therefore, an object of the present invention is to provide a more effective means for controlling iridovirus infections for fish species other than red sea bream, yellowtail fish, striped horse mackerel, yaito grouper, white grouper, yellowtail grouper, and yellowtail grouper.

The present inventors have demonstrated that by increasing the effective amount of antigen in the vaccine, even in fish species other than red sea bream, yellowtail fish, striped horse mackerel, yaito grouper, white grouper, yellowtail grouper, and mackerel, iridoviruses can be used for immunization based on humoral immunity. It has been newly discovered that infectious diseases can be effectively controlled.

Therefore, the present invention provides (1) a vaccine preparation for iridovirus infections in fish that contains an inactivated iridovirus as an antigen and is administered at least 10 ^7.0 TCID ₅₀ per dose; (2) an inactivated iridovirus as an antigen; The present invention provides a vaccine preparation for iridovirus infection in fish, which contains iridovirus at a concentration of 10 ^8.0 TCID ₅₀ /mL or more.

By administering inactivated iridovirus as an antigen at 10 ^7.0 TCID ₅₀ or more per dose, fish species other than red sea bream, yellowtail fish, striped trevally, yaito grouper, white grouper, mullet, and mackerel, such as porgy and mackerel. It can effectively prevent the occurrence, infection, propagation, and spread of iridovirus infections such as red sea bream iridovirus disease even in fish species such as the suborder Marlin. For example, by administering 0.1 mL or more of a vaccine formulation containing inactivated iridovirus at a concentration of 10 ^8.0 TCID ₅₀ /mL or more, 10 ^7.0 TCID _{50 or} more can be administered.

When a virus grown in cultured cells is used, that is, when the iridovirus is a virus grown in cultured cells, a monoclonal virus can in principle be used as the antigen. By propagating the virus in cultured cells, antigens of uniform quality can be prepared relatively easily and in large quantities.

However, even if iridovirus is grown in cultured cells, once the growth curve reaches a plateau, the amount of virus will no longer increase. It is extremely difficult to increase the dosage to a level where it is possible to administer 10 ^7.0 TCID ₅₀ or more.

On the other hand, the concentration of the virus can be increased, for example, by growing it in cultured cells that are highly sensitive to iridovirus. For example, the concentration of the iridovirus in the vaccine formulation can also be adjusted by containing, as the antigen, a concentrated solution of the culture supernatant obtained when the iridovirus is grown in cultured cells, or the culture solution and the cultured cells. can be increased to the extent that more than 10 ^7.0 TCID ₅₀ can be administered per dose, and for example, it can be used to prevent iridovirus infections such as red sea bream iridovirus disease, even for fish species such as the genus Irididae, the family Mackerel, and the suborder Marlin. can be effectively prevented.

According to the present invention, it is possible to effectively prevent iridovirus infections in fish species other than red sea bream, yellowtail fish, striped horse mackerel, yaito grouper, white grouper, yellowtail grouper, and mackerel, such as fish species of the genus Ishidae, family Mackerel, and suborder Marlin. .

<About the vaccine formulation according to the present invention>
The present invention provides (1) a vaccine preparation for iridovirus infections in fish, which contains an inactivated iridovirus as an antigen and is administered at least 10 ^7.0 TCID ₅₀ per dose; It includes all vaccine preparations for fish iridovirus infections that contain iridovirus at a concentration of 10 ^8.0 TCID ₅₀ /mL or higher.

This vaccine formulation uses an inactivated iridovirus as an antigen. A wide variety of known iridoviruses can be used, and there are no particular limitations. Examples of iridoviruses for fish include iridoviruses of the genus Megalocystisvirus of the family Iridoviridae, such as red sea bream iridovirus and infectious spleen and kidney necrosis virus (ISKNV). For example, a known isolate may be used, or an isolate isolated from fish that has developed an iridovirus infection may be used.

