KR20110081233A - Vaccine for treatment/prevention of scuticociliatosis in fish - Google Patents

Abstract

Provided is an effective preventive and therapeutic vaccine with infection protection against Scuticasis, especially Philasterides dicentrarchies with high mortality. The vaccine includes (1) inactivated strains showing the same serotype as P. dicentrarchi Iyo I strain, (2) strains showing inactivated strains as P. dicentrarchi Nakajima strain, and (3) As an active ingredient, inactivated compounds of the strain showing the same serotype as P. dicentrarchi Mie0301 strain are used as active ingredients.

Description

Vaccine for treatment / prevention of scuticociliatosis in fish}

The present invention relates to a vaccine effective for the prevention or treatment of scutica.

Skutikasis, one of the parasitic diseases of fish, was first seen in flounder in Japan for the first time in 1990 in Japan. In 2004, the value of farmed flounder in our country was about 9.3 billion yen. In Aime Prefecture's Fish Disease Guidance Center, which accounts for about 20% of this, the diagnosis rate of Skutikasis in the number of cases of flounder fish disease is 15-20 per year. As high as%, mortality from this condition is estimated to be 30-70%. There are no treatments or preventive measures for this condition, and there are some companies that inevitably close off the flounder culture due to the occurrence of this disease. Since then, cases of flounder farming have been confirmed not only in Japan but also in Korea. For this reason, it is an infectious disease which is most developed in the aquaculture site of the flounder. It is reported that the Scutika insects, which are the cause of the disease, have been Uronema marinum. However, various kinds of Scutika insects have been separated from the flounder of Scutika disease after that.

In recent years, as a result of morphological and genetic identification by the inventor group, it has been found that Philasterides dicentrarchi (this name is synonymous with Miamiensis avidus) is the main cause of the symptoms (Non-Patent Document 1). In addition, the inventors of the present inventors confirmed the high pathogenicity of the parasites to flounder by experimental infection using P. dicentrarchi (Non-Patent Document 2).

Various treatment methods have been tested in order to control Scutica caterpillars containing P. dicentrarchi. For example, the insecticidal effect (nonpatent literature 3) by immersion by formalin etc. was confirmed, and the effect by the treatment method by the bathing bath containing hydrogen peroxide (patent document 1) is also confirmed. However, P. dicentrarchi can not be said to have sufficient effects because it penetrates into the host fish's brain due to internal parasitics.

In such a situation, the formalin inactivating vaccine of this parasite is effective for sputicasis (Patent Documents 2, Non-Patent Documents 4 and 5) or prevention by a fresh water bath lacking a specific metal. The treatment method is reported (patent document 3).

[Patent Document 1] Patent No. 3882939 [Patent Document 2] International Publication No. WO 2008/084125 [Patent Document 3] Japanese Patent Application Laid-Open No. 2006-306834

[Non-Patent Document 1] A Collection of Lectures at the Japanese Society of Fish Diseases, Japan in 2004. 65, 2004 [Non-Patent Document 2] Diseases of Aquatic Organisms, 73, pp. 227-234, 2007 [Non-Patent Document 3] Aime Fisheries Experimental Research Report, No. 6, pp. 65-70, 1997 [Non-Patent Document 4] A Collection of Lectures at the Japan Society for Fish Diseases, 2005, A-29, 37, 2005 [Non-Patent Document 5] Journal of Fish Pathology, (Korea), Vol. 19, No. 2, pp. 173-181, 2006

However, even in the conventional vaccine known to be effective for skutika, the effect cannot be said to be sufficient, and development of the vaccine which is more effective is desired. In particular, the present inventors found that in the present invention, three serotypes exist in P. dicentrarchi, and that a single vaccine cannot prevent infection of the present parasites of other serotypes. Therefore, an object of the present invention is to provide a vaccine which can effectively prevent and treat Scutica, especially in the case of infection of P. odicentrarchi with high mortality.

The present inventors have diligently studied, and the mixed vaccine of the present invention, which is a combination of vaccines obtained by three different serotypes of the parasite, has dramatically improved the effectiveness against P. dicentrarchi. The present invention also found effective prevention and treatment against the infection of the parasite that could not be prevented. Thus, the mixed vaccine of the present invention was completed.

