Method of production of dry polyvalent virus-vaccine for marek's disease prevention and dry polyvalent virus-vaccine for marek's disease prevention

FIELD: medicine.

SUBSTANCE: method of production of dry polyvalent virus-vaccine includes separate infection of cell culture with strain PC-126 of turkey herpesvirus (virus of Marek's disease 3rd serotype) and one-day chicken infected with chicken herpesvirus (virus of Marek's disease 2nd serotype), incubation, turkey herpesvirus harvest, and sampling of double flag follicles of chicken herpesvirus infected chicken, protective medium addition, separate ultrasonic processing of virus cell mass and flag follicle mass, freezing and drying of end product followed with their mixing. At that chicken herpesvirus strain are sampled for (VMD 2nd serotype) strains "42", "50", "SB-1", inoculated in dosage 10000-50000 functional residual capacity (FRC) for chicken and grown in body within 12-25 days. Follicles processed with ultrasonic is removes, and protective medium processed with ultrasonic and containing released chicken herpesvirus is added equal proportion of processed with ultrasonic clean cell cultures of bird embryos grown within 24-72 hours. Dry polyvalent virus-vaccine contains cell-free lyophilized strain FC-126 of turkey herpesvirus - 3rd serotype of Marek's disease virus in protective medium. In addition virus-vaccine includes cell-free lyophilised strains of chicken herpesvirus - 2nd serotype of Marek's disease virus, produced by any cl.1-5, in protective medium at ratio 2000 FRC /units: 100-5 00 FRC /units, respectively.

EFFECT: vaccine has high immunogenic activity and storage stability.

10 cl, 3 tbl, 4 ex

 

The invention relates to Virology and biotechnology and can be used in the manufacture of dry polyvalent VirusWall against Marek's disease (BM) birds.

The Marek's disease (BM) is a malignant lymphoproliferative disease of birds of viral etiology. An important means of struggle against BM vaccines is. To prevent BM use liquid and lyophilized monovalent vaccine from strain FC-126 herpes virus of turkeys (UGI) - third serotype of the virus Marek's disease (VBM), which protect the bird from the disease (efficacy of vaccination 76-85%) [1]and liquid culture bi - and multivalent vaccine strain FC-126 why, "42", "50" herpes virus of chickens (WMC) - second serotype EBM [2] and/or attenuated strains VBM first serotype (the effectiveness of vaccination 82-100%) [5]. However, despite the high efficacy of polyvalent liquid culture vaccine BM, these drugs have significant drawbacks: high cost for storage and transportation of liquid nitrogen required, in addition, they are not easy to use.

Known dry monovalent vaccine on the basis of WHI [1, 3] do not have the necessary degree of protection of poultry against virulent and highly virulent strains of Marek's disease virus.

Known liquid culture polyvalently against BM, containing a combination of viruses, second and third serotypes, in particular strains of "42", "50" (serotype 2) and strain FC-126 (serotype 3) "Polyvalent vaccine for the prevention of Marek's disease and the method of its manufacture" Lukin VA, SP F.G., White DE, Shevyrev NS, Bezgin V.M., Gusev A.A., Barros Paloma E.V., Smolensk V., Panov B.C. - RF Patent 2059414, AK 39/255, 1996. A method of manufacturing a known vaccine is that pre-grown culture of cells separately infect WMC 2nd serotype, UGI 3rd serotype, growing the infected cells are harvested in a stabilizing environment, stored at -196°With, before use, the vaccine is thawed, diluted in diluent from the calculation that a single dose (0.2 cm3contains 2000 FOB UGI and 300 I shape. However, for this vaccine requires liquid nitrogen.

The task of the claimed group of inventions was to create a dry multivalent VirusWall against BM, stable, high immunogenic activity that does not require storage and transportation of liquid nitrogen, stored at a temperature of 4-25°With at least 2 years (time of observation), a convenient and economical to use, and also has a lower cost of preparation and method of its manufacture.

The claimed vaccine has a distinct advantage over the known compounds.

