RU2410117C1 - Viral vaccine against marek's disease - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: viral vaccine contains cell-associated turkey herpes virus (THV) of "Vladimir" strain, cattle blood serum (CBS) and dimethylsulfoxide (DMSO) in ratio, wt %: 60.080.0; 15.025.0 and 5.015.0 respectively. Strain is deposited in FGI VGNKI collection of microorganisms under registration number (reference) "Vladimir No 124-DEP" THV (further "Strain"). Strain is reproduced in culture of SPF-chicken embryo fibroblast cells (CFE) and reaches titre 3.5106 PFU/cm3, for vaccine manufacturing used is suspension of infected CFE cells with titre not lower than 5.5105 PFU/cm3. ^ EFFECT: viral vaccine possesses high immunogenic activity, protects immunised population of poultry against infection of epizootic MDV, circulating on the territory of Russia. ^ 6 cl, 5 ex, 3 tbl

Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the development and manufacture of means for the specific prevention of Marek's disease.

Marek's disease (BM) is a highly contagious, lymphoproliferative, malignant, viral disease of birds. BM is registered in all countries of the world where industrial poultry farming is developed, including in our country, causing large economic losses. BM is characterized by the formation of single (in the initial stage) and multiple tumors in the internal organs, skin, muscles, as well as changes in the central and peripheral nervous system. The Marek's disease virus (VBM) damages the immunocompetent organs (spleen, thymus, cloacal sac) and thus has immunosuppressive activity, which leads to a decrease in the general resistance of birds and an increase in their sensitivity to other diseases. In this regard, BM often occurs in association with other diseases. An important way to prevent BM and reduce losses from the disease is vaccination. For specific prophylaxis, three types of vaccines are used: 1) from attenuated VBM strains (serotype 1); 2) from a naturally attenuated non-pathogenic VBM (serotype 2) and 3) from strains of turkey herpes virus (serotype 3). The basis for the prevention of BM is the active immunization of chickens at the age of 24 with live mono-, bi - and multivalent vaccines. Most of the world-famous prophylactic drugs are based on the turkey herpes virus FC-126 strain (VGI) or its combinations with naturally known representatives of 1 and 2 VBM serotypes and reliably protect the vaccinated population from field VBM strains of moderate pathogenicity [1, 2, 3 , four].

The popularity of virus-based vaccine based on VGI (strain FC-126) is due to the fact that this virus does not require attenuation, is not dangerous in terms of reversion, and undergoes stabilization in the extracellular state. Two types of vaccines are prepared on the basis of VGI: cell-free (dry) and cell-associated (native).

Known virus vaccine against BM, containing a suspension of fibroblast cells of embryos of SPF-chickens (FEK) infected with cell-associated HVI strain FC-126, with infectious activity not lower than 1.0 · 10 ⁵ FOU / cm ³ , blood serum of cattle (cattle ) and dimethyl sulfoxide (DMSO) as a cryoprotectant in the ratio, wt.%:

Suspension of infected cells 80.0 ÷ 90.0 Bovine serum 5.0 ÷ 10.0 DMSO 5.0 ÷ 10.0 [5].

The disadvantage of this virus vaccine is that it does not provide satisfactory protection against VBM circulating in the Russian Federation.

In connection with the recent appearance in the poultry farms of highly pathogenic pathogens of BM, cases of not always sufficient effectiveness of known virus vaccines used to prevent BM have become more frequent. This circumstance forces us to constantly search for new strains of HBV suitable for the manufacture of effective vaccines against BM.

The closest to the invention according to the set of essential features is a virus vaccine against BM, containing a suspension of FEC cells infected with cell-associated HVI strain "ARRIAH / No. 110", with infectious activity not lower than 1.0 · 10 ⁵ FOU / cm ³ , cattle blood serum and DMSO as a cryoprotectant in the ratio, wt.%:

Suspension of infected cells 60.0 ÷ 80.0 Bovine serum 15.0 ÷ 25.0 DMSO 5.0 ÷ 15.0 [6].

A significant disadvantage of the prototype virus vaccine is that its use does not exclude the release of the pathogen BM into the environment by the vaccinated bird.

The problem to which the present invention is directed, is to obtain a vaccine against BM on the basis of a new strain of VGI, which creates effective protection of poultry against BM and prevents the release of the pathogen BM to the environment by the vaccinated bird.

The technical result from the use of the present invention is to expand the arsenal of highly immunogenic, harmless and areactogenic virus vaccines against BM that can protect poultry from highly pathogenic strains of the pathogen BM circulating in the Russian Federation.

The specified technical result was achieved by the creation of a virus vaccine against BM, characterized by the following set of features.