By infecting cultured cells with these isolates, the iridovirus isolates can be propagated. For example, a process in which a virus solution is added to the culture solution of cultured cells to infect the cultured cells with the virus, and a few days after infection, the cultured cells and culture solution are collected, and the culture supernatant is added to the cultured cells as a virus solution. By repeating this process, the virus strain can be passaged and propagated, and antigens for vaccines can be prepared. Known cell lines such as GF cells and BF-2 cells can be used as cultured cells.

For example, by propagating an iridovirus strain in cultured cells, it is possible to prepare a stock solution of a vaccine formulation that contains iridovirus as an antigen and in which the iridovirus is a virus propagated in cultured cells.

In order to administer 10 ^7.0 TCID ₅₀ or more (for example, 10 ^7.0 TCID ₅₀ to 10 ¹⁰ TCID ₅₀ , the same applies hereinafter) per dose, it is necessary to prepare a highly concentrated antigen. For this purpose, for example, a highly concentrated antigen may be prepared by propagating an iridovirus strain in cultured cells that are highly sensitive to iridovirus and expressing the virus at a higher level than usual. Cultured cells highly sensitive to iridovirus can be obtained using known methods, such as tissues collected from fish species that are highly susceptible to red sea bream iridovirus (e.g., sea bass such as red sea bream, rock bream, rock bream, barramundi, etc.). Cells are seeded and cultured, each cell is monocloned and passaged, each cultured cell is infected with an iridovirus strain, and a cultured cell line with high virus proliferation is selected. Highly sensitive cultured cell lines can be established relatively regularly. Furthermore, as the cultured cells highly sensitive to iridovirus, in vitro fertilized eggs (embryonic cells) of fish species highly sensitive to red sea bream iridovirus or cultured cells derived therefrom may be used.

Alternatively, a highly concentrated antigen may be prepared, for example, by concentrating the culture supernatant obtained when iridovirus is grown in cultured cells. As the concentration means, a wide variety of known means such as centrifugal ultrafiltration can be employed. For example, a vaccine preparation (undiluted solution) of 10 ^8.0 TCID ₅₀ /mL or more (for example, 10 ^8.0 TCID ₅₀ to 10 ¹¹ TCID ₅₀ , the same applies hereinafter) is prepared by concentrating the culture supernatant containing iridovirus, and the solution is By administering 0.1 mL or more of iridovirus, it is possible to administer 10 ^7.0 TCID ₅₀ or more per dose.

Furthermore, in order to prepare a highly concentrated antigen, a culture solution and cultured cells obtained by propagating iridovirus in cultured cells may be used as (undiluted solution of) a vaccine preparation. By containing not only a culture solution but also an effective amount of cultured cells, that is, by containing both a culture solution containing iridovirus and cultured cells containing infected iridovirus as effective antigens, It is possible to prepare a vaccine preparation (undiluted solution) of 10 ^8.0 TCID ₅₀ /mL or more, and by administering 0.1 mL or more of the solution, it is possible to administer 10 ^7.0 TCID ₅₀ or more of iridovirus per dose.

In this way, the antigen may be (1) a concentrated solution of the culture supernatant obtained when the iridovirus is propagated in cultured cells, or (2) the culture solution obtained when the iridovirus is propagated in cultured cells; By creating a vaccine preparation containing the above-mentioned cultured cells, it becomes possible to administer 10 ^7.0 TCID ₅₀ or more of iridovirus as a high-concentration antigen at a time.

The antigen according to the present invention may be an inactivated antigen. Iridovirus can be inactivated, for example, by physical treatment (ultraviolet irradiation, X-ray irradiation, heat treatment, ultrasonication, etc.), chemical treatment (treatment with formalin, etc., chloroform・This can be done by performing treatments such as organic solvent treatment with alcohol, acid treatment with weak acids such as acetic acid, treatment with chlorine, mercury, etc.).