Technical R is the result of the claimed invention is the proposed vaccine contains as a basic component lyophilized strain 3rd serotype FC-126 why, as well as enhancing and prolonging the effect of WHI - strains 2nd serotype herpes chickens "SB-1", "42", "50" or any other strains WMC also in dried form in an effective amount. The problem is solved by the fact that the claimed dry virusvaktsinu polyvalent and dried and developed the method of cultivation of herpes virus of chickens, getting it free from cells condition followed by lyophilization. Dry the polyvalent virusvaktsinu and method of its manufacture constitute a single inventive concept: one of the inventions is to get another.

The claimed vaccine contains free from cells UGI grown in cell culture of embryos of birds (basic component), and the herpes viruses of chickens (second serotype VBM), grown for 12-25 days in infected Chicks at day old and isolated from the epithelium of the feather follicles (reinforcing component), taken in an effective amount and placed in a protective environment, such as 7-12%sucrose solution in phosphate-buffered saline at pH 7,2-7,4 in the following ratio, wt.%:

Cell-free strain FC-126 Viru is and herpes turkeys 30,0-50,0
Cell-free strain "42"10,0-20,0
Cell-free strain "50"10,0-20,0
Cell-free strain SB-1"10,0-20,0
Protective environmentRest

The vaccine is prepared as follows.

Example 1.

To prepare the base component vaccine, why cultured on the cell culture of embryos of birds, and then produce the removal of infected cells in a protective environment and after ultrasonic disintegration released from infected cells, UGI subjected to drying (Temporary instruction of the manufacture and control of the dry culture virus-vaccine against Marek's disease from strain FC-126 herpes virus of turkeys, approved BS Ministry of agriculture of the USSR 01.06.1984) [4].

Enhancing the immunogenicity of the second component of the vaccine is free from cells herpes virus chicken is prepared as follows. For cultivation WMC use pathogen free chickens (SPF chickens) daily age, which inoculant virus uterine seed 2nd serotype of the strain, "42", "50", "SB-1" or any other strain WMC dose 10000-50000 fotosobraniyami units (I) on the chicken. Chickens kept in sterile conditions within 12-25 days. Then chick totally Deplete by decapitation, Teleut the wings from the body and aseptically collect feather follicles [10 chickens in 30 ml protective environment (7-12%sucrose in phosphate-buffered saline at pH 7,2-7,4)]. Received follicles treated with ultrasound on the cage in the frequency range from 12 to 35 kHz and density of sound in dezintegrarea the substrate in the range from 0.6 to 0.9 watts/cm3to free shape of the cells. Feather follicles are removed. After that, the processed ultrasound protective medium containing released shape, add an equal volume of processed ultrasound uninfected cell culture of embryos of birds grown in 24-72 hours. The resulting mixture was filled into vials 1-2 cm3, frozen and dried under the same conditions as WGI, after which the vials stoppered, covered with metal caps and running.

Prepared the main component on the basis of vgi and enhancing the immunogenicity of the second component of the vaccine is free from cells herpes virus of chickens control for sterility, safety and infectious activity by titration on a 24-hour culture of cells of chick embryos grown in vials with raskovoi surface of 20-25 cm2. The infectivity must be at least 5×105I/cm3for UGI and not less than 5×104I/cm3for WMC, respectively.

Bi - or polyvalent drug amount immediately before vaccination. To do this, 1 cm3the base component on the basis of UGI unite in the solution of the body for vaccines to enhance the immunogenicity of the second component of the vaccine on the basis of the shape in the amount of 0.5 cm 3ratio of 2000 I/goal UGI and 100-500 I/goal shape, respectively.

The vaccine is stable for 24 months (time of observation) at a temperature of 4-25°C.

Example 2. Test infectious activity of the claimed vaccine was conducted as follows.

Each component of the claimed vaccine bred SFGA serial 10-multiple dilutions from 10-1up to 10-5and brought in bottles of 50 ml with 24-hour monolayer cell culture of chicken embryos from SPF chickens (pre-drain them growth environment) 0.1 cm3dilutions from 10-1up to 10-5with each dilution for 4 bottles. After 1 hour, made a supportive environment.

