RU2246317C1 - Method for manufacturing vaccine against infectious laryngotracheitis in poultry - Google Patents

Abstract

FIELD: veterinary virology, biotechnology.

SUBSTANCE: the present innovation deals with infecting 9-11-d-aged embryos of SPF-hens with "O #112-DEP" viral strain of poultry laryngotracheitis (pcs), incubation of infected system, collection of virus-containing material, addition of protective medium to virus-containing material at the ratio of 1:19-3:17, correspondingly, drying the obtained mixture due to lyophilization followed by the control of the target product. As a result, one should obtain virus vaccine against infectious poultry laryngotracheitis at the content of virus-containing material at biological activity being, at least, 6.0 lg EID₅₀/cu. cm. The present method enables to increase immunogenic activity, safety and areactogenicity of the target product and widen the number of preparations for specific prophylaxis of infectious poultry laryngotracheitis being of high immunogenic activity, safety and areactogenicity at ocular and oral ways of immunization.

EFFECT: higher efficiency.

3 cl, 8 ex, 7 tbl

Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the development and production of means of specific prophylaxis of infectious laryngotracheitis (ILT) of birds.

ILT, an acute contagious disease of birds, is the cause of a high mortality rate (usual mortality is 10-20%, there are cases of high mortality, which is 50-70%), in cases of ill individuals the daily gain and egg production are reduced. ILT is caused by the herpes virus. One of the main means of combating ILT is vaccination with live and inactivated vaccines.

The advantages of the virus vaccines are that they are suitable for mass use by watering, aerosol spraying, etc., for the immunization of birds, a relatively small amount of the immunizing drug is required, since the live antigen continues to multiply in the body of the immunized bird, they are cheaper in production and storage, relatively little space is required in refrigeration units.

A known method of manufacturing a virus vaccine against bird ILT, including infection of a sensitive biological system with virus-containing material from an attenuated strain “G” of a homologous virus, incubation of the infected system, collecting virus-containing material, adding a protective medium to it, drying the mixture and controlling the target product (1).

A known method of manufacturing a virus vaccine against bird ILT, including infection of a sensitive biological system with virus-containing material from an attenuated strain of “TsNIIPP" of a homologous virus, incubation of the infected system, collecting virus-containing material, adding a protective medium to it, drying the mixture and controlling the target product (2).

A known method of manufacturing a virus vaccine against bird ILT, including infection of a sensitive biological system with virus-containing material from the NT clone of the attenuated strain “TsNIIPP” of a homologous virus, incubation of the infected system, collection of virus-containing material, adding a protective environment to it, drying the mixture and monitoring the target product (3).

The disadvantages of the known methods are that the virus vaccines obtained by these methods have low immunogenic activity. These vaccines form an immunity that cannot protect 100% of the poultry head from being infected with the epizootic ILT virus.

Closest to the proposed invention in terms of essential features is a method of manufacturing a virus vaccine against bird ILT, comprising infecting a sensitive biological system with virus-containing material from an attenuated strain of VNIIBP homologous virus, incubating the infected system, collecting virus-containing material, adding a protective environment to it, drying mixtures and control of the target product (4).

The disadvantages of the prototype method are that the vaccine virus produced by this method has low immunogenic activity and increased reactogenicity.

In this regard, the problem remains the development of a method of manufacturing a virus vaccine against bird ILT, which has both high immunogenic activity, harmlessness and areactogenicity in ocular and oral methods of immunization.

The objective of the present invention was to develop a method of manufacturing a highly immunogenic, harmless and areactogenic virus vaccine against bird ILT based on a new production strain of a homologous virus.

The technical result from the use of the invention is to increase the immunogenic activity, harmlessness and areactogenicity of the drug and expand the arsenal of means of specific prophylaxis of bird ILT, which have high immunogenic activity, harmlessness and arectogenicity with ocular and oral methods of immunization.

The specified technical result was achieved by creating a virus vaccine against bird ILT, characterized by the following set of features.

1. A method of manufacturing a virus vaccine against bird ILT.

2. Infection of a sensitive biological system with virus-containing material from the attenuated strain “About No. 112-DEPT” of the bird ILT virus.

3. Of the sensitive biological systems, 9-11 day old SPF chicken embryos are used.

4. The attenuated strain “O No. 112-DEPT” of the bird ILT virus is used in an effective amount.

5. For infection of 9-11-day-old embryos of SPF-chickens, virus-containing material from attenuated strain “O No. 112-DEPT” of bird ILT virus with biological activity of at least 6.0 lg EID ₅₀ / cm ^{3 is used} .