The present invention,

[1] (1) Pilatesterides decentrachi (Philasterides dicentrarchi) Inactivated substance of the strain showing the same serotype as Iyo I strain (2) Inactivated substance of the strain showing the same serotype as the Pilateris decentraci Nakajima strain, and (3) The same serotype as the strain Pilateris decentracchi Mie0301. Mixed vaccine for the prevention or treatment of skuticaosis which uses the main inactivating agent which shows the following as an active ingredient;

[2] A strain showing the same serotype as the Pilates decentraci Iyo I strain is selected from the group consisting of Iyo I strain, JF05To strain, RF05To strain, and SK05 Kyo strain. Phosphorus, the combination vaccine according to [1];

[3] A method for preventing or treating squaticosis, comprising administering the mixed vaccine according to [1] or [2] in an effective amount to a subject in need of prevention or treatment of squaticosis;

[4] (1) A strain that shows the same serotype as the Pilates decentraci Iyo I strain, (2) A strain showing the same serotype as the Pilates decentraci Nakajima strain, and (3) Use in the preparation of a mixed vaccine for the prevention or treatment of squaticosis, of the strain which shows the same serotype as the teredis decentralchi Mie0301 strain;

[5] (1) strains showing the same serotype as the Pilates decentraci Iyo I strain, (2) strains showing the same serotypes as the pilates decentraci Nakajima strain, and (3) pilas A step of inactivating the strains having the same serotype as the teriedes and decentralized Mie0301 strains, alone or in combination of two or more, and a step of mixing the obtained inactivating agent. It is related with the manufacturing method of the mixed vaccine as described in [1] or [2].

The present invention is a vaccine having high efficacy for the prevention and treatment of squaticosis including infection of P. dicentrarchi with high mortality. More specifically, according to the present invention, it is possible to provide a safe fish vaccine without any problem even when administered to aquaculture fish farmed on the premise of food. The inactivated vaccine of the present invention can be produced in a simple and low cost process because of the fact that P. dicentrarchi can be easily produced in the process of obtaining the antigen, and the inactivation method is also simple. Moreover, the vaccines produced can induce the ability to defend against infection against Scutica due to P. dicentrarchi. Therefore, it can contribute greatly to the improvement of productivity and quality in aquaculture industry and related industries, and the improvement of environmental hygiene in aquaculture.

1 shows Iyo I strain (lane 1), JF05To strain (lane 2), RF05To strain (lane 3), SK05 Kyo strain (lane 4), Nakajima strain (lane 5), Mie0301 strain (lane 6) and anti-Iyo I strain. Fig. 2 shows the results of western blotting using serum of SK05 Kyo, Nakajima, and Mie0301 strains. A, B, C, and D in the figure are response profiles of anti-Iyo I strain, SK05Kyo strain, Nakajima strain, and Mie0301 strain, respectively. In addition, the leftmost lane of FIG. 1 shows a molecular weight marker (マ-カ-).
Figure 2 is a graph showing the change in cumulative mortality of the challenge test by the Iyo I strain (4.03 × 10 ⁴ cells / fish) of the flounder inoculated with the three mixed vaccines. ■ shows three kinds of mixed vaccine inoculation ports (接種 區), and ◆ shows MEM control.
FIG. 3 is a graph showing the change in chronological cumulative mortality of the challenge test by Nakajima strain (4.03 × 10 ⁴ cells / fish) of flounder inoculated with the three mixed vaccines. ■ shows three kinds of mixed vaccine inoculation ports (接種 區), and ◆ shows MEM control.
Figure 4 is a graph showing the change in cumulative mortality of the challenge test by Mie0301 strain (4.03 × 10 ⁵ cells / fish) of the flounder inoculated with three mixed vaccines. ■ shows three kinds of mixed vaccine inoculation ports (接種 區), and ◆ shows MEM control.
FIG. 5 is a graph showing the change in chronological cumulative mortality of challenge tests by Iyo I strains (6.12 × 10 ⁵ cells / fish) of flounder vaccinated with each single vaccine of Iyo I, Nakajima and Mie0301. ●, ■, ▲, and ○ show Iyo I, Nakajima, Mie, and MEM controls.
FIG. 6 is a graph showing the change in chronological cumulative mortality of challenge test by Nakajima strain (6.12 × 10 ⁵ cells / fish) of flounder vaccinated with each single vaccine of Iyo I, Nakajima and Mie0301. ●, ■, ▲, and ○ show Iyo I, Nakajima, Mie, and MEM controls.
FIG. 7 is a graph showing the change in chronological cumulative mortality of the challenge test by Mie strains (6.12 × 10 ⁵ cells / fish) of flounder vaccinated with each single vaccine of Iyo I, Nakajima and Mie0301. ●, ■, ▲, and ○ show Iyo I, Nakajima, Mie, and MEM controls.
FIG. 8 is a graph showing the change in chronological cumulative mortality of the challenge test by Iyo I strain (3.06 × 10 ⁵ cells / fish) of halibut inoculated with the three mixed vaccines. ● shows three mix vaccines and ■ shows MEM control.