According to the invention, the proposed virus vaccine contains a suspension of FEC cells infected with cell-associated HVI strain "Vladimir" (copyright), with an infectious activity of not less than 5.5 · 10 ⁵ FOU / cm ³ , blood serum of cattle and DMSO as a cryoprotectant in the ratio wt.%:

Suspension of infected cells 60.0 ÷ 80.0 Bovine serum 15.0 ÷ 25.0, DMSO 5.0 ÷ 15.0

The viral isolate that served as the source for the Vladimir strain was isolated in 1999 from healthy private-sector turkeys in the Rostov Region. The production strain "Vladimir" VGI obtained by sequential passaging in cell culture FEC. Strain "Vladimir" is a pathogenic genetically homogeneous viral material, actively propagating in the cell culture of FEC with an infectious activity of 2.0 · 10 ⁶ FOU / cm ³ .

Strain "Vladimir" VGI was deposited July 19, 2005 in the All-Russian state collection of strains of microorganisms used in veterinary medicine and livestock, Federal State Institution "All-Russian State Center for the Quality and Standardization of Medicines for Animals and Feed" (FGU "VGNKI") under registration number (link): strain "Vladimir No. 124 - DEPT" VGI.

The possibility of its use for the manufacture of a vaccine against BM has been experimentally confirmed. The strain "Vladimir" provides a vaccine against BM, which protects poultry against highly pathogenic strains of the pathogen.

The present invention includes the following set of essential features that provide a technical result in all cases for which legal protection is sought:

1. The vaccine virus BM.

2. Suspension of FEC cells infected with cell-associated VGI of strain Vladimir in an effective amount.

3. Serum of cattle.

4. DMSO as a cryoprotectant.

The signs of the invention, characterizing the proposed virus vaccine and coinciding with the signs of the prototype, including the generic concept, reflecting the purpose, are:

1. Virus vaccine against BM.

2. Suspension of FEC cells infected with cell-associated VGI in an effective amount.

3. Serum of cattle.

4. DMSO as a cryoprotectant.

Compared with the prototype virus vaccine, an essential distinguishing feature of the proposed virus vaccine is that it contains a suspension of PEC cells infected with the cell-associated HVI strain Vladimir, which has high antigenic and immunogenic activity, in an effective amount.

The present invention is also characterized by other distinctive features expressing specific forms of execution or special conditions for its use:

1. Suspension of FEC cells infected with cell-associated VGI of the strain "Vladimir", with an infectious activity of at least 5.5 · 10 ⁶ FOU / cm ³ in an effective amount.

2. Suspension of FEC cells infected with cell-associated VGI of the strain Vladimir, with an infectious activity of at least 5.5 · 10 ⁶ IU / cm ³ in the amount of 60.0 ÷ 80.0 wt.%.

3. Serum of cattle in the amount of 15.0 ÷ 25.0 wt.%.

4. DMSO as a cryoprotectant in an amount of 5.0 ÷ 15.0 wt.%.

5. Suspension of FEC cells infected with cell-associated VGI, with infectious activity not lower than 5.5 · 10 ⁶ FOU / cm ³ , blood serum of cattle and DMSO as a cryoprotectant in the ratio, wt.%:

Suspension of infected cells 60.0 ÷ 80.0 Bovine serum 15.0 ÷ 25.0 DMSO 5.0 ÷ 15.0

The present invention extends the arsenal of highly immunogenic, harmless and reactive anti-BM virus vaccines capable of protecting poultry from highly pathogenic BM pathogen strains circulating in the Russian Federation.

The achievement of the technical result from the use of the invention is explained by the fact that it contains antigenic material from the Vladimir strain VGI, which has high antigenic and immunogenic activity.

Strain "Vladimir" VGI is characterized by the following features and properties.

Morphological features

The strain "Vladimir" belongs to the family Herpesviridae, the subfamily Gammaherpesvirinae, the genus Herpesvirus, serotype 3 and is a cell-associated virus. Based on a comparative electron-microscopic study of the production strain “ARRIAH” VGI and strain “Vladimir” VGI when reproducing them in cell cultures, the morphological characteristics of both strains are identical. These viruses went through the ontogenetic developmental cycle in the nucleoplasm, and in the cytoplasm - the final stages of morphogenesis. The intranuclear development cycle includes the stages of a viroplast, nucleoid, assembly of a nucleocapsid, and the formation of an external supercapsid membrane. The most common form present at all stages of cultivation is a nucleocapsid with a nucleoid represented by a dense formation enclosed in a capsid protein coat. During electron microscopic examination, the VGI strain “Vladimir” is represented by virions typical of VBM, which have a cubic symmetry type and an icosahedron shape. The virus has polymorphism, it can be with or without a shell. In the nucleus of the affected cell, the virion has a value of 90 ÷ 100 nm, in the cytoplasm its size increases due to the protein membrane to 180 ÷ 200 nm. The shell consists of 162 capsids located in cubic symmetry.