6. Incubation of the infected system.

7. Collection of virus-containing material.

8. Adding a protective environment.

9. A protective medium is added to the virus-containing material in a ratio of 1: 19-3: 17, respectively.

10. Drying the mixture.

11. Control of the target product.

12. The target product is obtained with the content of virus-containing material from the attenuated strain “About No. 112-DEPT” of the bird ILT virus with biological activity of at least 6.0 lg EID ₅₀ / cm ³ .

The present invention includes the following set of essential features, providing a technical result in all cases for which legal protection is requested.

1. A method of manufacturing a virus vaccine against bird ILT.

2. Infection of a sensitive biological system with virus-containing material from the attenuated strain “About No. 112-DEPT” of the bird ILT virus.

3. Incubation of the infected system.

4. Collection of virus-containing material.

5. Adding a protective environment.

6. Drying the mixture.

7. Control of the target product.

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

1. Of the sensitive biological systems using 9-11-day-old embryos of SPF-chickens.

2. For infection of 9-11-day-old embryos of SPF-hens, virus-containing material from attenuated strain “O No. 112-DEPT” of bird ILT virus with biological activity of at least 6.0 lg EID ₅₀ / cm ^{3 is used} .

3. A protective medium is added to the virus-containing material in a ratio of 1: 19-3: 17, respectively.

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

1. A method of manufacturing a virus vaccine against bird ILT.

2. Infection of a sensitive biological system with virus-containing material from an attenuated strain of avian ILT virus.

3. Incubation of the infected system.

4. Collection of virus-containing material.

5. Adding a protective environment.

6. Drying the mixture.

7. Control of the target product.

Compared with the prototype method, an essential distinguishing feature of the present invention is the use of a homologous virus in an effective amount in the manufacture of a virus vaccine against ILT of birds of virus-containing material from a new attenuated strain “About No. 112-DEPT”.

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

1. Of the sensitive biological systems using 9-11-day-old embryos of SPF-chickens.

2. For infection of 9-11-day-old embryos of SPF-hens, virus-containing material is used from the attenuated strain “About No. 112-DEPT” of the bird ILT virus with biological activity of at least 6.0 lg EID ₅₀ / cm ³ .

3. A protective medium is added to the virus-containing material in a ratio of 1: 19-3: 17, respectively.

The vaccine virus obtained by the proposed method has a higher immunogenic activity, harmlessness and areactogenicity compared to the virus vaccine made in accordance with the prototype method, and allows you to expand the arsenal of means of specific prophylaxis of bird ILT with high immunogenic activity, safety and areactogenicity with ocular and oral methods of immunization.

The achievement of the technical result from the use of the proposed method is explained by the fact that in the manufacture of the virus vaccine against bird ILT, virus-containing material is used from a new attenuated strain “O No. 112-DEPT” of a homologous virus with high biological, antigenic and immunogenic activity in its native form and after inactivation .

The strain "About No. 112-DEPT" virus ILT birds is a new, previously unknown, in its native form non-pathogenic.

The original virus for obtaining the strain "About No. 112-DEPT" was isolated from the larynx and trachea of a chicken with signs of a latent form of the disease in Kazakhstan in 1992.

The production strain "About No. 112-DEPT" was obtained by repeated consecutive passages on 9-11-day-old embryos of SPF-hens.

The strain "About No. 112-DEPT" was deposited in the All-Russian state collection of strains of microorganisms used in veterinary medicine and livestock, the All-Russian State Research Institute of Control, Standardization and Certification of Veterinary Medicines (VGNKI) 05.06.2000 under the registration name "About No. 112 -DEP ".

The strain "About No. 112-DEPT" has a high biological, antigenic and immunogenic activity in its native form and after inactivation.

The possibility of its use for the manufacture of a virus vaccine with high immunogenic activity and safety in ocular and oral methods of immunization has been experimentally confirmed.

The strain "About No. 112-DEPT" virus ILT birds characterized by the following features and properties.

Morphological properties

The strain "About No. 112-DEPT" virus ILT belongs to the family Herpesviridae. Electron microscopy (instrumental magnifications of 4.5 × 10 ⁵ and 5 × 10 ⁵ ) in the field of view of the microscope shows clearly distinguishable viral particles of ILT with their characteristic morphology. Virions have a spherical shape, their sizes vary from 200 to 250 nm (some up to 330 nm). ILT virions are composed of a spherical nucleotide surrounded by a supercapsid shell of complex shape. Virion has an icosahedral type of symmetry. The icosahedral capsid is covered with a lipoprotein membrane, which has peplomer units on its surface. The nucleocapsid dimer is 113 ± 2 nm, the average shell nucleocapsid diameter is about 350 nm.

Nucleocapsids 100-110 nm in size are located in the viral particle, usually eccentric, less often in the center of the particles. The genome of the ILT virus has a length of 155,000 polynucleotides and consists of a long unique sequence of I1 (120,000 polynucleotides) and a short unique sequence of Is (17,000 polynucleotides). The virus contains glycoproteins (205, 160, 115, 90, 85, 74, 60 and 50 kD). Glycoprotein B (gB) is the first structural protein of the ILT virus.