Each inactivated substance, ie, inactivated vaccine, used in the present invention can be prepared according to a conventional method, for example, can be prepared as follows. Fish Coin Cell CHSE-214 (ATCC No. CRL-1681; Marine Applied Life Sciences Division, Graduate School of Fisheries Science, Hokkaido University) Yoshimizu Mamoru, P. dicentrarchi (hereinafter referred to as this parasite) Intake by Professor Yoshino Yong-Ho, and then incubated at 20 ° C. for 5 days. Fish-coated cell CHSE-214 is a medium for CHSE-214 cells [Minimum Essential Medium (eagle) MEM medium prescribed with 10% fetal bovine serum (Nisui Pharmaceutical Products: Products Code 05900) was adjusted to pH 7.3 with sodium hydrogen carbonate) and incubated at 20 ° C. until monolayer. The parasites thus cultured are collected by centrifugation and inactivated by adding an inactivating agent, for example, formalin, glutaraldehyde, β-propiolactone, or the like. For example, in the example of formalin, 35% formalin liquid is added so that the end concentration may be 0.3%, and inactivated overnight at a temperature of 4 ° C. However, when antigenicity is impaired by inactivation, inactivation conditions are alleviated. For example, the weight loss of an inactivating agent, addition of a pH buffer, lowering inactivation temperature, etc. are mentioned.

Examples of protozoa used herein include (1) strains showing the same serotype as Philasterides dicentrarchi Iyo I strain, (2) strains showing the same serotype as Nakajima strain, and (3) strains identical to Mie0301 strain. Use the visible strains, that is, three strains showing different serotypes. As the strain showing the same serotype as Iyo I strain, for example, JF05To strain, RF05To strain, and SK05Kyo strain can be used.

These notes were isolated and identified by the group of the present inventors, and the details thereof are shown in Table 1.

In Iyo type I (serotype, serotype I), a protein that reacts strongly at 30 kDa is detected upon western blotting using anti-Iyo I strains of rabbit antibodies (FIGS. 1A and 1B). Lanes 1-4). On the other hand, such a protein is not detected in anti-Nakajima strain rabbit antibody (FIG. 1C) and anti-Mie0301 strain rabbit antibody (FIG. 1D).

In Nakajima type (serotype II), a protein that reacts strongly at 38 kDa is detected when western blotting is performed using anti-Nakajima strain and anti-Mie0301 strain. (Lane 5) .

In Mie0301 type (serotype III), a protein that reacts strongly at 34 kDa is detected upon western blotting using anti-Nakajima strain and anti-Mie0301 strain. .

The morphological features of the parasites used in the present invention are oval, about 30 × 400 μm, with cilia. Unlike other ciliary worms, it is histopathogenic and feeds not only bacteria but also animal cells (eg fish coin cells). In addition, the parasites are classified into anarchstrum (Ancistrum) (Scuticociliatida), Pilasta (Philasterina), Pilasta (Philasteridae).

Passage cultures of the parasites used in the present invention are passaged into fresh cells once a month under preservation at 20 ° C. At least once every few days, when the parasites are observed under a microscope and the carcass becomes small, passage is carried out within a month. It has been confirmed that the pathogenic change caused by the passage is stable for 5 years. In addition, the change of antigenicity by passage is also not recognized.