For example, by adding formalin to a highly concentrated antigen-containing solution at a volume concentration of 0.01 to 2.0%, more preferably 0.05 to 1.0%, and sensitizing the culture solution at 4 to 30°C for 1 to 10 days, Inactivation with formalin can be performed. Further, for example, after the inactivation treatment, an inactivating agent such as formalin may be removed by washing with a buffer solution or the like, or a neutralizing agent may be added for neutralization.

As mentioned above, for example, when administering a vaccine formulation at 0.1 mL per dose, by adjusting the concentration of iridovirus in the vaccine formulation to 10 ^8.0 TCID 50 /mL or higher, the iridovirus concentration can be adjusted to 10 ^7.0 TCID ₅₀ /mL or higher. TCID ₅₀ or more can be administered. For the concentration of iridovirus in the vaccine preparation according to the present invention, for example, the virus infectivity titer before inactivation may be used as an index, and the concentration of iridovirus in the vaccine preparation may be determined by directly measuring the amount of inactivated iridovirus in the vaccine preparation. The infectious titer corresponding to the value may be used as an index. Note that the amount of inactivated iridovirus can be estimated by direct measurement using, for example, ELISA method or protein assay method.

The vaccine preparation according to the present invention may have a total protein content other than viral antigens of 0.1% or less (1.0 mg/mL or less). When propagating iridovirus in cultured cells, serum or the like may be added to the culture solution. In that case, since these contaminant proteins remain in the vaccine preparation (undiluted solution), it is necessary to remove these contaminant proteins by known means such as ultrafiltration and use it as the vaccine preparation (undiluted solution). Good too. By excluding contaminant proteins such as those derived from serum and suppressing the total protein amount in the preparation to, for example, 0.1 mg/mL or less, it is possible to increase the antigen concentration and make the vaccine more effective. It can reduce the risk of side effects caused by proteins.

The vaccine formulation according to the invention may contain an adjuvant.

A wide variety of known adjuvants can be used. For example, animal oils (squalene, etc.) or their hydrogenated oils, vegetable oils (palm oil, castor oil, etc.) or their hydrogenated oils, anhydrous mannitol/oleate, liquid paraffin, polybutene, caprylic acid, oleic acid, higher fatty acid esters, etc. Oil-based adjuvants, including PCPP, saponins, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymers, methacrylic acid copolymers, maleic anhydride copolymers, alkenyl derivative polymers, oil-in-water emulsions, Water-soluble adjuvants such as cationic lipids containing quaternary ammonium salts, precipitating adjuvants such as aluminum hydroxide (alum) and sodium hydroxide, toxin components derived from microorganisms such as cholera toxin and Escherichia coli heat-labile toxin, others, bentonite, Examples include muramyl dipeptide derivatives and interleukins. Alternatively, a mixture of these may be used.

In addition, the vaccine formulation according to the present invention may include buffering agents, isotonic agents, soothing agents, preservatives, antibacterial agents, antioxidants, pH regulators, dispersants, aromatics, etc., depending on the purpose and use. A coloring agent, an antifoaming agent, etc. may be added as appropriate.

Suitable examples of the buffer include buffers such as phosphate, acetate, carbonate, citrate-tartrate, trishydroxymethylaminomethane, and HEPES.

Suitable examples of tonicity agents include sodium chloride, glycerin, D-mannitol, and the like.

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

Suitable examples of drugs for preservative purposes include thimerosal, paraoxybenzoic acid esters, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, various preservatives, antibiotics, and synthetic antibacterial agents. Agents and the like can be used.

Suitable examples of antioxidants include sulfites, ascorbic acid, and the like.

Suitable examples of pH regulators include acids such as hydrochloric acid, carbonic acid, acetic acid, citric acid, phosphoric acid, boric acid, and sulfuric acid, and alkali metals such as sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. hydroxides, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, monoethanolamine, diisopropanolamine, etc. can be used.

