KR101688960B1 - Vaccine against scuticociliated ciliate - Google Patents

Abstract

The present invention relates to a scuticacid vaccine composition.
As described above, according to the present invention, there is provided a vaccine composition for a Sucutika infestation which comprises an antibacterial Gram-positive bacterium, preferably an infectious bacterium belonging to the genus Streptococcus, In addition to being able to effectively prevent or cure the infestation of the fish with the Sucutica by showing an excellent immune effect against the Ticaca larvae, and considering the serious economic loss caused by the infection of the Sucutica infestation in the domestic cultured flounder, Which is effective in minimizing the economic loss due to the scutchaca insects.

Description

{VACCINE AGAINST SCUTICOCILIATED CILIATE}

The present invention relates to a scuticacid vaccine composition. More particularly, the present invention relates to a scuticacid vaccine composition, and more particularly, to a scuticacid vaccine composition which uses freezing and fluoridated scuticacid as an antigen and as an immunosuppressant, a gram- Which comprises administering an effective amount of the composition to a subject.

Olive flounder ( Paralichthys olivaceus ) is one of the most important fisheries in Korea. However, due to high density, environmental effects and infectious diseases, many forms of the disease occur. A number of infectious agents, including viral (hirame rhabdovirus, flounder herpesvirus, viral hemorrhagic septicemia virus, nervous necrosis virus), bacterial ( Vibrio ichthyoenteri , Flexibacter maritimus , Edwardsiella tarda , Streptococcus iniae ) and parasitic ( Cryptocaryon irritans , Scuticociliate ) It is exposed to disease.

Sutuca cordata are ciliated parasites and are classified as Ciliophora gate, Oligohymemnophora river, Scuticociliatia neck. Scuticaceae are found in the hippocampus erectus (Thompson and Moewus., 1964), southern bluefin tuna ( Thunnus maccoyii ) (Munday et al., 1997), Scophthalnus maxinus (Dykova and Figueras. (2005), and the occurrence of severe deaths in the flounder ( Paralichthys olivaceus ) (Kim et al., 2004a; Jung et al., 2005)

Sucutica is infected in skin, gills, muscles, intestines, and brain. Clinical signs of infected flounder include skin necrosis and hemorrhage, dermal lesions, and color darkening (Jung et al., 2007). In Jeju, it was reported that the flounder mortality rate in fish farms by sututa squid reached 30% (Jin et al., 2009). Formalin bathing method is suggested as a countermeasure against Sututika infectious disease, but it is effective only when the suture is infected with the body surface, and effective treatment method is not presented when the Sututika infested the body.

Fish that have survived an infection of a specific disease will have defense when they later become infected with the same disease, which can be called an adaptive immune response. The vaccine was developed using the principle of adaptive immune response. Vaccines are made from antigens derived from pathogens that have been pathogen-free in a variety of ways, which can be used to stimulate the immune system when the same pathogen is infected (Ellis, 1988).

Drugs and chemicals used to treat fish diseases can increase the resistance of pathogenic or non-pathogenic microorganisms, which can be harmful to humans and animals as well as to the environment. Vaccines are emerging as an important means of preventing disease in aquaculture. However, in general, vaccines that are inactivated only by pathogens are less resistant to disease and therefore the use of immunosuppressants is essential. Immune adjuvants in most mammalian vaccines are used as an important factor in the efficacy of the vaccine (Gudding et al., 1999). Immune supplements stimulate the production of antibodies, memory responses, and stimulation of cell-associated immune responses.

The study of immunoadjuvants suitable for vaccines against Sututika spp. Has been reported very little, and although there is a desperate need for such vaccines, there is no suitable adjuvant for Sutuca spp. Vaccines.

Korean Patent Publication No. 10-2004-0039938 (a fluoridated vaccine against fishes), Korean Patent No. 10-0735038 (a recombinant protein vaccine against a scuticaca, A composition for preventing and treating ticarcidae).

The present inventors carried out an attack experiment on a group of Sucutika spp injected with a vaccine used as an immunizing adjuvant by fluorinating Streptococcus parauberis belonging to gram-positive bacteria, The present inventors have completed the present invention by experimentally confirming that the defense force is obtained from the infection through the enhancement of the immune response.