In the three mixed vaccines of the present invention, three types of inactivated vaccines (preferably formalin inactivated vaccines) having different serotypes are combined. The ratio of the combination is not particularly limited, but, for example, for a minimum amount of a single vaccine, each single vaccine may be used in an amount of 1 to 5 times, preferably 1 to 3 times, with respect to the number of cells. In particular, preferably, each single vaccine is mixed in a ratio of 1: 1: 1.

The concentration of the mixed vaccine of the present invention does not need to be particularly limited. You can adjust the density according to your scene. In particular, if the total amount of each vaccine is 10 ⁵ cells / fish or more, sufficient effectiveness can be obtained.

In addition, the Applicant shall submit each of the states of Iyo I, Nakajima, and Mie0301 of the Philasterides dicentrarchi on July 24, 20, and the states of JF05To, RF05To, and SK05Kyo on the Philasterides dicentrarchi. On July 7, the application was made to the Patent Biological Depository Center of the Institute for Industrial Technology, an independent administrative corporation designated by the Minister of Patent Affairs (Japan), as of August 1, 20, and October 16, 20, He was notified by the Buddhist Ministry of Depositary of Trusteeship because he could not be entrusted. Therefore, the protozoan which can be used in the present invention is treated as a self-deposit, and the applicant will guarantee the sale of Article 27-3 of the Enforcement Rule of the Japan Patent Law.

The fish species to which the mixed vaccine of the present invention can be adapted are not particularly limited as long as they have a possibility of developing scutosis, but are not particularly limited to flounder, large flounder, sea bream, sea bream, sea bream, and sea bream. Gangdam Dome (Ishigakidai), flounder (Meitagarai), etc. are mentioned. In addition, any kind of fish can be used for fish of any age or any size that may be infected. Examples of the usage include methods such as oral administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, dipping treatment and the like. Among them, intraperitoneal administration can induce sufficient immunity with a low antigenic amount.

Examples of protozoa capable of effective prophylaxis and treatment by the mixed vaccine of the present invention include Philasterides dicentrarchi Iyo I strain, Nakajima strain, Mie0301 strain, JF05To strain, RF05To strain, and SK05Kyo strain.

(Example)

Hereinafter, although this invention is demonstrated further more concretely based on an Example, it does not limit this invention.

Reference Example 1

Non-assisted assays and western blotting were performed to compare the antigenicity of Philasterides dicentrarchies from different species and regions.

(1) assimilation assay

Rabbit isolates were selected from each isolate of Iyo I, Nakajima, Mie0301, and SK05Kyo from Philasterides dicentrarchi. Iyo I strain, Nakajima strain, Mie0301 strain, JF05To strain, RF05To strain, and SK05 Kyo strain were used for the test strain. In non-assisted assays, the diluted antiserum was diluted 20, 40, 80, 160, 320, 640, 1280, and 2560 times with each strain of Philasterides dicentrarchi (about 100 cells). The agglomeration and inactivation were observed under a microscope, and the inactivation potency was obtained.

The results are shown in Table 2. The anti-Iyo I strain (antibody) showed an aggregate antibody titer of 80-fold or more with respect to Iyo I strain, JF05To strain, RF05To strain, and SK05Kyo strain, respectively. However, in Nakajima and Mie0301 strains, it was 20 times or less. On the other hand, anti-Nakajima strain (antibody) showed 1280-fold aggregate antibody titer with respect to Nakajima strain, but it was 20 times or less with respect to the remaining 5 strains. In the anti-Mie0301 strain (antibody), as shown by the anti-Nakajima strain (antibody), high immobilization was recognized only in the homo-type Mie0301 strain.

(2) Western blotting

Western blotting was carried out by the following method by a regular method. Antisera and strain were the same as the above non-assisted assay.

This parasite is blended with 10% fetal bovine serum (FBS) in Eagle MEM medium Nisui (Nisui Pharmaceutical Co., Ltd.), and 7.5% sodium bicarbonate water (炭 酸水 素 ナ ト リ ウ ム 水) ) Was cultured on fish coin cells CHSE-214 cells cultured in a monolayer in a medium adjusted to pH 7.3 for 5 days.