Antigenic properties

The strain "About No. 112-DEPT" of the ILT virus is stably neutralized by homologous serum, is identified in the blood serum of chickens containing antibodies to this virus, in RDP, PH and ELISA. In RDP, it is identified by homologous hyperimmune sera in a titer of 1: 8. In pH, the neutralization index of homologous hyperimmune sera at dilutions of 1:10 and 1:20 averages 3.8 lg EID ₅₀ / cm ^3, respectively. In ELISA is identified by field and laboratory hyperimmune sera in the titer up to 1: 24579. Antigenic differences of the strain "About No. 112-DEPT" in relation to other strains of the ILT virus have not been established. The strain does not have hemagglutinating properties.

Biotechnological characteristics

Strain "About No. 112-DEPT" exhibits high biological, immunogenic and antigenic activity both in native form and after inactivation. Strain "No. 112-DEPT" is used for the manufacture of live and inactivated vaccines against bird ILT and diagnostic preparations. The virus has a pronounced tropism to the epithelial cells of the mucous membrane of the larynx, trachea and cloaca of birds. The strain is cultured in 9-11 day old SPF chicken embryos. Reproduction of the virus occurs in the tissue of the chorioallantoic membrane (HAO). The infectious virus titer in a 10-20% suspension of HAO on the 5th day of cultivation, subject to generalized distribution throughout the envelope, is at least 6.0 lg EID ₅₀ / cm ³ .

Chemo- and genotaxonomic characterization

Strain "No. 112-DEPT" is a DNA-containing virus.

Resistance to external factors

The virus is sensitive to lipolytic agents, heat and various disinfectants. A temperature of 55 ° C destroys the virus within 10-15 minutes, and 38 ° C in 48 hours. The virus remained active for more than 19 months in the frozen carcasses of sick and forcibly killed chickens, stored at minus 10, 18 and 28 ° С, on the shell surface in the conditions of the thermostat it is inactivated after 12 hours, and it lasts no more than 9 days in infected houses. Fully inactivated in a 1% solution of creolin for 30 seconds. It retains its activity for a long time in a lyophilized state or at minus 20-60 ° C. In a lyophilized HAO homogenate, the virus remains infectious for at least 1.5 years. In the frozen HAO tissue, the infectivity of the virus persists for at least 4 years.

Immunogenicity of the strain

When vaccinating chickens older than 15 days of age, the ocular method at a dose of 1000 EID ₅₀ and the oral method (drink) at a dose of 4000 EID ₅₀ induces the production of specific antibodies and creates immunity against ILT in 100% of vaccinated chickens. In the lyophilized HAO homogenate, the virus retains immunogenicity for at least 1.5 years.

Epizooticity of the strain

When the vaccinated and intact birds are kept together, the transmission of the vaccine virus of strain "No. 112-DEPT" by contact has not been established.

The condition for maintaining the strain

Lyophilized material from strain "About No. 112-DEPT" is stored at a temperature of 4 ° C, virus-containing HAO - at a temperature not exceeding minus 40 ° C. For resuspension of the lyophilized virus, an isotonic phosphate-buffered solution of pH 7.4-7.6 is used.

Genetic purity

After 20 passages on the Khao 9-11-day-old embryos of SPF-hens, no changes in the immunobiological properties of strain "About No. 112-DEPT" were found.

Additional features and properties

Reactogenicity is absent.

Pathogenicity is absent.

Virulence is absent.

Oncogenicity is absent.

There is no contagiousness.

The attenuation stability was observed during 4 passages on sensitive chickens.

Based on the data obtained, it can be argued that the strain "About No. 112-DEPT" of the bird ILT virus according to antigenic and immunological spectra is an original, taxonomically new, previously unknown variant of the bird ILT virus.

The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information, and the identification of sources containing information about analogues of the invention, it was established that the applicant did not find sources characterized by signs that are identical (identical) to all the essential features of the invention. The definition from the list of identified analogues of the prototype as the closest in the set of essential features of the analogue allowed us to establish a set of essential in relation to perceived by the applicant the technical result of the distinctive features of the proposed method of manufacturing a virus vaccine against bird ILT, set forth in the independent claim.

Therefore, the claimed solution meets the condition of patentability "novelty."

To verify the conformity of the proposed solution to the patentability condition "inventive step", an additional search of known solutions was carried out to identify features included in the distinctive part of the independent claim. The search results showed that the proposed solution does not follow explicitly for the specialist from the prior art set forth in the corresponding section of the description (no solutions having features matching the distinguishing features of the present invention have been identified), and the effect of the transformations provided for by the essential features of the proposed invention has not been identified to achieve a technical result.