In conclusion, it is an object of the present invention to provide a method for the treatment and / or prophylaxis of bacterial infections, which comprises the use of fluoridated Gram-positive bacteria, preferably of the bacterium of the genus Fluoridated Streptococcus, more preferably of the fluoridated Streptococcus parauberis And to provide a vaccine composition for scuticacid.

It is a further object of the present invention to provide a method for the treatment and / or prophylaxis of bacterial infections caused by fluoridated Gram-positive bacteria, preferably fluorinated streptococcus bacteria, more preferably streptococcus parauberis, And a method for producing a vaccine composition for a scuticacid.

In order to achieve the above object, the present invention provides a vaccine composition for a scuticacid comprising an antigen and an fluorinated Gram-positive bacterium. The gram-positive bacteria are preferably bacteria of the genus Streptococcus, more preferably streptococcus parauberis.

The antigen is characterized by freezing and fluorinating the Sutuca cordata.

The vaccine composition is characterized in that it comprises at least one selected from the group consisting of squalene, aluminum hydroxide, and the like.

The present invention also relates to a method for producing an antigen comprising the steps of: (1) preparing an antigen; And (2) mixing the Gram-positive bacteria with the antigen produced in the step (1). The gram-positive bacteria are preferably bacteria of the genus Streptococcus, more preferably streptococcus parauberis.

In the step (1), the antigen is characterized by being frozen and frozen to form a scutica sac.

And at least one selected from the group consisting of squalene, aluminum hydroxide, and the like in the step (2).

In addition, the present invention provides a method for preventing or treating the infection of a fish with Sucutika using the vaccine composition.

The fish is characterized in that it is selected from a flounder, a turbot, a perch, a dolphin, a pike, and a red sea bream.

According to the present invention as described above, fluoridated gram-positive bacteria, preferably streptococcus bacteria, more preferably, fluoridated streptococcus parauberis are included as an adjuvant. The present invention provides a vaccine composition for a scuticacid which is effective for preventing or treating infection of a scuticacid of a fish by showing an excellent immunity effect against a scuticacid, , It is possible to minimize the economic loss due to the Suktika burden in an economical way.

Figure 1 is a graph (shown an attack inoculation results carried out for 5 weeks after vaccination according to the present invention in a vaccine efficacy test Ⅰ ◆; Na DELTA ve, ■; not vaccinated control ( Control), ▲; vaccinated by M. avidus pellet (Pellet), ●; vaccinated by M. avidus culture medium (culture) □; vaccinated by M. avidus culture medium mixed with squalene and aluminum hydroxide emulsion (culture + Sq + Alum), △; vaccinated by M. avidus culture medium mixed with formalin inactivated Streptococcus parauberis (Culture + S. para), vaccinated by M. avidus culture medium mixed squalene, aluminum hydroxide and formalin inactivated S. parauberis (Culture + Sq + Alum + S. para).
Figure 2 is a graph showing the result of the attack inoculation performed 10 weeks after vaccination according to the present invention in a vaccine efficacy test Ⅰ (◆; Na DELTA ve, ■; not vaccinated control ( Control), ▲; vaccinated by M. avidus pellet (Pellet), ●; vaccinated by M. avidus culture medium (culture), □; vaccinated by M. avidus culture medium mixed with squalene and aluminum hydroxide emulsion (culture + Sq + Alum), △; vaccinated by M. avidus culture medium (Culture + Sq + Alum + S. para), which was mixed with formalin inactivated, Streptococcus parauberis (Culture + S. para), and vaccinated by M. avidus culture medium mixed with squalene, aluminum hydroxide and formalin inactivated S. parauberis .
FIG. 3 is a graph showing the results of the inoculation of the vaccine 5 weeks after the vaccination according to the present invention in the vaccine efficacy test II; (iv) vaccinated by M. avidus pellet (Pellet + Sq + Alum), □; vaccinated by M. avidus pellet mixed with formalin inactivated Streptococcus parauberis (Pellet + S. para), △ ; vaccinated by M. avidus pellet mixed with squalene, aluminum hydroxide and formalin inactivated S. parauberis (Pellet + Sq + Alum + S. para)).
FIG. 4 is a graph showing the results of the inoculation of the vaccine administered 18 weeks after the vaccination according to the present invention in the vaccine efficacy test II (3 weeks after booster vaccination) Vaccinated by M. avidus pellet (Pellet), • vaccinated by M. avidus pellet mixed with squalene and aluminum hydroxide emulsion (Pellet + Sq + Alum), □ vaccinated by M. avidus pellet mixed with formalin inactivated Streptococcus parauberis Pellet + S. para), △; vaccinated by M. avidus pellet mixed with squalene, aluminum hydroxide and formalin inactivated S. parauberis (Pellet + Sq + Alum + S. para).