Suitable examples of the dispersant include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polysorbate 80 (TWEEN80), and the like.

Suitable examples of the fragrance include citrus fragrances such as lemon, orange, and grapefruit, peppermint, spearmint, menthol, pine, cherry, fruit, yogurt, and coffee.

Suitable examples of coloring agents include caramel pigment, gardenia pigment, anthocyanin pigment, annatto pigment, paprika pigment, safflower pigment, red yeast rice pigment, carotene pigment, carotenoid pigment, flavonoid pigment, cochineal pigment, amaranth (red No. 2) , Erythrosin (Red No. 3), Allura Red AC (Red No. 40), New Coccin (Red No. 102), Phloxin (Red No. 104), Rose Bengal (Red No. 105), Acid Red (Red No. 106), Tartrazine ( Yellow No. 4), Sunset Yellow FCF (Yellow No. 5), Fast Green FCF (Green No. 3), Brilliant Blue FCF (Blue No. 1), Indigo Carmine (Blue No. 2), Copper Chlorophyll, Copper Chlorophyllin Sodium, etc. be able to.

Suitable examples of antifoaming agents include dimethicone, simethicone, silicone emulsion, sorbitan sesquioleate, nonionic substances, and the like.

In addition to the above, this preparation contains auxiliary ingredients, such as light-absorbing pigments (riboflavin, adenine, adenosine, etc.) that aid in preservation and efficacy, chelating agents and reducing agents (vitamin C, citric acid, etc.) for stabilization. ), carbohydrates (sorbitol, lactose, mannitol, starch, sucrose, glucose, dextran, etc.), casein digests, various vitamins, etc.

The dosage form of the vaccine preparation can be any known one and is not particularly limited. For example, it may be used as a liquid preparation.

In addition, this vaccine preparation may be a mixed vaccine preparation with one or more vaccines for other diseases.

<About the method for preventing iridovirus infections in fish according to the present invention>
The present invention broadly encompasses a method for preventing iridovirus infections in fish, which comprises administering inactivated iridovirus as an antigen at a dose of 10 ^7.0 TCID ₅₀ or more per dose.

By administering the above-mentioned highly concentrated antigen to fish, it is possible to effectively prevent the occurrence, infection, propagation, and spread of iridovirus infectious diseases such as red sea bream iridovirus disease in a wide range of fish species.

The present invention is widely applicable to fish species that can be infected with iridoviruses other than red sea bream, yellowtail fish, striped horse mackerel, yaito grouper, white grouper, yellowtail grouper, and mackerel grouper. Applicable fish include, for example, fish of the genus Ishidae (e.g., corpus bream, bream bream, etc.), fish of the mackerel family (e.g., fish of the genus Tuna such as tuna, yellowfin tuna, albacore, and bigeye, fish of the genus Suma, such as Japanese jack mackerel, mackerel, etc.) Examples include fish of the genus Mackerel, fish of the genus Bonito such as bonito), fish of the suborder Marlin (fish of the family Swordfish family such as swordfish, fish of the family Marlinidae such as marlin), and the like.

As a method for administering the inactivated vaccine preparation according to the present invention, for example, an injection method can be adopted.

In order to administer 10 ^7.0 TCID ₅₀ or more per dose, for example, 0.1 mL or more of an inactivated vaccine preparation prepared to have an iridovirus concentration of 10 ^8.0 TCID ₅₀ /mL or more before inactivation treatment is administered intramuscularly or intraperitoneally. May be administered. In addition, depending on the size of each fish species or individual fish, for example, the virus concentration may be adjusted appropriately so that 10 ^7.0 TCID ₅₀ or more can be administered per time, or the concentration of iridovirus before inactivation treatment may be adjusted to 10 7.0 TCID 50 or more. Once prepared to a concentration of ^8.0 TCID ₅₀ /mL or higher, the inactivated vaccine preparation may be administered in a range of 0.05 mL to 5.0 mL, more preferably 0.05 to 3.0 mL, and most preferably 0.05 to 1.5 mL. The method for adjusting the concentration of iridovirus before inactivation treatment to 10 ^8.0 TCID ₅₀ /mL or more is as described above.