Therefore, the proposed method of manufacturing a virus vaccine against bird ILT meets the condition of patentability "inventive step".

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

Example 1

For the manufacture of a virus vaccine, an avirulent for chickens and harmless to humans production strain “O No. 112-DEPT” of bird ILT virus with biological activity of at least 6.0 lg EID ₅₀ / cm ³ , which is stored in a lyophilized state, is used. For reproduction of the virus using 9-11-day-old embryos of SPF-chickens. Eggs for chicken embryos are obtained from farms that are safe for infectious diseases, including ILT, or from SPF laying hens unvaccinated against ILT. In order to obtain a mother seed brood, the contents of three ampoules of the production strain O No. 112-DEPT are diluted 1: 5 with a sterile physiological solution of pH 7.2-7.4 with the addition of antibiotics and the resulting virus is infected with well-developed 9-11-day-old embryos in the allantoic cavity in a volume of 0.2 cm ³ . Infected embryos are incubated at a temperature of 37.0 ± 0.5 ° C and a relative humidity of 60-70% for 5 days.

On day 6 determine the characteristic of the ILT virus lesions of Khao. A 15-30% suspension is prepared from the affected HAO, which is frozen and freed from coarse particles by low-speed centrifugation. The resulting virus-containing material with a biological activity of not less than 6.0 lg EID ₅₀ / cm ^{3 is} used as a matrate seedling for mass infection of 9-11-day-old SPF chicken embryos. Embryos are infected with a mother virus in a volume of 0.2 cm ³ . Infected embryos are kept in a thermostat at 37.5 ± 0.5 ° C and relative humidity of 60-70% for 5 days. All embryos and survivors who died after 72 hours of incubation are placed in cold stores at 2-6 ° C for 16-18 hours. After cooling, the infected embryos are opened, HAO is removed with characteristic changes (specific nodule plaques). Affected HAO is placed in a refrigerator at a temperature not exceeding minus 40 ° C. HAO is used for the preparation of virus-containing material. For this, affected HAOs are thawed and a homogeneous homogeneous mass is prepared from them. The resulting virus-containing material is mixed with a protective medium in a ratio of 19: 1-17: 3, which corresponds to the content of ingredients in the virus vaccine obtained by the proposed method, wt.%:

Virus-containing material 85.0-95.0

Protective medium up to 100.0

and is necessary to obtain a drug with a given immunogenic activity.

As the ingredients of the protective environment, substances widely used for these purposes (separately or in combination) are used: sucrose, gelatose, lactose, skim milk, etc. A mixture of virus-containing material and a protective environment is thoroughly mixed. The resulting mixture is monitored for sterility, hemagglutinating activity (HA activity) in a drop of DGA and for determining the titer of the virus before lyophilization. The biological activity of the semi-finished vaccine, which is going to prepare the vaccine, when titrated at the RCE should not be less than 6.0 lg EID ₅₀ / cm ³ . After that, the mixture is dried. To do this, after obtaining positive control results, the semi-finished vaccine is cooled to 2-8 ° C and packaged in sterile ampoules or penicillin vials so that the layer thickness of the spilled liquid semi-finished product is no more than 1/2 the height of the ampoule or vial. Ampoules and semi-finished vials are placed in special metal cassettes, which are then installed in a refrigerator with a temperature of minus (60 ± 5 ° C) for 8-24 hours, depending on the volume of the suspension in ampoules and vials. The frozen semi-finished product is lyophilized. The drying time for the semi-finished product is 56-72 hours, depending on the design of the sublimation unit and the level of material in the vial or ampoule. At the end of drying, the bottles with the dry vaccine are corked and sealed with aluminum caps. Dry vaccine ampoules are sealed. The dry virus vaccine against bird ILT from strain "About No. 112-DEPT" is a homogeneous porous in the form of a tablet or an amorphous mass of light brown color. Mass fraction of moisture - not more than 3%. The biological activity of the virus in the vaccine should be at least 6.0 lg EID ₅₀ / cm ³ .

The resulting vaccine against bird ILT has the optimal component composition (formulation), shown in table 1.

The content of virus-containing material in the range of 85.0-95.0 (wt.%) Represents an effective amount of antigen in the proposed vaccine.

Before use, the virus vaccine is diluted with a diluent, which is a 5% solution of glycerol in a phosphate-buffered solution, labeled with a neutral food coloring such as indigo carmine, and is intended for the preparation of a working dilution of the vaccine with the ocular method of immunizing birds and bringing the pH value of water close to neutral or slightly alkaline when using the vaccine by the method of evaporation.