Antigenic properties

According to its antigenic properties, the strain “Vladimir” VGI belongs to the 3rd serotype. When introduced into the body, poultry causes latent viremia. Antibodies to this virus are found in the blood of chickens by 14–21 days of life with a further increase by 40–60 days. During vaccination, the virus induces the formation of specific antibodies detected in the reaction of diffusion precipitation (RDP) and enzyme-linked immunosorbent assay (ELISA). Activity in the complement binding reaction (CSC) has not been established. The concentrated antigen from the Vladimir strain VGI reacts in RDP with specific serum to VGI and VBM. Common antigens with VBM are detected in the direct and indirect immunofluorescence reactions (RIF). When the cell is destroyed, it reacts with antibodies to VGI and VBM in the neutralization reaction (PH). The ELISA reacts with antibodies to HBV and VBM. In the reaction of delayed hemagglutination (RHCA) and the reaction of hemagglutination (RHA), the red blood cells do not agglutinate.

Biotechnological characteristics

Strain "Vladimir" VGI actively propagates in the cell culture FEC. The cultivation of fibroblasts is carried out according to generally accepted methods using a mixture of media based on the lactalbumin hydrolyzate (GLA), Needle and 199, taken in equal parts, with the addition of 10% fetal bovine serum. Fibroblast cells are grown in bottles with a capacity of 50 cm ³ and 100 cm ³ (Povitsky), in mattresses with a capacity of 1.5 dm ³ and in roller 3-liter vessels. The inoculum cell concentration is 0.6 ÷ 0.7 million / cm ³ for 50 cm ³ bottles and 1.2 ÷ 1.5 million / cm ³ for 3-liter roller vessels. When propagating in a monolayer of chicken fibroblasts, the virus exhibits pronounced cytopathic changes in the form of homogeneous foci with a size of 0.1 ÷ 0.2 mm. During consecutive passages in the cell culture of FEC, the infectious titer of the virus increased and reached 3.5 · 10 ⁶ FOU / cm ³ . The strain is not pathogenic for chicken embryos. In the embryos infected with the Vladimir strain of VGI, mild hepatitis was observed during the autopsy, and on HAO - gray-white star-shaped plaques with a size of 0.5–0.1 mm in the amount of 30–50 pieces. Strain "Vladimir" VGI is intended for the manufacture of a virus vaccine against BM.

Genotaxonomic characteristic

The strain "Vladimir" VGI is a DNA-containing virus with a molecular mass of 108 ÷ 120 · 10 ⁶ D. Nucleic acid is a double-stranded linear molecule. The content of G + C is 46%. The virion consists of a nucleoid, an icosahedral capsid asymmetrically located around the capsid of an electron-dense material designated as tegument, and a lipoprotein membrane. Virionic DNA is involved in the formation of virus precursor proteins. The main antigenic proteins are glycoproteins A (gA) and B (gB). Glycoprotein B plays an important role in the induction of protective immunity and is highly conserved among herpes viruses. It is a complex of three glycoproteins: gp 49, gp 60 and gp 100.

Using the polymerase chain reaction (PCR) using primers complementary to the conserved gene gB encoding glycoprotein B, the nucleotide sequence of the Vladimir strain VGI was analyzed.

The controls were: the feather follicle epithelium collected from chickens infected with the BM virus of the HPRS-16 strain of serotype 1; BM virus of strain SB 1, related to serotype 2, and samples of the anti-BM virus vaccine based on the ARRIAH strain of VGI, related to serotype 3. For primers M, universal for all virus types were flanked, which flank the 450 g nucleotide fragment of the gB gene. For typing, we used internal type-specific primers M, MS, and MF, which are complementary to the VBM genomes of the 1st, 2nd, and 3rd serotype, respectively. PCR results showed the presence of specific amplicons with primers M (421 N), which confirmed the belonging of the genome of the strain “Vladimir” VGI to the 3rd serotype of VBM. In this case, the MS primer (386 N.) interacted with the VBM genome of the 2nd serotype, and with the MF primer (380 N.) the VGI gene of the 3rd serotype, which testified to the methodically correctly conducted experiment and showed high specificity and sensitivity of PCR in relation to BM and GI virus models.

Resistance to external factors

The resistance of the strain "Vladimir" VGI to physical and chemical factors is characterized by the following data.

Centrifugation at 50,000 g precipitates the virus for 1 hour. In the range of pH 4 ÷ 10 the virus is sensitive to ether. The virus can withstand repeated freezing - thawing and exposure to ultrasound for 10 minutes. Complete thermal inactivation of the virus occurs at 4 ° C after 2 weeks of storage, at (+20) - (+ 25) ° C after 4 days, at 37 ° C after 18 hours and at 60 ° C in 10 minutes.