After culturing the parasite, the parasite was recovered by centrifugation (500 × g, 20 ° C., 5 minutes), washed with Eagle MEM medium containing no FBS, and then suspended in Tris-EDTA buffer. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing the same amount of 10% sodium dodecyl sulfate (SDS) was mixed into the suspension, and the mixture was heated on a heat block. It heated at 3 degreeC. The protein was separated from the suspension on the gel by SDS-PAGE. This gel was dyed with Coomassie Brilliant Blue (manufactured by Wako Pure Chemical Industries, Ltd.) containing 25% methanol-10% acetic acid.

Subsequently, the protein separated on SDS-PAGE was transferred to a polyvinylidene fluoride (PVDF) membrane, and the membrane was blocked for 2 hours with TBS-T containing 10% of skimmed milk. And TBS-T were washed.

Next, the blocked membrane was reacted with antiserum for 1 hour as a primary antibody, and washed with TBS-T (Tris-Buffered Saline-Tween). Furthermore, it was made to react for 1 hour using a peroxidase label goat anti-rabbit IgG as a secondary antibody. The film was washed with TBS-T, and the band was visualized with a horseradish peroxidase (HRP) conjugate substrate kit (Bio Rad Labradorless Co., Ltd.).

The result was a result of supporting an assimilation assay (FIG. 1). That is, 30 kDa protein of Iyo I strain (lane 1 in FIG. 1), JF05To strain (lane 2), RF05To strain (lane 3), and SK05Kyo strain (lane 4) is expressed as anti-Iyo strain I (Ia) and serum (FIG. 1A). In each antibody of SK05Kyo strain (FIG. 1B), strong reactivity was shown. However, this band was not recognized in Nakajima strains (lane 5) and Mie0301 strains (lane 6). Moreover, in anti-SK05Kyo antibody (B), a strong response was recognized at 34 kDa in Mie0301 strain.

On the other hand, when anti-Nakajima strain (FIG. 1C) and anti-Mie0301 strain (FIG. 1D) were used, strong bands were observed at Nakjima strain and Mie0301 strain at 38 kDa and 34 kDa, respectively.

From the results of these assimilation assays and western blotting, it became clear that the Philasterides dicentrarchi had at least three serotypes (serotype I: Iyo I, JF05To, RF05To, SK05Kyo, serotype II; Nakajima, serotype III; Mie0301)

Example 1 Validity Test of Three Combination Vaccines

(1) Production of vaccine

(a) Official words

200 flounder fry were fed for two weeks using a feed for marine seedlings (Marubini Nisin Feed Co., Ltd.). What was preliminarily raised to 10.3 g was used.

(b) Cultivation of this parasite

This parasite was mixed with 10% fetal bovine serum in Eagle MEM medium (Nisui) (manufactured by Nisui Pharmaceutical Co., Ltd.) and adjusted to pH 7.3 with 7.5% sodium bicarbonate water. On fish coin cells CHSE-214 cells cultured in monolayers, the cells were cultured for 5 days.

(c) Preparation of formalin inactivating vaccine

1.65 × 10 ⁶ to 9.85 × 10 ⁶ cells of the cultured parasites (Iyo I strain, Nakajima strain, and Mie0301 strain), and 35% formalin (Nakaraitesuku) to have a tip concentration of 0.3%. ), And three days of inactivation vaccines were prepared by inactivating overnight at 4 ° C. And the above three types of vaccines were added in the same ratio, and the mixed vaccine was manufactured.

(2) vaccination

100 μL of the formalin inactivating vaccine prepared in (1) was intraperitoneally inoculated to the concentration of 6.76 × 10 ⁵ cells / fish on the flounder (average body weight 10.3 g). After 2 weeks, immunization was performed at a concentration of 9.85 × 10 ⁵ cells / fish as additional immunity.

In addition, the MEM medium immediately used in the cell culture as a control was inoculated in 100 μL intraperitoneal.

(3) safety test

After vaccination, the seefish was housed in a 10-L tank and flown, and breeding observation was performed for 2 months, and safety was confirmed. During the breeding period, food was fed in the same manner as above.