Hereinafter, the present invention will be described in detail.

The present invention was carried out with the aim of developing an effective immunosuppressive agent as part of the enhancement of the effect of the Sucutika spp. Miamiensis avidus vaccine. Immunosuppressive agents such as Squalene (Sq), Aluminum hydroxide (Alum), and formalin - inactivated Streptococcus parauberis (S. para) were tested in two experiments using two types of antigen 'Pellet' Were used singly or in combination.

Antigens were used under the following assumptions; ['Pellet' is a whole cell that contains a squid surface antigen, which can regulate the immune response of the flounder. In addition, the protein secreted during the cultivation of the Sukutika cattle cell line as a food source will also act as an antigen, and thus the Sukutika culture medium will also enhance the immune response as an antigen. Thus, in the present invention, M. avidus cells, Pellet ', M. avidus cells, and' culture 'including culture medium. Inactivation of antigens was performed by freezing at -80 ° C instead of chemicals such as formalin, sulfoxide, doxycycline hyclate and albendazole. Frozen inactivation is more safe than the chemical substance, and the denaturation of the antigen protein can also be reduced.

For long-term immune formation, the antigen should be released slowly in the body and this effect can be expected through coating the antigen with oil. Incomplete Freund's adjuvant (mineral oil) is commonly used in fish and mammalian vaccines. However, these mineral oils cause side effects after vaccination such as intestinal adhesion, poor growth, granuloma, and poor appetite. Metabolizable natural oils can be used as a substitute for mineral oil and can eliminate the side effects of mineral oil.

Since squalene is effective for promoting humoral and cellular immune responses as metabolic and effective immunosuppressants, squalene was selected as an immunosuppressive agent in the present invention. Aluminum hydroxide enhances the response of T helper cell type 2 (Th2), which enhances the humoral immune response. In addition, aluminum hydroxide has proven to be safe for humans and animals for a long period of time. Therefore, the inclusion of aluminum hydroxide in squalene may serve as a more effective adjuvant.

S. parauberis is a gram-positive bacterium and is a causative agent of bacterial diseases in domestic cultured flounder. Gram-positive bacteria and their constituents play a role in activating macrophage function in mammals (Gupta et al., 1993). Macrophages play a role in stimulating the function of lymphocytes and other immune cells, along with the phagocytosis of pathogens (Surhone et al., 2010). Macrophages secrete many types of cytokines such as interleukin (IL) and Tumor Necrosis Factor (TNF). In addition, peptidoglycan and teichoic acid, the cell wall constituents of Gram-positive bacteria, stimulate the secretion of TNF-α, IL-1β and IL-6 (Mattsson et al., 1993; Heumann et al., 1994).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1. Experimental material

(1) Cultivation of Sututika spp . Miamiensis avidus

In this study, we investigated the effect of strain YS2 on the growth of the genus Sucutica , which was isolated from the brain of a flounder (Jung et al., 2005). Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), penicillin (50 IU / ml) and streptomycin (50 IU / ml) (DMEM) (Gibco, USA) to a Chinook salmon (Oncorhynchus tshawytscha) embryo ( CHSE-214) cell line culture was incubated with a food source in, M. avidus was maintained by subculturing in 10 ℃ incubator at 45 day intervals . In order to prepare the antigens and to test the attack, Sukutika was used to inoculate M. avidus , which was maintained in the laboratory, in another flask cultured with CHSE-214 cells. The CHSE-214 cells in the Flask were used as a feed source for Sututika, and the Flask was incubated at 15 ° C for 5-7 days.

(2) fluoridation of the antigen

Sucutica spp. Cultured as a feed source of CHSE-214 was harvested and inactivated for use as an antigen. The culture medium containing M. avidus was centrifuged at 15 ° C and 1,500 rpm for 10 minutes, and the supernatant was removed and inactivated by only freezing the scuticacid ('Pellet' antigen) at -80 ° C for at least 2 days. This antigen was named 'Pellet'. The culture medium containing M. avidus as another antigen was immediately inactivated by freezing at -80 ° C for at least 2 days without centrifugation ('Culture' antigen). The 'Culture' antigen was used to identify the possibility that the enzyme of the protease, which secretes into the medium in the process of growing up, acts as an antigen to increase the efficacy of the vaccine. The antigen inactivated at -80 ° C was again inoculated into CHSE-214 cells to confirm whether or not the inactivation was correct.