Storage and preservation

The virus is stored in a cell-associated form in a protective medium with 20% cattle serum and 10% DMSO in hermetically sealed ampoules in liquid nitrogen at -196 ° C for 24 months.

Pathogenic properties

Does not cause disease in embryos and chickens; contagiousness has not been established. Laboratory and domestic animals are not sensitive to the virus.

Immunogenicity of the strain

Strain "Vladimir" VGI protects against the development of tumors in a dose of 1000 IU in more than 93% of chickens in a production environment. Immunogenicity does not change for 5 consecutive passages.

Additional features and properties

Strain "Vladimir" VGI is free from contamination by bacterial and fungal flora, foreign viruses and mycoplasmas, harmless in a 10-fold dose for day old chickens.

The essence of the invention is illustrated by examples of its implementation and use, which do not limit the scope of the invention.

Example 1

The production strain "Vladimir" VGI obtained by sequential passaging in cell culture FEC. For this, under aseptic conditions, the kidneys were removed from the carcasses of the turkeys in Petri dishes, released from the capsule tissue and transferred to a flat-bottomed flask. Renal tissue was subjected to grinding with scissors, washed with Hanks solution with antibiotics (penicillin with streptomycin at 100 IU / cm ³ ). Then a 0.25% trypsin solution was introduced into the flask, a sterile magnet was lowered into it, and the suspension was dispersed on a magnetic stirrer for 3-7 minutes. After dispersion was completed, 2% of fetal serum of the suspension volume was added to the suspension. Then, the suspension obtained from the renal tissue was applied to the formed monolayer of the FEC cell culture. The infected cell culture FEC was transferred to a thermostat (37.5 ± 1) ° C for 2 hours in order to contact the inoculum. Subsequently, at the end of the contact period, the inoculum was removed, the monolayer was washed with Hanks solution and a nutrient support medium was introduced, consisting of equal parts of 0.5% lactalbumin hydrolyzate (GLA), Eagle and 199 media with 10% fetal serum. The culture was examined daily under a microscope. After 3 days, they held a “blind” passage, i.e. cells removed from one vial without signs of monolayer degeneration, 2 culture vials were inoculated. In a similar way, we conducted another “blind passage”. After 48 hours, the first foci of the cytopathic action (CPP) of the virus were noted in these vials, which were characterized by the appearance of single polykaryocytes throughout the monolayer. 24 hours later, a third passage was held. In this case, infection was carried out at a rate of 1: 5. At the third passage level, the entire virus monolayer was found to show the CPD of the virus in the form of plaques characteristic of VGI. When the virus CPD was reached, 60–70% of the monolayer area of the cell was removed from the glass using a dispersing solution according to the generally accepted method. Part of the cell suspension was used for the next passage. The other part was subjected to storage in liquid nitrogen according to the procedure modified by the Federal State Institution “ARRIAH”.

As a result of the work, a strain of VGI was isolated from the kidneys of healthy turkeys, to which the authors named the strain “Vladimir” VGI.

The resulting strain was subjected to comprehensive control in accordance with the OIE Guidelines for Standard Diagnostic Methods and Vaccines (1996) and at the level of the 3rd passage was stored in liquid nitrogen as a seed virus.

The production strain “Vladimir” VGI was tested by the following parameters: determination of species and strain affiliation; control for the absence of contamination by bacterial and fungal flora; control for the absence of mycoplasma contamination; control for the absence of contamination with foreign viruses; control of the infectious activity of the strain; strain safety control.

Determination of species and strain belonging was carried out by two methods:

- assessment of lesions of the chorioallantoic membrane (HAO) infected in the yolk sac of SPF chicken embryos.

When assessing lesions after 13 days, HAO found plaques of a grayish-white color with a star-shaped form in the amount of 15–25 on the membrane, which is characteristic of VGI.

When studying the nucleotide sequence of the genome of the strain "Vladimir" by sequencing according to Senger showed that the amino acid sequence of the virus corresponds to the VGI data presented in the international genetic bank.

Control for the absence of contamination by bacterial and fungal flora was carried out according to GOST 28085-89.

The growth of bacteria and fungal flora in all nutrient media was absent.

Control for the absence of mycoplasma contamination was carried out by sequential passage on Edward's medium and the MK-COM test system in PCR. The presence of mycoplasmas in the inoculum virus was not detected.

Control for the absence of contamination with foreign viruses was carried out by bioassay on embryos of SPF chickens of different ages and three infection methods.

Infected embryos were incubated at 36.5 ° C: in the yolk sac for 13 days, in the allantoic cavity for 6 days, and for 9 days in HAO. At the end of the incubation period, the embryos were opened, followed by possible pathologies. Additionally, electron-microscopic control was carried out by phase contrast method.