The vaccine virus against bird ILT from strain “About No. 112-DEPT” is subjected to control for solubility, determination of the mass fraction of moisture in the preparation, sterility, biological activity, safety and immunogenic activity.

To determine the solubility of the virus vaccine, take 3 vials (ampoules) and add the above diluent to them to the original volume of the vaccine. The dry mass of the vaccine completely dissolves within 2-3 minutes, forming a homogeneous suspension without flakes and sediment.

Mass fraction of moisture is determined according to GOST 24061-80, which does not exceed 3%.

Determination of the sterility of the drug is carried out according to GOST 28085-89.

Biological activity is determined by titration of a sample of the virus vaccine on the developing embryos of SPF hens of 10-day incubation. As a sample, take 2 bottles (ampoules) of the virus vaccine. The contents of the vials (ampoules) are diluted with a sterile physiological solution of pH 7.2 to the initial volume of the virus, and then mixed and prepared ten-fold dilutions from 10 ^-1 to 10 ^-8 . Each dilution infects 4 embryos in the allantoic cavity in a volume of 0.2 cm ³ . 10 embryos left uninfected as a control. Embryos are incubated for 120 hours at 36.5-37.5 ° C and relative humidity 60-70%. Ovoscopy of embryos is performed daily. Embryos that have fallen within 72 hours after infection are collected and destroyed, since their death during this period is not specific. The death of embryos in the following days is attributed to the action of the vaccine virus ILT in birds. After 5 days, the embryos are opened. At the same time, changes in the CAO characteristic of the action of the bird ILT virus (the presence of plaque nodules) are taken into account. The virus titer is calculated according to the method of Kerber in the modification of Ashmarin. The biological activity of the vaccine should be at least 6.0 lg EID ₅₀ / cm ³ .

The vaccine is tested for safety in 20 chickens older than 25 days old, free of antibodies to the ILT virus. To test for harmlessness, use 6 bottles of virus vaccine against bird ILT. The contents of the vials are diluted to the original volume with the above diluent, combined into a common sample and two dilutions of the vaccine are prepared: the first so that 10.05 EID ₅₀ (10-fold ocular dose) is contained in 0.05 cm ^{3 of the} dissolved vaccine, and the second so that 1 cm ³ diluted vaccines contained 40,000 EID ₅₀ (10-fold oral dose). In the first case, the vaccine is administered with an eye pipette to the naked conjunctiva of the eye to 10 chickens, one drop each, in the second case, 1 cm ^{3 is} drunk using a pipette to the same number of chickens. Chickens are monitored for 10 days. Chicks must remain alive and clinically healthy during the observation period. Some chickens are allowed to have mild suppression, conjunctivitis, rhinitis and a decrease in appetite for 3-5 days after the introduction of the vaccine. After 3-4 days, these phenomena disappear.

To determine the immunogenic activity of the obtained preparation, 6 bottles (ampoules) with a virus vaccine are used, the contents of each of which are diluted with the diluent to the initial volume equal to the volume of the vaccine before lyophilization, are combined and dilutions of the vaccine on the diluent are prepared from the total sample: for the ocular immunization method, so that 0.05 cm ³ (the volume of one drop) contains 1000 EID ₅₀ , and for the oral method, so that 1 cm ^{3 of the} preparation contains 4000 EID ₅₀ . In the experiment, 50 chickens were taken and 20 of them were immunized ocularly (1 drop per eye), 20 chickens were given orally (1 cm ^{3 was} drunk), and 10 chickens were left as a control. 18 days after vaccination, the experimental and control chickens are infected with the virulent strain “Bogatishchevsky” of the bird ILT virus. The virus is administered intratracheally at a dose of 500 ID ₅₀ in a volume of 0.5 cm ³ .

Chickens are observed for 10 days. Before infection, vaccinated chickens are taken blood, serum is obtained and examined in ELISA. A vaccine is considered immunogenic if it protects up to 16 chickens out of 20 vaccinated by each method against ILT. In the control group, at least 8 chickens out of 10 infected should be ill with clinical signs of ILT.

The proposed virus vaccine immunizes clinically healthy birds older than 15 days of age. Poultry up to 60 days of age is vaccinated twice with an interval of 14 days. Immunity is formed 21 days after vaccination. Revaccination is carried out every 6 months.

Example 2

Tests of the immunogenic activity of the virus vaccine against bird ILT, manufactured by the proposed method as described in example 1, and containing, wt.%:

Virus-containing material from attenuated strain

"About No. 112-DEPT" of the ILT virus of birds reproduced

in 9-11 day old SPF chicken embryos

with biological activity

6.0 lg EID ₅₀ / cm ³ 85.0

Protective environment up to 100.0.