(3) Production of vaccine

Ⅰ) Two types of M. avidus antigens, 'Pellet' and 'Culture', can be used to prepare vaccine by antigen alone or by immunizing with each immunosuppressant (squalene, squalene + aluminum hydroxide, formalin inactivated Streptococcus parauberis + squalene + And S. parauberis , which is inactivated as a control . The total volume of each combined vaccine was adjusted with DMEM without FBS.

Squalene (Sigma, Australia), glycerol (Amresco, USA) and Tween 80 (Sigma, USA) were used to prepare vaccine using squalene as an adjuvant. For brief preparation, 5% squalene, 20% glycerol and 0.5% Tween 80 were mixed for 10 minutes using a vortex machine. Inactivated antigens were added to the mixture for 15 minutes on ice using a Polytron PT 1200 E homogenizer (Kinematica, Switzerland) Homogenized. The vaccine was called 'Antigen + Sq'.

Iii) Preparation of a vaccine using squalene + aluminum hydroxide as an adjuvant is similar to the above-mentioned method of preparing a vaccine using squalene as an adjuvant. The difference was that 0.5% aluminum hydroxide (Sigma, Korea) was added before homogenization, and aluminum hydroxide was added to the mixture, followed by homogenization by adding antigen. The vaccine was called 'Antigen + Sq + Alum'.

Iv ) Preparation of vaccine using S. parauberis as an adjuvant was prepared by mixing formalin-inactivated S. parauberis (Daesung Microbiological Labs Co., Ltd., Korea) with antigen. 60 ㎎ / ㎖ of S. parauberis was added and mixed with M. avidus antigen and homogenized on ice for 10 minutes using Polytron PT 1200 E homogenizer. The vaccine thus produced was designated 'Antigen + S. para'.

V ) Vaccine using formalin-inactivated Streptococcus parauberis + squalene + aluminum hydroxide as an adjuvant is similar to that of vaccine using squalene + aluminum hydroxide as an adjuvant. In the step of adding antigen, 60 mg of S. parauberis / Ml were added together and homogenized. The vaccine thus produced was designated 'Antigen + S. para + Sq + Alum'.

Vi ) In the case of vaccine containing Formalin-inactivated Streptococcus parauberis as an adjuvant, S. parauberis was inoculated with 2.5 × 10 ⁹ colony forming units (CFU) per experimental animal at the time of vaccination.

(4) Preparation of Sutuchikana for attack infection

DMEM supplemented with 10% FBS and cultured with CHSE-214 cells as a food source were harvested just before attack infection. The culture medium containing M. avidus was centrifuged at 1500 rpm for 10 minutes, and the supernatant was removed. The supernatant was removed and the supernatant was removed by centrifugation under the same conditions. The concentration of inoculum was adjusted using DMEM without FBS. The number of Sucutika spp. Was counted using a hemocytometer, and the amount of Sucutika spp. Cultured in a 75 cm 2 tissue culture flask (Nunc, Denmark) was 7.0 to 9.0 × 10 ⁶ cells per flask.

(5) Experimental words

Two experiments were conducted to evaluate the effect of vaccines containing different adjuvants. The flounder used in the experiment was purchased from Jeonnam Yongkwang (Experiment Ⅰ) and Muan (Experiment Ⅱ) purchased from two fisheries. Purchased flounder was sampled at 1 ~ 2% per day of fish body in natural water temperature in water tank in Chungnam Univ. Ten test mice were randomly selected before the experiment, and then examined by microscopy to determine if the scutica parasites were detected in the gills, skin, and brain.

Example 2. Experimental Method

(1) Statistical analysis and relative percentage survival (RPS)

The results of all inoculations were statistically analyzed using rank (mantel-cox) test using Graph Pad Prism5 software (Version 5.02) and the relative survival rate (RPS) was calculated by Amend in 1981. The formula for RPS calculation is as follows.