The presence of foreign contaminant viruses in the seed virus was not detected.

The control of the infectious activity of the strain was determined by titration of the virus in an FEC cell culture.

The titer of the inoculum virus was 2.0 × 10 ⁶ IU / cm ³ .

The safety of the strain “Vladimir” VGI was determined by introducing a ten-fold dose of the virus to the daily SPF chickens. Observation of the chickens was carried out for 15 days, given their safety and physiological condition.

During the entire observation period, the chickens remained clinically healthy and were not detected at the injection site at the injection site.

As a result of the work carried out, the sowing herpes virus of the Vladimir strain was sterile, there was no contamination with mycoplasmas and foreign viruses.

Virus-containing material from the Vladimir strain is harmless to diurnal SPF chickens, is specific, the infectious activity of the strain was 2.0 · 10 ⁶ FOU / cm ³ .

Example 2

Liquid culture virus vaccine against BM is prepared on the basis of the cell-associated HVI of the pathogenic strain "Vladimir". Inoculum VGI and production series of virus vaccines are prepared in the cell culture of FEC and stored in liquid nitrogen. To obtain a primary cell culture, embryos of SPF hens of 11–12 days old are used. Trypsinization of embryos is carried out according to generally accepted methods. The resulting concentrated cell suspension is diluted to a seed concentration of 0.2 ÷ 0.5 million / cm ^{3 with a} nutrient medium containing 20% GLA, 30% 199 medium, 30% Needle medium and 10% cattle serum (growth medium). pH of the finished growth medium 7.0 ÷ 7.2. The finished suspension is poured into bottles with a capacity of 3 dm ³ at 400 ÷ 450 cm ³ . The cell culture is incubated at (38.0 ± 0.5) ° C for 24 ÷ 36 hours until a continuous monolayer is obtained on roller machines with a rotation speed of 9 ÷ 10 rpm. The cell culture for virus titration is grown in 50 cm ³ bottles (inoculum concentration of 0.5 ÷ 0.6 million / cm ³ ). After 24–36 hours, in the vessels with cell culture, the growth medium is replaced by a support medium. The support medium contains an equal amount of GLA, Eagle media and 199 with the addition of 2% cattle serum. To prepare the production volume of the inoculum VGI from the Vladimir strain, they are introduced into the vessels with the obtained cell culture, which are filled with a support medium. The number of seed virus ampoules used to infect the cell culture is determined based on the titer of the uterine strain “Vladimir” VGI. The contents of the ampoules are combined (if the titer of the virus allows, it is better to infect 1 ÷ 2 bottles with a cell culture from one ampoule) and dilute (or use without dilution) nutrient medium from the calculation so that each cm ³ contains 10,000 IU. For the first passage, it is necessary to infect at least 4 bottles. By the time the virus is harvested, typical focal lesions in cell culture should be clearly visible under a microscope on 30-50% of the monolayer area. The virus monolayer is removed with trypsin and a second passage is performed. For this, infected cells collected from one bottle infect a fresh monolayer at a rate of 1: 5-1: 10, i.e. 5 ÷ 10 bottles, and cultivate a monolayer before the virus is removed within 48 hours. The second passage virus infects a much larger part of the cells: in the cell culture, symplasts and polykaryocytes (large multinuclear cells) are formed that are easily visible under a microscope by 50–70% of the monolayer. The monolayer with the second passage virus is removed with trypsin. After removal of trypsin, a support medium with 10% serum of cattle and 10% DMSO is introduced into the bottles. The collected suspensions of infected cells are combined in a total volume, packaged in 1 ÷ 2 cm ³ into ampoules, which are subjected to freezing and stored in liquid nitrogen. The volume of production seed should be at least 800 ÷ 1000 cm ³ with a cell concentration before freezing of 5 ÷ 10 million / cm ³ .

The control of the obtained antigenic material for safety is carried out on 20 chickens of one day old.

Chickens are given a 10-fold immunizing dose. Control chickens were observed for 15 days.

Infectious activity of production seed is determined by titration on a 1 ÷ 2-day culture of cells of FEC grown in bottles with a capacity of 50 cm ³ . Infectious activity of the production virus virus seed should not be lower than 1.0 · 10 ⁵ IU / cm ³ .

To control sterility using 3 ÷ 4 ampoules. Sowing is done on meat-peptone broth (MPB), meat-peptone agar (MPA), thioglycol medium and Saburo medium, 5 tubes of each medium.

The uterine (inoculum) production series of the virus is controlled to prevent contamination with pathogens of BM, infectious encephalomyelitis, Newcastle disease, avian influenza, infectious laryngotracheitis, infectious bronchitis, and smallpox of birds.

The control is carried out by bioassay on developing chicken embryos (REC) 4 ÷ 6-, 8 ÷ 9- and 11 ÷ 12 days of age.