The vials were incubated in the conditions of thermostat within 5 days for UGI and 6-10 days for WMC. After incubation of the virus in monolayer bottles were stained with a solution amylovora black and counted tricks according to the approved methodology (TU 4621-157-80). Test data are shown in table 1.

Table 1
Infectious activity included in the inventive vaccine lyophilized strains in cell cultures of chicken embryo
StrainTime of cultivation, the dayInfectious activity, I/cm3
FC-WHI 55×105
42 WMC85,4×104
42 WMC101,2×105
50 WMC85,1×104
50 WMC107,0×104
SB-185,5×104
SB-1107,8×104

From table 1 it follows that infectious activity of lyophilized strains of vgi and shape in cell cultures of chicken embryo consistent.

Example 3. Test immunogenic activity of the claimed vaccine in laboratory conditions on a daily chickens were carried out in comparison with the known dry vaccine (WGI), which is a basic component (1), a known liquid polyvalent vaccine (2) and placebo (control group chickens without vaccination).

Daily chickens were injected intramuscularly known and proposed vaccine. 7 days after vaccination of Chicks of the experimental and control groups were infected by the virulent VBM strain Jm by intraperitoneal injection.

Analysis was performed 90 days after infection.

The test results presented in table 2.

Table 2
Test immunogenic vaccines against BM
no groupType of vaccineNumber of infected chickens (animals)Mortality (goal.) from BMQty opened 90 day ChicksThe safety of the chickens by the time of autopsy, %The anatomic picture of the BM at the opening 90 day
1UGI + shape (the claimed vaccine)96096100No
2UGI (dry preparation) HSBC96789of 92.7No
3UGI + shape (known polyvalent liquid vaccine)96096100No
4Control not vaccinated96514546,8720 BM

From table 2 it follows that the claimed dry polyvalent vaccine is no different for protective activity against known liquid polyvalent vaccine.

Example 4. Test immunogenic vaccines under field conditions was carried out on daily chickens in comparison with the known dry the liquid on Valentey vaccine and control group (chickens without vaccination).

Daily chickens have introduced the famous vaccines and claimed.

Monitoring chickens were over 230 days after vaccination.

The results of vaccine trials in field conditions are presented in table 3.

Table 3
The effectiveness of vaccines against Marek's disease in field conditions
VaccineThe number of ChicksThe number of cases of BM, % of the number of dead ChicksThe efficacy of vaccination, %
Declare250000100
Known (monovalent dry, UGI)8330010,1583,57
Known (polyvalent liquid)645000,5599,8
The control group2500030,86-

From table 3 it follows that in the field only claimed the vaccine had 100%efficiency.

Thus, the claimed dry the polyvalent virusvaktsinu for the prevention of Marek's disease high-tech, easy to use and transport, has 100%efficiency, stable, not less than two years (time of observation) and can store pritemperature 4-25° C.

Sources of information

1. USSR author's certificate No. 792643, AK 39/252, SC 7/00 - prototype.

2. RF patent № 2059414, AK 39/255.

3. RF patent № 2144376, AK 39/255, 12N 7/00.

4. Temporary instruction of the manufacture and control of the dry culture VirusWall against Marek's disease from strain FC-126 herpes virus of turkeys, approved BS Ministry of agriculture of the USSR 01.06.1984.

5. Sarma G. Field trial and immunogenesity studies on the polyvalent Marek′'s disease vaccines in chickens. // In: the 19-th World′'s Poultry Congress, V.1. World Poultry Science Association. Amsterdam. - 1992. - P.310-314.

1. A method of manufacturing a dry multivalent VirusWall for the prevention of Marek's disease, including separate infection of cell cultures with strain PC-126 herpes virus of turkeys (Marek's disease virus of a third serotype) and infection of day old Chicks by the herpes virus of chickens (Marek's disease virus of a second serotype), incubation, collect cell mass containing the herpes virus of turkeys, and the collection of feather follicles primaries infected chickens, containing the herpes virus of chickens, adding a protective environment, separate processing vaccinated cell mass and the mass of feather follicles ultrasound, freezing and drying the target product with further aggregation, characterized in that strains of the herpes virus of chickens (VBM second serotype) use the strains of "42", "50", "SB-I", inoculant their dose of 10000-FOE on chicken and grow in the body during 12-25 days and after processing follicles by ultrasound follicles are removed and processed by the ultrasound protective medium containing released herpes virus chicken, add an equal volume of processed ultrasound uninfected cell culture of embryos of birds grown in 24-72 hours

2. The method according to claim 1, characterized in that infect cell culture of fibroblasts SPF-quail embryos and SPF-the embryos of chickens.