"RPS = [1 - (% mortality of vaccinated group /% mortality in control] × 100"

(2) the antibody is tested

I) Blood collection

In the experimental group vaccinated and the control group, 3 mice were sampled per each experimental group through subarachnoid vascular system 1 day before each attack. The collected blood was allowed to stand at room temperature for 1 hour and then centrifuged at 4 ° C and 5000 rpm for 10 minutes to separate the supernatant (serum) only and stored at -20 ° C until use in the experiment.

Ii) Coagulation reaction

The serum used in the experiment was subjected to complement inactivation treatment at 42 ° C for 30 minutes and then diluted 2 times, 4 times, 8 times, 16 times, 32 times, 64 times, 128 times in the double dilution series using DMEM without FBS Fold, and diluted to 256-fold. The strain YS2 was cultured in CHSE-214 cells, centrifuged at 1,500 rpm at 15 ° C for 10 minutes, and the supernatant was removed. The supernatant was resuspended in DMEM and counted using a hemocytometer. Sutuca cordata were allowed to stand at 15 ° C for 30 minutes and then used in the experiment. Diluted sera diluted in the same volume as 20 μl (2 × 103 cells / well) of the prepared scuticacid was inoculated into each well of 96-well plates (Nunc, Denmark) and the plates were incubated at 20 ° C. incubator for 3 hours The reaction was observed. The titer of each serum was indicated by the final dilution step in which the aggregation reaction was observed.

Experimental Example 1. Vaccine Efficacy Test I

(1) Vaccination

The experimental fishes (6.5 ㅁ and 0.2 g) purchased from Yeonggwang and transferred to Chonnam National University Aquaculture Center were transported to a hydraulic tank containing 1 ton of seawater per each 50 fish. In Experiment I, 'Pellet', in which only the M. avidus cell pellet was inactivated, and 'Culture', in which the culture of M. avidus was inactivated. The 'culture' antigen was also used as a sole antigen vaccine and was mixed with each adjuvant to produce 'Culture + Sq + Alum', 'Culture + S. para', and 'Culture + Sq + Alum + S. para' . Each group of vaccines was inoculated into the experimental group by intraperitoneal inoculation with 50 μl of each vaccine. The concentration of Sucutika antigen in each vaccine was 2.9 × 10 ⁴ cells. The control group was inoculated intraperitoneally with 50 .mu.l / well of DMEM without FBS. The average water temperature during the experiment was 18 ℃.

[Table 1]

(2) attack infection

To test the effect of the vaccine, two attack infections were conducted at 5 and 10 weeks after vaccination. The flounder was transferred to the infection laboratory of Chonnam National University two days before the attack infection, and 20 litters of each flounder were placed in a tank containing 25 liters of UV treated seawater.

At 5 weeks after the vaccination, Sukutika sacrifice was prepared at 2.6 × 10 ⁵ cells / ㎖ in the attack experiment. In the attack experiment, 100 μl of the prepared Sukutika sac was inoculated into the flounder by intramuscular inoculation. The negative control (Navie) did not inoculate. The experiment was conducted for 20 days and the average water temperature was 20 ℃.

At 10 weeks after the vaccination, the scuticacid was prepared at 1.7 × 10 ⁶ cells / ㎖ in the attack experiment. In the attack experiment, 100 μl of the prepared scuticacid was inoculated into the flounder by intramuscular inoculation. The negative control (Navie) did not inoculate. The experiment was conducted for 24 days and the average water temperature was 18 ℃.

(3) Effectiveness of vaccine

This experiment was carried out to determine if formalin - inactivated S. parauberis was effective as an adjuvant to M. avidus . In order to confirm the effect, the offensive vaccination was carried out 5 and 10 weeks after the vaccination.

The results of the inoculation after 5 weeks of vaccination showed that the mortality rate was 4 days after the inoculation and the cumulative mortality rate was 'Control', 'Pellet', 'Culture', 'Culture + Sq + Alum' 30%, 40%, 45%, 35% and 25%, respectively, in Culture + S. para and Culture + Sq + Alum + S. para. The p value was 0.0012, 0.0260, 0.0033, and 0.005 in the 'Pellet', 'Culture + Sq + Alum', 'Culture + S. para' and 'Culture + Sq + Alum + 0.0003, respectively.

In the results of the inoculation 10 weeks after the vaccination, the subjects had the lowest mortality rate from 50% in the culture + S. para group to 100% in the other groups, (Fig. 2). Therefore, the results of the comparison with the control group were confirmed only in 'Culture + S. para' group by statistical analysis, and the p value was found to be <0.0001.

