KR20020012427A - Porcine Encephalomycarditis parvo virus combined oil vaccine - Google Patents

Abstract

PURPOSE: A method for preparing the titled mixed vaccines is provided by mixing the cultured encephalomyocarditis virus and porcine parvovirus of pigs with an oil adjuvant. Whereby, the vaccines inhibit infection, proliferation and excretion of the two viruses and abortion and prevent still-birth of a brood sow. CONSTITUTION: Encephalomyocarditis virus and porcine parvovirus of pigs are inoculated into a hamster kidney cell and a pig kidney cell respectively, cultured and then mixed with an oil adjuvant comprising 93% by weight of mineral oil, 5.7% by weight of aracel A and 1.3% by weight of tween after inactivation with formalin.

Description

Production method of porcine brain myocarditis virus and porcine parvovirus mixed oil vaccine {Porcine Encephalomycarditis parvo virus combined oil vaccine}

The present invention relates to a method for producing a mixed oil vaccine capable of simultaneously preventing swine propagation disorders caused by pig encephalomyocarditis viris and Porcine parvovirus infection.

Swine parvovirus is a well-known disease, showing symptoms of miscarriage, stillbirth, and mummies in early pigs, and no specific clinical symptoms in sows and piglets. It shows various symptoms such as deterioration, early delivery, miscarriage, stillbirth and mummies, and even normal birth has a huge effect on pig breeding regardless of birth and birth, with about 50 percent of the dead pigs dying. In addition, in September 1989, the inventors of the present invention have isolated and cultured the virus and found it to be swine brain myocarditis virus.

The present invention is not only an effective vaccine that can prevent the growth of sows similar to the sows common in hog farms, but also to suppress the proliferation and excretion of these two most important viruses. The application of the patent for swine brain myocarditis viremia (EMC) and swine parvovirus syndrome (PPV) mixed oil vaccine will be described below in detail by way of examples.

end. Passage and Preservation of Seed Viruses

When the seed virus was inoculated into the cells with the highest proliferative ability against the pig brain myocarditis virus, that is, the young hamstar kidney cells, and cultured for 72 hours at 37 ° C, it was taken when the cytopathic effect appeared about 80% or more. After inoculation into pig kidney cells, it is incubated at 36 ℃ for 7 days, and then the tissue culture solution is collected and stored in -80 ℃ cryogenic freezer or lyophilized for vaccine production.

I. Medium composition for production and inoculation of seed poison

Swine cerebral myocarditis virus venom inoculates 1/100 of the media when the young hamstar kidney cells have grown more than 80% in the Earls medium (Supplementary Note 1).

Porcine parvovirus venom is selected from healthy pigs 1 to 3 months of age and aseptically collected from the kidneys, cut to 1 mm ³ or less with scissors, digested with 0.25% trypsin solution, and the dispersed tissue cells are used as a medium for earth growth. Appropriate dilution is inoculated into the culture bottle and cultured at 37 ° C., when the kidney cells form 50% of the monolayer cells, inoculate 1/10 of the venom.

(Supplementary Note 1) Earth Growth Medium

Eagle MEM --------------------------- 830 ml

10% Lactalbmin ------------------------ 50ml

Fetal bovine serum --------------------- 100ml

7.5% NaHCO _{3 ----------------------------------------} 20ml

Penicilline G-Na ---------------------- 100unit / ml

Streptomycin --------------------------- 100µg / ml

Fungizone ------------------------------ 0.025 µg / ml

All. Extraction and disposal method

(1) extraction and inactivation

Pig brain myocarditis virus to yield jongdok to the infection the culture medium after 48 hours after inoculation was collected aseptically using a guinea pig blood by HA is measured (blood eungjipga) is at least 128 times or TCID ₅₀ 10 ^7.5 TCID ₅₀ / Collect only the infection solution of 0.1ml or more, add the final concentration of formalin to 0.2%, inactivate it by shaking for 3 hours at 25 ℃, and use it as the vaccine stock solution. Porcine parvovirus was incubated for 3 days after inoculation, and then the culture solution was collected and inactivated at 25 ° C for 5 days after adding formalin to the final concentration of 0.2% in infected culture solution with a hemagglutination of 512 times or more using guinea pig blood cells. It is then used as a vaccine solution.

(2) Treatment method

Inactivated vaccine solution, that is, pig brain myocarditis virus vaccine stock and inactivated pig parvovirus stock solution, were mixed in equal amounts each time, and then adjuvant oil adjuvant (Supplementary Note 2) was added and mixed in a small container. Illness

(Supplementary Note 2) Oil Adjuvant

Mineral Oil ------------------------------------- 93%

Aracel A --------------------------------------- 5.7%

Tween 80 -------------------------------------- 1.3%

la. Standardization of the product

1) Inactivation test

To investigate the survival of pig brain myocarditis virus and pig parvovirus, each inactivated culture was inoculated when porcine brain myocarditis virus cultured young hamstar kidney coin cells and formed more than 80% of monolayers. When porcine kidney cells were cultured and more than 80% of monolayers were formed, the cells were inoculated and cultured at 37 ° C. for 7 days. As a result, the cell degeneration and hemagglutination were observed.