(4) Attack test (determination of validity)

On day 4 after the last immunization, the flounder (vaccine inoculation) and the unimmunized flounder (control) were immunized with the mixed vaccine, respectively, divided into four spheres, and each sphere was tested with 15 mice. Infection was done by each of Iyo I, Nakajima and Mie0301. In addition, for comparison, the vaccinated and control groups received spheres not infected with the parasite. In Iyo I and Nakajima strains, they were intraperitoneally inoculated to 100 μL at 4.03 × 10 ⁴ cells / fish. In addition, in Mie0301 strain, since the pathogenicity was clearly lower than that of the other two strains from the preliminary test results, the intraperitoneal inoculation was performed at 4.03 × 10 ⁵ cells / fish.

The effectiveness of a vaccine was calculated | required as an effective rate by the formula (death rate of 1-vaccin inoculation / mortality of control) x100%.

(5) Test result

The results (change in cumulative cumulative mortality) are shown in FIG. 2 (attack by Iyo I strain), FIG. 3 (attack by Nakajima strain), and FIG. 4 (attack by Mie0301 strain).

The flounder vaccinated with the three mixed vaccines had 75%, 73.3%, and 92.9% of the 14-day attack days for each protozoan attack (Iyo I, Nakajima, and Mie0301). All showed good immune effects. Significant differences were found in Iyo I and Nakajima states with a risk of less than 5%. Significant differences were identified in Mie0301 with a risk of less than 1%.

Comparative Example 1: Validation of a Single Vaccine

(1) production of vaccine

Cultivation of empty fish and this parasite was carried out in the same manner as in Example. Preparation of the formalin inactivating vaccine was carried out using the above cultured parasites (Iyo I strain, Nakajima strain and Mie0301 strain) in 1.65 × 10 ⁶ to 9.85 × 10 ⁶ cells, and 35% formalin (0.3%) so that the end concentration was 0.3%. Nakaraitesuku) was added, inactivated overnight at 4 ° C, and three types of formalin inactivating vaccines were prepared.

(2) Vaccination

The three formalin inactivating vaccines produced in (1) above were inoculated intraperitoneally with a flounder (average body weight of 10.05 g) so as to have a concentration of 1.65 × 10 ⁵ cells / fish. After 2 weeks, immunization was performed at a concentration of 3 × 10 ⁵ cells / fish as additional immunization.

In addition, the MEM medium immediately used in the cell culture as a control was inoculated in 100 μL intraperitoneal.

(3) safety test

After vaccination, the seefish was housed in a 10-L water tank and flown for 2 months while observing safety, and safety was confirmed. During the breeding period, food was fed in the same manner as above.

(4) Attack test (determination of validity)

On day 4 after the final immunization, the flounder (vaccine inoculation) and the unimmunized flounder (control), each of which were immunized with three formalin inactivating vaccines, were divided into four and 16 bulbs, respectively. Viii) 15 animals were tested. Infected by Iyo I, Nakajima and Mie0301 strains. Infectivity value was inoculated intraperitoneally so that it might become 100 microliters in 6.12 * 10 <^5> cells / fish.

The effectiveness of the vaccine was determined as the effective rate by the formula (mortality rate of 1-vaccin inoculation / mortality rate of control) x 100%.

(5) test result

The results (change in cumulative cumulative mortality rate) are shown in FIG. 5 (attack by Iyo I strain), FIG. 6 (attack by Nakajima strain), and FIG. 7 (attack by Mie0301 strain).

The flounder vaccinated with Iyo I strains had an effective rate of 41.7%, 0%, and 0 for 13 days of attack against each protozoan attack (Iyo I strain, Nakajima strain, and Mie0301 strain, respectively). % And the effect was also low even with the same serotype. Only Iyo I strain was found to have a significant difference of 1% or less with respect to the control.

In addition, the flounder vaccinated with Nakajima strains had an effective rate of 0%, 21.4%, and 0 for 13 days of attack against each protozoan attack (Iyo I strain, Nakajima strain, and Mie0301 strain). %, And the same serotype was also less effective. There was no significant difference in all the strains with respect to the control.

In addition, the flounder vaccinated with Mie0301 strains had an effective rate of 33.3%, 25.0%, and 13 days of attack against each protozoan attack (Iyo I strain, Nakajima strain, and Mie0301 strain), respectively. 33.3%, and the same serotype was less effective. Only Mie0301 and Iyo I strains were found to have a significant difference of 5% or less for the control.