The relative survival rate was 60% for 'Pellet', 'Culture', 'Culture + Sq + Alum', 'Culture + S. para' and 'Culture + Sq + Alum + S. para' , 47%, 40%, 54% and 67%, respectively. After 10 weeks of vaccination, the survival rate of attack was 100% and the relative survival rate was 0% in the other groups except 'Culture + S. para' group. In the 'Culture + S. para' group, 50% Respectively. The results of the attack in Experiment I are summarized in Table 2.

[Table 2]

(4)

Whether or not the antibody was produced against the inoculated vaccine was confirmed by the coagulation reaction when the serum mixed with the serum taken from the flounder was mixed, and the aggregate was represented by the reciprocal of the maximum dilution factor in which the aggregation occurred. For the coagulation reaction, the strain Sututika ( M. avidus strain YS2), which is the same as that used for the vaccination, was used. The results of coagulation reaction are summarized in Table 3. After 5 and 10 weeks of vaccination, no aggregation reaction occurred in the serum of the control. The coagulation reaction did not occur in the 'Pellet' and 'Culuture' groups, which did not contain the adjuvant, and higher coagulation was observed in the coagulation reaction after 5 weeks of vaccination than after 10 weeks of vaccination. The highest titer was confirmed in 'Culture + Sq + Alum + S. para' among vaccine experiments.

[Table 3]

(4) Conclusion of vaccine efficacy Experiment I

The vaccine used in Experiment I was 'Pellet', 'Culture', 'Culture + Sq + Alum', 'Culture + S. para' and 'Culture + Sq + Alum + S. para'. Experimental animals were vaccinated by intraperitoneal inoculation. In order to confirm the effect of vaccine, attack experiment was carried out through intramuscular inoculation.

Pellet, Culture + Sq + Alum, Culture + S. para and Culture + Sq + Alum + S. para were compared with the control. 'Group had a p-value of less than 0.05.

The relative survival rate (RPS) was 67% (Culture + Sq + Alum + S. para), 60% (Pellet), 54% (Culture + S. para), 47% ) Showed the highest efficacy in the order of no immunosuppressant and no immunosuppressant. Thus, at the 5th week after immunization, most of the vaccines used in the experiment were found to be defensive against the insect.

In the attack experiment (10 weeks after vaccination), the vaccine was effective in 'Culture + S. para' group compared to the control group, showing a relative survival rate of 50% and 0% in the other groups. These results are probably due to the inoculation of Sutuca larvae at a concentration too high (1.7 × 10 ⁵ / fish) compared to the fish in the second attack inoculation experiment. In Experiment I, two antigens, 'Pellet' and 'Culture' were used as antigens, but unlike expectation, there was no effective difference between two antigens. Therefore, in Experiment Ⅱ, only 'Pellet', which is easy to control the amount of antigen, was used as an antigen. Experiments were conducted to evaluate the effectiveness of each adjuvant by using the same adjuvant as Experiment I and increasing the amount of the adjuvant.

Experimental Example 2: Vaccine efficacy test II

(1) Vaccination experiment

The experimental fishes (10 ㅁ 2 g) purchased from Muan and transferred to Chungnam University Cultivation Center were transported to a water tank at a rate of 50 fish per experimental group. In Experiment 2, the experiment was conducted using M. avidus cell 'Pellet' antigen as an antigen, and 'Pellet', 'Pellet + Sq + Alum', and 'Pellet + S. para' were mixed with a single antigen vaccine or each immunizing adjuvant. And four vaccines of 'Pellet + Sq + Alum + S. para' were prepared. Each group of vaccines was inoculated into the experimental animals by intraperitoneal inoculation with 100 μl per mouse. The concentration of Sucutika antigen mixed in each vaccine was 1.0 × 10 ⁶ cells / fish. The control group was inoculated intraperitoneally with 100 .mu.l / well of DMEM without FBS. The average water temperature during the experiment was 15 ℃.

(2) Vaccination Booster vaccination

Booster vaccination was administered 15 weeks after the initial vaccination. Each group of flounder was inoculated intraperitoneally with 100 μl of vaccine using the same antigen and adjuvant as the initial vaccine. One difference was that the vaccine was produced at a concentration of 5.6 × 10 ⁵ cells / fish in booster vaccination with the concentration of the scuticum antigen in the vaccine used in the booster vaccination.