2) Sterility Test

The test vaccine was inoculated in NUTRIENT broth (NA), NUTRIENT agar (NA) and liquid thio medium (Thio) as shown in Table 1, and cultured at 22 ° C. and 37 ° C. for 7 days to examine the incorporation of various bacteria. As shown in 1, the incorporation of various bacteria was not recognized.

Table 1. Sterility Test

Vaccine Lot No. 22 ℃ 37 ℃ Thio NA NB Thio NA NB One - - - - - - 2 - - - - - -

Example

1. Virus content test and hemagglutination prior to inactivation of test vaccine

For the virus content test before inactivation of the test vaccine 2Lot, EMC virus was inoculated into hamstar kidney cells and porcine parvovirus into porcine kidney cells and incubated at 37 ° C. for 7 days, and showed high titers as shown in Table 2.

Table 2. Virus content and blood cell count before inactivation of test vaccine

Coagulation test

Vaccine Lot No. Virus content (TCID ₅₀ / ml) Hemagglutination (HA) EMC PPV EMC PPV One 10 ^8.0 10 ⁸⁵ 128 ^× 2048 ^× 2 10 ⁸⁵ 10 ⁸⁵ 128 ^× 2048 ^×

2. Safety test on test animals of test vaccine

Ten mice (15g) were selected for the test vaccine, five were subcutaneously inoculated with 1.0ml each, and five were inoculated intraperitoneally with 0.5ml each. Seven guinea pigs (350g) were inoculated with 2.0ml each muscle for 10 days of clinical observation. As shown in Table 3, all the experimental animals survived without abnormality and were found to be excellent in safety.

Table 3. Safety test on test animals of test vaccine

Vaccine Lot No. Observation period (days) Laboratory Animals and Clinical Symptoms Mouse (15 g) Inoculation amount / site Clinical symptoms Guinea pig (350 g) Inoculation amount / site Clinical symptoms One 10 5 1.0ml / subcutaneous clear 7 2.0ml / subcutaneous clear 5 0.5ml / subcutaneous 〃 〃 2 10 5 1.0ml / subcutaneous 〃 7 2.0ml / subcutaneous 〃 5 0.5ml / subcutaneous 〃 〃

3. Immunogenicity test for pigs in test animals

end. Immunogenicity test for cerebral myocarditis virus in pigs was performed by injecting 2.0 ml into each of 5 antibody-negative pigs (100-150 kg) into the muscles and two weeks after the second inoculation. Hemagglutination inhibition (HI) was measured. As shown in Table 4, EMC showed high titers of 64 to 256 times and PPV of 512 to 2048 times.

Table 4. Immunogenicity Test in Pigs

Pig number HI antibody titer (hemagglutination inhibitory titer) EMCV PPV Before vaccination After inoculation Before vaccination After inoculation 1 2 3 4 5 <10 <10 <10 <10 <10 2561286464128 <10 <10 <10 <10 <10 512102410242048512

I. Immunogenicity Tests for Guinea Pigs

The immunogenicity test for porcine parvovirus was performed by guinea pigs (350 g) and subcutaneously inoculated with 2.0 ml each to measure neutralized antibody and HI antibody titers of blood collected 4 weeks later. The embryo and hemagglutination inhibitory values showed 80-320 times higher antibody titers.

Table 5. Immunogenicity Tests for Guinea Pigs

Guinea Pick Number Neutralizing antibodies Hemagglutination inhibition Before vaccination After inoculation Before vaccination After inoculation 1 2 3 4 5 <10 <10 <10 <10 <10 1601691280250100 <10 <10 <10 <10 <10 1608032016080

4. Preservation test of test vaccine

The test antibody was stored at 5 ° C, and the HI antibody titers of cerebral myocarditis virus in pigs and pig parvovirus in guinea pigs were measured at five-month intervals.

Table 6. Vaccine integrity test

Test animals Storage temperature HI antibodies 0 months 5 months 10 months 16 months 20 months 25 months Before vaccination After inoculation Before vaccination After inoculation Before vaccination After inoculation Before vaccination After inoculation Before vaccination After inoculation Before vaccination After inoculation pig 1 2 3 4 5 5 ℃ <10 <10 <10 <10 <10 12812825664128 <10 <10 <10 <10 <10 12812812825664 <10 <10 <10 <10 <10 646412864128 <10 <10 <10 <10 <10 6432326464 <10 <10 <10 <10 <10 3216163216 <10 <10 <10 <10 <10 16881616 Guinea pig 1 2 3 4 5 5 ℃ <10 <10 <10 <10 <10 32032016080160 <10 <10 <10 <10 <10 32016016016064 <10 <10 <10 <10 <10 64128643264 <10 <10 <10 <10 <10 64128646432 <10 <10 <10 <10 <10 3264323216 <10 <10 <10 <10 <10 16168168

As described above, the mixed vaccine of the present invention is a vaccine having a very good effect of preventing the sows of the sows commonly occurring in pig farms, as well as inhibiting the proliferation and excretion of two viruses such as myocardial myocarditis viru and parvovirus.

Claims (1)

Oil adjuvant adjuvant consisting of mineral oil 93%, Aracel A 5.7% and Tween80 1.3% by inoculating pig brain myocarditis virus and parovirus into hamstar kidney cells and primary pig kidney cells, respectively, and incubating them. Method for producing a swine brain myocarditis virus and parvovirus mixed oil vaccine, characterized in that by mixing the same amount of the inactivated vaccine prepared in.