The inactivated vaccine preparation may be administered once as long as its effect lasts, but it may be administered multiple times at intervals of 1 to 60 days depending on the size of the target fish, the degree of vaccine effectiveness, etc. For example, it is preferable to administer 10 ^7.0 TCID ₅₀ or more once or twice at an interval of 1 to 60 days.

<About the use of the present invention for producing a vaccine preparation against iridovirus infection of fish>
The present invention broadly encompasses the use of the iridovirus for the production of a vaccine preparation against iridovirus infections in fish, which contains an inactivated iridovirus as an antigen and is administered at 10 ^7.0 TCID ₅₀ or more per dose. do.

For example, by using inactivated iridovirus at a concentration of 10 ^8.0 TCID ₅₀ /mL or more, it is possible to manufacture a vaccine formulation against iridovirus infection in fish that is administered at a dose of 10 ^7.0 TCID ₅₀ or more per dose. . The iridovirus used and its concentration, the purpose, usage, dose, and ingredients other than the antigen of the vaccine preparation to be produced are the same as above.

In Example 1, we investigated the preventive effect against red sea bream iridovirus disease when the red bream iridovirus was inoculated and immunized with an inactivated antigen containing both the culture solution and the cultured cells when red sea bream iridovirus was grown in cultured cells. Verified.

As an antigen, a cell-contaminated antigen was prepared. After infecting adherent GF cells with red sea bream iridovirus RIE-124 strain, the cells and culture medium were separated and mixed. These cells contain red sea bream iridovirus during infection, and the culture solution contains red sea bream iridovirus. Formalin was added to the fractionated cell-containing culture solution at a concentration of 0.1% to inactivate it, and it was used as a sample of "cell-containing antigen." The concentration of iridovirus in this sample was 10 ^8.04 TCID ₅₀ /mL. The RIE-124 strain used was a monoclonal red sea bream iridovirus obtained by collecting the spleen of affected fish at a red sea bream farm in Ehime Prefecture in August 2012, and isolating and subculturing it in cultured cells. It is a stock.

As a positive control, normal culture supernatant antigen was prepared. In the same manner as above, GF cells were infected with red sea bream iridovirus RIE-124 strain, and then the culture supernatant was obtained. This culture supernatant contains red sea bream iridovirus. Formalin was added to the culture supernatant at a concentration of 0.1% to inactivate it, and it was used as a sample of "culture supernatant antigen." The concentration of iridovirus in this sample was 10 ^7.25 TCID ₅₀ /mL.

Ninety young Japanese rockfish with an average weight of 22.1 g were prepared, divided into three groups of 30 each, and each group was raised in a test tank.

Of the three groups, the first group of rockfish were immunized with cell-containing antigens, and the second group was immunized with 0.1 mL of normal culture supernatant antigen by intraperitoneal injection under non-anesthetized conditions, and then reared for 14 days. The animals were reared and observed at a water temperature of 25°C. In addition, in the third group, as a control, the animals were reared and observed under the same conditions without administering the antigen.

Subsequently, red sea bream iridovirus RIE12-1 strain was prepared as a challenge strain, and 14 days after immunization, ^100.5 TCID ₅₀ /fish was injected intraperitoneally to each rockfish under anesthesia. Rearing and observation continued under the same conditions for 28 days after the attack. Furthermore, the average weight of Ishidai at the time of attack was 31.0 g.