In the case of isolation of the contaminant virus, it is identified using specific sera by the method of formulation of RGA, RZHA, RDP or RN according to conventional methods.

For the production of the production series of the anti-BM virus vaccine from the Vladimir strain, VGI use the virus from 2 to 5 consecutive passages (from matrix production seedlings) in the cell culture FEC. The cell-associated virus infects the cell culture and after 72 ÷ 120 hours of incubation, in the presence of changes typical for the viruses on the monolayer (60 ÷ 80% of the CPD) in the thermal room at (30 ÷ 40) ° С, the collection of infected cells is performed. For this, infected monolayer cell cultures are treated with 0.125 ÷ 0.25% trypsin solution. After removal of trypsin, a support medium with 20% serum of cattle and 10% DMSO is introduced into the bottles. The volume of the medium for collecting and suspending cells is 0.02 ÷ 0.04 cm ³ / cm ^{2 the} area of the bottle. Collected suspensions of infected cells (antigenic material) are combined in a total volume. The concentration of cells in suspension should be 8-10 million / cm ³ . The resulting antigenic material is poured 1 cm ³ into ampoules with a capacity of 3 ÷ 5 cm ³ , which are sealed, frozen and stored in liquid nitrogen. The volume of the production series of the vaccine should be at least 500 ÷ 600 cm ³ .

The proposed virus vaccine against BM has the optimal component composition, wt.%:

Suspension of FEK cells infected cell-associated VGI strain "Vladimir", with infectious activity not lower than 5.5 · 10 ⁵ IU / cm ³ 60.0 ÷ 80.0 Bovine serum 15.0 ÷ 25.0 DMSO 5.0 ÷ 15.0

Technological control of the production of anti-BM virus vaccine includes control for the absence of contamination of the bacterial and fungal flora of culture media and solutions used at each stage of the technological process, the semi-finished product of the virus vaccine, and the control of the semi-finished product for infectious activity.

The control of the finished anti-BM virus vaccine includes:

- determination of the appearance of the drug;

- determination of contamination by bacterial and fungal flora;

- determination of mycoplasma contamination;

- determination of contamination with foreign viruses;

- determination of harmlessness and infectious activity.

The titer of the virus in the virus vaccine should not be lower than 5.5 · 10 ⁵ IU / cm ³ , 1000 IU should be taken for one vaccination dose. When diluting the vaccine with a diluent, the vaccination dose should be contained in 0.2 cm ³ . The ready-made virus vaccine ampoule contains 400 or 800 doses of the drug. The shelf life of the virus vaccine, provided that it is stored in liquid nitrogen, is 24 months from the date of manufacture. Virus vaccine liquid culture of the strain "Vladimir" VGI is used for prophylactic purposes. A clinically healthy one-day-old bird (in the first hours of life) should be vaccinated once. Immunity is formed 14 ÷ 21 days after vaccination. Virus vaccine at a dose of 1000 cfu / 0.2 cm ³ is administered intramuscularly to chickens in the upper part of the thigh or subcutaneously in the region of the upper third of the neck in a volume of 0.2 cm ³ .

Example 3

Tests of the immunogenic activity of the virus vaccine against BM, made as described in example 2, and containing, wt.%:

Suspension of FEK cells infected cell-associated VGI strain "Vladimir", with infectious activity 6.0 · 10 ⁵ FOU / cm ³ 70.0 Bovine serum 20,0 DMSO 10.0

The immunogenic activity of the virus vaccine from the Vladimir strain of VGI was determined by administering a single immunizing dose of the drug in an amount of 1000 IU to commercial chickens intramuscularly in a volume of 0.2 cm ³ . The chickens were delivered from the poultry farm "Verkhnevolzhskaya" Tver region.

The control of experience in the verification of the drug was:

- positive control - a group of chickens immunized with the MAREK-BIO vaccine manufactured by AviNova LLC;

- negative control - a group of chickens infected with viral VBM, strain "047";

- control of the purity of experience - a group of isolated intact chickens.

The experimental chickens were kept in isolated boxes for 90-120 days, taking into account their condition on a daily basis. They were provided with heating in accordance with technological requirements, a supply of complete feed was provided taking into account the breed and age.

Before the start of the experiment, the samples of biological products were commissioned anonymized and encrypted. Paint of various colors was used to group mark the chickens.

The studied drugs were injected intramuscularly in the upper third of the inner thigh at the doses indicated in the passports.

21 days after vaccination, the chickens of all groups, with the exception of the control group, were intramuscularly infected with virulent strain “047” VBM at a dose of 1000 FOU / 0.2 cm ³ .

The safety of the experimental bird was recorded daily. The corpses of dead chickens were frozen at minus 20 ° C and stored until commissioned.

