KR20040039938A - Killed vaccine against scuticociliated ciliate - Google Patents

Abstract

PURPOSE: Provided is a vaccine for inactivating scuticociliated ciliate to increase immunity to scuticociliated ciliate in the fish, and consequently to prevent the infection therewith as well as retard perish. CONSTITUTION: A vaccine for inactivating scuticociliated ciliate is characterized by inoculating scuticociliated ciliate into CHSE-214(chum salmon embryo), RTG-2(rainbow trout gonad), EPC(epithelioma papilosum carp) and FHM(fathead minnow) cells under the dark condition, followed by culturing at a low temperature and inactivation with formalin.

Description

Killed vaccine against scuticociliated ciliate

The present invention relates to a method for producing an inactivated vaccine of a Squatica insect which produces a vaccine by inactivating a Squatica parasite in a fish with formalin. In the present invention, a scutica worm parasitic in a fish is subjected to cancer's coin cells CHSE-214 (chum salmon embryo), RTG-2 (rainbowtrout gonard), EPC (epithelioma papilosum carp) and FHM (fathead minnow cells) cells under cancer conditions. The present invention relates to a method for preparing an inactivated vaccine for Scutika insects, which is inoculated into a large amount at low temperature, maintains the fragmentation of the larvae, and then inactivates the larvae with formalin.

The present invention relates to an inactivated vaccine of Scutica insects parasitic in fish, and when inactivated Scutica insects with formalin and then inoculated into fish, immunity is generated in the fish body, which shows the efficacy as a vaccine.

Scutika insects frequently occur in flounder, the representative fish of Korea, and the mortality rate is very high when it occurs in childhood, and the mortality rate is high when there is a lot of organic matter in the farming environment or when other diseases and complex infections occur. high. Scutika are parasitic in gills, body surface and fins, and tissues collapse. As symptoms progress, the muscles are exposed and penetrate the brain and various organs, causing necrosis of tissues.

Formalin is mainly used to control Scutika insects, but the concentration that can control more than 200 ppm of Scutica causes severe stress on the flounder, and the insects that penetrate the body are impossible to control. Fresh water baths and chitosan have also been tried, but there is no clear effect and there is no effective prevention and remedy for Scutika insects. As a countermeasure against bacterial and viral diseases, vaccines have been developed for the prevention of diseases along with antibiotics and antiviral substances.However, parasites are difficult to cultivate in vitro, making it difficult to obtain large amounts of antigens, and the antigenicity of the insects is unclear. Vaccines have been developed only in some limited human and livestock parasites, and there are no parasitic vaccines in fish.

As the safety of the drugs and drugs used for the treatment of fish diseases and the load on the environment have become a problem, interest and necessity for the development and use of the vaccine using the fish's own immune system are emerging. Vaccines against parasitic fish parasites have been studied in Ichthyophthirius multifiliis, Amyloodinium ocellatum and Cryptobia salmositica (Woo, Dev. Biol. Stand . 90: 223-241, 1997). . Scutica is a member of the same ciliary worm as Ichthyophthiriusmultifiliis , but because it is proliferated by dividing, the antigenic protein does not change according to life history, and it is possible to cultivate in vitro . .

The present inventors have a mass culture technology of Scutika insects, and based on this, the vaccine was inactivated with formalin to develop a vaccine, and the effectiveness thereof was revealed through infection experiments.

An object of the present invention is to prepare a vaccine against the Scutika insects and to verify the efficacy of the vaccine to prevent the Scutika insects and to reduce the death of farmed fish. To this end, the vaccine was prepared by mass culturing Scutika insects, and the produced vaccine was administered to the flounder to confirm that specific immune responses against Scutika insects were generated in the body of the flounder. It was found to be effective in preventing infection.

1 is a state in which the Scutika insects are infected under shading conditions in order to effectively infect the flounder.

Figure 2 shows ulcers and bleeding symptoms in the dorsal fin (2a) and the back muscle area (2b) of the flounder killed by Scutika insects.

Fig. 3 shows a large number of carcasses infected with the fins 3a and gills 3 of flounder killed by Scutika insects (arrows).