The immunogenic activity of the vaccine was determined in chickens. For this, dilution of the vaccine on a diluent was preliminarily prepared: for the ocular immunization method so that 0.05 cm ³ (the volume of one drop) contained 1000 EID ₅₀ , for the oral one, that 4000 EID _{50 were} contained in 1 cm ³ . In the experiment, 50 chickens were taken and 20 of them were immunized ocularly (1 drop per eye), 20 orally (they were fed 1 cm ³ ), and 10 chickens were left for control. Eighteen days after vaccination, the experimental and control chickens were infected with the virulent Bogatischevsky strain of avian ILT virus. The virus was administered intratracheally at a dose of 500 EID ₅₀ in a volume of 0.5 cm ³ . Chickens were observed for 10 days. A vaccine is considered immunogenic if it protects 16 chickens out of 20 vaccinated against ILT disease. In the control group, at least 8 chickens out of 10 infected should be ill with signs of ILT. The test results are shown in table 2, which indicate that all three series of virus vaccines against bird ILT from strain "About No. 112-DEPT" made according to the above recipe, in all respects meet the requirements laid down in the technical conditions for the drug and almost equally effective for ocular and oral use in birds. In the control groups of chickens, the incidence of ILT was 100%, while the mortality rate was 48.6%.

Example 3

Tests of the immunogenic activity of the virus vaccine against bird ILT, manufactured by the proposed method as described in example 1, and containing, wt.%:

Virus-containing material

from the attenuated strain "No. 112-DEPT"

reproduced ILT virus

in 9-11 day old SPF chicken embryos

with biological activity

6.0 lg EID ₅₀ / cm ³ 90.0

Protective environment up to 100.0.

This formulation of the proposed virus vaccine was tested as described in example 2. The experiments were carried out three times. The test results are shown in table 3, which indicate that all three series of virus vaccines made according to the above recipe against bird ILT from strain "About No. 112-DEPT", in all respects meet the requirements laid down in the technical conditions for the drug, and practically equally effective for ocular and oral administration in poultry. In the chickens of the control groups, the incidence of ILT was 100%, the mortality rate in 3 experiments was 45.8%.

Example 4

Tests of the immunogenic activity of the virus vaccine against bird ILT, manufactured by the proposed method as described in example 1, and containing, wt.%:

Virus-containing material from the attenuated strain "About No. 112-DEPT"

reproduced ILT virus

in 9-11 day old SPF chicken embryos

with biological activity

6.0 lg EID ₅₀ / cm ³ 95.0

Protective environment up to 100.0.

This formulation of the proposed virus vaccine was tested as described in example 2. The experiments were carried out three times. The test results are shown in table 4, which indicate that all three series of virus vaccines against bird ILT from strain "About No. 112-DEPT" made according to the above recipe, in all respects meet the requirements laid down in the technical conditions for the drug and almost equally effective for ocular and oral use in birds. In the control groups of chickens, the incidence of ILT was 100%, while the mortality rate in 3 experiments was 50.2%.

Example 5

Comparative tests of the immunogenic activity of the virus vaccine against bird ILT, manufactured by the proposed method as described in example 1, and containing, wt.%:

Virus-containing material

from the attenuated strain "About No. 112-DEPT"

reproduced ILT virus

in 9-11 day old SPF chicken embryos

with biological activity

6.0 lg EID ₅₀ / cm ³ 85.0

Protective medium up to 100.0

and virus vaccines against ILT of birds made on the basis of well-known domestic and foreign strains of a homologous virus: the Laryngo-vac virus vaccine (Holland), the virus vaccine from the NT clone of the TsNIIPP strain, and the virus vaccine from the VNIIBP strain . The study of the immunogenic activity of these vaccines was carried out on SPF chickens of 25 days of age. All chickens were orally and ocularly administered a single dose of the above vaccines. Evaluation of the effectiveness of vaccines was carried out according to the results of the control of infection with the virulent strain "Bogatishchevsky" virus ILT. The results of the studies are presented in table 5.

The data in table 5 indicate that during the control infection of vaccinated chickens with the virulent strain "Bogatishchevsky" of the ILT virus, only the virus vaccine from strain "About No. 112-DEPT" of this formulation induced immunity to ILT in 100% of vaccinated chickens.

Example 6

Comparative tests of the immunogenic activity of the virus vaccine against bird ILT, manufactured by the proposed method as described in example 1, and containing, wt.%:

Virus-containing material

from the attenuated strain "About No. 112-DEPT"

reproduced ILT virus

in 9-11 day old SPF chicken embryos

with biological activity

6.0 lg EID ₅₀ / cm ³ 90.0

Protective medium up to 100.0

and virus vaccines against bird ILT made on the basis of well-known domestic and foreign strains of a homologous virus: the Laryngo-vac vaccine virus (Holland), the virus vaccine from the NT clone of the TsNIIPP strain and the virus vaccine from the strain VNIIBP ". The studies were carried out as described in example 5. The research results are presented in table 6.