(3) Attack infection experiment

To test the efficacy of the vaccine, two attack infections were performed 5 and 18 weeks after vaccination (three weeks after Booster vaccination). The flounder was transferred to the infection laboratory of Chonnam National University two days before attack infection, and was placed in a tank containing 25 liters of UV - treated seawater for each experiment. The detailed number of attack infection test words are summarized in Table 5.

At 5 weeks after the vaccination, Sukutika sacrifice was prepared at 2.6 × 10 ⁵ cells / ㎖ in the attack experiment. In the attack experiment, 100 μl of the prepared Sukutika sac was inoculated into the flounder by intramuscular inoculation. The negative control (Navie) did not inoculate. The experiment was carried out for 20 days in a 15 ℃ constant temperature chamber.

After 18 weeks of vaccination (3 weeks after booster vaccination), the scuticacid was prepared at 6.9 × 10 ⁵ cells / ㎖ in an attack test. The test for attack infection was inoculated with 100 μl of prepared scuticacid per flounder Respectively. The negative control (Navie) did not inoculate. The experiment was carried out for 20 days in a 15 ℃ constant temperature chamber.

[Table 4]

(4) The efficacy of vaccine

This experiment was carried out in order to confirm the effect of Ajvant at high antigen levels. The flounder inoculated with each vaccine was tested for vaccine efficacy by two inoculations, five weeks after vaccination and three weeks after booster vaccination (18 weeks). In the previous experiments, the 'Pellet' antigen was more effective than or similar to the 'Culture' antigen between 'Pellet' and 'Culture', and the 'Pellet' antigen was more easily controlled than the 'Culture' have. Therefore, only 'Pellet' was used as an antigen in this experiment.

The cumulative mortality rate was 85% in 'Pellet', 'Pellet + Sq + Alum', 'Pellet + S. para' and 'Pellet + Sq + Alum + S. para' %, 55%, 15%, 0% and 90%, respectively (Fig. 3). Statistical analysis showed that there was a significant difference in p value between <0.0001 and <0.0001 in 'Pellet + Sq + Alum' group and 'Pellet + S. para' group, respectively.

Pellet + Sq + Alum, Pellet + S. para, and Pellet + Sq + Alum (Pellet + Sq + Alum) (Fig. 4) were 85%, 60%, 40%, 15% and 82.4%, respectively. Statistical analysis showed that there was a significant difference in p value between <0.0001 and <0.0001 in 'Pellet + Sq + Alum' and 'Pellet + S. para' when compared with the control.

The relative survival rate was 35.5%, 82.4% in Pellet, Pellet + Sq + Alum, Pellet + S. para and Pellet + Sq + Alum + S. para after 5 weeks of vaccination, 100%, and -5%, respectively. In the case of an attack of 18 weeks after the vaccination (after 3 weeks of Booster vaccination), 'Pellet', 'Pellet + Sq + Alum', 'Pellet + S. para' and 'Pellet + Sq + Alum + S. para' %, 53%, 82.4%, and 3.2%, respectively.

[Table 5]

(5)

For the coagulation reaction, the strain Sututika ( M. avidus strain YS2), which is the same as that used for the vaccination, was used. The results of coagulation reaction are summarized in Table 6. After 5 and 10 weeks of vaccination, no aggregation reaction occurred in the serum of the control. The results of the agglutination of serum isolated at 5 weeks of vaccination were summarized as 3 test specimens (1, 2, 3) for each test group. In the 'Pellet' group (32, 8, 16), 'Pellet + Sq + (8, 8, 4), 'Pellet + S. para' (16,32,16) and 'Pellet + Sq + Alum + S. para' Aggregation of isolated serum at 18 weeks of inoculation showed no aggregation in the serum isolated from all test fishes except for one test fish in the 'pellet + S. para' group.

[Table 6]

(6) Conclusion of vaccine efficacy Experiment II

Experiment Ⅱ was carried out based on Experiment Ⅰ. The vaccine used in Experiment Ⅱ consisted of four vaccines of 'Pellet', 'Pellet + Sq', 'Pellet + S. para' and 'Pellet + Sq + S. para' The experiment was carried out in the same manner as in Experiment Ⅰ, in order to test the effectiveness of the vaccine in two attack experiments (8 weeks after vaccination and 18 weeks after vaccination). Experiment II used more antigen than Experiment I in order to know more clearly the effect of vaccine. The flounder that had been vaccinated first was attacked at 5 weeks and the next flounder was booster vaccinated 15 weeks after the initial vaccination to enhance the immune response.