Figure 4 shows the preventive effect of the vaccine by artificially infecting Scutika after vaccinating the flounder. (-●-control,-▲-injection vaccine,-■-immersion vaccine,-×-vaccinated)

Figure 5 shows the preventive effect of the vaccine by artificially infecting Scutika after vaccinating the flounder. (-●-control,-▲-injection vaccine,-×-vaccinated)

Figure 6 shows the detection of antibodies against Scutica from flounder serum. (-●-Vaccination 1,-▲-Vaccination 2,-■-Vaccination 3,-◆-Vaccination 4, -○-Unvaccinated 1,-□-Unvaccinated 2)

Figure 7 shows the effect of the serum component on Scutika, flounder serum of the vaccination to stop the movement of Scutika, causing aggregation.

Figure 8 shows the phagocytosis of the halibut leukocytes showed that the vaccination was higher phagocytic activity than the control group not vaccinated.

Fig. 9 shows the chemotactic activity of halibut leukocytes, showing that leukocytes taken from vaccinated halibut had higher chemotactic activity against Scutica.

10 is a graph showing the efficacy of Scutica inactivated vaccine in aquaculture.

The present invention relates to a method for producing an inactivated vaccine of a Squatica insect which produces a vaccine by inactivating a Squatica parasite in a fish with formalin. In the present invention, the scutica worms parasitic in fish are cancer-coated cells of CHSE-214 (chum salmon embryo), RTG-2 (rainbowtrout gonard), EPC (epithelioma papilosum carp) and fathead minnow cells (FHM) cells under cancer conditions. The present invention relates to a method for preparing an inactivated vaccine for Scutika insects, which is inoculated into a large amount at low temperature, maintains the fragmentation of the larvae, and then inactivates the larvae with formalin.

The present invention provides a method of sustained culture of Scutica insects. In the present invention, the stable production of the vaccine requires the production of the same antigen every time, for this purpose it must be available when necessary while maintaining the same strain (strain). In the present invention, the Scutica caterpillar isolated from the flounder in 1998 has been maintained and cultured to the present state without contamination. The present invention is inoculated with Scutika insects in CHSE-214 (chum salmon embryo), RTG-2 (rainbowtrout gonard), EPC (epithelioma papilosum carp) and fathead minnow cells (FHM) cells of fish fish cells It is possible to maintain for 6 months, and if the passage at 20-25 ° C., active cell division begins.

The present invention also provides a technique for mass culturing Scutica. For the cultivation of Scutika insects, cultivation of up to 10 ⁴ cells / ml was achieved by mixing 2% proteose peptone, 1% yeast extract, glucose and minerals of various concentrations. (See Yoshinaga, T. and J. Nakazoe, Fish Pathol : 28: 131-134, 1993). In the present invention, when cultured using fish cells, cultured 5 × 10 ⁶ cells / ml of larvae, the number of larvae 500 times larger than before.

In the present invention, an inactivated vaccine was produced using a mass culture technique of Scutika insects, and the method of directly injecting the vaccine into the fish (injection vaccine) and the method of dissolving the vaccine in water (immersion vaccine) are effective as a vaccine.

The cellular immune response of leukocytes and the antibody components contained in the blood of the olive flounder after the administration of the vaccine produced specific immunity against Scutica insects and revealed the effect of immune enhancement as a vaccine. In addition, by verifying the efficacy as a vaccine through experiments in vivo and aquaculture, the present invention provides a breakthrough technology and method for the prevention of Scutika insects.

Hereinafter, embodiments of the present invention will be described in detail. However, the scope of the present invention is not limited only to these examples.

Example 1 Retention and Mass Culture of Scutica Insects

When the brains of flounder infected with Scutika insects are aseptically extracted, washed three times with sterile saline, inoculated into fathead minnow cells (FHM) and cultured at 20 ° C., the active carcass divides and actively divides cells. Some were transferred to a culture flask containing fresh FHM cells once every six months while incubating at 10 ° C. to maintain the same strain.

For mass cultivation of Scutika insects, incubate at the bottom of a 225 cm ² flask until the FHM cells are full, inoculate Scutica insects, and incubate at 20 ° C. do. In this way, 2-5 × 10 ⁶ cells / ml of Scutica insects can be recovered.