The data in table 6 indicate that during the control infection of vaccinated chickens with the virulent strain "Bogatischevsky" of the ILT virus, only the vaccine virus from strain "No. 112-DEPT" of this formulation induced immunity to ILT in 100% of vaccinated chickens.

Example 7

Comparative tests of the immunogenic activity of the virus vaccine against bird ILT, manufactured by the proposed method as described in example 1, and containing, wt.%:

Virus-containing material

from the attenuated strain "No. 112-DEPT"

reproduced ILT virus

in 9-11 day old SPF chicken embryos

with biological activity

6.2 lg EID ₅₀ / cm ³ 95.0

Protective medium up to 100.0

and virus vaccines against bird ILT made on the basis of well-known domestic strains of a homologous virus: the Laryngo-vac virus vaccine (Holland), the virus vaccine from the NT clone of the TsNIIPP strain, and the virus vaccine from the VNIIBP strain . The studies were carried out as described in example 5. The research results are presented in table 7.

The data in table 7 indicate that during the control infection of vaccinated chickens with the virulent strain "Bogatishchevsky" of the ILT virus, only the virus vaccine from strain O No. 112-DEPT of this formulation induced immunity to ILT in 100% of the vaccinated chickens.

Example 8

Tests of the immunogenic activity of the virus vaccine against bird ILT, manufactured by the proposed method as described in example 1, and containing, wt.%:

Virus-containing material

from the attenuated strain "About No. 112-DEPT"

reproduced ILT virus

in 9-11 day old SPF chicken embryos

with biological activity

6.0 lg EID ₅₀ / cm ³ 90.0

Protective environment up to 100.0.

The tests were carried out in the unsuitable for bird IFL GUP-APK "Nazarievo" of the Odintsovo district of the Moscow region. The vaccine virus against bird ILT from strain "No. 112-DEPT" immunized 240 thousand birds, of which 80 thousand broilers and 160 thousand laying hens and repair young calves Heidex white breed. Vaccination was carried out by the method of drinking according to the instructions for the use of the drug. Observation of the vaccinated livestock revealed that in a dysfunctional poultry house, the isolation of a sick bird continued for another 6 days, after which the clinical signs of the disease disappeared and the physiological state of the chickens returned to normal. In poultry houses, where there were no signs of ILT, no adverse reactions to vaccination were observed.

The well-being of the ILT in the economy is preserved to this day. Thus, the above information indicates the fulfillment when using the invention of the following set of conditions:

- a method of manufacturing a virus vaccine against bird ILT, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology;

- for the proposed invention in the form as described in the independent claim, the possibility of its implementation using the means and methods described in the application or known prior to the priority date is confirmed;

- when using the proposed method for the manufacture of a virus vaccine against ILT of birds based on the strain "About No. 112-DEPT", a technical result is achieved, provided for by the task of creating the invention.

Therefore, the present invention meets the condition of patentability "industrial applicability".

Sources of information

1. Babkin V.F. and other Veterinary medicine. Resp.mid.vid. thematic science. zb. Kiev, 1975, issue 42, 14-19.

2. Auth. testimonial. USSR No. 129791; 30h, 6; 1960 year

3. Dutko Yu.S., Shevchenko A.A. et al. Immunological efficacy of a vaccine against infectious laryngotracheitis of hens. Veterinary Medicine, 1991, 3, 32-34.

4. Malushko V.V., Sokolov V.D. et al. Experience in combating infectious laryngotracheitis. Poultry farming, 1983, 12, 21-23 (prototype).

Table 1
The optimal component composition (formulation) of the virus vaccine against ILTis strain "About No. 112-DEPT" made by the proposed method (wt.%) Ingredients Minimum Average Maximum Virus-containing material 85 90 95 Protective environment fifteen 10 5 table 2
Immunobiological characteristics of the virus vaccine against bird ILT from strain "About No. 112-DEPT" made by the proposed method №№ Sterility The biological activity of lgEID ₅₀ / cm ³ Vaccine Administration Method Harmlessness Immunogenicity The number of chickens in the experience Of these, responded to the vaccine. The number of chickens in the experience Of which are immune % ILT virus protection 1 experience Sterile 6.1 ± 0.17 Oral 10 0 twenty eighteen 90 Ocular 10 0 twenty twenty 100 2 experience Sterile 6.0 + 0.20 Oral 10 0 twenty 19 95 Ocular 10 0 twenty twenty 100 3 experience Sterile 6.20 + 0.14 Oral 10 0 twenty 17 85 Ocular 10 0 twenty eighteen 90 Table 3
Immunobiological characteristics of the virus vaccine against bird ILT from strain "About No. 112-DEPT" made by the proposed method №№ Sterility The biological activity of lgEID ₅₀ / cm ³ Vaccine Administration Method Harmlessness Immunogenicity The number of chickens in the experience Of these, responded to the vaccine. The number of chickens in the experience Of which are immune % ILT virus protection 1 experience Sterile 6.15 ± 0.12 Oral 10 0 twenty eighteen 90 Ocular 10 0 twenty twenty 100 2 experience Sterile 6.20 + 0.11 Oral 10 0 twenty 17 85 Ocular 10 0 twenty eighteen 90 3 experience Sterile 6.10 + 0.08 Oral 10 0 twenty eighteen 90 Ocular 10 0 twenty twenty 100