The slaughter of the entire bird, followed by autopsy and pathological anatomical examination was carried out at 108 days of age. At the same time, a pathological and anatomical dissection of chickens that died during the entire period of the experiment was performed.

At autopsy, the presence of changes characteristic of BM in the internal organs was recorded. In case of death of chickens without signs of BM, samples of the material were taken from them to confirm the specificity of the mortality according to the results of a histological examination.

The effectiveness of immunization was calculated by the formula:

,

,

where E is the effectiveness of immunization;

In -% of birds with signs of Marek's disease in the vaccinated group;

K -% sick bird with signs of Marek's disease in the control group.

The results are presented in table 1.

The data in table 1 indicate that the effectiveness of immunization of chickens with a virus vaccine against BM from the strain "Vladimir" VGI was 96.3%, which is 3.8% higher than the positive control. At the same time, 50.2% of the chickens in the unvaccinated control group fell ill and died with signs of BM.

Example 4

Tests of the immunogenic activity of the virus vaccine against BM, made as described in example 2, and containing, wt.%:

Suspension of FEK cells infected cell-associated VGI strain "Vladimir", with infectious activity 5.5 · 10 ⁵ FOU / cm ³ 75.0 Bovine serum 15.0 DMSO 10.0

The immunogenic activity of the anti-BM virus vaccine from the Vladimir strain of VGI was determined in comparative experiments conducted on the basis of the FSI VNIIZZh using commercial broiler chickens obtained from the Vladimir Broiler poultry farm. It was formed 5 experimental groups of 50 animals each, which were administered at the age of 24 hours:

- virus vaccine against BM from the strain "ARRIAH / No. 110" VGI at a dose of 1000 IU / 0.2 cm ³ (group No. 1);

- a prototype virus vaccine against BM from the strain "Vladimir" VGI at a dose of 1000 IU / 0.2 cm ³ (group No. 2);

- a prototype dry vaccine against BM from the strain "Vladimir" VGI at a dose of 2000 FOU / 0.2 cm ³ (group No. 3).

Group 4 chickens were not vaccinated and served as a control of the virulent virus. In group No. 5, the chickens were not vaccinated or infected. This group served as a control of chickens and was in an isolated room. After 14 days, the chickens of the first four groups were infected with virulent BM virus from strain Gm. For 210 days, the bird was monitored daily. Immunogenic activity was taken into account according to the results of the clinical, pathological, anatomical and histological manifestations of BM. In case of death of chickens without typical signs of BM, pieces of parenchymal organs, heart and sciatic nerve were taken from them, fixed in a 9% formalin solution, and histological examination of the obtained preparations was performed to confirm the diagnosis.

The effectiveness of immunization with test drugs was determined as described in example 3. The results of the studies are presented in table 2.

According to table 2, in group 4, 34 heads died from BM, which makes 65.4% of chickens, while 60% of them had a pronounced pathology of BM (exhaustion, tumors in the liver, spleen, glandular stomach). The specific death of chickens from this group was confirmed by histological studies of pathological material, which made it possible to evaluate the immunogenic activity of the tested preparations with a high degree of reliability.

In group No. 2 of chickens vaccinated with the anti-BM virus vaccine from the VGI strain Vladimir VGI, the immunization efficiency was the highest and amounted to 94.11%. In contrast, the immunogenic activity of the virus vaccine against BM from the strain "ARRIAH / No. 110" VGI was the lowest and amounted to 79.41%. This indicates that the HBV vaccine from the Vladimir strain of VGI has higher immunogenic activity compared to the prototype virus vaccine from the strain ARRIAH / No. 110. In group No. 5, which served as a control of chickens, no signs of BM were observed.

Example 5

Tests of the immunogenic activity of the proposed virus vaccine against BM, manufactured as described in example 2, and containing, wt.%:

Suspension of FEK cells infected cell-associated VGI strain "Vladimir", with infectious activity 6.0 · 10 ⁵ FOU / cm ³ 70.0 Bovine serum 20,0 DMSO 10.0

The immunogenic activity of the anti-BM virus vaccine from the Vladimir strain of VGI was determined in comparative experiments at the Verkhnevolotsk base of the VIEV using commercial broiler chickens obtained from the Verkhnevolzhskaya poultry farm in the Tver Region. 5 experimental groups of 50-55 goals each were formed, three of which were control and two experimental.

The first experimental group of broiler chickens was immunized with the Bimarek virus vaccine at a dose of 2000 IU / cm ³ . The second experimental group of broiler chickens was immunized with a vaccine against BM from the strain "Vladimir" VGI at a dose of 1000 IU / 0.2 cm ³ .