3. The method according to claim 1, wherein receiving the target product by combining 1 cm3strain of the herpes virus of turkeys and 0.5 cm3strain of the herpes virus of chickens at a ratio of 2000 I/goal : 100-500 I/bird, respectively.

4. The method according to claim 1, characterized in that the infectious activity of the vaccine is not less than 5×105I/cm3for herpes virus of turkeys and not less than 5×104I/cm3for herpes virus of chickens, respectively.

5. The method according to claim 1, characterized in that a protective environment take 7-12%sucrose solution in phosphate-buffered saline at pH 7,2-7,4.

6. Dry the polyvalent virusvaktsinu for the prevention of Marek's disease containing cell-free lyophilized strain FC-126 herpes virus of turkeys - third serotype of the virus Marek's disease in a protective atmosphere, characterized in that it further comprises a cell-free lyophilized strains viruscheck chickens - second serotype of the virus Marek's disease, obtained according to any one of claims 1 to 5, in a protective environment at a ratio of 2000 I/goal : 100-500 I/bird, respectively.

7. Dry the polyvalent virusvaktsinu according to claim 6, characterized in that the cell-free strains of the herpes virus of chickens (Marek's disease virus of a second serotype) it contains lyophilized strains "42", "50", "SB-1"obtained according to any one of claims 1 to 6.

8. Dry the polyvalent virusvaktsinu on any of PP and 7, characterized in that the cell-free strain FC-126 herpes virus of turkeys and protective environment is taken in the ratio, wt%:

Cell-free strain FC-126
herpes virus of turkeys30,0-50,0
Protective environmentrest

9. Dry the polyvalent virusvaktsinu on any of PP-8, characterized in that the cell-free strains of "42", "50", "SB-1" herpes virus of chickens and protective environment is taken in the ratio, wt.%:

cell-free strain "42"10,0-20,0
cell-free strain "50"10,0-20,0
cell-free strain SB-1"10,0-20,0
protective environmentrest

10. Dry p is levelentry virusvaktsinu on any of PP, 8 and 9, characterized in that a protective environment vaccine contains 7-12%sucrose solution in phosphate-buffered saline at pH 7,2-7,4.



 

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The invention relates to veterinary Virology and biotechnology

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The invention relates to veterinary Virology and biotechnology and can be used in the production of vaccines against Marek's disease (BM)

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SUBSTANCE: method of production of dry polyvalent virus-vaccine includes separate infection of cell culture with strain PC-126 of turkey herpesvirus (virus of Marek's disease 3rd serotype) and one-day chicken infected with chicken herpesvirus (virus of Marek's disease 2nd serotype), incubation, turkey herpesvirus harvest, and sampling of double flag follicles of chicken herpesvirus infected chicken, protective medium addition, separate ultrasonic processing of virus cell mass and flag follicle mass, freezing and drying of end product followed with their mixing. At that chicken herpesvirus strain are sampled for (VMD 2nd serotype) strains "42", "50", "SB-1", inoculated in dosage 10000-50000 functional residual capacity (FRC) for chicken and grown in body within 12-25 days. Follicles processed with ultrasonic is removes, and protective medium processed with ultrasonic and containing released chicken herpesvirus is added equal proportion of processed with ultrasonic clean cell cultures of bird embryos grown within 24-72 hours. Dry polyvalent virus-vaccine contains cell-free lyophilized strain FC-126 of turkey herpesvirus - 3rd serotype of Marek's disease virus in protective medium. In addition virus-vaccine includes cell-free lyophilised strains of chicken herpesvirus - 2nd serotype of Marek's disease virus, produced by any cl.1-5, in protective medium at ratio 2000 FRC /units: 100-5 00 FRC /units, respectively.

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