Table 4
Immunobiological characteristics of the virus vaccine against bird ILT from strain "O No. 112-DEPT" made by the proposed method №№ Sterility The biological activity of lgEID ₅₀ / cm ³ Vaccine Administration Method Harmlessness Immunogenicity Qty
CHICKENS
in experience Of these, responded to the vaccine. The number of chickens in the experience Of which are immune % ILT virus protection 1 experience Sterile 6.15 ± 0.12 Oral 10 0 twenty 17 85 Ocular 10 0 twenty twenty 100 2 experience Sterile 6.10 + 0.11 Oral 10 0 twenty eighteen 90 Ocular 10 0 twenty 19 95 3 experience Sterile 6.25 + 0.8 Oral 10 0 twenty eighteen 90 Ocular 10 0 twenty twenty 100

Table 5
Comparative evaluation of the immunogenic activity of avian ILT virus vaccines The name of the strain from which the virus vaccine is prepared Administration Methods The number of birds in the experience Control infection results infected of which are immune % ILT virus protection LARYNGO-VAC (Holland) Oral twenty twenty fifteen 75 Ocular twenty twenty eighteen 90 Clone NT PCNIIP Oral twenty twenty thirteen 65 Ocular twenty twenty 12 60 Strain VNIIBP Oral twenty twenty fifteen 75 Ocular twenty twenty fifteen 75 Strain
"About No. 112-DEP" (Russia) Oral twenty twenty eighteen 90 Ocular twenty twenty twenty 100 THE CONTROL Intratracheal 10 10 0 0

Table 6
Comparative evaluation of the immunogenic activity of avian ILT virus vaccines The name of the strain from which the virus vaccine is prepared Administration Methods The number of birds in the experience Control infection results infected of which are immune % ILT virus protection LARYNGO-VAC (Holland) Oral twenty twenty 14 70 Ocular twenty twenty eighteen 90 Clone NT PCNIIP Oral twenty twenty thirteen 65 Ocular twenty twenty 12 60 Strain VNIIBP Oral twenty twenty fifteen 75 Ocular twenty twenty 14 70 Strain
"O No. 112-DEP" (Russia) Oral twenty twenty 19 95 Ocular twenty twenty twenty 100 THE CONTROL Intratracheal 10 10 0 0 Table 7
Comparative evaluation of the immunogenic activity of avian ILT virus vaccines The name of the strain from which the virus vaccine is prepared Administration Methods The number of birds in the experience Control infection results infected of which are immune % ILT virus protection LARYNGO-VAC (Holland) Oral twenty twenty fifteen 75 Ocular twenty twenty 17 85 Clone NT PCNIIP Oral twenty twenty 12 60 Ocular twenty twenty eleven 55 Strain VNIIBP Oral twenty twenty fifteen 75 Ocular twenty twenty fifteen 75 Strain "O No. 112-DEPT" (Russia) Oral twenty twenty 19 95 Ocular twenty twenty twenty 100 THE CONTROL Intratracheal 10 10 0 0

Claims (4)

1. A method of manufacturing a virus vaccine against infectious laryngotracheitis of birds, comprising infecting a sensitive biological system with virus-containing material from an attenuated strain of a homologous virus, incubating the infected system, collecting the virus-containing material, adding a protective medium, drying the mixture and controlling the target product, characterized in that for infection sensitive biological system using virus-containing material from attenuated strain "O" of the infectious laryngo virus tracheitis of birds, collection VGNKI "About No. 112-DEPT", in an effective amount. 2. The method according to claim 1, characterized in that of sensitive biological systems using 9-11-day-old embryos of SPF-hens. 3. The method according to claim 1 or 2, characterized in that for the infection of 9-11-day-old embryos of SPF-hens, virus-containing material is used from the attenuated strain "About No. 112-DEPT" of the infectious laryngotracheitis virus of birds with biological activity of at least 6.0 lg EID ₅₀ / cm ³ . 4. The method according to claim 1, characterized in that the protective medium is added to the virus-containing material in a ratio of 1: 19-3: 17, respectively.