In the first attack trial, the cumulative mortality rate was 85% (Pellet + S. para) without vaccination, but the group survived all but died and showed excellent efficacy as a vaccine. In addition, the addition of squalene and alum to the pellet and the addition of the bacterium Streptococcus parauberis to the pellet increased the efficacy of the vaccine as compared to the pellet alone.

Immunization after 15 weeks of vaccination and immunization after 18 weeks showed a similar pattern to the results of the first attack. Therefore, it was confirmed that the vaccine against Pseudomonas aeruginosa in the Pellet + S. para group showed the strongest synergistic effect that the inactivated Streptococcus parauberis bacteria increased the efficacy of the Sucutica vaccine .

It was difficult to conclusively conclude that the antibody level of the flounder and the defense strength of the flounder obtained from the coagulation test were directly related to each other. However, in Experiment I, it was confirmed that the group containing the adjuvant in the vaccine showed higher aggregation value than the group not containing the adjuvant, and thus, it was confirmed that the adjuvant enhanced the specific antibody production in the flounder I could. In Experiment Ⅱ, all groups that were collected after 5 weeks of vaccination showed a similar value of aggregation, which is considered to be due to the fact that the amount of antigen in the vaccine of Experiment Ⅱ is about 35 times higher than that of Experiment Ⅰ. In the coagulation test, it was found that the production of a specific antibody can produce a specific antibody only with an antigen regardless of the presence or absence of an immunosuppressant when the concentration of the antigen is high. In the case of vaccine group that did not use immunoadjuvant while using low concentration of antigen, it was confirmed that generation of specific antibody was difficult. That is, even if the amount of the antigen in the vaccine is low, it can be confirmed that the vaccine added with the adjuvant can stimulate the production of the specific antibody.

In Experiment II, the amount of antigen in the vaccine was 1.02 × 10 ⁷ / ml, which is about 35 times the amount of antigen contained in the vaccine used in Experiment I. As a result of the experiment using the antigen only vaccine in Experiment I and Experiment II, it was confirmed that the vaccine having high antigen amount was more effective in the scutica vaccine. In Experiment I and Experiment II, although the antigen is different from 'Pellet' and 'Culture', 'S. the same immunosuppressive drug called &quot; para &quot; was used. The difference of the antigens was regarded as the result of the experiment Ⅰ, and there was no difference in the result analysis because the specific difference in the effect of the vaccine could not be confirmed. In the case of the culture + S. para group, which confirmed the efficacy in Experiment I, the cumulative mortality rate was higher than that of Pellet + S. para, which was confirmed in Experiment II, but this is considered to be the difference in the amount of antigen contained in the vaccine. Thus, we concluded that the Sukutika vaccine, which contains a high concentration of antigen, helps boost the immune response.

As a result of two experiments, 'S. Para 'group showed the highest defense against the attack of Sukhtika spp. In the group using' Squalene + alum 'as the immunosuppressant, the second highest defense against Sukutika spp. Respectively. In Experiment Ⅱ, 'Pellet + Sq + Alum' group showed 82.4% and 53% relative survival rate of two attack infections, respectively. 'Sq + Alum' was found to enhance the immune response to Sukitika Could.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

Antigens inactivated by inoculation of Sutuca cordata; And
A vaccine composition for a scuticacid comprising a fluorinated Gram-positive bacterium.
The method according to claim 1,
Wherein the gram-positive bacteria are streptococcus bacteria.
The method according to claim 1,
Wherein the gram-positive bacterium is Streptococcus parauberis.
delete delete (1) preparing an antigen inactivated by inoculation of a scuticacid; And
(2) mixing the antigen produced in the step (1) with a fluorinated Gram-positive bacterium.
The method according to claim 6,
Wherein the gram-positive bacteria are streptococcus bacteria.
The method according to claim 6,
Wherein the gram-positive bacterium is Streptococcus parauberis. delete delete A vaccine composition according to claim 1, wherein the vaccine composition is used to prevent or treat the infection of the fish with Sukitika.
12. The method of claim 11,
Wherein the fish is selected from the group consisting of flounder, turbot, perch, red sea bream, oyster, and red sea bream.

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