The results are shown in Figure 1. FIG. 1 is a graph showing the survival rate of Ishida bream when immunized with a cell-contaminated antigen. In the figure, the horizontal axis represents the number of days since the attack, and the vertical axis represents the survival rate (%). In the figure, "cell-contaminated antigen" is the result when immunized with the above-mentioned cell-contaminated antigen, "culture supernatant antigen" is the result when immunized with the above-mentioned normal culture supernatant antigen, and "no administration (control)" Each represents the result when immunization with the antigen was not performed.

As shown in Figure 1, the survival rate after virus challenge in the case of no administration was 0%, and even in the case of immunization with normal culture supernatant antigen, the survival rate was 6.7%; When immunized with the virus, the survival rate was 53.3%, which was significantly higher. These results showed that the cell-contaminated antigen was significantly more effective in preventing iridovirus disease in fish of the genus Iridium than ordinary culture supernatant antigens.

In Example 2, the preventive effect against red sea bream iridovirus disease was verified when the high concentration antigen of the inactivated red sea bream iridovirus was inoculated into rock bream to immunize them.

The following three types of antigens were prepared as follows.

(1) Normal antigen (1x): As in Example 1, infect GF cells with red sea bream iridovirus strain RIE-124, centrifuge to obtain the culture supernatant, and add formalin to the culture supernatant. was added at a concentration of 0.1% to inactivate it, and it was used as a "normal antigen (1x)" sample. The concentration of iridovirus in this sample was 10 ^7.34 TCID ₅₀ /mL.

(2) Concentrated antigen (5 times): The same culture supernatant as in (1) was concentrated five times by 100 kDa centrifugal ultrafiltration, and formalin was added at a concentration of 0.1% to inactivate it. (5 times)" sample. The concentration of iridovirus in this sample was 10 ^8.04 TCID ₅₀ /mL.

(3) Cell-contaminated antigen: As in Example 1, after infecting GF cells with red sea bream iridovirus RIE-124 strain, separate and mix the cells and culture medium, and add formalin to the cell-containing culture medium. It was added at a concentration of 0.1% to inactivate it and used as a sample of "cell-contaminated antigen." The concentration of iridovirus in this sample was 10 ^8.24 TCID ₅₀ /mL.

Sixty young Ishidai fish with an average weight of 9.4 g were prepared, divided into four groups of 15 each, and each group was kept in a test tank.

Of the four groups, the first group of Ishidai was given (1) the normal antigen, the second group was given the concentrated antigen (2), and the third group was given the cell-contaminated antigen (3), 0.1 mL each. The animals were immunized by intraperitoneal injection, and then reared and observed for 14 days at a rearing water temperature of 25°C. In addition, in the fourth group, as a control, the animals were reared and observed under the same conditions without administering the antigen.

Subsequently, red sea bream iridovirus RIE12-1 strain was prepared as a challenge strain, and 14 days after immunization, ^100.5 TCID ₅₀ /fish was injected intraperitoneally to each rockfish under anesthesia. Rearing and observation continued under the same conditions for 20 days after the attack. The average weight of the Ishidai at the time of attack was 15.6.

The result is shown in figure 2. FIG. 2 is a graph showing the survival rate of Ishida bream when immunized with each antigen. In the figure, the horizontal axis represents the number of days since the attack, and the vertical axis represents the survival rate (%). In the figure, "enriched antigen (5x)" indicates the result when immunizing with the above concentrated antigen, "cell-containing antigen" indicates the result when immunizing with the above-mentioned cell-containing antigen, and "normal antigen (1x)" indicates the result when immunizing with the above-mentioned cell-containing antigen. "No administration (control)" represents the results when immunized with the above-mentioned normal antigen, and "No administration (control)" represents the results when immunized with no antigen.