Example 2: Inactivation of Scutika insects

After centrifuging the culture medium of the cultured Scutika insects at 1000 g for 10 minutes, the phosphate buffer solution (0.01M, pH 7.4 PBS) was added to resuspend the Scutica insects that settled at the bottom of the centrifuge tube. It was added to the final 0.3% formalin as an inactivating agent and treated at 4 ° C for 24 hours. After inactivation is complete, wash the cells with 1000 grams of phosphate buffer solution twice for 10 minutes to remove formalin, and count the number of cells with a hemocytometer. Immersion vaccine is sonicated and crushed carcasses in order to improve the absorption into the body of the flounder to confirm that the carcasses are completely destroyed under the microscope. Injectable vaccines use inactivated cells as they are. The vaccine should be stored frozen until use.

Example 3: Prevention of Mortality by Injection and Immersion Vaccine

Flounder with an average body length of 8.6 cm (average weight: 6.3 g) was bred at a breeding water temperature of 18-22 ° C. while feeding oxygen with an oxygen supply device and feeding commercial flounder feed.

The vaccine was injected intraperitoneally at a concentration of 1.43 × 10 ⁵ cells per flounder and boosted at the same concentration 2 weeks later. The immersion vaccine was counted by counting the inactivated worms on a hemocytometer and sonicated, soaking the flounder for 5 minutes while supplying air to the seawater at a concentration of 2.4 × 10 ⁵ cells / ml. Two weeks later, boosters were given the same concentration. Two weeks after booster inoculation, 12 animals were taken from each experimental group to measure immune responses, and 20 animals were used for infection experiments.

As an infection test to measure the effect of the vaccine on the living body, live Scoochi caterpillars were infected with the flounder breeding tank to 2.0 × 10 ² cells / ml, and mortality was observed for 3 weeks. After artificially infecting Scutika insects, 25% of seawater was exchanged daily, and was bred in a state of blocking light by surrounding the breeding tank with a black film (FIG. 1). Lungfish was able to visually observe ulceration and bleeding in the periphery and back muscles, a specific symptom of Scutika insect infection (Fig. 2). 3).

The transition of the untreated and vaccinated groups is shown in FIG. 4. Survival rate was 75% for injection vaccine, 40% for immersion vaccine and 15% for vaccinated vaccine. This result shows that the vaccine vaccine has 71% mortality effect and the immersion vaccine has 47% mortality reduction effect. The present invention shows excellent efficacy in preventing and reducing mortality of Scutika insects in the flounder.

Example 4: Prevention of Death by Injection Vaccine

The flounder, with an average body length of 28.9 cm (average weight: 226 g), was fed at a temperature of 18-20 ° C while feeding oxygen with a oxygen supply device and feeding commercial flounder feed. A vaccine at a concentration of 6.12 × 10 ⁵ cells per flounder was injected intraperitoneally. After 4 weeks of immunization, 1 ml of blood was collected from the arrhythmia and live Sukuchika was infected with the flounder tank to reach 3.5 × 10 ² cells / ml. Observed. The results are shown in FIG.

As shown in FIG. 5, 81% of the vaccinated spheres survived and 42% of the vaccinated spheres survived, indicating that the vaccine was effective in preventing mortality by Scutica.

Example 5: Detection of Specific Antibodies Against Scutikatis by ELISA

Blood from vaccinated flounder and normal flounder were taken and measured by ELISA to determine whether specific antibodies against Scutica were generated from serum. The sonicated Scutica insects were used as antigens and attached to ELISA plates and blocked with 10% whole milk powder. Thereafter, the serum taken from the flounder was diluted by 2-fold and reacted with monoclonal antibodies against the immunoglobulin of the flounder. Thereafter, after reacting with a 2000 times diluted peroxidase attached anti-mouse chlorine antibody, color development was performed and the absorbance was measured at 492 nm. The results are shown in FIG.

Serum taken from the vaccinated flounder showed higher absorbance than normal serum, indicating that the vaccine induces a specific humoral immune response in the flounder, effectively producing antibodies against Scutica.

Example 6 Effect of Vaccination Flounder on Scutica

Serum from vaccinated flounder and normal flounder were diluted 1/4, 1/16, 1/64 and 1 / 256-fold, and then reacted with Scutica cultivated in FHM. The result is shown in FIG.