Example 2 Effectiveness Test of Three Mixed Vaccines by High-Dose Single Immunization

(1) Production of vaccine

(a) Official words

160 flounder fry are fed for two weeks using a feed for marine seedlings (product of Marubini Nisin Feed Co., Ltd.) for halibut flounder. Breeding was used.

(b) Cultivation of this parasite

This parasite contains 10% fetal bovine serum in Eagle MEM medium (Nisui) (manufactured by Nisui Pharmaceutical Co., Ltd.) and pH 7 with 7.5% sodium bicarbonate water. It was cultured for 5 days on fish coin cells CHSE-214 cells cultured in monolayers in the medium adjusted to .3.

(c) Preparation of formalin inactivating vaccine

The cultured parasites (Iyo I strain, Nakajim strain and Mie030 strain) 4.30 × 10 ⁶ to 8.85 × 10 ⁶ cells were treated with 35% formalin (Nakaraitesuku) to give a 0.3% end concentration. ), Was inactivated overnight at 4 ° C, and three types of formalin inactivating vaccines were prepared. And the above three types of vaccines were added in the same ratio, and the mixed vaccine was prepared.

(2) Vaccination

Various formalin inactivating vaccines prepared in (1) above were inoculated intraperitoneally with 100 μL so as to have a concentration of 2.15 × 10 ⁶ cells / fish.

In addition, the MEM medium immediately used in the cell culture as a control was inoculated in 100 μL intraperitoneal.

(3) safety test

After vaccination, the seefish was housed in a 10 L tank, and breeding observation was performed for two months in running water, and safety was confirmed. During the breeding period, food was fed in the same manner as above.

(4) Attack test (determination of validity)

On the 11th day of immunization, the flounder (vaccine inoculation) and the unimmunized flounder (control), which were immunized with the mixed vaccine, were divided into two spheres, and the test was carried out with 30 spheres. Iyo I strains were infected by intraperitoneal inoculation at 3.6 × 10 ⁵ cells / fish. In addition, the vaccinated and control groups were filled with the cells that did not infect the parasite.

The effectiveness of the vaccine was determined as the effective rate by the formula (mortality rate of 1-vaccin inoculation / mortality rate of control) x 100%.

(5) test result

The results (the change in the cumulative cumulative mortality rate) are shown in FIG.

The flounder vaccinated with the three mixed vaccines showed a good immune effect with an attack rate of 68.5% for 14 days of attack against Iyo I strains, and a significant difference of 5% or less was confirmed. The non-infected cells of the three mixed vaccines and the control were omitted from FIG. 8 because no mortality was identified.

As mentioned above, although this invention was demonstrated according to the specific aspect, the deformation | transformation and improvement which are obvious to those skilled in the art are included in the scope of the present invention.

Claims (5)

(1) Philanideides dicentrarchi (Philasterides dicentrarchi) Inactivated substance of the strain showing the same serotype as Iyo I strain, (2) Pilatesterides dicentrarchi (Nakajima strain) Scutica containing as an active ingredient a main inactivated substance showing the same serotype and (3) a state inactivated substance showing the same serotype as the strain of Pilatesterides decentraci Mie0301. Mixed vaccines for the prevention or treatment of symptoms. The mixed vaccine according to claim 1, wherein the strain showing the same serotype as the Pilates decentraci Iyo I strain is a strain selected from the group consisting of Iyo I strain, JF05To strain, RF05To strain, and SK05Kyo strain. The preventive or therapeutic method of the squeutica disease which comprises administering the mixed vaccine of Claim 1 or 2 to an object in need of the prevention or treatment of squeutosis. (1) a strain showing the same serotype as the Pilates decentraci Iyo I strain, (2) a strain showing the same serotype as the pilateris decentraci Nakajima strain, and ( 3) Use in the manufacture of a mixed vaccine for the prevention or treatment of squaticosis of a strain showing the same serotype as the strain of Pilatesterides decentrach Mie0301. (1) A strain showing the same serotype as the Pilates decentraci Iyo I strain, (2) A strain showing the same serotypes as the pilateris decentraci Nakajima strain, and (3) A step of inactivating a strain showing the same serotype as the strain of Pilatesterides decentralchi Mie0301, alone or in combination of two or more, and
The manufacturing method of the mixed vaccine of Claim 1 or 2 including the process of mixing the obtained inactivating agent.

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