As shown in Figure 2, the survival rate was 33.3% even in the case of no administration, and the survival rate was 53.3% even in the case of immunization with normal antigens, which is a slight survival rate compared to the no-administration group. However, the survival rate of the group immunized with concentrated antigen and cell-contaminated antigen was both 73.3%, which was significantly higher than that of the group immunized with regular antigen. . These results show that the concentrated antigen and cell-contaminated antigen are significantly more effective in preventing red sea bream iridovirus disease in fish belonging to the genus Iridia compared to ordinary culture supernatant antigens. It was suggested that by setting the effective antigen amount to 10 ^7.0 TCID ₅₀ or more, it is possible to effectively prevent red sea bream iridovirus disease in fish of the genus Ishida.

In Example 3, the preventive effect against red sea bream iridovirus disease was verified when the red seabream iridovirus disease was immunized by inoculation of a high concentration antigen of red sea bream iridovirus concentrated 50 times.

The culture supernatant obtained in Example 2 was concentrated 50 times by 500 kDa centrifugal ultrafiltration, buffer exchanged with PBS to remove contaminant proteins, and then formalin was added at a concentration of 0.1% to inactivate it. Antigen (50x magnification)" sample. The concentration of iridovirus in this sample was 10 ^9.04 TCID ₅₀ /mL.

Further, formalin was added to the culture supernatant obtained in Example 2 at a concentration of 0.1% to inactivate it, and a sample of "normal antigen (1x)" was prepared.

Sixty young Ishidai fish with an average weight of 8.4 g were prepared, divided into three groups of 20 fish each, and each group was kept in a test tank.

Of the three groups, the first group of parrotfish were immunized with concentrated antigen (50x) and the second group with normal antigen (1x) by intraperitoneal injection of 0.1 mL each under non-anesthetized conditions. The animals were reared and observed at a rearing water temperature of 25°C for days. In the third group, as a control, PBS was administered instead of the antigen, and the animals were reared and observed under the same conditions.

Subsequently, red sea bream iridovirus RIE12-1 strain was prepared as a challenge strain, and 14 days after immunization, 10 ^0.5 TCID ₅₀ /fish was injected intraperitoneally to each rockfish. Rearing and observation continued under the same conditions for 27 days after the attack.

The results are shown in Figure 3. FIG. 3 is a graph showing the survival rate of Ishida bream when immunized with a 50-fold concentrated antigen. In the figure, the horizontal axis represents the number of days since the attack, and the vertical axis represents the survival rate (%). In the figure, "Concentrated antigen (50x)" indicates the result when immunized with 50 times concentrated antigen, "Normal antigen (1x)" indicates the result when immunized with normal antigen (1x), and "PBS ( "Control)" respectively represents the results when PBS was administered instead of the antigen.

As shown in Figure 3, the survival rate when immunized with a normal concentration of antigen was 10%, which was similar to the negative control when administered with PBS (15%), whereas the survival rate was 50 times more concentrated. The survival rate when immunized with the high-concentration antigen was 85%, which was significantly high. These results showed that high-concentration antigens are significantly more effective in preventing iridovirus disease in fish of the genus Iridium than antigens at normal concentrations.

1 is a graph showing the survival rate of rock bream when immunized with a cell-contaminated antigen in Example 1. Graph showing the survival rate of rock bream when immunized with concentrated antigen or cell-contaminated antigen in Example 2. Graph showing the survival rate of rock bream when immunized with a 50-fold concentrated antigen in Example 3.

Claims (3)

Contains a culture solution obtained when iridovirus is grown in cultured cells and the cultured cells, and contains inactivated iridovirus as an antigen at a concentration of 10 ^8.0 TCID ₅₀ /mL or more, and 10 ^7.0 TCID ₅₀ per use. A vaccine preparation for iridovirus infection of fish, which is administered as described above. 2. The vaccine preparation according to claim 1, wherein the target is a fish species of the genus Ishida. Contains a culture solution obtained when iridovirus is grown in cultured cells and the cultured cells, and contains inactivated iridovirus as an antigen at a concentration of 10 ^8.0 TCID ₅₀ /mL or more, and 10 ^7.0 TCID ₅₀ per use. A method for preventing iridovirus infection in fish by administering the above.

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