The vaccinated flounder sera caused agglutination of 30-200 flocked masses, stopping the movement of the Scutika insects. Agglutination was observed in serum diluted up to 1/16 fold and Scutica insects stopped moving until 1/64 fold dilution. However, agglutination did not occur in serum taken from normal flounder, but movement was slightly slowed down in high serum but not completely lost. From this result, it can be seen that by vaccination, the substance of the sputum worms is prevented from moving and the aggregates of the larvae are generated to kill the squitices. In other words, a vaccine is produced in the body of the flounder effectively coping with the Scutika insects, which is effective in the prevention of Scutika insects.

Example 7 Measurement of Leukocyte Phagocytic Activity

In order to measure the activity of the vaccinated flounder leukocytes, leukocytes were separated from the heads of 12 flounder for each experiment, adjusted to a concentration of 2.0 × 10 ⁷ cells / ml, and attached to the cover glass. 0.3% latex beads were added thereto and reacted at 25 ° C for one hour. After the reaction, the dye was stained with Kimja staining solution, and the number of leukocytes in which latex beads were fed among 200 leukocytes was counted.

The result is shown in FIG. Feeding rate of the leukocytes in the immunized cells was 46.4% and that of the control was 21.4%. From this, it can be seen that halibut leukocyte activity was increased by vaccination.

Example 8 Measurement of Coin Activity

The ability of leukocytes on the flounder to migrate to the site of the caries to feed on Scutika insects was measured. Scotica opsonized with 10% flounder serum was placed under the chamber for measuring chemotactic activity, and a filter was placed in the center, followed by injection of 4.0 × 10 ⁶ cells of flounder leukocytes into the upper part of the chamber. After reacting at 25 ° C. for 3 hours, the leukocyte count of the filter was counted by 20 times with a 1000 × optical microscope. Twelve samples of each of the injection and control groups were measured, and FIG. 9 shows the results.

Compared with the non-immunized control group, the greater number of leukocytes taken from the flounder of the vaccinated group was shifted to Scutica, and the vaccination increased the leukocytes' chemotactic activity. In other words, vaccination allows leukocytes, which are responsible for the immunity of the flounder, to better recognize Scutica insects, and this enhancement of cellular immunity contributes to the vaccine effect.

Example 9 Efficacy Test of Scutica Vaccine in Aquaculture

Each year, two flounder farms identified with Scutika's outbreak were selected for field trials. The flounder in A farm was 12-15cm in size and housed in a 15 ton tank. The water level of the tank was lowered to 10 cm so as to have a concentration of 1-2 × 10 ³ cells / ml and immersed for 1 hour. The two untreated vaccines were placed as controls and boosted once again at the same concentration three weeks after the vaccine was administered. The flounder in B farm was 6-7cm in size and housed 25,000 animals in one tank. The two tanks were immersed for 1 hour to have a concentration of 3-4 × 10 ³ cells / ml, and boosted again at the same concentration after 3 weeks.

The results are shown in FIG. Scutica vaccines in A and B farms reduce the average mortality caused by Scutika insects by 72% and 86%, showing excellent effects in preventing Scutica in flounder farms.

As described through the above examples, the inactivated vaccine of Scutika insects is very useful for the fish farming industry, which can be effectively used as a preventive vaccine of Scutika insects that cause great damage every year in fish farms by enhancing immunity against Scutika insects of fish. Invention.

Claims (3)

Scutica parasites were inoculated into fish chemotactic cells CHSE-214 (chum salmon embryo), RTG-2 (rainbowtrout gonard), EPC (epithelioma papilosum carp) and fathead minnow cells (FHM) under cancer conditions. After aseptic mass culture at low temperature to maintain the cleavage of the cartilage and then inactivate the cartilage with formalin to prepare a vaccine for the inactivated vaccine of Skutika insects, characterized in that for producing a vaccine. The method of claim 1, wherein the vaccine is sonicated by Scutika insects and used as an immersion vaccine. An inactivated vaccine composition of a squatworm, characterized by containing an inactivated vaccine produced using an inactivated squatcatworm as an active ingredient.