The control of experience in testing a monovalent vaccine against BM from the strain "Vladimir" VGI served as:

- a group of broiler chickens immunized with the MAREK-BIO vaccine manufactured by AviNova LLC (positive control);

- a group of chickens infected with virulent VBM, strain "047" (negative control);

- a group of isolated intact chickens (control of the purity of the experiment).

The experimental chickens were kept in isolated boxes for 90-120 days, taking into account their condition on a daily basis. Heating was provided in accordance with technological requirements, a supply of complete feed was provided taking into account the breed and age.

Before the start of the experiment, the samples of biological products were commissioned anonymized and encrypted. Paint of various colors was used to group mark the chickens.

The test drugs were administered intramuscularly in the upper third of the inner thigh at the doses indicated above.

21 days after vaccination, the chickens of all groups, with the exception of the control group, were intramuscularly infected with virulent strain “047” VBM at a dose of 1000 FOU / 0.2 cm ³ .

The safety of the experimental bird was recorded daily. The troupe of dead chickens was frozen at minus 20 ° C until commissioned opening.

The slaughter of the entire bird, followed by autopsy and pathological anatomical examination was carried out at 108 days of age. At the same time, a pathological and anatomical dissection of chickens that died during the entire period of the experiment was performed.

At autopsy, the presence of changes characteristic of BM in the internal organs was recorded. In case of death of chickens without signs of BM, samples of the material were taken from them to confirm the specificity of the mortality according to the results of a histological examination.

The effectiveness of immunization with test drugs was determined as described in example 3. The results of the studies are presented in table 3.

The data shown in table 3 indicate that all the studied drugs have high immunogenic activity, reaching 93.9%. Moreover, in the control unvaccinated group, 63% of chickens fell ill and fell with signs of BM. It follows from this that a prototype of a monovalent anti-BM virus vaccine from the Vladimir strain of VGI has a high degree of protection against the virulent virus.

Information sources

1. Witter R.L. New serotype 2 and attenuated Marek's disease vaccine virues. Comparative efficacy. Avian Dis., 1987, v. 31, No. 4, 752-765.

2. Bivalent Marek's vaccine. Poultry Intern., 1989, v. 28, No. 8, 44-50.

3. Lukina V.A., Barros-Paloma E.V. et al. Development and implementation of industrial technology for the manufacture of a new polyvalent virus vaccine against Marek’s disease. Thes. doc. scientific production conf. "100 years of the Kursk Biofactory and Agrobiological Industry of Russia", Kursk, 1996, 182-184.

4. RF patent No. 2144377, A61K 39/255, C12N 7/00; C12N 5/06 (C12N 7/00, C12P 1:93); 01/20/2000

5. Temporary instructions for the manufacture and control of cell culture virus vaccine for BM from strain FC-126 VGI. Approved by the GUV Ministry of Agriculture of the USSR 06/04/1984

6. RF patent No. 2144378; A61K 39/255, C12N 7/00; C12N 5/06 (C12N 7/00, C12R1: 93); 01/20/2000 (prototype).

Claims (6)

1. Virus vaccine against Marek’s disease, containing the cell-associated turkey herpes virus (HVI), bovine serum (cattle) and dimethyl sulfoxide (DMSO) as a cryoprotectant, characterized in that it contains a suspension of fibroblast cells as a cell-associated HVI embryos of SPF-chickens (FEC) infected with cell-associated VGI of strain "Vladimir" this. Herpesviridae, subweed. Gammaherpesvirinae, genus Herpesvims, serotype 3, deposited in the collection of FGU "VGNKI" under the registration number (link) "Vladimir No. 124-DEPT", in an effective amount. 2. The virus vaccine according to claim 1, characterized in that it contains a suspension of PEC cells infected with cell-associated HVI strain "Vladimir", with an infectious activity of at least 5.5 · 10 ⁵ FOU / cm ³ in an effective amount. 3. Virus vaccine according to claim 2, characterized in that it contains a suspension of PEC cells infected with cell-associated HVI strain "Vladimir", with an infectious activity of at least 5.5 · 10 ⁵ IU / cm ³ in the amount of 60.0 ÷ 80 , 0 wt.%. 4. The virus vaccine according to claim 1, characterized in that it contains serum of cattle in an amount of 15.0 ÷ 25.0 wt.%. 5. Virus vaccine according to claim 1, characterized in that it contains DMSO in an amount of 5.0 ÷ 15.0 wt.%. 6. Virus vaccine according to any one of claims 1 to 5, characterized in that it contains a suspension of PEC cells infected with cell-associated HVI strain "Vladimir", with an infectious activity of at least 5.5 ÷ 10 ⁵ IU / cm ³ , blood serum Cattle and DMSO in the ratio, wt.%:
suspension of infected cells 60.0 ÷ 80.0 cattle blood serum 15.0 ÷ 25.0 DMSO 5.0 ÷ 15.0